Comparison of Cassava and Sugarcane Bagasse for Fuel Ethanol Production

Complimentary Contributor Copy Complimentary Contributor Copy

PLANT SCIENCE RESEARCH AND PRACTICES

HANDBOOK ON CASSAVA

PRODUCTION, POTENTIAL USES AND RECENT ADVANCES

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Complimentary Contributor Copy

PLANT SCIENCE RESEARCH AND PRACTICES

Additional books in this series can be found on Nova’s website under the Series tab.

Additional e-books in this series can be found on Nova’s website under the e-book tab.

Complimentary Contributor Copy

PLANT SCIENCE RESEARCH AND PRACTICES

HANDBOOK ON CASSAVA

PRODUCTION, POTENTIAL USES AND RECENT ADVANCES

CLARISSA KLEIN EDITOR

New York

Complimentary Contributor Copy

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher.

We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN.

For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected].

NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication.

This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data

Names: Klein, Clarissa, editor. Title: Handbook on cassava: production, potential uses and recent advances / editor: Clarissa Klein. Other titles: Plant science research and practices. Description: Hauppauge, New York: Nova Science Publishers, [2016] | Series: Plant science research and practices | Includes index. Identifiers: LCCN 2016044245 (print) | LCCN 2016045346 (ebook) | ISBN 9781536102918 (hardcover) | ISBN 9781536103076 Subjects: LCSH: Cassava. | Cassava--Utilization. | Cassava--Technological innovations. Classification: LCC SB211.C3 H36 2016 (print) | LCC SB211.C3 (ebook) | DDC 633.6/82--dc23 LC record available at https://lccn.loc.gov/2016044245

Published by Nova Science Publishers, Inc. † New York

Complimentary Contributor Copy

CONTENTS

Preface vii Chapter 1 Comparison of Cassava and Sugarcane Bagasse for Fuel Ethanol Production 1 Yessica Chacón Pérez, Daissy Lorena Restrepo Serna and Carlos Ariel Cardona Alzate Chapter 2 Cassava Production and Its Economic Potentials in Sub-Sahara Africa: A Review 29 Emmanuel Ukaobasi Mbah Chapter 3 Cassava Production and Utilization in the Coastal, Eastern and Western Regions of Kenya 41 C. M. Githunguri, M. Gatheru and S. M. Ragwa Chapter 4 Socio-Economic Determinants of Modern Technology Adoption and the Influence of Farm Size on Productivity and Profitability in Cassava Production: A Case Study from South-Eastern Nigeria 55 Chidiebere Daniel Chima and Sanzidur Rahman Chapter 5 Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods and Pastas 87 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto and Liz Pérez Chapter 6 Technological Aspects of Processing of Cassava Derivatives 105 Elisa Cristina Andrade Neves, Daniela Andrade Neves, Kleidson Brito de Sousa Lobato, Gustavo Costa do Nascimento and Maria Teresa Pedrosa Silva Clerici Chapter 7 Sustainable Management of Cassava Processing Waste for Promoting Rural Development 129 Anselm P. Moshi and Ivo Achu Nges Chapter 8 Wastewater from Cassava Processing as a Platform for Microalgae- Mediated Processes 149 Tatiele C. do Nascimento, Erika C. Francisco, Leila Queiroz Zepka and Eduardo Jacob-Lopes

Complimentary Contributor Copy vi Contents

Chapter 9 Cassava Wastewater as Substrate in Biotechnological Processes 171 Cristiano José de Andrade, Ana Paula Resende Simiqueli, Fabiola Aliaga de Lima, Juliana Bueno da Silva, Lidiane Maria de Andrade and Ana Elizabeth Cavalcante Fai Chapter 10 Technical, Cost and Allocative Efficiency of Processing Cassava into Gari in Delta State, Nigeria 201 Brodrick O. Awerije and Sanzidur Rahman Chapter 11 Status of Cassava Processing and Challenges in the Coastal, Eastern and Western Regions of Kenya 217 C. M. Githunguri, M. Gatheru and S. M. Ragwa Chapter 12 Cassava Waste: A Potential Biotechnology Resource 231 Aniekpeno I. Elijah Chapter 13 Potential Uses of Cassava Products and Its Future Challenging Opportunities 251 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won Chapter 14 Utilization of Modified Cassava Flour and Its By-Products 271 Setiyo Gunawan, Zikrina Istighfarah, Hakun Wirawasista Aparamarta, Firdaus Syarifah and Ira Dwitasari Chapter 15 Recent Advances in the Development of Biodegradable Films and Foams from Cassava Starch 297 Giordana Suárez and Tomy J. Gutiérrez Chapter 16 Cassava Cultivation, Processing and Potential Uses in Ghana 313 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre Chapter 17 Potential Uses of Cassava Bagasse for Bioenergy Generation by Pyrolysis and Copyrolysis with a Lignocellulosic Waste 335 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman Chapter 18 Trend in the Trade of Cassava Products in the Coastal, Eastern and Western Regions of Kenya 357 C. M. Githunguri, M. Gatheru and S. M. Ragwa Chapter 19 Wild Relatives of Cassava: Conservation and Use 373 Márcio Lacerda Lopes Martins, Carlos Alberto da Silva Ledo, Paulo Cezar Lemos de Carvalho, André Márcio Amorim and Dreid Cerqueira Silveira da Silva Index 407

Complimentary Contributor Copy

PREFACE

Cassava produces about 10 times more carbohydrates than most cereals per unit area, and are ideal for production in marginal and drought prone areas. Cassava, which originated from tropical South America, is a perennial woody shrub with an edible root, which today is grown in tropical and subtropical regions of the world where it provides energy food and serves as a veritable source of food and income for over a billion people. This handbook provides new research on the production, consumption and potential uses of cassava. Chapter 1 - During the last years, biofuels from different feedstocks have been studied and scaled up to industry to provide energy needing mainly for transport. Raw materials with high sugar content are mostly used in the production of biofuels as ethanol, butanol, hydrogen, etc. In the last years in tropical countries as Colombia, the ethanol production from starch and lignocellulosic biomass has been studied as a new alternative. Then, raw materials as cassava and sugarcane bagasse are presented as good options but more research is needed to understand the real advantages of these feedstocks. The amount of fermentable sugars obtained from the biomass is a decisive factor in the global yield of ethanol production. In this sense, different technological schemes can be proposed in the pretreatment step of the process followed by enzymatic hydrolysis to get the sugars. Starch-rich raw materials only require a milling and cooking as pretreatment. On the other hand, the pretreatment stage in some type of lignocellulosic materials must consider the reduction of particle size and specific technologies as dilute acid or alkaline methods. This chapter shows an analysis of ethanol production from cassava and sugarcane bagasse taking into account the availability, type and different pretreatment technologies to be applied to the raw material. Additionally, a techno-economic and environmental assessment is performed, in order to compare the proposed processes. Chapter 2 - Cassava (Manihot esculenta Crantz), which originated from tropical America and today a dietary staple to most people living in Sub-Sahara Africa is a perennial woody shrub with an edible root, which is rich in carbohydrates, calcium (50 mg 100-g), phosphorus (40 mg 100-g), vitamins B and C, as well as some essential minerals, while its tender leaves serve as a veritable source of lysine rich protein. The roots though poor in protein and other minerals, their nutrient compositions differ depending on the variety and age of the harvested crop, as well as soil conditions, climate, and other environmental factors under which the crop is grown. The stem of cassava is used as planting material and can serve as a standard substrate in mushroom production as well as fuel wood when dried. Cassava is characterized as one of the most drought tolerant crop that is capable of growing on marginal soils. The

Complimentary Contributor Copy viii Clarissa Klein crop is rarely cultivated as a mono-crop because of its physiological growth habit and duration, which makes it to stand out as an excellent component crop in most intercropping systems. Hence, it is usually intercropped with most vegetables, yam, sweet potato, melon, maize, sorghum, millet, rice, groundnut, sesame, soybean, cowpea and other legumes, as well as plantation crops (such as oil palm, kola, rubber, cocoa, cashew and coffee) among other field crops grown in the tropical regions of the world. Roots of cassava may be due for harvesting between six months and three years (36 months) after planting. Apart from food, cassava root tubers are very versatile, hence its derivatives and qualitative starch are effectively used in a number of products such as foods, confectionery, sweeteners, paper, glues, textiles, plywood, biodegradable products, monosodium glutamate, and pharmaceuticals. Cassava chips and pellets are used in animal feed and alcohol production as well as ethanol and bio-diesels. Crop improvements associated with cassava is tailored on developing genotypes that can effectively correlate the end product with its utilization at the industrial level. The impact of research on cassava development ranging from biotec- breeding, genetics and selection to production, value-chain addition and utilization is immerse, hence, high quality improved cassava varieties which are disease- and pest-resistant, low in cyanide content, drought-resistant, early bulking, high starch content, high dry matter content, and high yielding are being cultivated by most farmers in the tropical regions today where cassava thrives. Annual world production of cassava (184 million tonnes) with Nigeria being the leading producer has continued to increase due to the development of improved varieties with high yield, excellent culinary qualities and resistance to pests and diseases among other invaluable properties. In this review therefore, scientific findings by a number of scholars on cassava are discussed with the aim of making the information a veritable tool for researchers in this field. Chapter 3 - Cassava is the second most important food root crop in Kenya. Despite its high production in the coastal and western regions, utilization is limited to human consumption. A situational analysis on cassava production was carried out to determine its current status in the western, coastal and eastern regions of Kenya. A sample of farmers was randomly selected from each region and interviewed using a structured questionnaire. Off- farm activities were undertaken by 37% in eastern and western and 32% in the coastal regions. Access to extension services was 50% in the coast, 65% in eastern and 88% in western regions. Relative to other food crops, 66.7% of respondents ranked cassava 2nd at the coastal region while 37.5% and 57% of respondents in eastern and western regions ranked it 5th and 1st, respectively. At the coastal, western and eastern regions, 92%, 67% and 65% of the respondents intercrop cassava with other crops, while 8%, 33% and 35% grow it as a sole crop, respectively. On adoption of improved cassava varieties, western region was leading with 77% followed by coast (30%) and eastern (13%). At the coast, 23% considered lack of market as the major constraint followed by pests and diseases (16%) and destruction by large mammalian pests (11%). In eastern, 15% reported drought as the major constraint followed by lack of market (13%) and pests and disease (42%). In western, the major constraints were large mammalian pests (12%), weeds (12%), lack of planting materials (8%) and insect pests (3%). At the coastal, eastern and western regions cassava was ranked second, fifth and first respectively relative to other food crops. The western region had more improved cassava varieties than the other regions. In the coastal region, the major constraint to production was lack of market while in the eastern region, the major constraint was drought and in western, the major constraints were wild animals and weeds. Cassava was utilized more as family food Complimentary Contributor Copy Preface ix in western than in coastal and eastern regions. On processing of cassava and cassava based products, western region was leading followed by coastal and eastern region last. The western region was leading in the processing of dried cassava chips and composite flour. The coastal region was leading in the processing of fried cassava chips, crisps and pure flour. The eastern region was ranked least in processing with a few respondents making fried cassava chips and pure cassava flour. Chapter 4 - The chapter investigates the influence of socio-economic factors on the adoption of individual components of modern agricultural technology (i.e., HYV seeds and inorganic fertilizers) in cassava and also examines farm size–productivity and farm size– profitability relationships of cassava production in South-eastern Nigeria including a discussion of constraints in the cassava sector. The hypotheses of the study are that farmers selectively adopt components of modern agricultural technology depending on their socio- economic circumstances and inverse farm size–technology adoption, size–productivity and size–profitability relationships exist in cassava production. The research is based on an in- depth farm-survey of 344 farmers from two states (243 from Ebonyi and 101 from Anambra states) of South-eastern Nigeria. The results show that the sample respondents are dominated by small scale farmers (78.8% of total) owning land less than 1 ha. The average farm size is small estimated at 0.58 ha. The study clearly demonstrated that inverse farm size–technology adoption and farm size–productivity relationships exist in cassava production in this region of Nigeria but not inverse farm size–profitability relationship. The level of modern technology adoption is low and mixed and farmers selectively adopt components of technologies as expected and use far less than recommended dose of fertilizers. Only 20.35% of farmers adopted both HYV cassava stem and fertilizers as a package. The bivariate probit model diagnostic reveals that the decision to adopt modern technologies are significantly correlated, implying that univariate analysis of such decisions are biased, thereby, justifying use of the bivariate approach. The most dominant determinant of modern technology adoption in cassava is farming experience and remoteness of extension services depresses adoption. A host of constraints are affecting Nigerian agricultural sector, which includes lack of extension agents, credit facilities, farm inputs, irrigation, value addition and corruption, lack of support for ADP staff and ineffective government policies. Policy implications include investment in extension services, provision of credit facilities and other infrastructures (e.g., irrigation, ADP staff), training of small farmers in business skills, promotion of modern technology as a package as well as special projects (e.g., Cassava Plus project) in order to boost production of cassava at the farm-level in Nigeria. Chapter 5 - Celiac disease is an immune disorder in which people cannot tolerate gluten because it damages the inner lining of their small intestine and prevents it from absorbing nutrients. Gluten is a protein found in wheat, rye, and barley and occasionally in some other minor products. A lot of foods; such as, baked food and pastas are manufactured using flour from wheat, rye, barley and oats, in which the gluten defines its functional properties. People who want to manufacture products containing gluten, have been looking for alternatives to solve this problem and to insure gluten-free products for the celiac population. Because, the cassava flour does not have gluten; the foods made with this flour could be one of the solutions for the development of food for gluten-intolerant consumers. Some research has been done in regard to substitute the gluten totally in order to produce baked goods, and pastas, quite similar in its functional properties, to those produced by wheat flour. The research was initiated producing flour from the edible portion of the cassava roots. Native and Complimentary Contributor Copy x Clarissa Klein modified flour from cassava roots were made using different treatments of heat, water concentration, as well as the use of salt, emulsifier, hydrocolloids or enzymes. All of the flour obtained were characterized in their chemical composition, physical, and functionality. The research suggests that is feasibility to use these types of flour in the production of numerous gluten-free baked goods, bread, and pastas, because they showed a wide spectrum of nutritional and functional properties which is causes by the effect of the additives and the treatments applied. Therefore, at a pilot phase, a second research experiment has started to produce pastas, and formulation of mix flour for cake, and pancakes, all of them gluten-free. The formulations and procedures that were implemented, as a function of the cassava flour, are discussed in this chapter. Chapter 6 - Cassava (Manihot esculenta Crantz) is a tuberous root grown in all regions of Brazil, mainly in the North region, and the state of Pará Pará is one of the largest producers. It is considered a high-energy food, rich in starch and fiber, but highly perishable, with moisture content of around 67.5%, used for direct human consumption or as raw material to produce cassava-derived products, by using the water activity principle for food conservation. Various products can be produced by artisanal or industrial processes, such as different types of cassava flour, cassava gums, fermented and native starch, tapioca flour, tucupi, among others. Flour is one of the main cassava products, and its use is widespread throughout the country as part of Brazilian eating habits, especially in the North and Northeast regions, consumed by rural, riverine, and urban populations of all social classes. However, the quality of cassava-derived products is very heterogeneous, often out of the standards established by Brazilian law, once they are produced by small producers following their decision-making processes. This chapter describes the technological differences in the manufacture of cassava- derived products, considering cassava varieties and processing stages, such as cassava fermentation before drying and drying process, as well as their effects on the physicochemical characteristics of the products, including moisture, pH, acidity, particle size, color of the products and gel, helping to spread the potential of cassava and enhancement of regional products. Chapter 7 - Cassava is the third-most important food source in the tropics after rice and maize. Cassava is the staple food for about half a billion people in the World. It is a tropical crop grown mainly in Africa, Asia, and South America. It can be cultivated on arid and semi- arid land where other crops do not thrive. During the processing of cassava into chips, flour or starch, enormous amount of wastes are generated ca. 0.47 tons for each ton of fresh tubers processed. This waste consists of peels, wastewater and pulp that contain between 36 to 45% (w/w) of starch and from 55 to 64% (w/w) of lignocellulosic biomass. An innovative processing system is therefore essential to take into account the transformation of this waste into value added products. This will address both the environmental pollution and inefficient utilization of these resources. The starch and lignocellulosic cassava processing waste can be converted into renewable energy carriers such as biogas through anaerobic digestion (AD), bio-ethanol through fermentation and bio-hydrogen through dark fermentation. In the case of AD, the waste can be used directly as substrate while for fermentation; the waste must be pre-treated to release monomeric sugars, which are substrates for bio-ethanol and bio-hydrogen production. There is possibility of sequential fermentation for either bio-ethanol or bio-hydrogen and AD for biogas production thereby making use of all the fractions of the cassava waste.

Complimentary Contributor Copy Preface xi

Generation of renewable energy from cassava waste could benefit rural populations where access to electricity is very poor. This would also reduce the dependence on firewood and charcoal that are known to provide almost 90 percent of domestic energy requirements. Such a development could help save trees, lower emissions that cause climate change and reduce the fumes from millions of tons of firewood that threaten human health, especially the health of women and children. Although deforestation and land degradation are well-known, the charcoal and firewood consumption that causes them is still on the rise. This chapter, therefore, explores the use of cassava waste for production of fuel energy with a focus for use as domestic cooking fuel. It also proposes an efficient approach to cassava processing to ensure efficient resource utilization in which every part of the tuber is converted to value added products mitigating environmental pollution and improving human health. Chapter 8 - Cassava is widely produced worldwide, and it is a suitable source of carbohydrates (roots), proteins and minerals (leaves). Because of perishability in fresh form, it is widely marketed in the form of gums and flour. Often, its roots have high amounts of cyanohydrin that emanates cyanide, which is highly toxic to human health. This toxic molecule is significantly present in the wastewater from the cassava processing. For this reason, the resulting wastewater, also known as manipueira, when dumped in the environment, causes huge damage to soil and to water sources. The environmental problem can be avoided by advances in industrial biotechnology, which offer potential opportunities for economic utilization of agro-industrial residues. Manipueira has high levels of organic matter and nutrients, which can serve as an ideal platform for bioprocesses mediated by microorganisms, especially microalgae, to obtain products with a high value, such as, carotenoids, phycobilins, polysaccharides, vitamins, fatty acids, and several natural bioactive compounds, which are applicable to foods, pharmaceutical products and bioenergy. This chapter describes the use of the wastewater from cassava processing as a platform for microalgae-mediated processes aiming to obtain bioproducts of commercial value. Divided into five parts, the chapter covers topics on cassava processing, the characteristics of waste from cassava, the impact of cassava waste on the environment, the potential industrial processes for wastewater conversion and the bioproducts from microalgae, summarizing a range of useful techno-economic opportunities to be applied on cassava processing plants. Chapter 9 - Progresses in biotechnological processes offer a vast array of possibilities for economic use of agro-industrial residues, such as cassava wastewater. Due to its chemical composition, cassava wastewater is an interesting substrate for microbial processes for the production of value-added bioproducts. Cassava wastewater comes from the manufacture of cassava (Manihot esculenta spp. esculenta) flour which has up to 90% of starch in its root (w/w) and is easily cultivable. The main producers of cassava in 2014 - Nigeria, Thailand, Indonesia and Brazil - were responsible for 48.61% of the total world production of 27.03 × 107 metric tons of the raw crop, which is mainly used as food and feed, but also as feedstock for biofuels and biochemicals. However, the industrial manufacturing of cassava roots generates a large amount of liquid (cassava wastewater – 2.5 liters/10 kg of cassava) and solid (bagasse) residues, in which are usually burned or disposed incorrectly. Cassava wastewater has a high content of nutrients including carbohydrates (9.6-37 g/L), protein (2.3 g/L), nitrogen (0.1-1.3 g/L) and minerals as phosphorous, potassium, calcium, magnesium, sulphur, iron, zinc, cooper, etc in pH value 5.5. Therefore, due to the plenty availability, non-market value, high content of nutrient and the continuous supply throughout the year (perennial Complimentary Contributor Copy xii Clarissa Klein crop), there is an interesting potential for the utilization of cassava wastewater as an alternative substrate in biotechnological processes, which would be in consonance with biorefinery approach. In this sense, during the past years, several biotechnological processes using cassava wastewater as substrate have been described, which are an alternative to reduce the production costs and the environmental impact. The various products which have been obtained from cassava waste water include biofuels (hydrogen, ethanol, butanol, methane), biosurfactants; organic acids (citric acid, lactic acid and succinic acid), volatile fatty acids (acetic, propionic, butyric and valeric acids), aromatic compounds, enzymes and prebiotics. Chapter 10 - The present study examines productivity, technical, cost and allocative efficiencies of processing cassava into gari by applying Data Envelopment Analysis (DEA) of 278 farmers/processors from three regions of Delta State, Nigeria. Results revealed that the mean levels of technical, cost and allocative efficiencies of gari processing is low estimated at 0.55, 0.35 and 0.64, respectively, implying that gari production can be increased substantially by reallocation of resources to optimal levels, given input and output prices. Inverse size– productivity and size–efficiency relationships exist in gari processing. In other words, marginal and small processors are significantly more productive and efficient relative to large processors. Availability of credit significantly improves technical and cost efficiencies. Extension contact significantly reduces efficiencies which is counterintuitive. Female processors are technically efficient relative to male processors while both perform equally well with respect to allocative and cost efficiencies in processing gari. Significant differences in efficiencies exist across regions as well. Processors located in Delta North and Delta South is relatively more efficient than processors located in Delta Central. A host of constraints affect gari processing which include lack of transportation, information, processing equipment and infrastructure and high cost of raw materials. Policy implications include investment in education targeted at small farmers/processors, improving agricultural credit services, processing equipments, infrastructure and transportation facilities and reforming extension services in order to make it effective in disseminating information regarding cassava processing. Chapter 11 - Whether cassava can be relied upon as a low cost staple food in urban centres and a source of steady real income for rural households will to a larger extent depend on how well it can be processed and presented to urban consumers in safe and attractive forms at competitive prices to those of cereals. A study was conducted in the coastal, eastern, central, and western regions of Kenya where only the major processors were visited and interviewed randomly using a structured questionnaire. At the coast, 62.5% of the processors were sole proprietors while 37.5% were in partnership. In the eastern region, 66.7% of the processors were sole proprietors while 33.3% were in partnership. In the western region, the only processor interviewed was a company based in Busia. At the coast, 75% of respondents had their own initial capital while in eastern 33% of respondents reported the same. Only 25% and 33% of respondents at the coast and eastern regions, respectively, had acquired their initial capital on credit. In western, the respondent had acquired initial capital through own resources and credit. In the study regions, all processors (100%) met their operating costs. In the coastal region (Mombasa), among the respondents interviewed, 50% made cassava crisps, 17% chapatti and 8% bhajia. In eastern region (Kibwezi), 50% made Nimix (composite flour) and 50% boiled cassava. In western region (Busia), 100% of respondents made composite flour (cassava mixed with other cereals). The major products reported were crisps, fried chips, composite flours (cassava mixed with cereals, legumes, leaves etc). Golden coloured crisps, Complimentary Contributor Copy Preface xiii fiber free cassava and sweet taste were preferred by consumers. Even though processors maintained high standards, none of the processors had their products patented. Processing of cassava products showed a rising trend which were marketed in supermarkets, direct consumers, retailers and wholesalers. Except for the eastern region, most processors could access raw materials throughout the year. Only a few processors in the coastal region had contractual arrangements with suppliers, whereas there was none in the other regions. Processing equipment were locally fabricated except in the eastern region where they were imported. The processors had reliable sources of power and water. The major constraints included market fluctuations, inadequate supply of cassava, city council regulations, competition from other related products like maize and sweetpotatoes, lack of credit facilities, market and capital, and processing equipment. Chapter 12 - Although cassava waste may pose serious environmental challenges if not properly disposed of, it could constitute important potential resource if properly harnessed especially by adopting modern biotechnology approach. In this study, plasmids extracted from bacterial isolates associated with cassava waste were explored, using molecular tools, in order to identify genes encoded on the plasmids as well as determine the industrial potentials of the genes borne on the plasmids. Bacterial species isolated from cassava peel (CP) and cassava wastewater (CW) from cassava processing centres in Abeokuta, Nigeria, were identified by aligning their 16S rRNA gene sequences with sequences in the GenBank. Plasmid DNA was extracted from the bacterial isolates, using the Pure Yield Plasmid Miniprep System (Promega, USA) and sequenced. The Open Reading Frame (ORF) Finder was used to identify ORFs on the plasmid DNAs. ORFs were translated and searched against publicly available archives [a non-redundant protein database of GenBank proteins, SWISS- PROT and cluster of orthologous groups (COG)] using the BLAST-P algorithm. Putative genes borne on the plasmids, as well as their products, were deduced from the plasmid nucleotide sequences. Plasmids were found on 14 bacterial isolates. Eight of the isolates (Lactobacillus plantarum, L. brevis, Bacillus coagulans, B. circulans, B. licheniformis, B. pumilus, Enterococcus faecalis and Pediococcus pentosaceus) were from CP while 6 isolates (Lactobacillus fallax, L. fermentum, L. delbruckii, Weisella confusa, Bacillus subtilis and Leuconostoc mesenteroides) were from CW. The gene, tanLpl - encoding tannase was detected on Lactobacillus plantarum plasmid while the gene (bgl1E) which encodes beta- glucosidase was found on Bacillus coagulans and Bacillus circulans plasmids. Other genes detected were hydroxynitrile lyase (HNL) gene on Bacillus licheniformis and Lactobacillus fermentum plasmids; poly-glutamic acid (PGA) synthesis regulator gene on Lactobacillus fermentum plasmid; glutamate synthase gene on Bacillus substilis plasmid; bacteriocin related genes on Lactobacillus fermentum, Lactobacillus fallax and Weisella confusa plasmids as well as some hypothetical proteins. These enzymes and accessory proteins are all well known for their importance in the food industry. Furthermore, the hypothetical proteins may turn out to be hitherto unknown enzymes for important metabolites or structural proteins. The plasmids could constitute an easy source of genes for mass production of the enzymes and their products. This study, therefore, shows that cassava waste has potentials as an important biotechnology resource, especially for the food industry. Chapter 13 - Cassava is the third largest source of food carbohydrates in the tropics after rice and maize. Cassava is a major staple food in the developing world, providing a basic diet for over half a billion people. Cassavas are multipurpose commercial products that have many potential uses, such as in bio-fuels, animal feed, medicines, bio-composite, food packaging Complimentary Contributor Copy xiv Clarissa Klein and so on. Apart of from these uses, processed cassava serves as an industrial raw material for the production of adhesives, bakery products, dextrin, dextrose, glucose, lactose and sucrose. This chapter elucidates the uses of cassava products and its future challenging opportunities. Chapter 14 - Cassava is an important component in the diets of more than 800 million people around the world. It is kind of tropic and sub-tropic plant. It is able to grow in less- nutrition soil. In a dry land, cassava sheds its leaves to keep it damp and produces new leaves in the rainy season. Otherwise, cassava can not survive in cold weather but it can grow very well in the area with pH 4-8. Cassava needs at least 5 months in the summer for producing ripe cassava. The aim of this chapter is to discuss the proximate composition, production, application, and modification process of cassava roots as well as their future perspective. The typical important parameters for proximate composition of cassava are protein, lipids, fibre, starch, cyanide acid and ash contents. The carbon to nitrogen ratio (C/N ratio) of dried fresh cassava roots is also important parameter for microbial activities within fermentation process. The development of new utilization techniques of cassava roots has gained increasing importance in chemical, food, and pharmaceutical industries, due to their content of economically-valuable compounds, the necessity of environmental friendly process, global food and energy security. There are several different methodologies for enhancing detoxification and improving the quality of cassava flour, such as fermentation process (liquid, solid state, submerged, culture and spontaneous fermentations), different microorganisms (yeast, fungi and bacteria) and different additional nutrients (with and without nutrients). Moreover, lactid acid is produced as by-product during the fermentation. This is also interesting topic due to the potential application of lactic acid for the production of biodegradable polymers. Another, the analysis methods of the compounds in cassava roots are also a challenging work. Few analytical methods are available to provide a detailed and simpler analysis. It is of great interest if new utilization of cassava roots and analysis methods of the compounds in cassava roots are available to establish all products during the fermentation. Chapter 15 - Currently eco-friendly polymeric materials are made from different biopolymers. In this sense, special attention has brought the use of starch at industrial level, since can be processed as conventional polymers. In the same way, one of the starches most used for developing biodegradable films and foams for use as packing material has been cassava (Manihot esculenta) starch, due to its high production and performance, which makes it be a promising material for replacement of polymers obtained from the petrochemical industry. At regard, in this chapter will be reviewed and discussed recent advances related to the development of biodegradable films and foams made from cassava starch. Chapter 16 - This review highlights the traditional and improved methods of cassava production and processing in Ghana. It also explains the geographical distribution of cassava production and utilisation. Facts and figures from agricultural production in Ghana is used to analyse production trends as well as the contribution of cassava to Agricultural Gross Domestic Production. Most importantly, cassava is a staple food crop and accounts for about 152.9 kg per capita consumption. Making it one of the most processed crop into gari, fufu powder and kokonte to increase its shelf life. Additionally, it can be used as an industrial crop because of its high starch content. These process technologies have contributed to the reduction of post-harvest losses in cassava production in Ghana. The residue generated from cassava processing has a huge potential in biorefinery. The review also brings into focus current research works in cassava residue utilisation, reviewing technologies for converting Complimentary Contributor Copy Preface xv this valuable feedstock which is a mixture of cassava peels, trimmings and cuttings into sugar platform in a biorefinery for the production of major products such as ethanol, lactic acid and protein. Chapter 17 - Cassava (Manihot esculenta) bagasse is a fibrous by-product generated in the tuber processing. After washing and peeling, the cassava is grated and then water is added in order to extract the starch. The mixture is filtered such that a rich starch solution and a wet solid residue can be separated. This slurry, known as bagasse, comprises up to 20% of the weight of the processed cassava. In addition, as the extraction of starch from cassava is less efficient than those based on processing of potato or maize, the bagasse contains around 50- 70% of starch on a dry basis. As it has no important use, with the exception of animal feed, the bagasse is usually rejected to water courses increasing the environmental pollution. Therefore, several strategies are being studied to find useful applications for this by-product. Pyrolysis of the bagasse and copyrolysis, namely the thermal degradation of mixtures of the bagasse and lignocellulosic biomass in inert atmosphere, could be an appealing possibility to employ this waste in order to generate green energy and/or other value-added products. In particular, growing attention is paid to the liquid products arising from pyrolysis/copyrolysis, commonly known as bio-oils, since they show many of the advantages of liquid fuels, such as inexpensive storage and transportation, and high energy density. In this scenario, the processes of pyrolysis of cassava starch, the major constituent of dry cassava bagasse, and of copyrolyisis of the starch with peanut hulls, an abundant lignocellulosic residue, were studied by performing experiments in a fixed-bed reactor at different process temperatures (400ºC – 600ºC). The pyrolysis of the starch led to a higher maximum yield of bio-oils that took place at a lower temperature than the copyrolysis (57 wt% at 400ºC vs. 49 wt% at 500ºC). Physichochemical characterization of the three kinds of pyrolysis/copyrolysis products with emphasis on the bio-oils was carried out mainly by proximate and ultimate analyses, Karl- Fischer titration, Fourier-transformed infrared spectroscopy, N2 adsorption, scanning electronic microscopy, and gas chromatography (GC-TCD and GC-MS). While the pyrolysis of the starch resulted in bio-oils with less nitrogen content, the copyrolysis produced bio-oils with lower content of oxygen and higher carbon percent. Water content of the bio-oils increased with rising process temperatures and it was lower for the liquids resulting from the pyrolysis of the starch. Also, the bio-oils arising from the pyrolysis of the starch presented more sugar compounds and fewer phenols. Besides, the pyrolysis of the starch led to a lower yield of solid products (bio-chars) than the copyrolysis. They showed greater high heating values (up to 35 MJ/kg) than those arising from the latter process in agreement with their larger carbon content and lower presence of ash. In addition, the bio-chars produced at the highest process temperature presented an incipient pore development, suggesting their possible use as rough adsorbents or as intermediary for further upgrading to activated carbons. Furthermore, the pyrolysis of cassava starch and copyrolysis with peanut hulls generated gases, principally CO2, CO, CH4 and H2, that could help to sustain the processes. Chapter 18 - The potential to increase cassava products utilization is enormous if the available recipe range can be increased. A marketing survey was conducted in Mombasa, Nairobi and Busia urban centres. In Mombasa and Nairobi, marketing of cassava products was done daily. In Busia, daily marketing accounted for 22% while 78% was through a local market that opens twice a week. In Mombasa, 100% of cassava products were mainly sold at the main market (Kongowea). In Nairobi, 94% of respondents sold their products in local markets (Gikomba and Kibera) and 6% to hotels. In Busia, 50% sold their products at the Complimentary Contributor Copy xvi Clarissa Klein main market and 50% in secondary markets. Sale of cassava products in Mombasa, Busia and Nairobi dates as early as 1956, 1962 and 1987, respectively. In Mombasa, cassava crisps and fried fresh cassava constituted 8% and fresh roots 92%. In Nairobi, boiled cassava constituted 6%, flour 25% and dried chips 69% of products being traded in. In Busia cassava flour constituted 33% and dried chips (for milling) 67% of the products sold. In Mombasa, the average price of a fresh root was 13 shillings during scarcity and 8 shillings during abundance. In Nairobi, a 2-kg tin (gorogoro) was sold at 69 and 55 shillings during scarcity and abundance, respectively. In Busia, the average price of a gorogoro was 35 and 31 shillings during scarcity and abundance, respectively. In Mombasa, the majority of those marketing cassava products were males while in Nairobi and Busia females dominated. The main products sold in Mombasa were crisps, fried chips, and fresh roots. In Nairobi, the main products were boiled cassava, flour and dry chips. In Busia, flour and dried chips were the main products. In Mombasa the major customers were final consumers, retailers and processors. In Nairobi major customers were final consumers, wholesalers, retailers and millers. In Busia customers were final consumers, wholesalers, retailers and processors. In Mombasa and Busia the principal suppliers of cassava products were both male and female while in Nairobi it was women. One of the main supply constraint reported was lack of cassava during scarcity. Competition from maize was cited in Mombasa and Nairobi. Costly transport was reported in Mombasa and Busia. In Mombasa, lack of credit was also cited. In Busia, other important constraints recorded were lack of sorghum and finger millet for blending cassava, and unfavourable weather for drying of cassava chips. Chapter 19 - The genetic improvement of cassava is directly related to the increase of productivity of culture, this has an important role in feeding in developing countries. Therefore, knowledge about the biology, distribution and conservation status of their wild relatives is essential, because it allows the harvest and conservation efforts to be directed to those unfamiliar species of which there are more severe threats. These data become even more relevant since some of their wild relatives are resistant to common diseases, such as whitefly. This chapter discusses the closest conservation of the wild relatives of cassava from the evaluation of biological collection, as well as recent collections by authors in Brazil and their cultivation in Germplasm banks. This work is part of a program of study of wild species of Manihot developed in partnership with the Federal University of Bahia Recôncavo (UFRB) and Cassava and Fruits National Research Center (CNPMF) of the Brazilian Agricultural Research Corporation (EMBRAPA) both located in Cruz das Almas, Bahia, Brazil. The program, started in 2010 aims to harvest and cultivate wild species of the genus with taxonomic, conservation and agronomic purposes, especially with regard to improving the cassava (M. esculenta Crantz). Harvests were made during the first six years of the project in four Brazilian regions encompassing 14 states and over 150 municipalities mainly from the central and eastern South America region. About 60 of the 80 south American species of Manihot in various environments were seen and harvested. Thirteen species phylogenetically close to cassava were selected to discuss their conservation status based on their occupation Area (AOO), Occurrence Extension (EOO), and potential use for the improvement of this culture. According to the International Union for Conservation of Nature (IUCN) criteria, all species showed some degree of threat, two considered Critically Endangered and the other Endangered according to AOO. The EOO analysis showed different results with only three endangered species, which can indicate subsampling of natural populations of these species. In preliminary studies among the analyzed species only three presented suggest valuable Complimentary Contributor Copy Preface xvii features to cassava improvement as resistance to pests and diseases, such as African cassava mosaic virus, bacterial blight, anthracnose, green mite and caterpillar ‘mandarová,’ or high dry matter content and protein in roots. However, the fact that some species were not included in the analysis, because they do not appear in the same M. esculenta clade, which also presents important features for improvement, suggests that they may also be the subject of breeding programs due to the ease of hybridization verified gender. Regular expeditions of harvest of wild species of Manihot, that were conducted since 2010 have helped to increase the distribution of data and also to broaden the panorama of each species ‘in loco,’ allowing the verification of their habitat conservation status, the number of individuals of each population, etc. However, expeditions have not been made yet specifically aimed at the closest relatives of cassava, covered in this study. It is emphasized that maintaining wild relatives of cassava germplasm bank is a practice of fundamental importance for the improvement of this culture, because the programs rely on the introduction of alleles with valuable agronomic traits contained in these species to minimize the limitations found in culture as pests and diseases.

Chapter 1

COMPARISON OF CASSAVA AND SUGARCANE BAGASSE FOR FUEL ETHANOL PRODUCTION

Yessica Chacón Pérez, Daissy Lorena Restrepo Serna and Carlos Ariel Cardona Alzate* Instituto de Biotecnología y Agroindustria Laboratorio de Equilibrios Químicos y Cinética Enzimática Departamento de Ingeniería Química Universidad Nacional de Colombia sede Manizales, Manizales, Colombia

ABSTRACT

During the last years, biofuels from different feedstocks have been studied and scaled up to industry to provide energy needing mainly for transport. Raw materials with high sugar content are mostly used in the production of biofuels as ethanol, butanol, hydrogen, etc. In the last years in tropical countries as Colombia, the ethanol production from starch and lignocellulosic biomass has been studied as a new alternative. Then, raw materials as cassava and sugarcane bagasse are presented as good options but more research is needed to understand the real advantages of these feedstocks. The amount of fermentable sugars obtained from the biomass is a decisive factor in the global yield of ethanol production. In this sense, different technological schemes can be proposed in the pretreatment step of the process followed by enzymatic hydrolysis to get the sugars. Starch-rich raw materials only require a milling and cooking as pretreatment. On the other hand, the pretreatment stage in some type of lignocellulosic materials must consider the reduction of particle size and specific technologies as dilute acid or alkaline methods. This chapter shows an analysis of ethanol production from cassava and sugarcane bagasse taking into account the availability, type and different pretreatment technologies to be applied to the raw material. Additionally, a techno- economic and environmental assessment is performed, in order to compare the proposed processes.

* Corresponding author: [email protected] (Carlos A. Cardona); Phone: (+57) (6) 8879300 ext. 55354. Complimentary Contributor Copy 2 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

Keywords: first and second-generation fuel ethanol, pretreatment step, cassava, sugarcane bagasse

1. INTRODUCTION

1.1. Worldwide Ethanol Production

In the last decade, almost 73.33% of ethanol production in the world has been used as fuel in the transport sector [1], [2]. The interest in its production has increased due to its use as a fuel that reduces the greenhouse gases emission. However, this product cannot be fully used in internal combustion engines. Therefore, it has been used as an additive in oil, whose concentration varies between 3-10% depending on the policies of each country [3], [4]. In this sense, fuel ethanol has been produced in different countries around the world, as shown in Figure 1. This figure shows the distribution of fuel ethanol production in the world for the year 2015, with a global production around 74,847 thousand tonnes of oil equivalents [5]. United States is the largest producer of fuel ethanol in the world with a production of 14,700 million gallons with a sale cost of $ 1.52 USD per gallon. Brazil and the European Union are in the following places. Besides, in the last three years the exportation in United States increased from 200 to 800 million gallons of fuel ethanol, which has been destined to Canada (30%), Brazil (15%), China (8%), South Korea (8%), Philippines (8%), United Arab Emirates (3%), Tunisia (3%), Netherlands (3%), India (6%), Mexico (4%) and in the rest of the world (11%) [6].

Figure 1. World distribution of fuel ethanol production in 2015. Taken from: [5].

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 3

1.2. Fuel Ethanol Feedstocks

Fuel ethanol or bioethanol can be produced from different types of biomass as source of fermentable sugars [2]. The use of renewable biomass derived from agricultural crops instead of petroleum-based compounds reduces greenhouse gas emissions [7], [8]. Biomass can be classified into sugar-containing, starchy and lignocellulosic biomass [9]–[11]. Table 1 takes into account this classification to present the potential for fuel ethanol production of different agricultural crops and residues. As it is shown, the starchy biomass has a high potential and actually, raw materials as corn, wheat, barley and cassava are used due to their high content of starch for further production of fermentable sugars. This criterion has not been the only fact to select one or other biomass for fuel ethanol production. It is also influenced by the technology employed to obtain fermentable sugars, crop productivity, logistics, production cost, food security and others [12], [13]. However, the use of agricultural crops as feedstocks for the production of fuel ethanol represents a risk in food security, because population growth results in the need for more land to supply the human food chain. This is the main reason to use lignocellulosic biomass as an interesting raw material.

Table 1. Yields of ethanol production with different feedstocks

Ethanol yield Feedstock Ref. (L/ton feedstock) Sugar-containing biomass Sugar cane 70 [3], [10], [15], [16] Sugar beet 100-110 [3], [17], [18] Sweet sorghum 60-80 [3], [15], [16] Starchy biomass [3], [6], [10], [15], Corn 418.60*, 360-410 [16]  Dry mill 421.58 [6]  Wet mill 388.80 [6] Rice 430 [3], [16] Wheat 340-390 [3], [15]–[19] Sweet potatoes 125 [3], [16] Potatoes 91-110 [3], [16], [17] Cassava 150-182 [3], [12], [15], [16] Barley 250-298 [3], [16], [17] Lignocellulosic biomass Straw (based on the content of cellulose) 183 [17] Grass (based on the content of cellulose) 38 [17] Wood chips (based on the content of cellulose) 237 [17] Wood chips (based on the content of cellulose and xylose) 340 [17] Wheat Straw 261.3 [10] Sugarcane Bagasse (acid hydrolysis process) 183-236 [20], [21] Switchgrass 253.62-416.40 [22] * Average in the U.S. Industry for 2015 [6].

Agricultural residues, forest biomass, herbaceous grass and some byproducts of agro- industrial supply chains are feedstocks of high availability in the world that are not used in food supply [11]. Furthermore, these raw materials have similar ethanol yields with respect to cassava. However, lignocellulosic biomass has a complex matrix formed by cellulose,

Complimentary Contributor Copy 4 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate hemicellulose and lignin that creates some challenges when obtaining fermentable sugars compared to other biomass. This situation leaded to an intensive the search for more efficient and economical technologies [14].

1.2.1. Sugar-Containing Biomass Feedstocks based on sugar-containing crops are related mainly to high sucrose content. Some examples are sugar cane and sugar beets with an average content of 13.50%wt and 12- 15%wt, respectively [10], [23]. Although ethanol yields are not as high compared to starch (see Table 1), these plants are the most important crops in tropical and subtropical countries with large harvested areas destined to obtain refined sugar. Figure 2 shows the production of the sugar-containing crops (sugarcane and sugar beet) in the main countries that produce fuel ethanol. Sugar beet production is representative for the European Union using 11.2 million metric tonnes (MMT) for bioethanol production [24]. Czech Republic and some countries of Northwestern of Europe are the most representative countries, taking advantage of their high productivity of sugar beet crops. On the other hand, France and Germany have respectively 40% and 45% of fuel ethanol production based in the sugar content of this plant [24], [25].

Figure 2. Comparison of sugar-containing crops in different countries in *2013 and 2014. Based on: [25].

Sugarcane is used mainly by Brazil, India, Colombia and Argentina in the production of bioethanol, either from the extracted juice or molasses [26]–[29]. In the case of Brazil, the production is carried out from fresh sugarcane juice. Meanwhile, other countries as Colombia use the clarified syrup and byproducts from the evaporation and crystallization processes involved in the sugar refining [10], [30]. In 2014, Colombia used 24 MMT of the 38 MMT of sugar cane available (see Figure 2) to produce 2.39 MMT of sugar, 406 million liters (ML) of ethanol and 0.28 MMT of molasses [31]. Moreover, only 5% of China's bioethanol production is based in molasses (from cane or beet sugar plants), in contrast to Thailand where 66.66% of the installed plants depend on molasses, using approximately 4 MMT [28], [32], [33].

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 5

1.2.2. Starchy Biomass Cereals, roots and tubers crops present a high starch content since it is a reserve compound for most vegetables. Starch is a polysaccharide used in the field of syrups and biofuels. For using the starch to produce biofuels, it is necessary to perform a hydrolysis to break down the carbohydrate bonds and then to obtain fermentable sugars. This process has a theoretical yield of 111 grams of glucose per 100 grams of starch [19]. Glucose is present in the chains of amylose and amylopectin responsible for the functional properties of starch. Amylose represents nearly 25% of starch, it has a straight chain of glucose joined by glycosidic alpha (1,4) bonds and it is responsible for starch gelation. The composition of amylose in cereal grains varies from 26-28%, while in the roots and tubers accounts for 17- 23%. The other 75% of starch is the amylopectin, which helps to thicken but not in gel formation [34]. Figure 3 shows the production of starch crops in the main countries producing fuel ethanol. It is evidenced that the highest production of cereals is distributed between corn and wheat in the United States, China, Brazil and European Union. Currently, there is a huge predilection for producing fuel ethanol from corn since the starch content in corn kernel (i.e., 60.59% wt. wet basis) is higher than other grain crops [10]. In the United States, corn is used for fuel ethanol while sugar beet is used to obtain refined sugar. The high ethanol productivity from corn in United States is related to the great technological and genetic development, which has enhanced the hydrolysis of starch into fermentable sugars or increasing the starch content in corn crops. In 2015, the ethanol production in the United States was 473 gallons per acre of corn, from which 90% were produced by dry mill and the remaining 10% by wet mill [6]. In contrast, the fuel ethanol production of other countries come from corn and other grain cereals, roots and tubers with the exception of Argentina who use also molasses or juice [28]. Some countries as Canada, China, Thailand and some in the European Union use wheat, barley, rye, rice or cassava, to supply part of their transport sector requirements. In 2014, the 24% of fuel ethanol production of Canada was derived from wheat using 1 MMT of this crop [35]. In 2015, 5,250 ML of fuel ethanol were produced in the European Union using 10.1 MMT of its cereals production as corn in Central Europe and Spain, wheat in Northwestern Europe, barley and rye in Germany, Poland, Baltic Region and Sweden [10], [24]. On the other hand, 70% of the ethanol production in China was based on corn and cassava. The use of Cassava in China and other countries has increased due to fuel alcohol government policies, which provide several economic benefits to improve energy production and reduction in CO2 emissions [32]. In Thailand, approximately 10 MMT of fresh cassava tubers were consumed annually as a starchy staple in natural or fermented forms [36] and 0.97 MT are destined to supply six ethanol plants with a daily production of 1.5 ML fuel ethanol in 2014. The main objective of the Thailand government is to increase the ethanol production up to1.9 ML per day using 0.5 MMT per year of rice and the implementation of other ethanol plant based on cassava [33]. The high productivity of this crop with respect to other countries shown in the Figure 3 and its yield of 223 ton of cassava per harvested hectare makes this raw material the more viable for fuel ethanol production in this country, taking into account the availability and low cost [25]. On the other hand, the production of Cassava in Colombia (3 MMT) is not at the same level with respect to other countries as Thailand (30 MMT).

Complimentary Contributor Copy 6 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

Figure 3. Comparison of starch crops in different countries in *2013 and 2014. Based on: [25].

1.2.3. Lignocellulosic Biomass The energy outlook in recent years have led to identify a new generation of biofuels. These are known as second generation biofuels, which are derived from cheaper raw materials that are not used in the food sector and have a large availability like lignocellulosic biomass [30], [37], [38]. The glucose present in the cellulose is the fermentable sugars available for producing the so known cellulosic ethanol. The yield of the cellulosic ethanol production from these materials can vary according to the composition of the lignocellulosic biomass due to the difference of grow conditions in the crops, the performance of the technological route used to obtain fermentable sugars and the strains used to consume hexoses or both hexoses and pentoses.

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 7

Table 2. Companies around the world producing cellulosic ethanol

Capacity Company Level Location Feedstock Process (MGy*) Biochemical processes Patented Process Proesa®: Wheat straw, pretreatment with steam, Province of rice straw and Crescentino Commercial 20 reduction of viscosity, SSCF Vercelli Arundo donax process and distillation [40], (giant cane) [44]. Demonstra- Vonore, Corn stover and Pretreatment with dilute 0.25 tion Tennessee switchgrass ammonia (low temperature and DuPont pressure), saccharification, Nevada, Commercial 30 Corn stover fermentation with Zymomonas Iowa mobilis and distillation [45]. Technology GreenPower+®: Pretreatment with hot water, American Alpena, Woodchip concentration and hydrolysis of Pilot 0.8 Process Inc. Michigan waste hemicellulose with dilute-acid sulfuric, fermentation and distillation [46], [47]. Thermochemical pretreatment, Wheat straw, Hugoton, enzymatic hydrolysis, Abengoa Commercial 25 corn stover and Kansas fermentation and distillation grass crops [40], [46]. Acid pretreatment, enzymatic Project Emmetsburg, hydrolysis, fermentation with LIBERTY Commercial 20 Corn cobs Iowa GMO yeast and distillation [6], (Poet-DSM) [48]. Corn stover and Pretreatment with their Visalia, Pilot 0.05 sugarcane crushing technology, enzymatic California bagasse hydrolysis with proprietary Edeniq additives to boost and stabilize São Paulo Sugar cane Pilot 0.2 enzyme activity, fermentation State bagasse and distillation [40], [49]. Pretreatment with technology São Miguel Sugarcane Approx. Proesa®, enzymatic hydrolysis, GranBio Commercial dos Campos, straw and 22 fermentation with CF process Alagoas bagasse and distillation [50]. Thermochemical processes Sorted MSW, residual Gasification to obtain synthesis Edmonton, biomass and gas, purification, catalytic Enerkem Commercial 10 Alberta other non- synthesis and purification [40], homogeneous [51]. waste Thermochemical/biochemical processes Gasification to obtain synthesis Vero Beach, Vegetative and gas, gas conditioning for the Ineos Bio Commercial 8 Florida wood waste fermentation with Clostridium ljungdahlii and distillation [52]. * Million U.S. Gallons of fuel ethanol per year. Based on [40]–[43], [46].

There is a special interest in the agricultural residues derived from corn and sugar cane to be used for fuel ethanol production. The main reasons are the residue to product ratio (RPR) and the crop productivity as seen in Figures 2-3 [25], [39]. The RPR for residues of corn crop

Complimentary Contributor Copy 8 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate are 0.273 for cobs, 2 for stalk and 0.2 for husk. In the United States, this represents 99 MMT, 722 MMT and 72 MMT, respectively. In sugar cane processing, the main residue is sugarcane bagasse with a RPR of 0.29, which represents 214 MMT for Brazil and 11.07 MMT for Colombia. Using the sugarcane bagasse as hexoses and pentoses feedstock for producing fuel ethanol in Colombia it would be increased the production around thirteen times with respect to the 406 ML of fuel ethanol produced in 2014. Nowadays, the most representative countries in the production of cellulosic ethanol are United States, Brazil and some of Europe countries with pilot and commercial scale plants, taking advantage of the availability and proximity of lignocellulosic biomass to the processing location [15], [40]. These have been endorsed by state programs or by great leaders in fuels production companies. Some of these companies are presented in Table 2. In Europe, one of the largest biorefineries with very advanced technology has been implemented. Located mainly in Italy, Crescentino currently is dedicated to the cellulosic ethanol production. However, the company is aiming also to produce n-butanol, an alcohol of great interest for oil companies due to its similarity with gasoline. Furthermore, United States has several projects focused on cellulosic ethanol production or a precursor of this (i.e., syngas) from lignocellulosic biomass like corn stover (corn cobs, leaves and stalks), switchgrass or other sources of non-food material as paper and municipal solid wastes. In Brazil, the production of ethanol has focused on the use of fermentable sugars present in sugarcane bagasse and straw through a biochemical process in the GranBio’s Bioflex I industrial unit [40]. Also, Canada is using municipal solid wastes through gasification to produce synthesis gas and subsequently, ethanol by catalytic synthesis [41]–[43].

1.3. Stages of the Fuel Ethanol Production

1.3.1. Pretreatment and Hydrolysis Stage As shown in Table 2, in biochemical processes, different pretreatments are proposed to dissociate the cellulose-lignin complex present in lignocellulosic biomass and the same happens to treat the starchy biomass only that it is a well-established technology. This stage is only a conditioning of raw material, for which it is necessary to carry out the hydrolysis of cellulose and starch to obtain fermentable sugars, respectively [14], [53]. For both cases, the pretreatment begins with a milling step to increase the contact area for the next stages of the process (Figure 4). The other technologies used to pretreat the biomass aim to break down the intermolecular bonds of starch and improve the cellulose accessibility [54]. After the pretreatment step and depending on the material, the hydrolysis process is performed using enzymes or chemical agents but it is better to use enzymes since the use of chemicals involves the presence of toxic compounds for fermentation and the utility cost is low compared with acid or alkaline hydrolysis given the low operation cost [14], [55]–[57]. Then, the pretreatment and hydrolysis stage determine the differences in yields and production costs for obtaining fuel ethanol from these two materials. In the starchy materials, the process involved to obtain fermentable sugars begins with heating to solubilize the starch. Here occurs the starch gelatinization and the conditions depends on gelatinization temperature that varies according to the starch biomass but generally a temperature of 80°C is used [57]–[59]. Then the cooked material is partially hydrolyzed with α-amylase and viscosity decreases [10]. This first hydrolysis is known as Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 9 liquefaction, which is carried out at temperatures between 80 to 90°C with the appropriate amount of alpha amylase and an uniform agitation [10], [60], [61]. The partially hydrolyzed starch is treated with amyloglucosidase to obtain a glucose-rich solution to be used in ethanol fermentation [62]. In order to obtain a solution of fermentable sugars from the polysaccharides presents in lignocellulosic biomass different technologies have been distinguished over time. Some of these are described in Table 3. The enzymatic hydrolysis of cellulose is carried out by cellulases, which are highly specific. This process is usually conducted at mild conditions (i.e., pH 4.8 and temperatures between 45-50°C) [14], [55], [56].

Lignocellulosic biomass Starchy biomass

Milling Milling

Pretreatment technology Cooking

Liquefaction with α - amylase Saccharification Acid hydrolysis with cellulases Saccharification with amyloglucosidase

Fermentable sugars

Pretreatment Hydrolysis

Figure 4. Pretreatment and hydrolysis stage to obtain fermentable sugars.

1.3.2. Fermentation Stage In addition, some configurations are designed for reducing the operation times and avoiding the inhibition of enzyme activity due to the accumulation of hydrolyzed sugars in the fuel ethanol production. This is the case of Separate Hydrolysis and Fermentation (SHF) [71]. The hydrolysis and fermentation in one stage, known as Simultaneous Saccharification and Fermentation (SSF) is presented in Figure 5 [72]. In this scheme the sugars produced during hydrolysis are immediately fermented into ethanol and then the problems associated with sugar accumulation and enzyme inhibition as well as contamination can be avoided [72]. Furthermore, this reduces the fermentation times, lowers enzyme requirement and increases productivity. Given that SSF process can use a single reactor and the same temperature for saccharification and fermentation process, this decreases capital costs [73]. It has been applied in some starch based commercial ethanol processes [74]. Other configuration is a variation of the SSF process referred to as Simultaneous Saccharification and Co- Fermentation (SSCF), which is applied mainly to the use of lignocellulosic materials [10]. In this scheme, pentose fermentation is included using a modified microorganism capable of metabolizing it, thus taking place a simultaneous fermentation of pentoses and hexoses [71].

Complimentary Contributor Copy 10 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

Table 3. Pretreatment technologies used to pretreat lignocellulosic biomass

Type pretreatment Process description Observation Ref. The biomass is pretreated with The process causes high-pressure saturated steam hemicellulose degradation and and then, the pressure is swiftly lignin transformation due to reduced, which makes the [55], high temperature, thus Steam materials undergo an explosive [56], increasing the potential of explosion decompression. The process is [63], cellulose hydrolysis. The factors typically initiated at a [64] that affect this process are temperature of 160-260°C for residence time, temperature, several seconds to a few Physico- chip size and moisture content. minutes. chemical The lignocellulosic materials are The AFEX process was not very exposed to liquid ammonia at effective for the biomass with high temperature and pressure high lignin content and does not for a period and then, the produce inhibitors for the [55], AFEX pressure is swiftly reduced. The downstream biological [65] dosage of liquid ammonia is 1-2 processes, so water wash is not Kg ammonia/Kg dry biomass, necessary. AFEX pretreatment temperature 90°C and residence does not require small particle time 30 min. size for efficacy It is responsible for solubilize It can be carried out with partial hemicelluloses and mineral acids (H2SO4, HCl, improve the accessibility of HNO and H PO ) generally it is enzymes to cellulose. Its cost is 3 3 4 [55], used H SO a diluted usually higher than some Acid 2 4 [65]– concentrations between 0.5-5% physicochemical pretreatment [67] (w/v) at temperatures low than processes. A neutralization of 160°C and solids loading pH is necessary for downstream Chemical between 10-40%. enzymatic hydrolysis or fermentation processes. Alkali pretreatment processes This process depends on the use lower temperatures and lignin content of the materials. pressures than other Compared with acid processes, [55], Alkaline pretreatment technologies. alkaline processes cause less [66], However, the residence time are sugar degradation, and many of [68] in the order of hours or days the caustic salts can be rather than minutes or seconds. recovered and/or regenerated. In biological pretreatment processes, microorganisms such as brown-, white- and soft-rot fungi are used to degrade lignin The advantages of biological and hemicellulose in waste pretreatment include low energy [55], materials. Brown rots mainly requirement and mild [66], Biological attack cellulose, while white and environmental conditions. The [69], soft rots attack both cellulose rate of hydrolysis in most [70] and lignin. White-rot fungi are biological pretreatment the most effective processes is very low basidiomycetes for biological pretreatment of lignocellulosic materials

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 11

Figure 5. Differences between schemes of SSF and SHF processes.

2. METHODOLOGY

Although the formation of fermentable sugars from starchy and lignocellulosic biomass are identified for requiring intense conditions of pretreatment, it should be noted that the technologies used have some differences in energy consumption, equipment requirements, among others, that affect the ethanol yield and production cost. These differences have been the starting point for different laboratory and simulation research aiming to provide the best conditions for the implementation of these fermentations at commercial level. Considering this, in this chapter a techno-economic and environmental analysis of fuel ethanol production was carried out using the software Aspen Plus V8.2®. The analysis is carried out in Colombia context with cassava and sugarcane as different feedstocks that have been studied as a new alternative for ethanol market. The process proposed for each feedstock involves conventional pretreatment technologies presented in Figure 4 to avoid technological limitations. For the fermentation stage, the SHF scheme was considered to ferment the hexoses derived of starch and a co-fermentation for the hexose and pentose derived from sugarcane bagasse. Given that different feedstocks and technologies have been used to obtain the fermentable sugars and fuel ethanol, it is necessary to define the feed flow as the point of comparison. The feed was 9.87 ton of biomass per hour in both processes with the composition presented in Table 4. The amount of fermentable sugars is varying given the composition of each raw material. In the case of a complete conversion of starch of cassava, 2616.34 kg of fermentable sugars per hour could be obtained, compared to 3529.59 kg of fermentable sugars per hour from the cellulose and hemicellulose of sugarcane bagasse. However, the technologies limitations in this process allow having 1794.81 kg/h and 2181.29 kg/h of fermentable sugars, respectively. According with this behavior, the amount of fermentable sugars available for ethanol fermentation are higher for sugarcane bagasse than in cassava.

Complimentary Contributor Copy 12 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

Table 4. Composition in weight percentage in wet basis of the raw materials used for studying fuel ethanol production

Component Cassavaa Sugarcane bagasseb Moisture 71.40 50.00 Cellulose 0.26 23.70 Hemicellulose 0.33 12.05 Lignin 0.01 11.70 Protein 0.80 2,40 Starch 26.50 . Ash 0.70 1.15 Source: a [75], b[21].

2.1. Process Design

2.1.1. Cassava Case The production process of fuel ethanol consists of the following steps: Conditioning and pretreatment, biotransformation, separation and purification. The design of the ethanol production process is based on the process developed for corn by Cardona and Quintero et al. [10], [75], which is presented in Figure 6. The fresh cassava is subjected to a process of chopping and sieving, to reduce its size up to 4 mm. Then, a gelatinization process is applied to dissolve polysaccharides aiming to improve the enzymatic hydrolysis step. This process is carried out at temperatures higher than gelatinization (i.e., 63°C) with continuous agitation to decrease viscosity and prevent the formation of gel when it is cooled [76]. To obtain a partial starch hydrolysate (liquefied starch), cooked starch is subjected to a treatment with α- amylase. This treatment takes place in a bioreactor at 88°C, obtaining a hydrolysate of cassava. Hydrolysate is then sent to a bioreactor where amyloglucosidase is added to convert starch fragments into glucose. The glucose solution is sent to another bioreactor in which the sugar is converted into ethanol using Saccharomyces cerevisiae at 31°C. The yeast biomass is separated by conventional sedimentation. The liquor obtained contains a concentration of 8 – 10% in weight of ethanol. This is destined to a conventional separation process identified for first generation fuel ethanol process. This process begins with distillation followed by rectification. The distillation was performed at 1 bar obtaining an ethanol concentration of 56.7% and subsequently, the rectification process increased the concentration up to 86.7%. Finally, the ethanol from the rectification stage was preheated and then, it is sent to an adsorption stage using molecular sieves. The adsorption process was carried out in two columns which comprises the pressurization of the column (using the preheated distillate from the rectification column), adsorption of water (the product is continuous removed), and desorption of water. Desorption of water was carried out at 0.14 atm [77]. Vapors resulting from the desorption process were recycled back to the rectification column where the ethanol was recovered. From one of the adsorption columns, ethanol was recovered at 99.5%v/v, whereas the other column is regenerated.

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 13

Cassava Water CO2

α-amylase S. cerevisiae and Glucoamylase nutrients Water solution CO2 and Absorber ethanol Sieve Dextrins

Impurities Cooking Liquefaction Saccharification Fermenter reactor reactor Sedimentation Regenerate

Yeast

Molecular

sieves

Rectification Distillation

Mixer Evaporator Dehydrated Ethanol Waste water Stillage

Figure 6. Flowsheet of fuel ethanol production for the Cassava case. Rectangles concerns to raw materials (blue), intermediaries (purple), product (green) and byproducts (red) of the process.

2.1.2. Sugarcane Bagasse Case The process scheme implemented in the simulation for this case is shown in Figure 7 and it is based on previous works [30], [78], [79]. First, the particle size reduction to a 16 mesh screen with the assistance of milling and sieving equipment is carried out. Then, a pretreatment step with chemical reagents is implemented aiming to increase the accessibility of the cellulose by means of the hemicellulose solubilization [67], [80]. Pretreatment using dilute sulfuric acid (0.9%wt.), 160°C and a solid load of 10%wt. was selected based on previous reports [81]. After the dilute acid pretreatment, it is necessary to wash the solid fraction in order to recover the hydrolyzed sugars and neutralize the solid fraction. The separation of the solid and liquid fractions was done with a filter. The liquid fraction has toxic compounds from the pretreatment step as furfural, HMF and acetic acid which are fermentation inhibitors [82], [83]. For this reason, it is necessary to remove inhibitors from the liquid fraction using temperature and chemical agents. Detoxification is a well-known method to remove these toxic compounds using calcium hydroxide at 60°C and then, the pH is adjusted for the co-fermentation process [84]. During neutralization, calcium sulfate (gypsum) is formed and precipitated by the pH change and it is removed by filtration. From this procedure, it is obtained a xylose liquor that is used in the co-fermentation. The solid fraction from the acid pretreatment can be denominated as cellulignin (i.e., fiber without hemicellulose) and it is an intermediary in the global process that can be easily digested by cellulase and β-glucosidase in a citrate buffer. Enzymes hydrolyze the glycosidic bonds of cellulose to obtain glucose and cellobiose units, a disaccharide composed of two glucose

Complimentary Contributor Copy 14 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate molecules linked by (1-4)-β bonds, which subsequently are broken to obtain monosaccharides. Saccharification reactor is operated at 50°C with a solids loading of 10% and then, the temperature is increased up to 90°C during five minutes for enzyme denaturalization. The remaining solid composed mainly of lignin is removed with the aid of a filter and the liquid fraction is concentrated to obtain the glucose liquor that it is mixed with the xylose liquor to carry out the co-fermentation. Co-fermentation process used a recombinant bacterium Z. mobilis with a plasmid pZB5. The plasmid is responsible for gene expression of xylose isomerase, xylulokinase, transketolase involved in the metabolic pathway to digest xylose and produce ethanol at a temperature of 30°C [85]. Separation and purification steps are the same procedures mentioned before in Section 2.1.1.

Sugarcane bagasse

Sulfuric acid Water Steam

Crusher Sulfuric acid Filter

Ca(OH)2 Evaporator Washing Hemicellulose Filter hydrolysis reactor Cellulignin Neutralization Gypsum Detoxification reactor reactor

Enzyme solution Steam Xylose liquor

Filter

Regenerate Evaporator Lignin Mixer Saccharification Glucose liquor reactor

CO2

Sedimentation Molecular

sieves

Rectification Distillation Co-fermentation Z. mobilis and reactor nutrients Mixer Bacterium

Dehydrated Ethanol Evaporator Waste water Stillage

Figure 7. Flowsheet of fuel ethanol production for the Sugarcane bagasse case. Rectangles concerns to raw materials (blue), intermediaries (purple), product (green) and byproducts (red) of the process.

2.2. Simulation Procedure

Simulation of the presented processes schemes is based on reports of different authors. Aspen plus V8.2 (AspenTech, USA) commercial software has a wide content of physicochemical properties, thermodynamic models and equipment that allows handling solids, liquids and gases in order to design processes and determine their material and energy balances. It is highly important to consider the thermodynamic properties of organic and

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 15 inorganic compounds present in biomass such as proteins (lysine), hemicellulose, lignin and ash presented by the National Renewable Energy Laboratory (NREL) [86]. During simulation procedure, the thermodynamic models used to represent the behavior of liquid and vapor phases were NRTL (Non-Random Two Liquid) and Hayden O’Connell EOS to obtain the activity coefficient and fugacity. A feed of 9.87 ton/h of raw material are pretreated and hydrolyzed using stoichiometric conversion of starch to dextrins and then to glucose [87]. The kinetic model used to describe the dilute acid pretreatment of the lignocellulosic biomass was proposed by Esteghlalian et al. [81] and Quintero et al. [21], [30]. The conversion of cellulose through enzymatic hydrolysis is based on the stoichiometry reaction presented by Da Silva Martins et al. [82]. For co-fermentation, user model is used to describe non-structured and non-segregated models of Z. mobilis with kinetic parameters reported by [85]. Finally distillation columns were simulated considering the methodology mentioned by Quintero et al. [38], [75].

2.3. Economic Analysis

The economic evaluation is carried out using the mass and energy balances from the software Aspen Plus® aiming to determine the size and amount of utilities required by the equipment involved in the process. Sizing and profitability of the process schemes were calculated in the complementary software Aspen Process Economic Analyzer V8.2. The depreciation of capital was calculated based on the straight line method for a project life of 10 years. From this assessment, the ethanol production cost was evaluated. Economic parameters in the Colombian context (tax rate and interest rate), raw material and utilities costs reported in previous works [75], [78], [79], [88], [89] were considered in this evaluation. Table 5 summarizes the data used in the economic assessment of the proposed process schemes.

Table 5. Investment parameters and prices used in the economic analysis

Item Unit Value Ref. Investment Parameters Tax rate % 25 [88] Interest rate % 17 Raw materials Cassava USD/kg 0.038 [75] Sugarcane bagasse USD/kg 0.010 [89] Sulfuric acid USD/kg 0.094 [88] Calcium hydroxide USD/kg 0.056 Cellulase USD/kg 1.0 [89] Utilities LP steam USD/tonne 1.57 MP steam USD/tonne 8.18 [78] HP steam USD/tonne 9.86 Potable water USD/m3 1.25 Fuel USD/MMBTU 7.21 [88] Electricity USD/kWh 0.10 Operation Operator USD/h 2.14 [88] Supervisor USD/h 4.29

Complimentary Contributor Copy 16 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

2.4. Environmental Analysis

The environmental analysis of the proposed cases is evaluated using the software developed by the Environmental Protection Agency (EPA): Waste Algorithm Reduction (WAR GUI). This software determines the potential environmental impact (PEI) per kilogram of product from the generated impact by inhalation and concentration of components in the output streams of the process, and the energy process according to power source [90]. The software evaluates the environmental impact based on eight categories: Human Toxicity Potential by Ingestion (HTPI), Human Toxicity Potential by Exposure (HTPE), Terrestrial Toxicity Potential (TTP), Aquatic Toxicity Potential (ATP), Global Warming Potential (GWP), Ozone Depletion Potential (ODP), Smog Formation Potential (PCOP) and Acidification Potential (AP) [90]. Besides, it was considered three different sources of conventional energy in the process schemes in order to analyze the relation between the impact categories and the generated contamination from these energy sources.

3. RESULTS AND DISCUSSION

3.1. Fuel Ethanol Production

Based on the composition of raw material presented in Table 4, the yields of ethanol per ton of feedstock expected were 190.13 L/ton of cassava and 258.7 L/ton sugarcane bagasse (considering the cellulose and hemicellulose content). However, the proposed processes schemes only achieves between 73 to 53% of the theoretical yield and even lower values than reported for stand-alone processes presented in Table 6. In the sugarcane bagasse case, the difference can be attributed to the physicochemical composition of the raw material, especially the moisture content that reduces the quantity of available cellulose and hemicellulose based on the information reported by Quintero et al. [30]. In the Cassava case, the different yield obtained in this work compared with that from Cardona et al. [91] can be attributed to the enzymes efficiency. This behavior reflects the influence of different existing technologies for obtaining fermentable sugars from this biomass. In this sense, different technologies such as simultaneous saccharification and fermentation (SSF) or even more innovative process known as simultaneous liquefaction, saccharification and fermentation (SLSF) can be considered for the cassava case aiming to improve yields. Moreover, the behavior of the process yield can be attributed to the microorganisms used in the fermentation step because of the different metabolic pathways for consuming the substrates. In the lignocellulosic biomass case, the results evidence that the fuel ethanol production (33,099 liters of ethanol per day) was very close to the yield obtained from cassava (32,654 liters of ethanol per day). The use of this raw material present a great opportunity for fuel ethanol production instead of Cassava; despite the great productivity of this crop in the world (i.e., 102.26 tons of cassava per Ha [25]). Due to the cassava is mainly used in the food industry, its availability is limited to be destined for biofuels production. This behavior is reflected in the use of 60% of cassava world production in the food industry, 33% animal feed and only 7% in the industry of textile, paper, food and fermentation [92]. On the other hand,

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 17 the amount of generated sugarcane bagasse in the world annually is close to 151.2 MMT for 540 MMT of sugarcane dry processed [63]. In the case of based-fuel ethanol production from cassava, it should be considered the use of bitter species that do not affect the market prices and food security, especially in developing countries.

Table 6. Ethanol production yields using as feedstock cassava and sugarcane bagasse

Yield Feedstock Reference (L/ton) Cassava Solid-state ethanol fermentation 361 [93] Liquefaction and SHF with yeast 164.47 Liquefaction and SSF with yeast 184.07 [75] Liquefaction and SSF with bacterium Z. mobilis 181.03 Liquefaction and SHF with yeast 166.80 [91] Liquefaction and SHF with yeast 139.69 This work Sugarcane bagasse Stand-alone From cellulose and hemicellulose in co-fermentation 74.55 [30] From cellulose and hemicellulose fermented with different 323.19 [89] microorganisms From cellulose and hemicellulose in co-fermentation 137.81 This work Biorefinery context (from cellulose) 56.37 [94] (from hemicellulose) 92.78

3.2. Economic Assessment

Table 7 presents the results of the economic analysis. From these results, the cost of fuel ethanol production and the profit margin are determined, for each process, assuming a sale price in Colombia of 1.24 USD/L [78]. Additionally, it is presented the share of the operating and financing costs. The economic feasibility of process schemes has a profit margin of 65.16% for Cassava and 47.66% for sugarcane bagasse. Production cost of fuel ethanol from Cassava is lower than the sugar cane in Brazil (0.47 USD/L) and it is higher compared with other countries as Thailand (0.18 USD/L) and other raw material such as corn in United States (0.40 USD/L) and wheat in Europe (0.42 USD/L) [6], [13], [95]. This difference is attributed to the market price of Cassava in countries such as Thailand, where the crop productivity is higher than the demand, which allows the reduction in the cost of raw material and at the same time, the fuel ethanol production cost. Based on this statement, the logistic of the Cassava supply chain must be considered aiming to reduce the cassava purchase price in the fuel ethanol production. Currently, the distilleries are located near to the sugarcane supply chain in order to mitigate the economic impact of the logistics issues.

Complimentary Contributor Copy 18 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

Table 7. Fuel ethanol production cost from cassava and sugarcane bagasse case

Cassava case Sugarcane bagasse case Item Share of total Share of total USD/L USD/L cost (%) cost (%) Raw Materials 0.272 62.91 0.309 47.7 Utilities 0.027 6.34 0.178 27.4 Operating Labor 0.008 1.80 0.008 1.2 Maintenance 0.005 1.13 0.007 1.0 Operating Charges 0.002 0.45 0.002 0.3 Plant Overhead 0.006 1.46 0.007 1.1 General and administrative 0.026 5.93 0.041 6.3 Depreciation of Capital 0.086 19.99 0.097 15.0 Production cost (total) 0.432 100.00 0.649 100.0 Profit margin (%) 65.16 47.66

In sugarcane bagasse case, the production cost of fuel ethanol is lower than the first generation fuel ethanol since high equipment in pretreatment stage are required. However, the production cost from sugarcane bagasse is similar to that from sugar beet in France (0.60 - 0.68 USD/L) [95]. With respect to other lignocellulosic biomass, the fuel ethanol production cost was similar to that from empty fruit bunches 0.57 USD/L, rice husk 0.63 USD/L, coffee cut-stems 0.68 USD/L and lower for Plantain Pseudostem 2.49 USD/L due to the differences in physicochemical composition and raw material costs [30], [79]. Besides, the production cost was similar to that reported in United States when corn stover is used as raw material [95]. This behavior represents an advantage for Colombia because, as mentioned in Section 1.2.3, both lignocellulosic biomass are considered main wastes of each country. This can enhance the market of fuel ethanol if it is implemented different pretreatment technologies that improve the formation of fermentable sugars. The utilities share for the ethanol production from sugarcane bagasse is higher than the Cassava case because of the amount of required energy in the pretreatment steps. Another parameter that has the highest influence in the production costs is the depreciation due to the high number of corrosion-resistant equipment required. Due to the high energy requirements of both processes, it is necessary the implementation of alternatives in order to reduce them. An alternative of energy and steam production could be the use of wastes from cassava crop (i.e., stems and leaves) or the produced lignin in the sugarcane bagasse case as raw material for gasification, pyrolysis or combustion technologies. Therefore, it would be achieved a better use of biomass and reducing wastes generated in the production process. The biggest impact would be reflected in the decrease of the production costs because of the reduction in the utilities costs with the implementation of this alternative.

3.3. Environmental Assessment

Figure 8 presents the results of the potential environmental impact (PEI) calculated using the software WAR. In this figure is shown the comparison of three sources of energy (coal, gas and oil) for both feedstocks. In the Cassava and sugarcane bagasse case, the fuel that

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 19 generated the lowest environmental impact per kilogram of product was the natural gas. In the case of the ethanol production from cassava and using natural gas as fuel, a negative PEI was obtained, which means a reduction of pollution in the environment. In other words, the generated wastes in the process are less polluting than the raw material used in the process scheme. The highest environmental potential is evidenced in the acidification potential because of the amount of CO2 release from the fermentation process. The environmental impact of this indicator changes based on the energy source that is implemented in the process since each fuel generates different amounts of CO2 in the combustion process. It is noteworthy that a previous analysis of the energy source used in each process scheme is required. The microorganism and the pretreatment procedure for each raw material are the most important differences in the evaluated processes. As a consequence, the amount of generated wastes varies. Although, the production of ethanol from the two processes does not present great difference. The ethanol purification generates a large quantity of wastes, mainly by the metabolism of the microorganism.

Figure 8. Potential environmental impact per kg of fuel ethanol using cassava and sugarcane bagasse as raw material.

Table 8 presents the amount of generated residues from both processes. The ethanol production from sugarcane bagasse generates more than three times the amount of stillage Complimentary Contributor Copy 20 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

when cassava is used as feedstock. The same behavior is evidenced in the liberation of CO2 from the process, which is 1.2 times higher than that generated with cassava. When sugarcane bagasse is used as raw material, other wastes, besides to those already mentioned, are obtained, which are the result of the acid pretreatment and detoxification process. The use of sugarcane bagasse as raw material increases the amount of generated wastes in comparison to cassava.

Table 8. Wastes obtained in the ethanol production

Waste Process [L/h] Cassava Sugarcane bagasse Wastes 28.72 60428.35 CO2 emission 1334.25 1701.76 Stillage 13671.52 46346.79

Table 9 presents the generated wastes during the pretreatment with dilute acid using sugarcane bagasse. The pretreatment of the sugarcane bagasse uses different reagents and it is also necessary to carry out detoxification processes in order to remove toxic compounds for both enzymatic hydrolysis as for fermentation.

Table 9. Products generated during dilute acid pretreatment of sugarcane bagasse

Component Flow [L/h] Sulfuric Acid 0.76 Calcium Oxide 0.12 Calcium Hydroxide 80.97 Protein 28.99 Glucose 127.80 Xylose 92.43 Furfural 0.29 Cellulose 1,399.04 Xylan 37.67 Lignin 1,110.96 Calcium Sulfate 1,538.43 Ash 32.56 Total 4,450.02

Table 10 presents the composition of stillage obtained from both processes. In the cassava case, it is evidenced that other components are presented such as ash, cellulose, dextrin, glucose, hemicellulose, protein and biomass. Compared with cassava, the stillage obtained from sugarcane bagasse present different components in smaller proportions.

Table 10. Stillage stream composition of the ethanol production

Component Cassava (%) Sugarcane bagasse (%) Ash 0.50 0.00 Cellulose 0.11 0.00 Dextrin 0.19 0.00 Ethanol 0.00 0.23 Glucose 0.56 0.00 Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 21

Component Cassava (%) Sugarcane bagasse (%) Hemicellulose 0.14 0.00 Protein 0.58 0.46 Water 97.57 98.06 Yeast 0.31 1.23

3.3.1. Energy Analysis Table 11 presents the energy consumption for each stage of the ethanol production using as feedstock cassava and sugarcane bagasse. The stages considered in the energy analysis are: milling and pretreatment, hydrolysis, fermentation, separation, purification and the concentration of stillage. In this sense, it is observed that when cassava is used as a feedstock 40.52 MJ per liter of ethanol are required. The ethanol production using sugarcane bagasse required 241.10 MJ/L of ethanol, which is higher than the cassava case. The hydrolysis and fermentation stage have the highest contribution to the energy consumption in the case of sugar cane bagasse. This behavior is evidenced since in this process scheme was considered the neutralization and evaporator of xylose and glucose to obtain the liquors rich in fermentable sugars. This process is high energy consumption in comparison to liquefaction, saccharification of starch and fermentation stage in both cases. Despite both processes have the same separation and purification stage, a significant difference in the energy consumption of each case is evidenced.

Table 11. Energy consumption per stage in the production of ethanol

Feedstock Cassava Sugarcane bagasse Stage MJ/h MJ/L Percentage MJ/h MJ/L Percentage Milling and 2,103.71 1.53 3.76 64,369.49 47.31 19.62 pretreatment Hydrolysis and 8,761.07 6.35 15.68 124,165.17 91.26 37.85 fermentation Separation and 16,016.05 11.61 28.66 37,371.69 27.47 11.39 purification Stillage 29,005.26 21.03 51.90 102,125.13 75.06 31.13 concentration Total 55,886.09 40.52 100.00 328,031.47 241.10 100.00

3.4. Food vs Fuel Production

The use of cassava as food for human consumption generates a controversy when it is used as a feedstock for the fuel generation because of the availability of this food for the human being would be diminished. With the growing demand for fuels, croplands are destined to meet these energy needs. The use of cassava as a feedstock for ethanol production is not feasible in countries that have low production; but in countries where its production is high and has the ability to increase croplands of this raw material, this would be an alternative for biofuel production. In comparison with the sugarcane bagasse considered as residue of sugar production without important food security problems (in terms of competition with food uses). According to this, it would be given and added value to a residue. But after analyzing the Complimentary Contributor Copy 22 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate results of the environmental assessment, it is necessary the development of techniques for improving existing processes aiming to reduce emissions into the environment.

CONCLUSION

The large technological development that presents the production of ethanol from cassava has been presented as an alternative for its production in Colombia, because its environmental impact is lower than the case when sugarcane bagasse is used as feedstock. The reason of this difference is the pretreatment and detoxification stages involved in sugarcane bagasse processing that produces a huge amount of wastes. On the other hand, the high energy and economic requirements for the production of fuel ethanol from sugarcane bagasse evidences the preference for first generation raw materials (food crops). Another uses of lignocellulosic biomass can be considered to produce different value added products. The previous statement considers that lignocellulosic biomass is a source of sugars, protein, oils and phenolic compounds. However, it is necessary to develop more sustainable processes that take advantage of these compounds. In this sense, more research related to the breakdown of the internal structure of this type of materials, avoiding the increase of the production costs and environmental impacts, is necessary.

ACKNOWLEDGMENTS

The authors express their acknowledgments to project “Development of modular small- scale integrated biorefineries to produce an optimal range of bioproducts from a variety of rural agricultural and agroindustrial residues/wastes with a minimum consumptions of fossil energy - SMIBIO” from ERANET LAC 2015.

Conflict of Interest

The authors confirm that this chapter has not conflict of interest.

REFERENCES

[1] The Statics Portal, “Global ethanol production for fuel use from 2000 to 2015 (in million cubic meters,” Statista, 2016. [Online]. Available: http://www.statista.com/ statistics/274142/global-ethanol-production-since-2000/. [Accessed: 01-Jul-2016]. [2] Ó. J. Sánchez and C. A. Cardona, “Conceptual design of cost-effective and environmentally-friendly configurations for fuel ethanol production from sugarcane by knowledge-based process synthesis,” Bioresour. Technol., vol. 104, pp. 305–314, 2012. [3] M. Balat, H. Balat, and C. Öz, “Progress in bioethanol processing,” Prog. Energy Combust. Sci., vol. 34, no. 5, pp. 551–573, 2008.

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 23

[4] R. Suarez-Bertoa, A. A. Zardini, H. Keuken, and C. Astorga, “Impact of ethanol containing gasoline blends on emissions from a flex-fuel vehicle tested over the Worldwide Harmonized Light duty Test Cycle (WLTC),” Fuel, vol. 143, pp. 173–182, 2015. [5] British Petroleum, “BP Statistical Review of World Energy. Fuel ethanol Production,” Knoema, 2016. [Online]. Available: https://knoema.com/BPWES2016/bp-statistical- review-of-world-energy-2016-main-indicators. [Accessed: 01-Jul-2016]. [6] Renewable Fules Association, “Fueling a high octane future.” pp. 1–40, 2016. [7] O. J. Sánchez, C. A. Cardona, and D. L. Sánchez, “Análisis de ciclo de vida y su aplicación a la producción de bioetanol: Una aproximación cualitativa,” Rev. Univ. EAFIT, vol. 43, no. 146, pp. 59–79, 2012. [8] J. A. Quintero, M. I. Montoya, O. J. Sánchez, O. H. Giraldo, and C. A. Cardona, “Fuel ethanol production from sugarcane and corn: Comparative analysis for a Colombian case,” Energy, vol. 33, no. 3, pp. 385–399, 2008. [9] C. A. Cardona Alzate and O. J. Sanchez Toro, “Energy consumption analysis of integrated flowsheets for production of fuel ethanol from lignocellulosic biomass,” Energy, vol. 31, no. 13, pp. 2111–2123, 2006. [10] C. A. Cardona, Ó. J. Sánchez, and L. F. Gutiérrez, Process Synthesis for Fuel Ethanol Production, vol. 2010, no. 10. United States: Taylor & Francis Group, 2010. [11] N. K. Mekala, R. Potumarthi, R. R. Baadhe, and V. K. Gupta, “Current Bioenergy Researches: Strengths and Future Challenges,” in Bioenergy Research: Advances and Applications, V. K. Gupta, M. G. Tuohy, C. P. Kubicek, J. Saddler, and F. Xu, Eds. Great Britain: Elsevier B.V., 2014, pp. 1–21. [12] A. Duarte Castillo and W. A. Sarache Castro, “La cadena agroindustrial de Biocombustibles: sus particularidades en el caso Colombian,” in Logística y Cadenas de Abastecimiento Agroindustrial, 1st ed., C. E. Orrego Alzate and G. Hernández Pérez, Eds. Manizales, Colombia: Universidad Nacional de Colombia - Sede Manizales, 2012, pp. 83–106. [13] S. I. Mussatto, G. Dragone, P. M. R. Guimaraes, J. P. A. Silva, L. M. Carneiro, I. C. Roberto, A. Vicente, L. Domingues, and J. A. Teixeira, “Technological trends, global market, and challenges of bio-ethanol production,” Biotechnol. Adv., vol. 28, no. 6, pp. 817–830, 2010. [14] P. Alvira, E. Tomás-Pejó, M. Ballesteros, and M. J. Negro, “Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review,” Bioresour. Technol., vol. 101, no. 13, pp. 4851–4861, 2010. [15] M. Balat and H. Balat, “Recent trends in global production and utilization of bioethanol fuel,” Appl. Energy, vol. 86, no. 11, pp. 2273–2282, 2009. [16] N. Patni, S. G. Pillai, and A. H. Dwivedi, “Wheat as a promising substitute of corn for bioethanol production,” Procedia Eng., vol. 51, no. NUiCONE 2012, pp. 355–362, 2013. [17] M. Enguídanos, A. Soria, B. Kavalov, and P. Jensen, “Techno-economic analysis of Bio-alcohol production in the EU: a short summary for decision makers,” Eur. Comm., no. May, p. 9; 27, 2002. [18] E. Poitrat, “The potential of liquid biofuel in France,” J. Chem. Inf. Model., vol. 16, pp. 1084–1089, 1999.

Complimentary Contributor Copy 24 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

[19] O. J. Sánchez, C. A. Cardona, and I. C. Paz Astudillo, “Fuel ethanol from sugar cane and starch,” in Renewable Fuels Developments in Bioethanol and Biodiesel Production, 1st ed., C. A. Cardona Alzate and J.-S. Lee, Eds. Manizales, Colombia: Universidad Nacional de Colombia - Sede Manizales, 2008, pp. 35–55. [20] T. Botha and H. von Blottnitz, “A comparison of the environmental benefits of bagasse-derived electricity and fuel ethanol on a life-cycle basis,” Energy Policy, vol. 34, no. 17, pp. 2654–2661, 2006. [21] C. A. Cardona Alzate, J. A. Posada Duque, and J. A. Quintero Suarez, “Bagazo de Caña: Uso Actual y Potenciales Aplicaciones,” in Aprovechamiento de subproductos y residuos agroindustriales: Glicerina y Lignocelulósicos, 1st ed., Manizales, Colombia: Universidad Nacional de Colombia - Sede Manizales, 2010, pp. 137–169. [22] A. Bansal, P. Illukpitiya, S. P. Singh, and F. Tegegne, “Economic competitiveness of ethanol production from cellulosic feedstock in tennessee,” Renew. Energy, vol. 59, pp. 53–57, 2013. [23] C. A. Cardona, C. E. Orrego, and I. C. Paz, “The Potential for Production of Bioethanol and Bioplastics from Potato Starch in Colombia,” Fruit, Veg. Cereal Sci. Biotechnol., pp. 102–114, 2009. [24] B. Flach, S. Lieberz, M. Rondon, B. Williams, and C. Teiken, “GAIN Report: EU-28 Biofuels Annual 2015,” 2015. [25] Food and Agriculture Organization of the United Nations (FAO), “FAOSTAT,” 2014. [Online]. Available: http://faostat3.fao.org/browse/Q/QC/E. [Accessed: 01-Jul-2016]. [26] S. Barros, “GAIN Report: Brazil Biofuels Annual 2015,” 2015. [27] A. Aradhey, “GAIN Report: India Biofuels Annual 2015,” 2015. [28] K. Joseph, “GAIN Report: Argentina Biofuels Annual 2015,” 2015. [29] J. Moncada, L. G. Matallana, and C. A. Cardona, “Selection of process pathways for biorefinery design using optimization tools: A colombian case for conversion of sugarcane bagasse to ethanol, poly-3-hydroxybutyrate (PHB), and energy,” Ind. Eng. Chem. Res., vol. 52, no. 11, pp. 4132–4145, 2013. [30] J. A. Quintero, J. Moncada, and C. A. Cardona, “Techno-economic analysis of bioethanol production from lignocellulosic residues in Colombia: a process simulation approach,” Bioresour. Technol., vol. 139, pp. 300–7, Jul. 2013. [31] Asociación de Cultivadores de Caña de Azucar de Colombia (Asocaña), “Balance azucarero colombian Asocaña 2000-2016 (TONELADAS).” [Online]. Available: http://www.asocana.com.co/modules/documentos/5528.aspx. [Accessed: 22-Jul-2016]. [32] A. Anderson Sprecher and J. Ji, “GAIN Report: China Biofuels Annual 2015,” 2015. [33] S. Preechajarn and P. Prasertsri, “GAIN Report: Thailand Biofuels Annual 2015,” 2015. [34] V. A. Vaclavik, “Almidones en los alimentos,” in Fundamentos de ciencia de los alimentos, Spain: Acribia S.A., 2002, pp. 45–61. [35] D. Dessureault, “GAIN Repor: Canada Biofuels Annual 2014,” 2014. [36] A. Kosugi, A. Kondo, M. Ueda, Y. Murata, P. Vaithanomsat, W. Thanapase, T. Arai, and Y. Mori, “Production of ethanol from cassava pulp via fermentation with a surface- engineered yeast strain displaying glucoamylase,” Renew. Energy, vol. 34, no. 5, pp. 1354–1358, 2009.

Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 25

[37] F. Scott, J. Quintero, M. Morales, R. Conejeros, C. Cardona, and G. Aroca, “Process design and sustainability in the production of bioethanol from lignocellulosic materials,” Electron. J. Biotechnol., vol. 16, no. 3, pp. 1–16, 2013. [38] J. A. Quintero and C. A. Cardona, “Process simulation of fuel ethanol production from lignocellulosics using aspen plus,” Ind. Eng. Chem. Res., vol. 50, no. 10, pp. 6205– 6212, 2011. [39] A. Koopmans and J. Koppejan, “Agricultural and forest residues - Generation, utilization and availability,” Reg. Consult. Mod. Appl. Biomass Energy, pp. 6–10, 1997. [40] European Biofuels Technology Platform, “Cellulosic Ethanol (CE).” [Online]. Available: http://biofuelstp.eu/cellulosic-ethanol.html#inbicon. [Accessed: 01-Jul- 2016]. [41] T. R. Brown and R. C. Brown, “A review of cellulosic biofuel commercial-scale projects in the United States,” Biofuels, Bioprod. Biorefining, vol. 7, no. 3, pp. 235– 245, 2013. [42] R. Janssen, A. F. Turhollow, D. Rutz, and R. Mergner, “Production facilities for second-generation biofuels in the USA and the EU - current status and future perspectives,” Biofuels, Bioprod. Biorefining, vol. 7, no. 6, pp. 647–665, 2013. [43] M. Galbe and G. Zacchi, “Pretreatment: The key to efficient utilization of lignocellulosic materials,” Biomass and Bioenergy, vol. 46, pp. 70–78, 2012. [44] BETARENEWABLES, “Proesa,” 2016. [Online]. Available: http://www. betarenewables.com/proesa/what-is. [Accessed: 01-Jul-2016]. [45] S. Francois Bazzana, C. E. Camp, B. Curt Fox, S. Schiffino, and K. Dumont Wing, “Ammonia pretreatment of biomass for improved inhibitor profile,” WO 2011/046818 A2, 2011. [46] Office of Energy Efficiency & Renewable Energy, “Integrated Biorefineries.” [Online]. Available: http://energy.gov/eere/bioenergy/integrated-biorefineries. [Accessed: 01-Jul- 2016]. [47] American Process, “GreenPower+.” [Online]. Available: http://www. americanprocess. com/GreenPowerPlus.aspx. [Accessed: 01-Jul-2016]. [48] L. Ward, “2015 DOE Bioenergy Technologies Office(BETO) IBR Project Peer Review,” POET-DSM Advanced Biofuels, 2015. [Online]. Available: http://www. energy.gov/sites/prod/files/2015/04/f22/demonstration_market_transformation_ward_3 433.pdf. [Accessed: 01-Jul-2016]. [49] Edeniq, “Pilot plants.” [Online]. Available: http://www.edeniq.com/pilot-plants/. [Accessed: 01-Jul-2016]. [50] GranBio, “Bioflex I.” [Online]. Available: http://www.granbio.com. br/en/conteudos/ biofuels/. [Accessed: 01-Jul-2016]. [51] Enerkem, “Exclusive thermochemical process,” 2015. [Online]. Available: http://enerkem.com/about-us/technology/. [Accessed: 01-Jul-2016]. [52] J. L. Gaddy, D. K. Arora, C.-W. Ko, J. R. Phillips, R. Basu, C. V. Wilkstrom, and E. C. Clausen, “Methods for increasing the production of ethanol from microbial fermentation,” US 8 642 302 B2, 2014. [53] P. Reinikainen, T. Suortti, J. Olkku, Y. Mälkki, and P. Linko, “Extrusion Cooking in Enzymatic Liquefaction of Wheat Starch,” Starch - Stärke, vol. 38, no. 1, pp. 20–26, 1986.

Complimentary Contributor Copy 26 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

[54] C. A. Cardona Alzate, O. J. Sánchez Toro, J. A. Ramírez Arango, and L. E. Alzate Ramírez, “Biodegradación de residuos orgánicos de plazas de mercado,” Rev. Colomb. Biotecnol., vol. VI, no. 2, pp. 78–89, 2004. [55] Y. Sun and J. Cheng, “Hydrolysis of lignocellulosic materials for ethanol production : a review,” Bioresour. Technol., vol. 83, no. 1, pp. 1–11, 2002. [56] P. Kumar, P. Kumar, D. M. Barrett, D. M. Barrett, M. J. Delwiche, M. J. Delwiche, P. Stroeve, and P. Stroeve, “Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production,” Ind. Eng. Chem. (Analytical Ed., pp. 3713–3729, 2009. [57] P. Atthasampunna, P. Somchai, A. Eur-aree, and S. Artjariyasripong, “Production of fuel ethanol from cassava,” MIRCEN J. Appl. Microbiol. Biotechnol., vol. 3, no. 2, pp. 135–142, 1987. [58] M. Bhattacharya and M. A. Hanna, “Kinetics of Starch Gelatinization During Extrusion Cooking,” J. Food Sci., vol. 52, no. 3, pp. 764–766, May 1987. [59] P. Taggart, “Starch as an ingredient: manufacture and applications,” in Starch in Food, A.-C. Eliasson, Ed. 2004, pp. 363–392. [60] P. V Aiyer, “Amylases and their applications,” African J. Biotechnol., vol. 4, no. 13, pp. 1525–1529, 2005. [61] P. M. de Souza and P. de Oliveira Magalhães, “Application of microbial α-amylase in industry - A review.,” Braz. J. Microbiol., vol. 41, no. 4, pp. 850–61, Oct. 2010. [62] S. Pervez, A. Aman, S. Iqbal, N. Siddiqui, and S. Ul Qader, “Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process,” BMC Biotechnol., vol. 14, no. 1, p. 49, 2014. [63] C. A. Cardona, J. A. Quintero, and I. C. Paz, “Production of bioethanol from sugarcane bagasse: Status and perspectives.,” Bioresour. Technol., vol. 101, no. 13, pp. 4754–66, Jul. 2010. [64] D. P. Maurya, A. Singla, and S. Negi, “An overview of key pretreatment processes for biological conversion of lignocellulosic biomass to bioethanol,” 3 Biotech, vol. 3, no. 5, pp. 415–431, 2013. [65] N. Mosier, C. Wyman, B. Dale, R. Elander, Y. Y. Lee, M. Holtzapple, and M. Ladisch, “Features of promising technologies for pretreatment of lignocellulosic biomass,” Bioresour. Technol., vol. 96, no. 6, pp. 673–686, 2005. [66] P. Kumar, D. M. Barrett, M. J. Delwiche, and P. Stroeve, “Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production,” Ind. Eng. Chem. Res., vol. 48, no. 8, pp. 3713–3729, 2009. [67] Y. H. Jung and K. H. Kim, “Acidic Pretreatment,” in Pretreatment of Biomass, A. Pandey, S. Negi, P. Binod, and C. Larroche, Eds. Elsevier, 2014, pp. 27–50. [68] A. Das, C. Mondal, and S. Roy, “Pretreatment Methods of Ligno - Cellulosic Biomass: A Review,” J. Eng. Sci. Technol. Rev., vol. 8, no. 5, pp. 141–165, 2015. [69] V. B. Agbor, N. Cicek, R. Sparling, A. Berlin, and D. B. Levin, “Biomass pretreatment: Fundamentals toward application,” Biotechnol. Adv., vol. 29, no. 6, pp. 675–685, 2011. [70] M. Saritha, A. Arora, and Lata, “Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility,” Indian J Microbiol., vol. 52, no. 2, pp. 122–130, 2012. Complimentary Contributor Copy Comparison of Cassava and Sugarcane Bagasse … 27

[71] D. Pinaki, W. Lhakpa, and S. Joginder, “Simultaneous Saccharification and Fermentation (SSF), An Efficient Process for Bio-Ethanol Production: An overview,” BIOCIENCES Biotechnol. Res. ASIA, vol. 12, no. 1, pp. 1–14, 2015. [72] F. Talebnia, D. Karakashev, and I. Angelidaki, “Production of bioethanol from wheat straw: An overview on pretreatment, hydrolysis and fermentation,” Bioresour. Technol., vol. 101, no. 13, pp. 4744–4753, 2010. [73] D. Dahnum, S. O. Tasum, E. Triwahyuni, M. Nurdin, and H. Abimanyu, “Comparison of SHF and SSF processes using enzyme and dry yeast for optimization of bioethanol production from empty fruit bunch,” Energy Procedia, vol. 68, pp. 107–116, 2015. [74] J. H. Lee, R. J. Pagan, and P. L. Rogers, “Continuous simultaneous saccharification and fermentation of starch using Zymomonas mobilis.,” Biotechnol. Bioeng., vol. 25, no. 3, pp. 659–669, 1983. [75] A. Q. Julian, A. C. Carlos, F. Erika, M. Jonathan, and C. H. Juan, “Techno-economic analysis of fuel ethanol production from cassava in Africa: The case of Tanzania,” African J. Biotechnol., vol. 14, no. 45, pp. 3082–3092, 2015. [76] J. Aristizábal and T. Sánchez, “Guía técnica para producción y análisis de almidón de yuca,” Food and Agriculture Organization of the United Nations (FAO), vol. 163. Roma, pp. 36–39, 2007. [77] J. A. Quintero, M. I. Montoya, Ó. J. Sanchez, and C. A. Cardona, “Evaluation of Fuel Ethanol Dehydration Through Process Simulation,” Fac. Ciencias Agropecu., vol. 5, no. 2, pp. 72–83, 2007. [78] J. Moncada, V. Hernández, Y. Chacón, R. Betancourt, and C. A. Cardona, “Citrus Based Biorefineries,” in Citrus Fruits. Production, Consumption and Health Benefits, D. Simmons, Ed. Nova Publishers, 2015, pp. 1–26. [79] L. V. Daza Serna, J. C. Solarte Toro, S. Serna Loaiza, Y. Chacón Perez, and C. A. Cardona Alzate, “Agricultural Waste Management Through Energy Producing Biorefineries: The Colombian Case,” Waste and Biomass Valorization, pp. 1–10, Jun. 2016. [80] S. Sun, S. Sun, X. Cao, and R. Sun, “The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials,” Bioresour. Technol., vol. 199, pp. 49–58, 2016. [81] A. Esteghlalian, A. G. Hashimoto, J. J. Fenske, and M. H. Penner, “Modeling and optimization of the dilute-sulfuric-acid pretreatment of corn stover, poplar and switchgrass,” Bioresour. Technol., vol. 59, no. 2–3, pp. 129–136, 1997. [82] L. H. da Silva Martins, S. C. Rabelo, and A. C. da Costa, “Effects of the pretreatment method on high solids enzymatic hydrolysis and ethanol fermentation of the cellulosic fraction of sugarcane bagasse,” Bioresour. Technol., vol. 191, pp. 312–321, 2015. [83] X. Guo, A. Cavka, L. J. Jönsson, and F. Hong, “Comparison of methods for detoxification of spruce hydrolysate for bacterial cellulose production,” Microb. Cell Fact., vol. 12, p. 93, 2013. [84] R. Purwadi, C. Niklasson, and M. J. Taherzadeh, “Kinetic study of detoxification of dilute-acid hydrolyzates by Ca(OH)2,” J. Biotechnol., vol. 114, no. 1–2, pp. 187–198, 2004. [85] N. Leksawasdi, E. L. Joachimsthal, and P. L. Rogers, “Mathematical modelling of ethanol production from glucose/xylose mixtures by recombinant Zymomonas mobilis,” Biotechnol. Lett., vol. 23, pp. 1087–1093, 2001. Complimentary Contributor Copy 28 Y. Chacón Pérez, D. L. Restrepo Serna and C. A. Cardona Alzate

[86] R. J. Wooley and V. Putsche, “NREL/MP-425-20685 Development of an Aspen Pus property database for biofuels components,” National Renewable Energy Laboratory, 1996. [87] J.-L. Wertz and O. Bédué, “Features of First Generation Biorefineries,” in Lignocellulosic Biorefineries, Taylor & Francis Group, 2013, p. 13–19; 83. [88] J. A. Dávila, V. Hernández, E. Castro, and C. A. Cardona, “Economic and environmental assessment of syrup production. Colombian case,” Bioresour. Technol., vol. 161, pp. 84–90, 2014. [89] S. H. Duque, C. A. Cardona, and J. Moncada, “Techno-Economic and Environmental Analysis of Ethanol Production from 10 Agroindustrial Residues in Colombia,” Energy Fuels, vol. 29, no. 2, pp. 775–783, 2015. [90] D. Young, R. Scharp, and H. Cabezas, “The waste reduction (WAR) algorithm: environmental impacts, energy consumption, and engineering economics,” Waste Manag., vol. 20, no. 8, pp. 605–615, Dec. 2000. [91] C. A. Cardona, Ó. J. Sanchez, M. I. Montoya, J. A. Quintero, and L. F. Gutierrez, “Energy and environmental impact of using lignocellulosic biomass or starch in bioethanol production,” in Renewable Fuels Developments in Bioethanol and Biodiesel Production, 1st ed., C. A. Cardona Alzate and J.-S. Lee, Eds. Manizales, Colombia: Universidad Nacional de Colombia - Sede Manizales, 2008, pp. 63–75. [92] A. Pandey, C. R. Soccol, P. Nigam, V. T. Soccol, L. P. S. Vandenberghe, and R. Mohan, “Biotechnological potential of agro-industrial residues. II: cassava bagasse.,” Bioresour. Technol. Technol., vol. 74, pp. 81–87, 2000. [93] K. Sato, K. Nakamura, and S. Sato, “Solid-State Ethanol Fermentation by Means of Inert-Gas Circulation,” Biotechnol. Bioeng., vol. 27, no. 9, pp. 1312–1319, 1985. [94] V. Aristizábal M., Á. Gómez P., and C. A. Cardona A., “Biorefineries based on coffee cut-stems and sugarcane bagasse: Furan-based compounds and alkanes as interesting products,” Bioresour. Technol., vol. 196, pp. 480–489, 2015. [95] A. Gupta and J. P. Verma, “Sustainable bio-ethanol production from agro-residues: A review,” Renew. Sustain. Energy Rev., vol. 41, pp. 550–567, 2015.

Chapter 2

CASSAVA PRODUCTION AND ITS ECONOMIC POTENTIALS IN SUB-SAHARA AFRICA: A REVIEW

Emmanuel Ukaobasi Mbah* Department of Agronomy, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria

ABSTRACT

Cassava (Manihot esculenta Crantz), which originated from tropical America and today a dietary staple to most people living in Sub-Sahara Africa is a perennial woody shrub with an edible root, which is rich in carbohydrates, calcium (50 mg 100-g), phosphorus (40 mg 100-g), vitamins B and C, as well as some essential minerals, while its tender leaves serve as a veritable source of lysine rich protein. The roots though poor in protein and other minerals, their nutrient compositions differ depending on the variety and age of the harvested crop, as well as soil conditions, climate, and other environmental factors under which the crop is grown. The stem of cassava is used as planting material and can serve as a standard substrate in mushroom production as well as fuel wood when dried. Cassava is characterized as one of the most drought tolerant crop that is capable of growing on marginal soils. The crop is rarely cultivated as a mono-crop because of its physiological growth habit and duration, which makes it to stand out as an excellent component crop in most intercropping systems. Hence, it is usually intercropped with most vegetables, yam, sweet potato, melon, maize, sorghum, millet, rice, groundnut, sesame, soybean, cowpea and other legumes, as well as plantation crops (such as oil palm, kola, rubber, cocoa, cashew and coffee) among other field crops grown in the tropical regions of the world. Roots of cassava may be due for harvesting between six months and three years (36 months) after planting. Apart from food, cassava root tubers are very versatile, hence its derivatives and qualitative starch are effectively used in a number of products such as foods, confectionery, sweeteners, paper, glues, textiles, plywood, biodegradable products, monosodium glutamate, and pharmaceuticals. Cassava chips and pellets are used in animal feed and alcohol production as well as ethanol and bio-diesels. Crop improvements associated with cassava is tailored on developing genotypes that can effectively correlate the end product with its utilization at the industrial level. The impact of research on cassava development ranging from biotec-

* E-mail: [email protected]; Phone: +234 803 460 8421. Complimentary Contributor Copy 30 Emmanuel Ukaobasi Mbah

breeding, genetics and selection to production, value-chain addition and utilization is immerse, hence, high quality improved cassava varieties which are disease- and pest- resistant, low in cyanide content, drought-resistant, early bulking, high starch content, high dry matter content, and high yielding are being cultivated by most farmers in the tropical regions today where cassava thrives. Annual world production of cassava (184 million tonnes) with Nigeria being the leading producer has continued to increase due to the development of improved varieties with high yield, excellent culinary qualities and resistance to pests and diseases among other invaluable properties. In this review therefore, scientific findings by a number of scholars on cassava are discussed with the aim of making the information a veritable tool for researchers in this field.

Keywords: cassava, production, intercropping, genotype, utilization

INTRODUCTION

Cassava, which originated from tropical South America, is a perennial woody shrub with an edible root, which today is grown in tropical and subtropical regions of the world where according to Bokang (2001), Ceballos et al. (2006) and Nuwamanya et al. (2009) it provides energy food and serves as a veritable source of food and income for over a billion people (FAO, 2007; Sis, 2013). Cassava, Manihot esculenta is the only one of 98 species in the family of Euphorbiaceae that is widely cultivated for food production. Currently, it is a dietary staple in most countries in Sub Sahara Africa (Hahn and Keyser, 1985; Kawano, 2003; Amani et al., 2005) where it is grown under subsistence farming by small scale farmers because the crop grows well in poor soils and requires limited labour. The crop is well adapted within latitudes 30 °north and south of the equator, at altitudes between sea level and 2,500 meters above sea level, in equatorial climes with rainfalls of 200 mm to 2,700 mm annually. Today it has been given the status of a cultigen with no wild forms of the species being known (Cock, 1986; Hulugalle and Ezumah, 1991; Akoroda, 2005; Mbah and Ogidi, 2012). Cassava root is rich in carbohydrates, calcium, vitamins B and C, and essential minerals. Its nutritional profile indicates 60 – 65, 20 – 31, 1 – 2 per cent moisture, carbohydrate and crude protein contents, respectively and a relatively low amount of vitamins and minerals while starch obtained from the crop contains 70 and 20 per cent amylopectin and amylose substances, respectively. However, nutrient composition differs according to variety and age of the harvested crop, and soil conditions, climate, and other environmental factors during cultivation. In terms of food calories produced per hectare per day, cassava gives food calories that are far more than 250,000, cal-1 hectare-1 day-1 relative to rice and maize with 176,000 and 200,000, cal-1 hectare-1 day-1, respectively (FAO, 2012).

CASSAVA IN INTERCROPPING

Intercropping, which is a type of mixed cropping entails the agricultural practice of cultivating two or more crops within a micro-ecological zone at the same time so that the component crops share the same ecological niche thereby enhancing the biological efficiency of the system relative to monocropping (Ofori and Stern, 1987; Adetiloye, 1989; Fininsa, Complimentary Contributor Copy Cassava Production and Its Economic Potentials 31

1997; Khan and Khaliq, 2004; Mbah and Muoneke, 2007). A number of advantages such as the reduction of weed pressure on the farm (Trenbath, 1993), effective control of soil erosion through appropriate canopy coverage and good root development (Alves, 2002), improved economical and environmental performance of the production system, (Hauggaard-Nielsen et al., 2001, Adjei-Nsiah, et al., 2007), reduction of excessive leaching of nitrate (Corre-Hellou, 2005) and improved crop yield stability (Ngendahayo. and Dixon, 2001; Njoku et al., 2009) among others can be adduced to positive intercrop association. According to Willey (1979), Mead and Willey (1980), Beeching et al. (2000), Mbah and Muoneke (2007) as well as Adrien et al. (2012) cassava fits with a great number of other crops in various cropping systems in the tropics and subtropics. Cassava is a long duration crop that matures between 6 and 36 months after planting, hence features prominently in different types of intercropping systems involving cereals, legumes, vegetables and even plantation crops such as maize, rice, groundnut melon, oil palm, and coffee coconut under a plant population that oscillates between 8,000 and 16,000 plants per hectare (Trenbath, 1993; Mbah et al., 2003).

PRODUCTION TECHNIQUES

Nigeria is the world's largest producer of cassava while Thailand is the largest exporting country of dried cassava root tubers followed by Vietnam (FAO, 2012). In terms of productivity, cassava farms in India are the highest ranking with an average fresh root tuber yield of 34.8 tonnes per hectare. However, productivity depends on a number of factors such quality stem cuttings employed during planting because cassava stem cuttings are not only bulky but are highly perishable depending on cultivar such that they dry up within a couple of days after harvesting if not properly preserved under shade in a slightly humid environment to prevent desiccation (Lozano et al., 1977; Eke-Okoro et al., 2005).

SELECTION AND PREPARATION OF PLANTING MATERIAL

Cassava is normally planted using stem cuttings obtained from a mother plant that is 8 - 12 months old. Eze and Ugwuoke (2009) reported that cassava stakes derived from the middle and lower (20 cm above the soil level) parts of the stem exhibit significant (P < 0.05) higher germination rates compared to those derived from the upper part of the stem and those obtained from the portion below 20 cm from the soil surface. Longer matured cassava stakes (15 - 20 cm) obtained from the middle portions of mature stem exhibit higher germination percentage compared to shorter stakes of 5 - 10 cm length (Cock et al., 1986; Eke-Okoro, et al., 2005). According to Ezulike, et al. (1993), Fauquet and Farguette (1990), Makumbi-kidza et al. (2000), Egesi et al. (2004) as well as Echendu (2006) the selection of healthy, disease- free and pest-free stakes is essential to ensure higher productivity. Cassava planting can be done manually or mechanically in moist, well prepared ridges or mounds or even flat in friable loose soils. It is achieved by burying the lower half of the stake (cutting) in a slanted position (45), or in an upright position (90) or where the soils are too shallow and friable cuttings are laid flat and covered with 2 - 3 cm soil. Good observation of

Complimentary Contributor Copy 32 Emmanuel Ukaobasi Mbah the polarity of the cutting is very important to ensure even and successful establishment of a cassava farm (Lozano et al., 1977; Alves, 2002; Imo, 2006; Uguru, 2011). Typical plant spacing for cassava is 1 m by 1 m to give a plant population of 10,000 plants per hectare. The crop can also be planted at 90 cm by 100 cm, 80 cm by 100 cm.

CULTIVARS

Currently a wide range of cassava cultivars have been developed since the on set of national and international breeding programmes and most of the released clones are highly resistant to many of the major diseases and pests of the crop. The cultivars exhibit strong variations not only in fresh root tuber yield but also in root diameter and length, disease and pest resistance levels, bulking rate, maturity period, being able to adapt to different environmental conditions, levels of dry matter content, cooking quality and garrification, as well as colour of root flesh among other traits (Braima et al., 2000; Okonkwo, 2002; Githunguri et al., 2004, El-Sharkawy 2006; Ekwe et al., 2008). In terms of physiological growth, cassava is a short day plant, hence tuberization is usually under photoperiodic influence such that when the day length is greater than 10 to 12 hours, root tuber formation is greatly impaired and yields are invariably low. However, shoot weight ratio value is higher. In contrast, when the crop is exposed to short day lengths, root tuber yield is enhanced (Imo, 1995).

WEEDING

Cassava is characterized by a relatively slow crop growth rate during the first three months after planting, hence, could be highly susceptible to the menace of weeds, which could lead to low root tuber yield. The efficacy of weeds in cassava farms depend on their growth rate or vigour, degree of density and growth period of weeds relative to the cassava crop (Akinpelu, et al., 2006). Akobundu (1980) as well as Liebman and Dyck (1993) reported that critical period for weed control in cassava is 12 to 16 weeks after planting. Manual weeding with hoe or the use of appropriate herbicides may be employed to control weeds in cassava farms effectively.

SOIL REQUIREMENTS AND FERTILIZER APPLICATION

The crop requires well-drained, light to medium soils with soil pH in water between 4.5 and 7.5. The crop is well adapted to acidic soils with high levels of exchangeable aluminium (Al), low levels of available phosphorus (P) and relatively high levels of potassium (K). Cassava responds well to P and K fertilization. The crops also benefits from the scavenging activities of vesicular-arbuscular (VA) mycorrhizae, which tap phosphorus in the soil and ramify it around the roots of cassava for effective utilization (Sieverding and Leihner, 1984). A number of Studies on fertilizer requirements of cassava by Howeler, (1981), IITA (1985), Carsky and Toukourou (2005), Aderi et al. (2010) as well as Byju et al. (2012)

Complimentary Contributor Copy Cassava Production and Its Economic Potentials 33 revealed that insufficient and unbalanced fertiliser use widens cassava yield gaps in terms of productivity. Furthermore, Njoku et al. (2009), Sayre et al. (2011) and Ezui et al. (2016) in their studies on fertiliser requirements for balanced nutrition of cassava submitted that potassium (K) is the most limiting nutrient relative to nitrogen (N) and phosphorus (P) to achieve fresh storage root yields of up to 8 Mg dry matter ha−1 in the West African sub region. Therefore, for enhanced nutrient use efficiency in both sole and intercropping systems, appropriate fertiliser recommendations based on balanced nutrition may lead to a reduction in cassava yield gaps. Depending on soil analysis, soils in humid tropics that are acidic require liming to the tune of 500 to 1,000 kg ha-1 and 400 to 600 kg ha-1 of NPK fertilizer in cassava cultivation (Eke-Okoro, 2000).

HARVESTING

Harvesting of cassava involves serious manual operations, which entails cutting the upper portion of the stem with the leaves at a height of 30 to 50 cm from the ground level and then with the aid of the stump the roots are carefully pulled out of the ground. Harvested root tubers are then neatly chucked out from the attachment base of the plant with the sharp machete. Cassava harvesting demands great care as to minimize damage to the roots and enhance the shelf life of the root tubers. According to Maini et al. (1977), Ashoka et al. (1984), Ikpi et al. (1986) and Njoku, et al.(2014a), root tubers of cassava mature and can be harvested between the age of 8 and 36 months after planting (MAP) depending on felt need, variety, insect pest attack, environmental factors among other factors. However, the appropriate harvesting age is 12 MAP.

ECONOMIC IMPORTANCE OF CASSAVA AND ITS POTENTIAL USES

The economic value for cassava products is the dry matter content which is the chemical potential of the crop and reflects the true biological yield of the crop (IITA, 1985) and according to Hahn et al. (1979), Lain (1985) and Kawano et al. (1987) dry matter content is controlled by polygenic additive factor as well as other factors such as age of the plant, variety, cropping season, location and efficiency of the canopy to intercept solar radiation. Barima et al. (2000) in his studies reported that dry matter content of cassava varies depending on accessions and ranges from 17 to 47 per cent. However, dry matter content above 30 per cent is considered high. Studies by Hahn et al. (1979), Ntawuruhunga et al. (1998) and Ngendahayo and Dixon (2001) indicated that optimal growth and productivity of cassava is related to its harvest index and the desirable indices range from 0.5 to 0.7. Potential fresh root tuber yield of cassava under favourable controlled environment can reach 90 t ha-1 while average yields from subsistence agricultural systems are about 10.0 t ha-1.

CASSAVA PROCESSING

Complimentary Contributor Copy 34 Emmanuel Ukaobasi Mbah

Cultivars of cassava are generally categorised as either sweet or bitter, signifying levels of toxic cyanogenic glucosides, linamarin and lotaustralin present in them (Okpara et al., 2014). Linamarase, a naturally occurring enzyme acts on the glucosides when the cells are ruptured and convert them to hydrogen cyanide. All parts of the plant contain this toxic substance. However, the leaves have the highest concentrations while the root peels (exoderma) have higher concentrations than the interior fleshy part (Aregheore and Agunbiade, 1991; White, 1998; Nuwamanya, et al., 2009). Sweet cassava cultivars can produce as little as 20 milligrams of cyanide (CN) per kilogram of fresh roots, while bitter cultivars may produce as much as 1 g kg-1 of fresh roots. Note, cassava grown under drought conditions, are highly prone to have more of these toxins in their roots. Fresh root tubers of cassava undergo post-harvest physiological deterioration (Njoku, et al., 2014b), which involves the activities of coumaric acids that initiate within 15 minutes after damage, and continues until the entire tuber is oxidized and blackened within 24 to 72 hours after harvesting. Thereafter, the roots are rendered completely unpalatable and useless. This implies that cassava root tubers immediately after harvesting require appropriate processing such as grating, sun drying, frying and soaking in water to ferment aimed at reducing the cyanide content in the enlarged roots before it can be fit for human consumption. Fresh cassava roots are usually peeled, grated and washed with water to extract the starch and can be used to make breads, crackers, pasta and pearls of tapioca while unpeeled roots can be grated and dried for use as animal feed. Also, cassava leaves can be used to fortify the level of protein content in animal feed. In industrial settings, cassava can be employed in the manufacture of products such as paper-making, textiles, adhesives, high fructose syrup and alcohol. Dried roots can be milled into flour and used for baking breads and other confectionaries (Nuwamanya, et al., 2009; Njoku, et al., 2014b). Apart from food, cassava is very versatile and its derivatives and starch are applicable in many types of products such as foods, confectionery, sweeteners, glues, plywood, textiles, paper, biodegradable products, monosodium glutamate, and drugs. Cassava chips and pellets are used in animal feed and alcohol production. Cassava leaves can be used to make soup or as feed for livestock, the stems can be used as planting materials, for mushroom production or as fuel wood while the root tubers can be cooked and eaten straight or processed (FAO, 2007).

SOME CRITICAL ECONOMIC IMPORTANCE OF CASSAVA

Cassava-Based Ethanol (Biofuel)

Current programs in a number of countries have shown significant research across board to assess the use of cassava as a veritable source of ethanol, biofuel or gasohol hence, cassava chips are gradually becoming a major source for the production. This is so because a ton of fresh root tuber of cassava yields about 150 litres of ethanol higher relative to other biological sources.

Complimentary Contributor Copy Cassava Production and Its Economic Potentials 35

Animal Feed

Cassava tubers and hay (dried cassava leaves) are used as animal feed. Cassava hay is prepared by harvesting juvenile cassava plants that are about three to four months (when the plant height is about 30 – 45 cm from the surface of the ground). The plants are then sun- dried for one to two days until it has final dry matter content of less than 85%. Nutritionally, cassava hay contains high protein (20 – 27% crude protein) and condensed tannins (1.5 – 4% CP). It is valued as a good roughage source for ruminants such as goats, sheep, dairy or beef cattle and even buffalo. It can be fed to the animals directly or as a protein source in concentrate mixtures. Also, cassava chips, pellets, root meal, ensilage and cassava foliage flour serve as veritable livestock feeds.

Industrial Uses of Cassava

Cassava comes in handy as raw material in a number of industries and can be used to make a number of products such as laundry starch, gums, glues, yeast, binders, commercial caramel, malt beer, pharmaceutical products- syrup, vitamins, monosodium glutamate, dextrins, butyl alcohol, proply alcohol, dextrose, acetone, glucose syrup, among others.

Cassava Confectionaries

High quality cassava flour (HQCF) can be used in the production of bread and cake as well as other secondary products such as biscuits, pies, croquette and noodles.

SOME PESTS, DISEASES AND CONSTRAINTS OF CASSAVA

Two principal pests affecting cassava production in Sub-Sahara Africa are the cassava green mite and the variegated grasshopper while major diseases are cassava mosaic disease (CMD), cassava bacterial blight (CBB), cassava anthracnose disease (CAD), and root rot (Trenbath, 1993; Echendu, 2006). Pests, diseases and poor management practices combined are responsible for crop yield losses as high as 50 per cent. Series of research studies by scientists in International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria and National Root Crops Research Institute, (NRCRI) Umudike, Nigeria have led to the development and release of a number of improved cassava varieties that are not only disease- and pest-resistant, low in cyanide content, drought-resistant but are also early maturing and high yielding. In general, disease-resistant varieties give sustainable fresh root tuber yields of about 50% more than local varieties. Also, according to Makumbi- kidza et al. (2009), a wide range of plant parasitic nematodes have been reported associated with cassava of which Scutellonema spp. and Meloidogyne spp., have been identified to have greater economic impact on the crop in the field.

Complimentary Contributor Copy 36 Emmanuel Ukaobasi Mbah

CONCLUSION

Cassava has a major role to play in the industrial development of Nigeria as well as other developing countries in tropical and subtropical regions of the world. However, the current average yield level is low relative to what is obtainable under standard management practices due to use of local or low yield potential varieties, poor soil fertility and nutrient management, pests and diseases effect, poor extension services, among others. Therefore, a good understanding of appropriate management practices coupled with the adoption of improved variety that would act as a enzyme in cassava production chain would boost fresh root tuber yield for better nutrition and enhance industrial development as well as serve as a good source of foreign exchange for the country. As part of the underlying efforts, current research activities centred on a cassava project code-named BioCassava Plus aimed at developing cassava varieties with lower cyanogen glucosides and fortified with vitamin A, iron and protein to help the nutritional status of people living the region is still on course with promising results having made some significant achievements by releasing some pro-vitamin A cassava varieties for multiplication by farmers in the sub-region.

REFERENCES

Aderi, O.S., Ndaeyo, N.U. and Edet, U.E. (2010). Growth and yield responses of four cassava morphotypes to low levels of NPK fertilization in humid agroecology of southeastern Nigeria. Nigeria Journal of Agriculture, Food & Environment, 6 (3 & 4):14 - 18. Adetiloye, P.O. (1989). A review of current competition indices and models for formulating component proportions in intercropping in cassava based cropping systems. Research, 11:72 – 90. Adjei-Nsiah, S., Kuyper, T., Leeuwis, C., Abekoe, M., Giller, K. (2007). Evaluating sustainable and profitable cropping sequences with cassava and four legume crops: effects on soil fertility and maize yields in the forest/savannah transitional agro- ecological zone of Ghana. Field Crops Research, 103, 87–97. Adrien, N.1., Nzola, M.1., Antoine, F.1. and Adrien, M. (2012). Enhancing yield and profitability of cassava in the savannah and forest zones of Democratic Republic of Congo through intercropping with groundnut. Journal of Applied Biosciences, 89:8320 – 8328. Akinpelu, A.O., Ogbonna, M.C., Asumugha, G.N., Ogbe,af.o. and Emehute, J.K.U. (2006). Socio-economic evaluation of improved cassava production (NR 8082) in Umudike. Root Crops Research Division, Annual Report, NRCRI, Umudike, Nigeria. Akobundu, I.O. (1980). Weed Control Strategies For Multiple Cropping Systems Of The Humid And Sub-Humid Tropics. Weeds and their control in the humid and sub-humid tropics. International Institute for Tropical Agriculture (IITA), Ibadan, pp.80 - 101. Akoroda, M.O. (2005). Root Crops, Institute of Agricultural Research, Ngaounudere, Cameroon, pp. 537.

Complimentary Contributor Copy Cassava Production and Its Economic Potentials 37

Alves, A.A.A. (2002). Cassava botany and physiology. In: Hillocks, Thresh, R.J., Bellotti, A.C. (eds.). Cassava Biology, Production and Utilisation. CABI. International Oxford. pp. 67 – 89. Amani, N.G., Kamenan, A., Rolland-Sabate, A. and Colonna, P. (2005). Stability of yam starch gels during processing. African Journal of Biotechnology, 4(1): 94 - 101 Aregheore E.M. and Agunbiade O.O. (1991). "The toxic effects of cassava (Manihot esculenta Crantz) diets on humans: A review. Veterinary Human Toxicology, 33 (3): 274 – 275. Ashoka PV, Nair SV, Kurian, TM (1984). Influence of stages of harvest on the yield and quality of cassava (Manihot esculenta Crantz). Mandraos Agricultural Journal, 71:447 - 449. Beeching, J.R., Niger, T and Tohman, J. (2000). Post harvest physiological deterioration of cassava. Proceedings of 12th symposium of International Society of Tropical Root Crops held in Tsukuba, Japan, September, pp.10-16. Bokanga M. 2001. Cassava: Post-harvest operations International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. pp. 220. Braima, J., Neuenschwamder, H., Yaninek, F., Cudjoe, J.P., Echendu, N. and Toko, M. (2000). Pest Control in Cassava farms: IPM Field Guide for Extension Agents. Wordsmiths Printers, Lagos, Nigeria. Pp.36. Byju, G., Nedunchezhiyan, M., Ravindran, C.S., Mithra, V.S.S., Ravi, V. and Naskar, S.K. (2012). Modeling the response of cassava to fertilizers: a site-specific nutrient management approach for greater tuberous root yield. Commun. Soil Science Plant Analysis, 43, 1149 –1162. Carsky, R.J. and Toukourou, M.A. (2005). Identification of nutrients limiting cassava yield Agro-ecosystem, 71, 151 – 162. Ceballos, H., Sanchez, T., Morante, N., Fregene, M., Dufour, D., Smith, A., Denyer, K., Perez, J., Calle, F. and Mestres, C. (2006). Discovery of an Amylose-free starch mutant in cassava (Manihot esculenta Crantz). Journal of Agriculture and Food Chemistry. 55: 7469-7476. Cock, J.H. (1986). Cassava: Its calories can overcome malnutrition. International Center for Tropical Agriculture (CIAT), Cali Colombia. Tropic, 147:30 - 33. Corre-Hellou, G. and Crozat, Y. (2005) Assessment of root system dynamics of species grown in mixtures under field conditions using herbicide injection and N-15 natural abundance methods: A case study with pea, barley and mustard. Plant Soil, 276:177 - 192. Echendu, T.N.C. (2006). Regional survey of cassava fields to assess cassava green mite pest damage, infestation and the distribution of the predator, T. aripo. National Root Crops Research Institute (NRCRI), Umudike, Nigeria. Annual Report and 2006 Research Proposals. Egesi, C.N., Ogbe, F.O., Dixon, A.G.O., Akoroda, M.O., Ukpabi, U.J., Eke-Okoro, O.N. and Ubalua, A. (2004). Multilocational trials of cassava mosaic disease resistant varieties. NRCRI., Umudike, Nigeria, Annual Report. Eke-Okoro, O.N. (2000). Effects of altitude and NPK fertilizer on photosynthesis efficiency and yield of cassava varieties in Nigeria. Journal of Sustainable agriculture and Environment, 2 (2):165 – 170.

Complimentary Contributor Copy 38 Emmanuel Ukaobasi Mbah

Eke-Okoro, O.N., Ekwe, K.C. and Nwosu, K.I. (2005). Cassava Stem and Root Production: A Practical Manual, National Root Crops Research Institute, Umudike, 51p. Ekwe, K., Nwachukwu, C.I. and Ekwe, C.C. (2008). Determinants of improved garri processing technologies utilization and marketing profile among rural households in southeastern Nigeria. Nigerian Journal of Rural Sociology, 8(1):1 - 8. El-Sharkawy, M.A. (2006). International research on cassava photosynthesis, productivity, eco-physiology, and responses to environmental stresses in the tropics. Photosynthetica, 44: 481 – 512. Eze, S.C. and Ugwuoke, K.I. (2009). Evaluation of different stem portions of cassava (Manihot esculentus) in the management of its establishment and yield, In: Olojede, A.O., Okoye, B.C., Ekwe, K.C., Chukwu, G.O., Nwachukw, I.N., Alawode, O. (Eds). Proceedings of the 43rd Annual conference of Agricultural Society of Nigeria, held at National Universities Commission Auditorium and RMRDC, Abuja, Nigeria, Tues. 20th – 23rd October, 2009, pp. 120-123. Ezuia, B., Frankeb, A.C., Mandoa, C.A., Ahiabord, B.D.K., Tettehe, F.M., Sogbedjia, J., Janssenb, B.H. and Giller, K.E. (2016). Fertiliser requirements for balanced nutrition of cassava across eight locations in West Africa. Field Crops Research, 185: 69–78. Ezulike, T.O., Udealor, A., Anebunwa, F.O. and Unamma, R.P.A. (1993). Pert damage and productivity of different varieties of yam, cassava and maize in intercrop. Agricultural Science and Technology, 3(1): 99 – 102. FAO (2007). Food and Agriculture Organisation, Statistics Division, FAO., Rome. http://Faostat.org. FAO (2012). Food and Agriculture Organisation, Statistical database, FAO., Rome. http://Faostat.org Fauquet, C. and Fargette, D. (1990). African Cassava Mosaic Virus: Etiology, Epidemiology, and Control. Plant Disease 74 (6): 404 –411. Fininsa, C. (1997). Multiple cropping potentials of beans/maize. Horticultural Science, 113: 12 - 17. Githunguri, C.M., Kanayake, I.J. and Waithaka, K. (2004). Effect of the growth environment and bulking rate on cyanogenic potential of cassava tuberous roots. Agricultural Research Institute, Nairobi (Kenya). Proceedings of the 8th KARI biennial Scientific Conference, held on 11th – 15th November, pp. 151-157. Hahn, S.K., Terry, E.R., Leushner, K., Akobundu, I.O., Okali, C. and Lal, R. (1979). Cassava improvement in Africa. Field Crops Research, 23:193 - 226. Hahn, S.K. and Keyser, J. (1985) Cassava. A basic food of Africa. Out look on Agriculture 14(2): 95 - 100. Howeler, R.H. (1981). Mineral Nutrition and Fertilization of Cassava (Manihot esculenta Crantz).Centro Internacional Agricultura Tropical (CIAT). Hauggaard-Nielsen, H., Ambus, P. and Jensen, E.S. (2001). Interspecific competition, N use and interference with weeds in pea-barley intercropping. Field Crops Research, 70:101 - 109. Hulugalle, N.R. and Ezumah, H.C. (1991). Effects of cassava-based cropping systems on physico-chemical properties and earthed or in casts in a tropical Alfisol. Agricultural Ecosystems and Environment, 35:55 - 63. IITA (International Institute of Tropical Agriculture). (1985). Cassava in Tropical Africa. A Reference Manual. pp. 12-13, 24-25. Complimentary Contributor Copy Cassava Production and Its Economic Potentials 39

Ikpi, A.E., Gebremeskel, T., Hahn, N.D, Ezumah, H.C. and Ekpere, J.A. (1986). Cassava: a crop for household food security. IITA-UNICEF Collaborative Program Report, IITA, Ibadan, Nigeria, pp. 110-113. Imo, V.O. (1995). Comparative evaluation of the effects of day-length and GA on early early growth and development of two cassava cultivars. Journal of Institute of Tropical Agriculture, Kyushu University, Japan, 18:1 – 7. Imo, V.O. (2006). Cassava Production. Barloz publishers Inc., Owerri, Nigeria. 102 p. Kawano, K. (2003). Thirty years of cassava breeding for productivity: biological and social factors for success. Crop Science, 43: 1325 - 1335. Khan, M.B. and Khaliq, A. (2004). Study of mungbean intercropping in cotton planted with different techniques. Journal of Research, Pakistan, 15 (1) 23 - 31. Lain S.L. (1985). Selection for yield potential. In: Cork, J. H. and Reyes, J. A (Eds.). Cassava: Research, Production and Utilization. Cali, Columbia: UNDP/CIAT. Liebman, M. and Dyck, E. (1993). Crop rotation and intercropping strategies for weed management. Ecological Applications, 3 (1): 92 – 122. Lozano, J.C., Toro, J.C., Castro, A. and Bellotti, A.C. (1977). Production of cassava planting material. Cassava Information center, Centro Internacional de Agricultura Tropical (CIAT). Series GE-17, 29 pp. Maini, S.B., Indira, P. and Mandal, R.G. (1977). Studies on maturity index in cassava. Journal of Root Crops, 3(2):33 - 35. Makumbi-kidza, N., Speijer, N. and Sikora, R. A. (2000). Effects of Meloidogyne incognita on growth and storage-root formation of cassava (Manihot esculenta). Journal of Nematology, 32 (4S):475 – 477. Mbah, E.U., Muoneke, C.O. and Okpara, D.A. (2003). Evaluation of cassava/soybean intercropping system as influenced by cassava genotype. Nigerian Agricultural Journal, 33:11 – 8. Mbah, E. U. and Muoneke, C. O. (2007). Productivity of cassava/okra intercropping systema as influenced by okra planting density. African Journal of Agricultural Research, 2: 223 – 231. Mbah, E.U. and Ogidi, E.O. (2012). Effect of soybean plant populations on yield and productivity of cassava and soybean grown in a cassava based intercropping system. Tropical and Subtropical Agroecosystems, 15:241 – 248. Mead, R. and Willey, R.W. (1980). The concept of a land equivalent ratio and advantages. Experimental Agriculture, 16: 217- 226. Ngendahayo, M. and Dixon, A.G.O. (2001). Effect of varying stages of harvest on tuber yield, dry matter, starch and harvest index of cassava in two ecological zones in Nigeria. In: Akoroda, M.O. and Ngeve, J.M (eds.). Root Crops in the 21st Century. Proceedings of the 7th Triennial Symposium of the International Society for Tropical Root Crops – African Branch, held at Centre International des Conference, Cotonou, Benin, 11 – 17 October, pp. 661 - 667. Njoku, D.N., Egesi, C.N., Asante, I., Offei, S.K. and Vernon, G. (2009). Breeding for improved micronutrient cassava in Nigeria: Importance, constraints and prospects. Proceedings of the 43rd Annual Conference of the Agricultural Society of Nigeria held on 20th – 23rd October, 2009 at the National Universities Commission Auditorium and RMRDC, Abuja, Nigeria. pp. 210 - 214.

Complimentary Contributor Copy 40 Emmanuel Ukaobasi Mbah

Njoku, D.N., Vernon, G.E., Offei, S.K., Asante, I.K., Egesi, C.N. and Danquah, Y. (2014a). Identification of pro-vitamin A cassava (Manihot esculenta Crantz) varieties for adaptation and adoption through participatory research. Journal of Crops Improvement, pp. 112 – 120. Njoku, D.N., Amadi, C.O., Njoku, J.C., and Amanze, N.J. (2014b). Strategies to overcome post-harvest physiological deterioration in cassava (Manihot esculenta) roots. A review. The Nigerian Agricultural Journal, 45 (1 &2):67 – 89. Ntawuruhunga, P., Ojulong, H. and Dixon, A.G.O. (1998). Genetic variability among cassava genotypes and its growth performance over time. In: Root Crops and Poverty alleviation. Proceedings of the 6th Symposium of the ISTRC – African Branch. IITA, Ibadan, Nigeria, pp. 242 - 248. Nuwamanya, E., Baguma, Y., Kawuki, R.S. and Rubaihayo, P.R. (2009). Quantification of starch physicochemical characteristics in a cassava segregating population. African Crop Science Journal, 16: 191 - 202. Ofori, F. and Stern, R. (1987). Relative sowing time and density of component crops in a maize/cowpea intercrop system. Experimental Agriculture, 23:41 – 52. Okonkwo, J.C. (2002). Evaluation of cassava genotypes for yield and response to biotic stress in Jos Plateau, Nigeria. Journal of Sustainable Agriculture & Environment, 4(9):29 - 35. Trenbath, B.R. (1993). Intercropping for the management of pests and diseases. Field Crops Res. 34:381 – 405. Okpara, D.A., Mbah, E.U. and Ojikpong, T.O. (2014). Association and path coefficients analysis of fresh root yield of high and low cyanide cassava (Manihot esculenta Crantz) genotypes in the humid tropics. Journal of Crop Science and Biotechnology, 17 (2): 1 ~ 7. Sayre, R., Beeching, J. R., Cahoon, E. B., Egesi, C., Fauquet, C., Fellman, J., Fregene, M., Gruissem, W., Mallowa, S., Manary, M., Maziya-Dixon, B., Mbanaso, A., Schachtman, D. P., Siritunga, D., Taylor, N., Vanderschuren, H. and Zhang, P. (2011). The BioCassava Plus program: biofortification of cassava for sub-Saharan Africa. Annual Review of Plant Biology 62: 251–272. Sieverding, E. and Leihner, D.E. (1984). Influence of crop rotation and intercropping of cassava with legumes on VA mycorrhizal symbiosis of cassava. Plant Soil, 80:143 – 146. Sis, I. (2013). How Non-GM cassava can help feed the world. Food plants-perennial, food shortages, GMOs, global warming/climate change. The Permaculture Research Institute, Australia 2013 International Project, Bulletin (1):1 - 2. Uguru, M.I. (2011). Crop Production, Tools, Techniques and Practice. Fulladu Publishing Company, Nsukka, Nigeria. Pp. 48 – 54. Willey, R. (1979). Intercropping–its importance and research needs. Part 1. Competition and yield advantage, Field Crops, Abstracts, 32:1 – 10. White, W.L.B., Arias-Garzon, D.I., McMahon, J.M. and Sayre, R.T. (1998). Cyanogenesis in Cassava, The Role of Hydroxynitrile Lyase in Root Cyanide Production. Plant Physiology, 116 (4):1219 – 1225.

Chapter 3

CASSAVA PRODUCTION AND UTILIZATION IN THE COASTAL, EASTERN AND WESTERN REGIONS OF KENYA

C. M. Githunguri1,*, M. Gatheru2 and S. M. Ragwa2 1Kenya Agricultural and Livestock Research Organization (KALRO) Food Crops Research Centre Kabete, Nairobi, Kenya 2KALRO Katumani, Machakos, Kenya

ABSTRACT

Cassava is the second most important food root crop in Kenya. Despite its high production in the coastal and western regions, utilization is limited to human consumption. A situational analysis on cassava production was carried out to determine its current status in the western, coastal and eastern regions of Kenya. A sample of farmers was randomly selected from each region and interviewed using a structured questionnaire. Off-farm activities were undertaken by 37% in eastern and western and 32% in the coastal regions. Access to extension services was 50% in the coast, 65% in eastern and 88% in western regions. Relative to other food crops, 66.7% of respondents ranked cassava 2nd at the coastal region while 37.5% and 57% of respondents in eastern and western regions ranked it 5th and 1st, respectively. At the coastal, western and eastern regions, 92%, 67% and 65% of the respondents intercrop cassava with other crops, while 8%, 33% and 35% grow it as a sole crop, respectively. On adoption of improved cassava varieties, western region was leading with 77% followed by coast (30%) and eastern (13%). At the coast, 23% considered lack of market as the major constraint followed by pests and diseases (16%) and destruction by large mammalian pests (11%). In eastern, 15% reported drought as the major constraint followed by lack of market (13%) and pests and disease (42%). In western, the major constraints were large mammalian pests (12%), weeds (12%), lack of planting materials (8%) and insect pests (3%). At the coastal, eastern and western regions cassava was ranked second, fifth and first respectively relative to other food crops. The western region had more improved cassava varieties than the other regions. In the coastal region, the major constraint to production was lack

* Email: [email protected]. Complimentary Contributor Copy 42 C. M. Githunguri, M. Gatheru and S. M. Ragwa

of market while in the eastern region, the major constraint was drought and in western, the major constraints were wild animals and weeds. Cassava was utilized more as family food in western than in coastal and eastern regions. On processing of cassava and cassava based products, western region was leading followed by coastal and eastern region last. The western region was leading in the processing of dried cassava chips and composite flour. The coastal region was leading in the processing of fried cassava chips, crisps and pure flour. The eastern region was ranked least in processing with a few respondents making fried cassava chips and pure cassava flour.

INTRODUCTION

Cassava (Manihot esculenta Crantz) produces about 10 times more carbohydrates than most cereals per unit area, and are ideal for production in marginal and drought prone areas, which comprise over 80% of Kenya’s land mass (Githunguri et al., 1998; Githunguri, 2002; Nweke et al., 2002). A cassava plant possesses several growth parameters and physiological processes which can be used to measure its ability to produce adequate yield under various abiotic and biotic stresses (Ekanayake et al., 1997a; Ekanayake et al., 1997b; Ekanayake, 1998; IITA, 1982, 1990a; Osiru et al., 1995). According to these authors, some of these parameters include long fibrous roots, shedding of leaves, leaf area index, leaf water potential, moderate stomatal conductance, transpiration rate, water use efficiency, crop growth rate and dry matter accumulation in the tuberous roots. Cassava can reach its production potential only where the attributes of the environment best match the crop requirements. Breeding and selection of varieties according to prevailing environmental characteristics can ensure optimal performance (IITA, 1990b). The cassava commodity system has four main components: production, processing, marketing and consumption. Linking them is the key to successful cassava products development. Strong ties with both public and private institutions engaged in research, extension and social development are essential in the accomplishment of this linkage. The exact character of these linkages will vary according to the stage of the project in technology generation and transfer (Githunguri et al., 2006). Plant breeders can contribute to better productivity and quality, agronomists to improvements in cultural practices and cropping systems, and agro-ecologists to the proper analysis of resource management issues. In order to enhance the commercial achievement of Economic Recovery Strategy goals, the Government of Kenya in collaboration with development partners established funding for enhancing agricultural commodity projects (Ministry of Agriculture (MoA), 2005). In this regard, the cassava value chain project was such a project supported to enhance cassava production, processing and marketing in Kenya and beyond our borders, especially the Common Market for Eastern and Southern Africa (COMESA) region and Europe (Kadere, 2002; Mbwika, 2002). In Eastern Kenya cassava is eaten either raw or boiled (Githunguri, 1995). Despite its great potential as a food security and income-generating crop among rural poor in marginal lands, its utilization remains low. The potential to increase its utilization is enormous with increased recipe range (Githunguri, 1995) and provision of adequate clean planting material. One of the major constraints to cassava production in the arid and semi-arid areas includes lack of adequate disease and pest free planting materials (Obukosia et al., 1993) exacerbated by the slow multiplication rates of 1:10. The Kenya Agricultural and Livestock Research Organization (KALRO) has bred cultivars tolerant to cassava mosaic Complimentary Contributor Copy Cassava Production and Utilization in the Coastal … 43 disease and acceptable to end-users (Githunguri et al, 2003). Other constraints to cassava production in Kenya including semiarid eastern include lack of adequate disease and pest free planting materials, poor cultural practices, lack of appropriate storage and processing technologies, poor market infrastructure (Githunguri and Migwa, 2003; Lusweti et al., 1997). KALRO has developed cassava varieties that are widely adapted to diverse agro-ecological zones, high yielding, early bulking, drought resistant/tolerant, resistant to major biotic and abiotic stresses and have good root quality (Githunguri et al., 2003; Githunguri, 2004). KALRO has recognized the importance of involving farmers in their selection and breeding research programmes as suggested by Bellon (2001) and Fliert and Braun (1999). Cassava is a major factor in food security across sub-Saharan Africa. In Kenya cassava is grown in over 90,000 ha with an annual production of about 540,000 tons. It is estimated that Africa produces about 42% of the total tropical world production of the crop (FAO, 1990). Cassava can grow in marginal lands, requires low inputs, and is tolerant to pests and drought (Githunguri et al., 1998; Nweke et al., 2002). Despite its great potential as a food security and income generation crop among rural poor in marginal lands, its utilization remains low in Kenya. In addition, it can be safely left in the ground for a period of 7 to 24 months after planting and then harvested as needed. Cassava is the second most important food root crop after Irish potato in Kenya. However due to its narrow production base it is ranked number 36 out of 50 in KARI’s 1991 priority setting exercise (KARI, 1995). Available statistics on cassava production in the country show a slow but steady increase in production. Cassava production in the country is concentrated in three main regions; Coastal, Central and Western region. Western and Coastal regions are the main cassava producing areas, producing over 80% of the recorded cassava output in the country (MoA, 1999). The importance of cassava as a food and cash crop in the central Kenya is however increasing. Cassava tubers are used as human food as well as animal feed. The leaves are also popular vegetable among the locals. The roots are either boiled or fried before consumption. The western (Western and Nyanza Provinces), coastal (Coast Province) and eastern (Central and Eastern Provinces) regions of Kenya account for 60%, 30% and 10% of production, respectively. Figure 1 shows a mature cassava crop grown by small holder poor households for subsistence. Despite being an important food security crop, cassava utilization in Kenya is limited to roasting and boiling of fresh roots for consumption in most growing areas. However, in Nyanza and Western provinces of Kenya, roots are also peeled, chopped into small pieces (cassava chips), dried and milled into flour for ugali. This is normally in combination with a cereal (maize or sorghum). In the Coast province cassava leaves are used as vegetable while in Eastern Province (Machakos and Kitui), raw cassava roots are chewed as a snack. Though cassava is considered to be a food security crop in the sub-Saharan Africa, its production in Kenya is low compared to other crops like maize, beans and sorghum. Its consumption is low especially in the central region of Kenya where it is considered a poor man’s crop and is usually consumed during periods of food scarcity. Despite its high production in the coastal and western regions of Kenya, utilization is limited to human consumption. In order to promote production which has been decreasing in recent years, there is need to explore and identify other uses of cassava. To achieve this, a situational analysis on cassava production, marketing, utilization and processing was carried out in three representative regions to determine the current status of the cassava value chain in Kenya. Complimentary Contributor Copy 44 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Figure 1. A mature Cassava Field ready for harvesting.

STUDY METHODOLOGY

The study was conducted in the western (Western and Nyanza Provinces), coastal (Coast Province) and eastern (Central and Eastern Provinces) regions of Kenya. A sample of 100 farmers was randomly selected from each province and interviewed using a structured questionnaire. Figure 2 shows a farmer being interviewed. The selection of survey sites was determined by intensity of cassava production and information acquired from the County Agricultural Officers within the respective regions. Data collected included information on farmers’ socioeconomic circumstances, agronomic practices, cassava varieties, marketing, utilization and processing at household level. The data collected were analysed using the Statistical Package for Social Sciences (SPSS).

Figure 2. A farmer in his farm showing one of the project officer problems they sometimes face with cassava farming.

Complimentary Contributor Copy Cassava Production and Utilization in the Coastal … 45

RESULTS AND DISCUSSION

Demographic and Socioeconomic Characteristics of Sample Farmers Growing Cassava in Coast, Eastern and Western Provinces

Demographic and socioeconomic characteristics of the sample cassava farmers are shown in Table 1. The mean age of head of household was 48 years at the Coast and Eastern Provinces, and 35 years in Western Province though, the differences were statistically not significant. The average household size was 10, 6 and 9 in Western, Eastern and Coast Provinces respectively. However, the difference was not statistically significant. On average, the number of shoats (sheep and goats) owned was higher in Eastern (5) than in Coast (3) and Western (2) Provinces. The number of cows owned was significantly (p=0.01) lower in the coastal region (1) than the other two regions. Average cassava growing experience was higher in Western Province (22 years) than in Coast (17 years) and Eastern (16 years) Provinces. At the coast, 61% of the respondents were males while 39% were females. In eastern 51% and 49% respondents were males and females respectively while in western, respondents comprised 75% males and 25% females. The results indicate that there were more male headed than female headed households though the difference was not statistically significant.

Table 1. Demographic and socioeconomic characteristics of sample farmers growing cassava in Coast, Eastern and Western Provinces of Kenya

Province Coast Eastern Western Characteristic Mean Std. Dev. Mean Std. Mean Std. χ2 Dev. Dev. Age of household head (years) 48 13 48 16 35 16 98.773NS Size of household (no.) 6 3 6 3 9 4 35.217NS Number of shoats owned 3 4 5 7 2 2 31.235NS Number of cows owned 1 2 3 3 3 2 46.646*** Cassava growing experience 17 14 16 15 22 14 62.298NS (years) Number Percent Number Percent Number Percent χ2 of of of of of of farmers farmers farmers farmers farmers farmers Gender of household head 1.708NS Male 19 61 18 51 6 75 Female 12 39 17 49 2 25 Education level of Household 1.589NS head None 10 32 8 23 2 25 Primary 14 45 21 60 4 50 Secondary 7 23 6 17 2 25 Off-farm income 0.194NS Yes 10 32 13 37 3 37 No 21 68 22 63 5 63 Access to extension services Yes No 15 50 22 65 7 88 4.807NS 15 50 12 35 1 12 NS=Non-significant; ***=Significant at p=0.01.

Complimentary Contributor Copy 46 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Literacy level was lowest at the coastal region where 32% had no formal education, 45% had primary and 23% had secondary education. In the eastern region, 17%, 60%, and 23% had secondary education, primary education and no formal education respectively. In western region, 25%, 50%, and 25% had secondary, primary, and no formal education respectively. Off-farm activities were undertaken by 32% in the coastal region, 37% in eastern and western regions. Access to extension services was 50% in the coast, 65% in eastern and 88% in western regions even though the differences were not significant.

Cassava Production

Production and consumption of cassava at the Coast, Eastern, and Western Provinces of Kenya was recorded in 1950, 1957 and 1960, respectively. Cassava growing and consumption may have an earlier history of introduction into these regions, but the survey could only capture when the farmer started growing cassava. This does not rule out an earlier introduction and history of cassava in Kenya. From the survey, a cumulative curve showed that there was a slow increase in cassava cultivation in the periods between 1950 and 1997, after which rapid cassava cultivation was recoded up to 2006 (Figure 3). This could be attributed to food security campaigns, which were initiated by then and conservation of indigenous food crops. Each region showed a different trend in cassava cultivation increments, interest and production. At the coastal region, cassava production started in 1950, picked up slowly until 1993, and then there was a rapid adoption rate up to 2006 (Figure 4). A similar trend was observed in eastern region but in western, there was a steady increase in adoption rate of cassava cultivation since its introduction (Figures 5 and 6). The importance of cassava relative to other food crops across the three regions was assessed. Relative to other food crops, 66.7% of respondents ranked cassava 2nd at the coastal region while 37.5% and 57% of respondents in eastern and western regions ranked it 5th and 1st respectively.

Cropping Systems and Cassava Varieties

At the coastal, western and eastern regions, 92%, 67% and 65% of the respondents intercrop cassava with other crops as is depicted in Figure 7, while 8%, 33% and 35% grow it as a sole crop respectively. The commonly used cassava varieties at the coast were Kibandameno (55%) and Kaleso (34%). In Eastern region, 78% of the varieties grown were unknown though there were a few farmers (6.3%) growing an improved variety locally known as Mucericeri. In western Kenya, many of cassava varieties were recorded with Migyera (23%) and SS4 (23%) being more preferred in the region. Other varieties available in the region were Magana (12%), Mucericeri (8%) and Adhiambo Lera (8%). The presence of more varieties in the western region is attributed to the cross border trade with Uganda. On adoption of improved cassava varieties, western region was leading with 77% followed by coast with 30% and eastern with 13%. At the coast, the main source of planting material was from own fields (44%) and other farmers (29%). In the eastern region, the main source of planting material was from other Complimentary Contributor Copy Cassava Production and Utilization in the Coastal … 47 farmers (53%) and from own fields (23%) while in the western region, the main source was from the Ministry of Agriculture (77%). At the coast 91% of the respondents, plant cassava during the April rains while 72% in eastern, plant cassava during the October rains. In the western region, cassava is planted in both seasons.

Figure 7. Cassava intercropped with other cereals. This is a common practice with farmers in all the cassava growing zones.

30 Number of farmers of Number

2000 2003

2001 2005

2002 2006

1950 1964 1969 1980 1985 1989 1993 1996

1957 1966 1975 1982 1987 1990 1994 1995 1997

1960 1968 1977 1983 1988

Year

Figure 3. Overall trend in cassava growing in Coast, Eastern and Western Provinces of Kenya for a period of 56 years.

Complimentary Contributor Copy 48 C. M. Githunguri, M. Gatheru and S. M. Ragwa

15 Number of farmers of Number

1950 1957 1968 1975 1980 1983 1987 1988 1989 1993 1997 2001 2002 2003 2005 Year

Figure 4. Overall trend in cassava growing in Coast Province of Kenya for a period of 55 years.

20 Number of farmers of Number

2000

2001

2002

2003

2005

2006

1957

1964

1966

1977

1982

1983

1985

1988

1990

1994

1995

1996

1997

Year

Figure 5. Overall trend in cassava growing in Eastern Province of Kenya for a period of 51 years.

4 Number of farmers of Number

1960 1969 1980 1988 1990 1994 1995 2001 Year

Figure 6. Overall trend in cassava growing in Western Province of Kenya for a period of 41 years.

Complimentary Contributor Copy Cassava Production and Utilization in the Coastal … 49

Main Reasons for Growing Specific Cassava Varieties

In the coastal region, farmers preferred high yielding varieties. Other preferred parameters were maturity period, taste (sweet taste), pests and disease resistance. In the eastern region farmers consider marketability (42%) as the most important parameter followed by taste 17%. High yielding varieties were also preferred. In western, 55% of respondents considered marketability as the most important parameter followed by resistance to pests and diseases and, earliness in maturity.

Major Constraints to Cassava Production

At the coast, 23% of the respondents considered lack of market as the major constraint followed by pests and diseases (16%) and destruction by large mammalian pests (11%). In eastern, 15% of the respondents reported drought as the major constraint followed by lack of market (13%) and pests and disease (42%). In western, the major constraints were large mammalian pests (12%), weeds (12%), lack of planting materials (8%) and insect pests (3%).

Pests and Disease Control Measures

Only 10% of respondents used mechanical methods to control termites at the coast. Except for eastern region where 7% of respondents used chemicals to control termites, there were no chemical control methods in the other regions. In the western region, 50% and 25% of respondents use biological control for cassava green mite and whiteflies respectively. For control of diseases, 18% of respondents at the coast used mechanical methods to control cassava mosaic virus while in eastern and western regions, there were no control measures taken.

Cassava Utilization

One hundred percent, 22% and 13% of respondents at the coast, eastern and western regions, respectively, use cassava leaves as vegetable. Besides being used as vegetable, 100% of respondents in western and 67% in eastern use cassava leaves as livestock feed. At the coast, 36% of respondents use cassava stems as firewood and 32% sell stems as planting materials to other farmers. In eastern, 33% of respondents use cassava stems as firewood while 30% use stems as planting materials. In western, 50% of respondents sell cassava stems as planting materials while 50% use it as firewood. Figure 7 shows a popular method of preserving clean planting materials by farmers in the coastal region. It was noted that 100% of respondents at the coast use cassava roots as family food, for sale and as gifts while 19% uses it purely as family food. In eastern, 100% of respondents use cassava as family food and for sale in local markets while 19% give cassava as gifts. In western Kenya, 100% of respondents use cassava as family food and for sale in local markets.

Complimentary Contributor Copy 50 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Figure 7. This is one method of preserving clean planting materials by farmers mostly in the coast region.

Sale of Cassava Roots

At the coast, 46%, 32% and 21% of respondents making decision on the sale of cassava were men, women and both sexes respectively. In eastern, 67% of respondents reported that decision on sale of cassava is made by women, 24% by men and 10% by both. In western, 25% of respondents reported that decision on sale of cassava was made by men, 25% by women and 50% by both. After the decision on sale had been made, 71% of respondents at the coast reported that actual sales were done by women, 4% by men and 25% by both. In eastern, 86% of cassava sale was by women, 10% by men and 4% by both sexes. In western Kenya 100% of respondents reported that cassava sale is done by women. Figure 8 shows a cassava trader narrating his mixed fortunes and misfortunes in the cassava business. At the coast 12% of respondents sold their cassava at the farm gate, 65% at the local markets, 15% to other places (e.g., Tapioca in Mazeras) and 8% at both farm gate and local markets. In eastern Kenya, 29% of respondents sold their cassava at farm gate, 65% at the local markets, 6% to different destinations while in western Kenya 25% sold their cassava at farm gate and 75% at the local markets. Ninety three percent (93%) of farmers at the coast sold their produce on cash basis and 7% on credit (mainly to big processors/factories). In eastern, 85% sold their cassava on cash basis, 5% on credit and in kind. In western Kenya, 100% of respondents reported that sales were on cash basis. At the coast, the main dealers in cassava sales were wholesalers (21%), retailers (25%) and both wholesalers and retailers (25%). In eastern, the main buyers were local consumers (53%) while 32% were both retailers and consumers. In western Kenya, 63% of both retailers and local consumers were the main buyers of cassava followed by both wholesalers and consumers at 13%.

Complimentary Contributor Copy Cassava Production and Utilization in the Coastal … 51

Figure 8. Cassava trader narrates fortunes and misfortunes in cassava business. There is no exact weight measure for a 90-kg sack during peak season hence sales are at random.

Cassava Processing

Major cassava products processed at the coast were fried cassava chips (cassava French fries) (21%) and cassava flour (11%). Other processed products included cassava crisps, half- cakes and composite flour (a mixture of cassava and other cereals). In eastern province, 3% of processors make cassava chips and 10% cassava flour. In western region 38%, of processors make cassava chips (dried chopped and sun dried cassava) and 38% composite flour (cassava mixed with other cereals). Other products include crisps, chapati and starch at 13%.

Quality Characteristics Mostly Preferred for Cassava Products

At the coast, 19% of respondents preferred white colour as the most important characteristics. Fiber-free cassava varieties and good taste were preferred by 8% of the respondents while size and colour were preferred by 8% of others. In eastern region, white colour and texture were preferred by 38% and taste by 13% of the respondents respectively. In western, moisture content (properly dried cassava chips) was preferred by 17% white colour by 67% of the respondents. At the coast region, 31% and 26% of respondents preferred Kibandameno and Kaleso varieties respectively for processing cassava into various products. In eastern, 67% of respondents preferred all varieties for processing while in western, 51% preferred Migyera followed by SS4 and Magana at 17% each.

Complimentary Contributor Copy 52 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Farmers’ Knowledge of Other Products Processed

In western, 78% of respondents were aware of other cassava products made elsewhere through their local markets while 18% had learnt about them through seminars organized by the Ministry of Agriculture and Farmer Field Schools. In eastern, 29% of respondents had learnt about the products from KARI and 24% through NGOs and community-based organizations. In the coastal region, 44% of respondents had learnt about other products from supermarkets at Mombasa.

Constraints in Processing

In the coastal region, 33% of farmers lacked appropriate equipment to process various cassava products. Other reasons for not processing cassava included lack of capital (22%) and knowledge (22%). In eastern, 56% of the respondents reported the major reason for not processing as lack of knowledge, while 18% attributed it to non-availability of cassava for processing. Other reasons included lack of appropriate equipment (6%). In western Kenya, 29% of respondents faced challenges of new technology adoption in processing. Other reasons identified included lack of knowledge and expensive processing oil.

CONCLUSION

The study showed that the importance of cassava relative to other food crops differed across the three regions. At the coastal, eastern and western regions it was ranked second, fifth and first respectively. In the western region, there were more improved cassava varieties than in the other regions. This can be attributed to access to extension services and exchange of varieties across the Ugandan border. In the coastal region, the major constraint to production was lack of market while in the eastern region, the major constraint was drought and in western, the major constraints were wild animals and weeds. There was more utilization of cassava as family food in western than in coastal and eastern regions. In all the regions, the sale of cassava roots and cassava-based products was carried out by women and on cash basis. On processing of cassava and cassava based products, western region was leading followed by coastal and eastern region last. The western region was leading in the processing of dried cassava chips and composite flour. The coastal region was leading in the processing of fried cassava chips, crisps and pure flour. The eastern region was the last in processing with a few respondents making fried cassava chips and pure cassava flour. The quality characteristics that were preferred for cassava and cassava-based products were mainly white colour, fibre-free cassava roots and sweet taste. There was more awareness on processed products in western where most respondents had heard about products processed elsewhere and a few had learnt through seminars organized by the Ministry of Agriculture. At the coast, the main constraint in processing was lack of appropriate equipment and capital. In eastern, the main constraint in processing was lack of knowledge and enough cassava. In western, the main constraint was lack of modern processing equipment.

Complimentary Contributor Copy Cassava Production and Utilization in the Coastal … 53

REFERENCES

Bellon, M.R. 2001. Participatory Research Methods for Technology Evaluation. A Manual for working with Farmers. Mexico, D.F.: CIMMYT, 93p. Ekanayake, I.J., D.S.O. Osiru and M.C.M. Porto. 1997a. Morphology of cassava. IITA Research Guide 61: 1 - 30. Ekanayake, I.J., D.S.O. Osiru and M.C.M. Porto. 1997b. Agronomy of cassava. IITA Research Guide 60: 1 - 30. Ekanayake, I.J. 1998. Screening for abiotic stress resistance in root and tuber crops. IITA Research Guide 68: 46pp. FAO, 1990: Roots, Tubers, Plantains and Bananas in human nutrition. FAO, Rome, Italy. Goering, T.J. 1979. Tropical root crops and rural development. World Bank Staff working Paper No. 324. Washington, D.C., World Bank. Fliert, E. van de and A.R. Braun. 1999. Farmer Field School for Integrated Crop Management of Sweetpotato. Field Guides and Technical Manual. Andi Offset, Yogyakarta, Indonesia: CIP,III-101p. Githunguri, C.M. 1995. Cassava food processing and utilization in Kenya. In: Cassava food processing. T. A. Egbe, A. Brauman, D. Griffon and S. Treche (Eds.) CTA, ORSTOM, pp119-132. Githunguri, C. M., I. J. Ekanayake, J. A. Chweya, A. G. O. Dixon and J. Imungi. 1998a: The effect of different agro-ecological zones on the cyanogenic potential of six selected cassava clones. Post-harvest technology and commodity marketing, IITA, 71-76pp. Githunguri, C.M., I.J. Ekanayake, J.A. Chweya, A. G. O. Dixon and J.K. Imungi. 1998b. The effect of different agroecological zones and plant age on the cyanogenic potential of six selected cassava clones. In: (R.S.B. Ferris Ed.) Post-harvest technology and commodity marketing. Proceedings of a postharvest conference held on 2 November – 1 December 1995, Accra, Ghana. IITA, Ibadan, Nigeria, 71 - 76pp. Githunguri, C.M. 2002. The influence of agro-ecological zones on growth, yield and accumulation of cyanogenic compounds in cassava. A thesis submitted in full fulfilment for the requirements for the degree of Doctor of Philosophy in Crop Physiology, Faculty of Agriculture, University of Nairobi, 195pp. Githunguri, C.M., Y.N. Migwa, S.M. Ragwa and M.M. Karoki. 2003. Cassava and sweetpotato agronomy, physiology, breeding, plant protection and product development. Root and Tuber Crops Programme in KARI-Katumani. Paper presented at the Joint Planning meeting organized under the Eastern Province Horticulture and Traditional Food Crops Project, held at Machakos, Kenya on 5th 7th March 2003, 5p. Githunguri, C.M. and Y.N. Migwa. 2003. Sweetpotato Feathery Mottle Virus Resistance and Yield Characteristics of Different Sweetpotato Cultivars in Machakos and Makueni Districts of Kenya. In: Githunguri C.M., Kwena, K., Kavoi, J., Okwach, E.W., Gatheru, M. and Abok, J.O. (Eds.). Kenya Agricultural Research Institute, KARI Katumani Research Centre annual report 2002. Pp. 91 - 95. Githunguri, C.M. 2004. Farmers’ Participatory Perspectives on Sweetpotato Cultivars in Kathiani Division of Machakos District, Kenya. In: Book of Abstracts of the 9th Triennial Symposium of the International Society for Tropical Root Crops- Africa Branch (ISTRC-AB), Mombasa, Kenya, 31st October – 5th November 2004. 84pp.

Complimentary Contributor Copy 54 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Githunguri, C. M, E. G. Karuri, J. M. Kinama, O. S. Omolo, J. N. Mburu, P. W. Ngunjiri, S. M. Ragwa, S. K. Kimani and D. M. Mkabili. 2006. Sustainable Productivity of the Cassava Value Chain: An Emphasis on Challenges and Opportunities in Processing and Marketing Cassava in Kenya and Beyond. KAPP Competitive Agricultural Research Grant Fund, pp. 106. IITA. 1982. Management practices for production of cassava planting materials. IITA tuber and Root crops production Manual series, 244pp. IITA. 1990a. Cassava in Tropical Africa. Reference Manual IITA, 176pp. IITA. 1990b. Targeting cassava Breeding and Selection. In: Proceedings of the fourth West and Central Africa Root Crops workshop, held in Lome, Togo, 12-16 December 1988. IITA Meeting Reports Series 1988/6, pp. 27-30. Kadere, T.T. 2002. Marketing opportunities and quality requirements for cassava starch in Kenya. In Proceedings of regional Workshop on improving the cassava sub-sector, held in Nairobi, Kenya, April 2002, 8-18, pp. 81 - 86. KARI, 1995: Cassava Research Priorities at the Kenya Agricultural Research Institute, Cassava Priority Setting Working Group. Lusweti, C.M., W. Kiiya, C. Kute, A. Laboso, C. Nkonge, E. Wanjekeche, T. Lobeta, S. Layat, A. Kakuko, and E. Chelang. 1997. The farming systems of Sebit: In: Summary from PRA activities. Pp. 54 - 67. Mbwika, J.M 2002. Cassava sub-sector analysis in the Eastern and Central African region. In Proceedings of regional Workshop on improving the cassava sub-sector, held in Nairobi, Kenya, April 2002, 8-18, pp. 8-18. Ministry of Agriculture, 1999: Provincial Annual Reports. Ministry of Agriculture (MoA). 2005. Strategy for Revitalizing Agriculture 2004 – 2014. Ministry of Agriculture, 23pp. Nweke, F. I., D. S. C. Spencer and J. K. Lynam. 2002: Cassava transformation. International Institute of Tropical Agriculture. 273p. Obukosia, S.D., Muriithi, and R.S. Musangi. 1993. Biotechnological approach to the improvement of root, tuber and horticultural crops in Kenya. Production constraints and potential solutions. Proceedings of the national agricultural biotechnology workshop, Nairobi, PP. 92-106. Osiru, D.S.O., M.C.M. Porto, and I.J. Ekanayake. 1995. Physiology of cassava. IITA, Research Guide 55: 3 - 19.

Chapter 4

SOCIO-ECONOMIC DETERMINANTS OF MODERN TECHNOLOGY ADOPTION AND THE INFLUENCE OF FARM SIZE ON PRODUCTIVITY AND PROFITABILITY IN CASSAVA PRODUCTION: A CASE STUDY FROM SOUTH-EASTERN NIGERIA*

Chidiebere Daniel Chima† and Sanzidur Rahman‡ School of Geography, Earth and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth, UK

ABSTRACT

The chapter investigates the influence of socio-economic factors on the adoption of individual components of modern agricultural technology (i.e., HYV seeds and inorganic fertilizers) in cassava and also examines farm size–productivity and farm size– profitability relationships of cassava production in South-eastern Nigeria including a discussion of constraints in the cassava sector. The hypotheses of the study are that farmers selectively adopt components of modern agricultural technology depending on their socio-economic circumstances and inverse farm size–technology adoption, size– productivity and size–profitability relationships exist in cassava production. The research is based on an in-depth farm-survey of 344 farmers from two states (243 from Ebonyi and 101 from Anambra states) of South-eastern Nigeria. The results show that the sample respondents are dominated by small scale farmers (78.8% of total) owning land less than 1 ha. The average farm size is small estimated at 0.58 ha. The study clearly demonstrated that inverse farm size–technology adoption and farm size–productivity relationships exist in cassava production in this region of Nigeria but not inverse farm size–profitability

* The chapter was developed from the first author’s PhD thesis submitted at the School of Geography, Earth and Environmental Sciences, University of Plymouth, UK in 2015. The data required for this project was generously funded by the Seale-Hayne Educational Trust, UK. All caveats remain with the authors. † Address for correspondence: Dr. Chidiebere Daniel Chima, School of Geography, Earth and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, Phone: +44-7883005944; +44-1752- 585911, Fax: +44-1752-584710, E-mail: [email protected]. ‡ Phone: +44-1752-585911, Fax: +44-1752-584710, E-mail: [email protected]. Complimentary Contributor Copy 56 Chidiebere Daniel Chima and Sanzidur Rahman

relationship. The level of modern technology adoption is low and mixed and farmers selectively adopt components of technologies as expected and use far less than recommended dose of fertilizers. Only 20.35% of farmers adopted both HYV cassava stem and fertilizers as a package. The bivariate probit model diagnostic reveals that the decision to adopt modern technologies are significantly correlated, implying that univariate analysis of such decisions are biased, thereby, justifying use of the bivariate approach. The most dominant determinant of modern technology adoption in cassava is farming experience and remoteness of extension services depresses adoption. A host of constraints are affecting Nigerian agricultural sector, which includes lack of extension agents, credit facilities, farm inputs, irrigation, value addition and corruption, lack of support for ADP staff and ineffective government policies. Policy implications include investment in extension services, provision of credit facilities and other infrastructures (e.g., irrigation, ADP staff), training of small farmers in business skills, promotion of modern technology as a package as well as special projects (e.g., Cassava Plus project) in order to boost production of cassava at the farm-level in Nigeria.

Keywords: modern technology adoption, farm size categories, profitability, bivariate probit model, cassava production, Nigeria

1. INTRODUCTION

Agriculture has been the mainstay of the economy of Nigeria and many other African countries, providing employment, food and source of livelihood for their rural and increasing population (Nwa, 2003). In Nigeria, the agricultural sector is the major employer, with nearly 70% of the country’s labour force engaged in one form of agriculture or the other (Abolagba et al., 2010). The sector is still characterised by small scale farmers using traditional farming methods with very low level of mechanization and modern technologies leading to low level of productivity (Chima, 2015). In sub-Saharan Africa, cassava is very important not just as a food crop but as a major source of cash income for a large population (NISER 2013). It is grown in over 90 countries and is the third most important source of calories in the tropics, after rice and maize (Tsegia et al, 2002). It is a staple for half a billion people in Africa, Asia and Latin America. Cassava is grown mainly by poor farmers, many of them women and often grown in marginal lands. For these people and their families, cassava is vital for food availability and income generation and it is a major source of commercial feed, fibre for paper and textile manufacturers and starch for food and pharmaceutical industries (Tsegia et al, 2002 and CGIAR, 2011). According to Westby (2008), world cassava demand is projected to reach 275 million tonnes by 2020 while Africa now produces about 62% of the total world production with Nigeria being the largest producer with 54 million tonnes of output in 2013 (FAOSTAT, 2015). Despite this, less than 5% of the output produced in Nigeria is used for industries while 95% is used for human consumption (NISER, 2013). In spite of the position of Nigeria as the leading producer of cassava in the world, the country still imports significant quantities of cassava products and by-products, such as starch, flour and sweeteners (Olukunle, 2016). This constituted a drain on the foreign exchange resources of the country given the recent collapse of world crude oil market. What is more worrisome is that a good proportion of these raw materials can be sourced from agricultural produce locally. For instance, in 2008 and 2011, raw material imports into Nigeria averaged $8.3 billion (18.9%) and $8.2 billion Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 57

(12.5%) respectively. As a proportion of total raw materials imported into the country, industrial agricultural raw materials accounted for 26.6% ($2.2 billion) in 2008 and rose sharply to 69.8% (£5.7 billion) in 2011 (Sanusi 2012). This trend is unsustainable given the declining economic condition of the country and hence the urgent need to diversify the economy and allowing the agricultural sector to play its role as a main source of foreign exchange. The average yield level of cassava in Indonesia is 19 mt/ha which is much higher than that in Nigeria which is estimated at 14.7 mt/ha (Nang’ayo et al., 2007). In contrast Thailand is the largest exporters of cassava products, exported a little under $1 billion USD of cassava products in 2009, has an efficient cassava value-added chain (APFCTN, 2014). A comparison of the Nigeria and Thai cassava sectors reveals that the cassava sector in Nigeria is plagued by low productivity; with average yields of 11.7 ton/ha, compared to 22 ton/ha in Thailand. Also Thai cassava yields have increased @ 1.7% per year over the last 15 years while yields in Nigeria have stagnated during the same period (FAOSTAT, 2009). The low productivity of the cassava sector in Nigeria has led to high costs per unit of production. The cost of cassava root production per ton is USD 10 higher in Nigeria than in Thailand. This has made Nigerian cassava products unable to compete with imported substitutes leading to a lack of demand for cassava by industrial users who prefer to import cheaper raw materials (APFCTN, 2014). Although both are tropical countries with similar production constraints such as low level of input use, high variability in commodity price and lack of adequate infrastructure (Sugino and Mayrowani, 2009), higher productivity in Thailand is mostly due to higher incidence of fertilizer use and mechanization. While in Nigeria, there is little or no use of chemical fertilizer in cassava production and farming is done by manual labour; especially weeding operation (Chima, 2015). Given this backdrop, this chapter is aimed at investigating the influence of socio- economic factors on the adoption of individual components of modern agricultural technology (i.e., HYV cassava stem and inorganic fertilizers) in cassava and to examine farm size–productivity and farm size–profitability relationships in cassava production at the farm- level in South-eastern Nigeria including a discussion of constraints in the cassava sector. The hypotheses of the study are that farmers selectively adopt components of modern agricultural technology depending on their socio-economic circumstances and inverse farm size– technology adoption, size–productivity and size–profitability relationships exist in cassava production. Bivariate probit model were used to determine the socio-economic determinants of modern agricultural technology adoption in cassava production given its advantage over univariate probit of allowing the evaluation of more than one technology (HYV stem and inorganic fertilizer) at the same time (Rahman, 2003 and Chirwa, 2005). The rest of the chapter is divided into seven sections. Section 2 presents the methodology, including study area, source of data and analytical framework. Sections 3, 4 and 5 present the results of farm-size technology adoption, farm-size productivity and farm-size profitability relationships respectively. Section 6 presents the results of the determinant of modern agricultural technology adoption in cassava. Section 7 provides discussion of the constraints of cassava production and modern agricultural technology adoption in the study area. Finally, Section 8 provides conclusions and policy implications.

Complimentary Contributor Copy 58 Chidiebere Daniel Chima and Sanzidur Rahman

2. METHODOLOGY

2.1. Study Area and Data

The primary study area is south-eastern Nigeria. Two states were chosen, Ebonyi and Anambra states. Ebonyi with 13 Local Government Area (LGA) is a rural/agrarian state was created on 1st October 1996 from Enugu and Abia states; and has a total landmass of 5,935 sq km of which 80% is rich in arable land (Nwibo, 2012). It has an estimated population of 2,173,501 with a growth rate of 3.5% per annum (NPC, 2006). The 70% of population are rural and the economy is primarily dependent on agriculture, which contributes about 90% to Gross Domestic Product (GDP). About 75% of its people are engaged in one form of farming or another and are mostly subsistence farmers (Ebonyi Agricultural Policy 2010). Anambra is more urban and was carved out of the old Anambra state in 1991 and has a land area of 4,415.54 sq km and population of 4.18 million; 70% of the land is rich and suitable for agricultural production (Nkematu, 2000 and NPC, 2006). The state has 21 Local Government Areas (LGA), consisting of 177 autonomous communities. The climate can generally be described as tropical with two identifiable seasons, rainy or wet and dry seasons. Farming is the predominant occupation of the rural people, the majority of whom are small holder subsistence farmers. Data used for the study were drawn from the two states; Ebonyi and Anambra states of Nigeria. Based on the cell structure developed by Agricultural Development Programme in Nigeria, three local government areas (LGAs) from each state were randomly selected. Then, 10 communities/villages from each of the LGA were then chosen randomly. Next farmers were chosen from these communities using a simple random sampling procedure. The total number of farm households in each village formed the sample frame. Then the sample size (n) of the household units in the study area is determined by applying the following formula (Arkin and Colton, 1963):

Nzpp2 (1) n  Ndzpp22(1)

Where n = sample size; N = total number of farm households; z = confidence level (at 95% level z = 1.96); p = estimated population proportion (0.5, this maximizes the sample size); and d = error limit of 5% (0.05). Application of the above sampling formula with the values specified in fact maximizes the sample size and yielded a total sample of 344 cassava farmers (Ebonyi State = 243; Anambra State = 101) in the study areas used for this study.

2.2. Profitability and Benefit Cost Ratio (BCR) Analytical Framework

This section discusses the framework to analyse profitability and Benefit Cost Ratio. This is done by analysing Total Variable Cost (TVC); Total Fixed Cost (TFC), Total Revenue (TR), Gross Margin (GM), Net Profit and Benefit Cost Ratio (BCR) for cassava farm enterprise. The key variables that are used to determine profitability of farm enterprise in this Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 59 study are defined and explained in this section. Also BCR are defined and explained. The key variables are: Variable Cost (VC): This is the cost that changes with the level of production of the farmer. i.e., if the farmer increases his/her farming activities or scale up his/her farming then the variable cost is likely to increase too. In this study, the variable cost is the sum total of total material input cost, total labour cost and transportation cost (Table 5.1, Section A). The services of farm equipment and tools are not captured in the variable cost because none of the farmers have access to farm machinery or tools; all farmers still use crude farm implements like hoes and cutlasses. Also there is no specific farm house. Instead farmers store their farm products in their residential house or local barns. Unit price of output: The unit price used to determine the Total Revenue (TR) is the actual selling price for the farmers who sold their farm output. For the farmers who did not sell their farm produce, the mean selling price of those who sold theirs were imputed to determine their TR. Total Revenue (TR): This is the total output of the farm enterprise multiplied by their market unit selling price for the farmers who sold their farm produce and the mean market unit selling price for the farmers who did not sell their farm produce. The TR varies from one farm enterprise to the other. Gross Margin (GM): This is the difference between the Total Revenue (TR) of each farm enterprise and the Total Variable Cost (TVC). (Note: GM=TR-TVC). Fixed Cost (FC): These are the costs associated with farm production but are fixed, which means that they remain the same throughout the production period. For this study the fixed costs are the mean cost for farmers renting-in land for farm production and mean interest paid on any loan acquired for farm production by the farmers who have loan. It is important to note that the mean cost of renting-in land and loan interest payment are just for the farmers who rented-in land or had any loan, to avoid distort comparisons with farmers that do not use these facilities. Net Profit (NP): This is the difference between Gross Margin (GM) and the Total Fixed Cost (TFC) for each farming enterprise (Note: NP=GM-TFC) Benefit Cost Ratio (BCR): This is the Total Revenue (TR) for each farming enterprise divided by their Total Cost (TC). It is a ratio and implies the return for every Naira (Nigeria currency) invested in the farm enterprise. The BCR value is good if it is positive and has the value of 1 or more. Therefore, the higher the BCR value, the better the return on every additional naira invested on that farm enterprise (Note: BCR=TR/TC).

2.3. The Theoretical Framework: Bivariate Probit Model

Many studies have analysed the determinants of adopting modern/improved agricultural technologies (including HYVs of rice, wheat and/or maize, cassava) by farmers in Nigeria and other developing countries. These studies are largely univariate probit or Tobit regressions of technology adoption on variables representing the social economic circumstances of farmers (e.g., Hossain 1989; Ahmed and Hossain 1990; Shiyani et. al 2002; Rahman 2003, Floyd et. al. 2003; Ransom et. al. 2003; Barrett 2004, Chirwa 2005). The implicit theory underpinning such modelling is the assumption of utility maximization by rational farmers which is described below. Complimentary Contributor Copy 60 Chidiebere Daniel Chima and Sanzidur Rahman

We denote the adoption of HYV as dv and the adoption of fertilizer as 푑푓; where 푝 = 1 for adoption and 푝 = 0 for non-adoption. The underlying utility function which ranks the preference of the 푖푡ℎ farmer is assumed to be a function of farmer as well as farm-specific characteristics, Z (e.g., family size, farming experience, farm size, extension contact etc.) and an error term with zero mean.

푈𝑖1 (푍) = 훽1푍𝑖 + 휀𝑖1 For adoption and

푈𝑖0 (푍) = 훽0푍𝑖 + 휀𝑖0 For non-adoption

Since the utility derived is random, the 푖푡ℎ farmer will adopt an agricultural system if and only if the utility derived from the adoption is higher than non-adoption; i.e., 푈𝑖1 > 푈𝑖0 Thus, the probability of adoption of the 푖푡ℎ farmer is given by (Nkamleu and Adesina 2000; Ajibefun, et al. 2002 and Rahman 2008):

푝 (퐼) = 푝(푈𝑖1 > 푈𝑖0)

푝(퐼) = 푝(훽1푍𝑖 + 휀𝑖1 > 훽0푍𝑖 + 휀𝑖0)

푝(퐼) = 푝(휀𝑖0 − 휀𝑖1) < 훽1푍𝑖 − 훽0푍𝑖) 푝(퐼) = 푝(휀𝑖 < 훽푍𝑖)

푝(퐼) = ∅(훽푍𝑖)∅

Where ∅ is the cumulative distribution function for 휀 the functional form of ∅ depends on the assumption made for the error term 휀, which is assumed to be normally distributed in a probit model. Thus for the 푖푡ℎ farmer, the probability of the adoption of a diversified HYV and fertilizer respectively is given by:

훽푍 1 −푡2 ∅ (훽푍 ) = ∫ 푖 푒푥푝 { } 푑푡 (1) 푑푣 𝑖 훼 √2휋 2

훽푍 1 −푡2 ∅ (훽푍 ) = ∫ 푖 푒푥푝 { } 푑푡 (2) 푑푓 𝑖 훼 √2휋 2

The two equations can each be estimated consistently with the single-equation probit method but such a commonly used approach is inefficient because it ignores the correlation between the error terms 휀푑푣 푎푛푑 휀푑푓 of the underlying stochastic utility function of HYV and fertilizer respectively. We apply the bivariate probit model in order to circumvent this limitation. Therefore, the bivariate probit model which is based on the joint distribution of the two normally distributed variables and is specified as follows: (Greene 2003 and Rahman 2008):

2 2 2 (3) 1    )2( /(  )1(2 f dfdv ),(  e  dv df dfdv  2 2 dfdv 1 

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 61

푑푣−휇푑푣 푑푓−휇푑푓 휀푑푣 − 푎푛푑 휀푑푓 − 휎푑푣 휎푑푓

Where 푝 is the correlation between dv and df, the covariance is 𝜎푑푣,푑푓 = 𝜌𝜎푑푣𝜎푑푓; 푤ℎ푖푙푒 휇푑푣, 휇푑푓, 𝜎푑푣 푎푛푑 𝜎푑푣 are the means and standard deviations of the marginal distributions of dv and df respectively. The distribution is independent if and only if 푝 = 0. The full maximum likelihood estimation procedure is utilized using the software program NLOGIT-4 (Economic software, Inc. (ESI) 2007). Therefore, the bivariate probit model is developed to empirically investigate the socioeconomic factors underlying the decision to adopt HYV seed and/or inorganic fertilizer. The dependent variable is whether the farmer adopts HYV seed and/or inorganic fertilizer; for HYV represented by dv, the variable takes the value 1 if the farmer adopts HYV and 0 if otherwise. Similarly, for inorganic fertilizer represented by 푑푓; the variable takes the value 1 if the farmer adopts fertilizer and 0 if otherwise.

3. AGRICULTURAL TECHNOLOGY ADOPTION AND FARM-SIZE RELATIONSHIPS

This section discusses the results of agricultural technology adoption patterns by farm size categories of the respondents. It evaluates whether agricultural technology is being adopted as a package and/or whether the inverse farm size-technology adoption relationship exists in the study area. Taken as a whole, Table 3.1 shows that only 20.35% of the respondents adopted agricultural technology as a package (fertilizer and HYV stem) in the study area, of which most of the adopters are small scale farms (80%) and the others are medium (14.29%); large (5.71%) scale farms respectively. This finding is consistent with Madukwe, et al. (2002) and Agwu, (2004) who noted a low adoption of agricultural technology among cowpea farmers in his study of factors influencing adoption of improved cowpea production technology in Nigeria.

Table 3.1. Agricultural technology adoption pattern by farm-size

Farm Agricultural Technology Adoption Pattern in Percentage Category Non Only Fertilizer Only HYV Adopters of Total Adopters of Adopters Adopters Adopters Both Technology Small 50.55 9.23 19.56 20.66 49.45 (137) (25) (53) (56) (271) Medium 43.90 4.88 26.83 24.39 56.10 (18) (2) (11) (10) (41) Large 62.50 9.38 15.63 12.50 37.50 (20) (3) (5) (4) (32) Total 50.87 8.72 20.06 20.35 (175) (30) (69) (70) (344) Source: Field Survey 2011 (NB: the parentheses are predicted estimated frequency).

Similarly, only 8.72% and 20.06% of respondents adopted only one element of the technology package, which are inorganic fertilizer technology and HYV stem technology Complimentary Contributor Copy 62 Chidiebere Daniel Chima and Sanzidur Rahman respectively. Also most of the farmers who adopted either of the elements of the technology package are small scale farmers, and largely adopted HYV stem (20.06%) than fertilizer (8.72%). This may be because of the cost of HYV stem relative to that of fertilizer (Chima, 2015). Table 3.1 also reveals that high numbers of the respondents (50.87%) did not adopt any of the agricultural technology. This finding is consistent with studies such as Ajayi, (1996), Madukwe, et al. (2002) and Agwu, (2004) that noted low level adoption of agricultural technology in Nigeria in their respective studies. Also, across the farm categories, almost a third of large scale farmers and half of small and medium scale farmers did not adopt any technology. This highlights the main issue of low agricultural productivity in Nigeria and this finding is consistent with studies such as Obasi, et al. (2013), Igwe, (2013) and Agwu, (2004) that noted low productivity, low profitability of farm enterprises; low and non- adoption of agricultural technologies in their respective studies. Across the farm categories, most of the farmers who adopted both technologies (80%) and either of the technologies (83.33% and 76.81%, respectively) are small scale farmers. The table clearly shows that across the farm categories, small farmers are more likely to adopt HYV cassava stem than inorganic fertilizer. Also, only 49.45% of the small scale farmers, 56.10% and 37.50% of the medium and large scale farmers adopted any kind of agricultural technology in the study area. Adoption of agricultural technology as a package is the main principal behind the success of Green Revolution in Asia. This principal is not being applied in the study area and this may be due to the constraints (see section 7 for details) associated with the adoption of agricultural technology in the study area (Chima, 2015). The table clearly demonstrates the key finding, i.e., an inverse farm-size agricultural technology adoption exists in the study area. In other words, the small scale farmers tend to adopt agricultural technology relative to medium and large scale farmers.

4. CROP PRODUCTIVITY AND FARM SIZE RELATIONSHIP AND THEIR INFLUENCING FACTORS

This section discusses the result of one of the key hypothesis of inverse farm size– productivity relationship in the study area. It also assesses crop productivity in relation to key influencing factors. This is done with a view to identify the variables that will be included in the bivariate probit model. Yield was chosen as the main yardstick to measure crop productivity level because of the direct effect of agricultural technology adoption has on this variable (Rahman, 2011).

4.1. Crop Productivity by Farm Size

The productivity of cassava grown by farmers in the study area and how they relate to their farm-size category is presented in Table 4.1. The table shows that 70.64% of the respondents are in Ebonyi and 29.36% in Anambra states. Analysis of farm-size categories shows that 78.8% are small scale farmers with farm size of 0.1-2.0ha, 11.9% medium scale (2.01-3.0ha) and 9.3% are large scale farmers with ≥ 3.01ha farm size.

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 63

The mean yield for cassava in all areas is 12424.58kg/ha, with 12330.81kg/ha and 12650.18kg/ha for Ebonyi and Anambra states, respectively. The yield level is similar to those estimated by National Programme on Agricultural and Food Security (NPAFS) 2009 Crop Yield Report (NPAFS, 2010) and the reasons for the differences in the states may be related to the level of farm input usage and production practices as noted in Chima, 2015. The one-way ANOVA result shows an inverse relationship between farm size-productivity with small scale farms producing the maximum yield and the large scale farms producing the least. This confirms the hypothesis of inverse farm size-productivity relationship of cassava production in the study area (Chima, 2015). Overall, Anambra state has a higher yield per hectare over Ebonyi state; this may be because farmers in Anambra state have higher mean farm input usage (fertilizer, pesticide, ploughing labour etc.) than those from Ebonyi state and this is reflected in their yield per hectare. This indicates that a higher level of input usage (agricultural technology adoption) given other factors may lead to higher productivity level. Also, as mentioned before, the table shows that small scale farmers have the best yield per hectare in the study area which is consistent with the literature and similar studies like Rahman, (2011); Fabusoro et al, (2010) and Igwe, (2013) who showed that the small scale farms are better managed and productive in developing countries.

Table 4.1. Productivity of respondents by farm-size

Study Area Farm Size Category Cassava Yield (Kg/ha) % of Farmer Ebonyi (243) Small 12405.13 74.49 (181) Medium 12244.92 15.64 (38) Large 11906.31 9.88 (24) All 12330.81 70.64 (243) Anambra (101) Small 12724.15 89.11 (90) Medium 12000.00 2.97 (3) Large 12061.81 7.92 (8) All 12650.18 29.36 (101) All Areas Small 12511.08 78.78 (271) (344) Medium 12227.00 11.92 (41) Large 11945.18 9.30 (32) All 12424.58 (344) ANOVA F-value d.f 1.84*** (2, 341) Source: Field Survey 2011 (Chima, 2015) One- way ANOVA using generalised linear mode Note: *** significant at 1% level (p <0.01) and Figures in parentheses are estimated frequency.

4.2. Productivity by Years of Farming Experience

Tentatively, it might be expected that greater years of farming experience would have a beneficial influence on the production parameter such as yield per hectare (Table 4.2). Mean years of farming experience of farmers in the study area is 19 years. The table reveals that across all areas, farmers with the most experience have the highest yield per hectare except in Ebonyi state where those with the least experience have the highest yield. The inconsistency

Complimentary Contributor Copy 64 Chidiebere Daniel Chima and Sanzidur Rahman in Ebonyi state may be due to the ineffectiveness of extension services in the state as discussed in Chima, 2015. This finding is consistent with literature and study such as Obasi et al. (2013) that showed similar findings in their respective study. Also across the farm categories in the study area, the relationship between yield and years of farming experience is inconsistent. Overall, for cassava farmers, the relationship between yield and years of farming experience is inconsistent especially among farmers in Ebonyi state. Some of the reasons for this have already been discussed above. Therefore, years of farming experience is included as one of the variables in the bivariate probit to ascertain if it has any significant effect or not.

4.3. Productivity by Educational Level of Respondents

As the previous section has revealed, there is a relationship between years of farming experience of farmers and production yield in some cases. However, there have been examples where small scale farmers with less experience produces better yield than those with more experience. This will be explored more in this section. The mean years of education of respondents is 8 years and about 17.4% are illiterate, 41.3% have had some form of primary education, 28.8% have had secondary education and 12.5% have had above secondary education respectively. The table shows that in all areas, farmers with secondary school level education have the highest yield (12537.85 kg/ha). This seems to be an anomaly since it is expected that educational level will have a positive effect on yield. Similarly, the trend in both states follows the same pattern as that observed in all areas only that the inconsistences observed in the states are more than those in all areas. The anomaly observed in cassava farms may be due to the constraints associated with farm input availability, extension services and other barriers that are discussed in Chapter 9 of Chima, 2015.

Table 4.2. Productivity by respondent years of farming experience

Study Area Farm Size Cassava Yield (Kg/ha) according to respondent years of Category farming experience ≤10 11 - 25 ≥ 26 All Ebonyi Small 12424.83 12246.49 12580.94 12405.13 Medium 12314.75 12504.41 12040.88 12244.92 Large 12083.33 11723.81 11975.09 11906.31 All 12415.05 12237.09 12246.21 12330.81 Anambra Small 12693.59 12454.17 12924.96 12724.15 Medium 12000.00 11600.00 12400.00 12000.00 Large - 12116.67 12028.89 12061.81 All 12644.05 12402.31 12826.82 12650.18 All Areas Small 12451.91 12337.53 12805.30 12511.08 Medium 12279.78 12434.84 12059.78 12227.00 Large 12083.33 11841.67 11987.90 11945.18 All 12438.12 12299.05 12517.87 12424.58 Source: Field Survey 2011 (Chima, 2015).

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 65

Table 4.3. Productivity by respondent’s educational level

Study Area Farm Size Cassava Yield (Kg/ha) according to respondents Category educational level Illiterate Primary Secondary Above Sec All Ebonyi Small 12687.90 12306.33 12448.78 12376.22 12405.13 Medium 12320.59 12199.19 12194.29 12491.67 12244.92 Large 11710.29 11920.07 12089.68 - 11906.31 All 12426.67 12218.17 12412.99 12389.80 12330.81 Anambra Small 12158.97 12881.37 13443.06 11603.70 12724.15 Medium - 12000.00 - - 12000.00 Large - 12061.81 - - 12061.81 All 12158.97 12744.05 13443.06 11603.70 12650.18 All Areas Small 12423.44 12579.23 12584.36 12197.95 12511.08 Medium 12320.59 12174.29 12194.29 12491.67 12227.00 Large 11710.29 11965.42 12089.68 - 11945.18 All 12327.24 12429.15 12537.85 12225.27 12424.58 Source: Field Survey 2011 (Chima, 2015).

The trend seems to be an anomaly where farmers with the greatest level of education produce the least yield. This may be due to interference by other factors as mentioned above. As observed before, small scale farms consistently produce the highest yield in the study area, highlighting their importance in the food availability of the study area. Generally, the relationship between yield and educational level in the study area is observed to be highly inconsistent. Therefore it is included as a variable in the econometric analysis in to observe if it has any significant effect.

4.4. Productivity by Family Size of Respondents

Family size plays an important role in providing labour towards farm production in the study area. Since the mean family size in the study area is 4, the family size of respondents was divided into two sub groups (≤ 4 & ≥ 5 size families) to observe its effect on yield. Taken as a whole, 61.3% of the respondents have a family size of four or less (≤ 4); with 38.7% having a family size of five or more (≥ 5). Table 4.4 shows that farmers with less family size (≤ 4) produce the highest yield in all areas and across the farm categories; this indicates a negative relationship between yield and family size in the study area. The trend observed for both states is similar to that in all areas and the same trend follows up even across the farm categories except for large scale farms in Ebonyi state. This is not expected and just as mentioned before, detailed analysis indicates that the differences in yield is not enough to warrant much discussion on it. Overall, small scale farms consistently produce the highest yield as observed before in the study. This finding is in line with Obasi et al. (2013) observation in his study that family size has a positive relationship with yield as long as the family members are directly engaged in the farming activities. Therefore this variable is included in the econometric analysis to observe if they have any significant effects.

Complimentary Contributor Copy 66 Chidiebere Daniel Chima and Sanzidur Rahman

Table 4.4. Productivity by respondent family size

Study Area Farm Size Cassava Yield (Kg/ha) according to respondent family size Category ≤ 4 ≥ 5 All Ebonyi Small 12490.32 12224.47 12405.13 Medium 12304.37 12185.47 12244.92 Large 11590.56 12064.18 11906.31 All 12418.78 12188.93 12330.81 Anambra Small 12820.98 12548.65 12724.15 Medium 12000.00 12000.00 12000.00 Large 12500.00 11915.74 12061.81 All 12796.99 12426.28 12650.18 All Areas Small 12596.28 12339.74 12511.08 Medium 12289.15 12167.80 12227.00 Large 11772.45 12023.70 11945.18 All 12528.12 12260.31 12424.58 Source: Field Survey 2011 (Chima, 2015).

4.5. Productivity by Respondent Distance to Extension Office

Tentatively, it is expected that the closer a farmer is to the extension centre/office, the more likely he/she is to come into contact with an extension agent; and the most likely the farm yield is to increase as a result of this. Taken as a whole, 71.5% of the respondents stated their distance to the nearest extension office. The respondents were then divided into two sub groups, those located ≤ 9 km from the extension office and others located ≥ 10 km in line with the mean (5.11km) distance. The result is presented in Table 4.5. It shows that across all areas, cassava farmers located with ≥ 10 km from their extension office have the better yield. This is not expected but a more detailed analysis shows that the difference in yield is not significant enough to warrant much discussion. Also across the farm categories, the relationship between yield and distance to extension office is not consistent, but small scale farmers that are closer to the extension office have the better yield. The trend observed in Anambra state is similar to that across the all areas, but in Ebonyi state, farmers that are closer to the extension office have the better yield. This may be due to the disparities in the two states as discussed in Section 2.1. Generally, distance to an extension office has a positive relationship to yield for cassava farmers across all areas and Anambra state except for Ebonyi state. The difference in the two states may be because of the level of development in the states (Chima, 2015). Also small farm size farmers consistently have the highest yield per hectare; this implies just as observed before that they are the most productive and efficient farm category. Therefore, distance to extension centre was included in the bivariate probit model.

4.6. Productivity by Extension Contact of Respondents

Similar to the Section above, extension contact indicates how often the farmer comes in contact with extension agents. Tentatively, it is expected that the more the contact farmers have with an extension agent, the more likely they are to adopt agricultural technology.

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 67

Therefore, it is expected to have a positive effect on yield. Taken as a whole, just 7% of the respondents said that they had contact with extension agents in the previous year, while the rest did not respond to the question. This underlines the key issue (lack of contact) with agricultural extension services and agricultural technology adoption in Nigeria. Given the low level of response, one has to be careful in drawing inference from this, but this is in line with studies such as Ayansina, (2011) that observed more contact between farmers and private extension organisations than with public extension organisation (ADP) in his study.

Table 4.5. Productivity by respondent distance to extension office

Study Area Farm Size Cassava yield (Kg/ha) according to respondent distance to Category extension office (Km) ≤ 9 ≥ 10 All Ebonyi Small 12428.06 12277.97 12414.42 Medium 12255.76 12866.67 12290.67 Large 11938.04 11176.47 11906.31 All 12349.56 12281.97 12344.09 Anambra Small 12359.80 12691.67 12423.02 Medium 11800.00 - 11800.00 Large 12070.63 - 12070.63 All 12238.89 12691.67 12299.26 All Areas Small 12421.51 12360.71 12415.33 Medium 12229.72 12866.67 12264.15 Large 11968.98 11176.47 11943.41 All 12337.67 12353.22 12339.02 Source: Field Survey 2011 (Chima, 2015).

The table below (Table 4.6) shows that across all areas, cassava farmers with contact have the better yield (12468.38 kg/ha). Likewise, across the farm categories, farmers with extension contact have the better yield except for medium scale farmers; but the differences in yield are not significant. This indicates a positive relationship between yield and extension contact. The trend in Ebonyi state is similar to that observed across all areas, but in Anambra state, those with no extension contact have the better yield. The difference in the states may be due to the apparent differences in the two states already discussed in section above and in Chapter 4 of Chima, 2015. Generally, one has to be careful in drawing any conclusive inference from this because of the low level of response to the question but just as observed before small scale farms consistently have the better yield. It is important to note that the low level of extension contact observed in this study is in line with similar studies such as Ayansina, (2011) and research interviews with ADP program managers in Chima, 2015, which stated that the ratio of extension agents to farmers is very low in the study area. Therefore, extension contact is included as one of the variables in the bivariate probit.

Complimentary Contributor Copy 68 Chidiebere Daniel Chima and Sanzidur Rahman

Table 4.6. Productivity by respondent contact with extension workers

Study Area Farm Size Cassava yield (Kg/ha) according to respondent contact Category with extension worker Contact No Contact All Ebonyi Small 12713.89 12383.21 12405.13 Medium 12127.78 12254.96 12244.92 Large 12255.56 11856.42 11906.31 All 12539.81 12314.09 12330.81 Anambra Small 12916.67 12719.77 12724.15 Medium 12000.00 12000.00 12000.00 Large 12125.56 11955.56 12061.81 All 12307.64 12679.64 12650.18 All Area Small 12742.86 12498.45 12511.08 Medium 12095.83 12241.18 12227.00 Large 12174.31 11868.81 11945.18 All 12468.38 12421.00 12424.58 Source: Field Survey 2011 (Chima, 2015).

4.7. Productivity by Training of Respondent

It is expected that farmers with more extension training are more likely to adopt agricultural technology. Therefore, it is expected that extension training will have a positive relationship with yield. As with the case of extension contact above, only 8.5% of the respondent’s stated that they had any agricultural extension training in the previous year, while the remaining did not respond to the question. Due to the low level of response, one has to take this into consideration when drawing conclusive inference from this finding. Also, the finding of a low level of extension training among farmers in this study is consistent with similar study such as Ayansina, (2011), that identify low levels of extension training among farmers from public extension organisations (ADP) in his study. The table reveals that in the study area, cassava farmers with no training have the better yield, even across the farm size categories. This indicates a negative relationship between cassava yield and extension training in the study area. This is not expected, but just like before, a closer analysis shows that differences in yield are not very significant and may be due to noise in the data set. This also underlines the issues around agricultural extension services in Nigeria and highlights the problem of lack of extension agents and the quality of extension training (Ayansina, 2011, Igwe, 2013 and Chima 2015). Therefore, extension training was included as one of the variables in the econometrics analysis to observe whether it has any significant effect on adoption and food production in the study area.

4.8. Productivity by Fertilizer Application of Respondents

This section discusses how respondents’ inorganic fertilizer usage (application) relates to their yield. The threshold of fertilizer usage was grouped into low level usage (≤ 100 kg/ha) Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 69 and high level usage (≥ 101 kg/ha). This was done in line with the mean (125.21kg/ha) fertilizer application in the study area and the literature. It is expected that fertilizer application will have a positive relationship with yield (Rahman, 2011) and the result is presented in Table 4.8. It reveals that 71% did not use any fertilizer, 11% used liquid fertilizer and 18% used inorganic fertilizer. Interestingly, of those that used fertilizer, 85% used the lower level of fertilizer and 15% the higher level of fertilizer. Those that used liquid fertilizer had a decent level of yield (12132.22 kg/ha) when compared to the other levels of fertilizer application. It also indicates that in all areas, farmers that applied the higher level of fertilizer have the better yield in the study area, except in Ebonyi state. It may be because the soil in Ebonyi state is good for cassava farming as noted by Nwibo, (2012) in his study. Overall, none of the farmers in Ebonyi state used any liquid fertilizer for their farming. This may be because of their unavailability and knowledge of how to use it (Nwibo, 2012 and Chima, 2015). Liquid fertilizer users and farmers who applied a higher level of fertilizer produced better yield in the study area. This finding is consistent with Guodong, et al. (2012) who evaluated the active ingredients in liquid fertilizer and revealed that since the ingredients are already in liquid form it is easier for plants to absorb them.

Table 4.7. Productivity by training of respondents

Study Area Farm Size Cassava yield (Kg/ha) according to respondent Category training Training No Training All Ebonyi Small 12321.89 12416.06 12405.13 Medium 11800.00 12269.63 12244.92 Large 11788.89 11923.08 11906.31 All 12220.24 12344.06 12330.81 Anambra Small 12580.95 12736.22 12724.15 Medium 11600.00 12200.00 12000.00 Large - 12061.81 12061.81 All 12458.33 12666.68 12650.18 All Area Small 12386.65 12525.42 12511.08 Medium 11733.33 12265.97 12227.00 Large 11788.89 11961.35 11945.18 All 12276.26 12440.85 12424.58 Source: Field Survey 2011 (Chima, 2015).

There is no significant difference in yield between those who did not apply any fertilizer and those who applied fertilizer. This indicates that the level of fertilizer application in the study area is significantly low and that its use does not make much difference to yield. This may be because the level of usage is far below the Recommended Fertilizer Rates (RFR) for cassava (NB: The Recommended Fertilizer Rates (RFR) for cassava is dependent on the soil type, nutrient level in the soil and the type of fertilizer available). The RFR are NPK 15:15:15 (600kg/ha); NPK 20:10:10 (450kg/ha) and NPK 12:12:17 (750kg/ha) depending on the fertilizer available to the farmer (IITA, 2013). Generally, Fertilizer application has a positive relationship with yield in the study; and small scale farms consistently have the best yield in

Complimentary Contributor Copy 70 Chidiebere Daniel Chima and Sanzidur Rahman the study area. Therefore, fertilizer application was included as one of the variables in the bivariate probit model.

Table 4.8. Productivity by respondent fertilizer application (kg/ha)

Study Area Farm Size Cassava yield (kg/ha) according to respondent fertilizer Category application (kg/ha) NO Fertilizer ≤ 100 ≥ 101 All Ebonyi Small 12417.84 12429.69 11920.00 12405.13 Medium 12298.03 12009.72 - 12244.92 Large 11808.85 12012.50 13333.33 11906.31 All 12345.05 12259.00 12155.56 12330.81 Anambra Small 12592.26 13500.00 13946.67 12724.15 Medium 11800.00 - 12400.00 12000.00 Large 12212.50 11488.89 12333.33 12061.81 All 12500.98 12695.56 13739.13 12650.18 All Areas Small 12443.82 12598.68 13541.33 12511.08 Medium 12267.84 12009.72 12400.00 12227.00 Large 11879.05 11837.96 12666.67 11945.18 All 12366.78 12327.21 13411.49 12424.58 Source: Field Survey, 2011 (Chima, 2015).

Table 4.9. Productivity by respondent HYV cassava stems usage

Study Area Farm Size Cassava yield (kg/ha) according to respondents use of Category HYV NO HYV HYV All Ebonyi Small 12407.84 12400.06 12405.13 Medium 12240.13 12248.39 12244.92 Large 11992.31 11734.31 11906.31 All 12345.63 12306.91 12330.81 Anambra Small 13184.06 12243.33 12724.15 Medium 12000.00 - 12000.00 Large 12215.74 11600.00 12061.81 All 13013.84 12215.36 12650.18 All Areas Small 12625.56 12335.61 12511.08 Medium 12202.22 12248.39 12227.00 Large 12053.24 11707.45 11945.18 All 12524.90 12276.62 12424.58 Source: Field Survey, 2011 (Chima, 2015).

4.9. Productivity by High Yield Variety (HYV) Usage

The relationship between HYV seed and crop yield is discussed in this section; farmers were sub-divided into users of HYV and non-users of HYV and the result is presented in Table 4.9. The constraints affecting the use of HYV are discussed in section 7; but the use of HYV is expected to have a positive effect on crop yield (Chima, 2015). Interestingly, the

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 71 table reveals that 60% of respondents did not use any HYV and 40% used HYV cassava stem in the study area. This finding is consistent with Madukwe, et al. (2002) and Ayansina, (2011) that noted low levels of HYV usage in their respective studies. The result shows that farmers that did not use HYV have the better yield in the study area, even though the difference in yield is not much. This indicates a negative relationship between the use of HYV and cassava yield in the study area. This is not expected and underlines/highlights the issue of the non-adoption of agricultural technology as a package in the study area (Chima, 2015, Madukwe, et al. 2002 and Ayansina, 2011).

5. PROFITABILITY AND FARM SIZE RELATIONSHIPS

5.1. Benefit Cost Ratio (BCR) of Cassava Production

The net profit and BCR is evaluated and discussed in this section. Key components like total variable cost, total revenue, gross margin, total fixed cost, net profit and BCR was evaluated. The total variable costs are costs associated with farm production that vary with output within the production period. The variable cost associated with farm production in this study is shown in section A of Table 5.1. It is made up of the material input cost (seed, ploughing; fertilizer, manure and pesticide costs); labour cost (hired labour and own labour) and transportation cost (handling and transportation). The cassava stem cost (own and bought) has been calculated using the real stem cost and the mean cost per kilogram of the bought stem for farmers who own cassava stem. Cassava stem cost is relatively cheap because it is readily available in the study area. Cassava ploughing cost is relatively high when compared to other crops such as rice. This is because of the nature of ploughed farm bed needed by cassava. Rice land preparation is less intensive than that of cassava that need large farm beds because they are root tuber crops and their ploughing is more labour intensive (Chima, 2015). Family labour is imputed as an opportunity cost of the hired labour; some studies like Junankar, (1989) criticized the use of the same market wage rate for family and hired labour as a gross simplification, while others like Sevilla-Siero, (1991) suggested an alternative view that farmers, by segmenting the labour market, may turn a negative farm profit to a positive one. For crops like yam and rice, they have higher hired labour ratio than cassava; this is because they are both labour intensive (Chima, 2015). The total revenue (total output by unit price) of the crop is shown in Section B. It is the total gross yield value of the farm enterprise. The gross margin is the difference between the total revenue and the total variable cost of the crop. The total fixed cost (section C) is the cost associated with farm production that does not vary with output and remains the same throughout the production period. In this study, it is the mean total of loan cost and land rent cost for farmers that have access to those facilities since none of the farmers has a designated farm house or has access to farm machines like tractors (Chima, 2015). The total cost is also shown in this section and it is the sum of the total variable cost and total fixed cost. Farm net profit (section D) is the difference between the gross margin and the total fixed cost.

Complimentary Contributor Copy 72 Chidiebere Daniel Chima and Sanzidur Rahman

Benefit cost ratio (BCR) is a ratio that determines the return for each additional naira invested in the farm enterprise. It helps in identifying the relationship between the cost and benefit of farming the crop. It is derived from dividing Total Revenue (TR) by Total Cost (TC) for the farm enterprises. It reveals the value of 1.95 for cassava. This implies that every Naira invested into cassava production will give a return of N 1.95.

Table 5.1. BCR of cassava production

Section A Farm Enterprise Variable Cost Factors Cassava Material input cost Unit Mean Seed #/ha 1146.36 Ploughing #/ha 38140.14 Fertilizer #/ha 17741.65 Manure #/ha 2194.29 Pesticide #/ha 5773.23 Hired Labour #/ha 11371.88 Own Labour #/ha 34798.96 Labour #/ha 46170.84 Hired Labour as % Of Total Labour % 24.63 Transportation #/ha 5865.87 Total Variable cost(TVC) #/ha 117032.40 Section B Total Revenue (TR) #/ha 248491.60 Gross Margin (GM) #/ha 131459.20 Section C Fixed cost Loan #/ha 1568.96 Rent #/ha 9074.87 Total Fixed Cost (TFC) #/ha 10643.83 Total Cost (TC) #/ha 127676.23 Section D Net Profit #/ha 120815.37 Benefit Cost Ratio 1.95 Source: Field Survey 2011 (Chima, 2015) Note: GM = TR–TVC; TC = TVC+TFC; Net Profit = GM– TFC; Benefit cost ratio =TR / TC; # = Naira (Nigeria currency); ha= hectare

5.2. Profitability by Farm Size

The result of the profitability of the farm size categories is presented in Table 5.2. It reveals that medium scale farms have the best net profit. This is not expected since Table 4.1 shows that small scale farms produced the better yield; but the profitability analysis reveals that they also have the highest variable cost of production. This indicates that whereas the small scale farms treat farming as a way of life, the medium and large scale farms approach it as a business (Igwe, 2013). The BCR indicates the return to investment for any additional one Naira invested in the enterprise. The table shows a positive return to investment (1.95) and reveals that medium

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 73

(3.12) scale farmers have the best return followed by large (2.65) and small (1.83) scale farmers respectively.

Table 5.2. Farm size categories profitability and benefit cost ratio

Farm Size Category Respondents Farm Size Categories Net Profit and Benefit Cost Ratio Cassava Net Profit BCR Small 113216.60 1.83 Medium 166187.70 3.12 Large 148659.00 2.65 All 121053.30 1.95 Source: Field Survey, 2011 (Chima, 2015).

Overall, all the farm size categories are profitable and have a positive BCR. It is important to note that a good proportion of the labour and ploughing costs are own or family provided; if this is taken into account, then the small scale farms will be the most profitable farm size category. It is very important to note that the table shows that inverse-farm size profitability relationships do not hold in the study area since the medium and large scale farms give the best return to investment.

6. DETERMINANTS OF AGRICULTURAL TECHNOLOGY ADOPTION IN CASSAVA: A BIVARIATE PROBIT ANALYSIS

Several studies such as (Shiyani et al. 2002, Floyed et al. 2003, Ransom et al. 2003 and Rahman 2008) have analysed the determinants of adoption of modern/improved technologies such as High yield variety (HYV), irrigation and fertilizer by farmers in their respective studies. These are largely univariate probit or Tobit regressions of technology adoption on variables representing the socio-economic characteristics or circumstances of farmers. The implicit theoretical underpinning of such modelling is discussed in section 2.3. A bivariate probit model in this study is developed to empirically investigate the socio- economic factors underlying the decision to adopt HYV and/or inorganic fertilizer usage. The dependent variable is whether the farmer adopts HYV and/or fertilizer; for HYV represented by dv, the variable takes the value of 1 if the farmer adopts HYV and 0 if otherwise. Similarly, for fertilizer, represented by df, the variable takes the value of 1 if the farmer adopts fertilizer and 0 if otherwise. Therefore, in a bivariate probit model, there are four possibilities and they are:

1. The non-adoption of both technologies (dv=0, df=0) 2. The adoption of fertilizer only (dv=0, df=1) 3. The adoption of HYV only (dv=1, df=0) 4. The adoption of both technologies (dv=1, df=1)

Variables were chosen, representing the socioeconomic circumstances of farmers in the study area and based on existing literature of technology adoption, which offer similar

Complimentary Contributor Copy 74 Chidiebere Daniel Chima and Sanzidur Rahman justification (Rahman, 2008). Also, the analysis of how some key socio-economic circumstances and production practices of farmers relate to their crop yield (section 4), influenced the choice of variable included in the bivariate probit analysis. Therefore, the socio economic variables selected to explain the adoption decisions are: family size, farming experience, farmer’s educational level, farm size, distance to extension office, gender of household head, main occupation of household head, farmer’s training, proportion of rented-in land; number of extension contacts and ranks of decision to adopt HYV such as high yield, high profit, high quality and ready market. The age of the respondents was not considered as a variable because the effects of age as a variable is inherent in the years of farming experience of the farmers; which was considered to be a more reliable variable given the main objectives of the study. Also the quadratic effects of age or any other variables (e.g., education) are not considered. This is because we are estimating a complex model, i.e., bivariate probit; therefore, only simple direct effects of individual variable regression were considered. Educational level is commonly used as an explanatory variable in many adoption studies such as Adesina and Baidu-Faoson (1995) and Nkamleu and Adesina (2000). The educational level is chosen as a variable for a number of reasons: at technical level, access to information and the capacity to understand the technical aspects and profitability may influence the crop production decision. The decision to include farming experience is straightforward; experienced farmers may be more likely to be open to adopting agricultural technology and have more access to land, especially in a developing country like Nigeria. Distance to extension centre and number of extension contacts can be singled out as one of the important sources of information dissemination directly relevant to agricultural production practices, especially in counties like Nigeria where farmers have limited access to information. This fact is reinforced in studies like Adesina and Zinnah (1993) that find a significant influence of extension education on the adoption of land-improving technology. Also the distance to extension centre reflects the distance to input purchase centre, since this is where most of the farmers go to buy farm inputs like fertilizer and HYV seed from ADP. In addition, the ranking of the reasons for adopting HYV were included as variables to account for their influence in the decision to adopt HYV. The ranking identifies the revealed preference of the farmers on factors that will influence their decision to adopt HYV seeds.

6.1. Summary Statistics of the Variables

A summary statistics of the variables used in the bivariate probit analysis is presented in Table 6.1, classified by adoption category. It provides a summary of the characteristics of the farms and shows that 51% of the respondents said that their main occupation is farming while the rest (49%) have other main or part-time occupation. Likewise, across the adoption categories, most of the adopters of both technologies and fertilizer and fewer of the non- adopters and HYV adopters have farming as their main occupation. The average years of educational level of respondent (8.10) is above the national average for farmers. This may be because of 49% of respondents do not have farming as their main occupation and therefore, are more likely to have some kind of education (Igwe, 2013). The mean years of farming experience is 19 years and across the adoption categories, the adopters of technology have a much higher level of farming experience than non-adopters. Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 75

Most of the respondents are male and the mean family size is 4; while the average farm size in the study area is 0.58 hectares. The mean distance to an extension centre is 5.11 km in difficult rural terrain and adopters of both technologies are closer to the extension centre. Only 10% and 11% of respondents have any agricultural technology adoption training and any contact with extension agents in the study area respectively; while the mean farm land rented-in is 0.53ha. The ranking of reasons for adopting HYV were used as variables to reflect how they influence the decision to adopt agricultural technology, the high yield rank (0.90) has the highest influence, while the high profit rank reason (0.54) has the least influence on the decision to adopt agricultural technology. Table 6.1 also shows the distinct features of farms, based on their adoption status. The F- test results show that except for a proportion of rented-in land, extension contact, high yield rank and high quality rank, significant differences exist across the farm adoption category with respect to the socio-economic circumstances of these farm households. Just as stated before, for example, the years of farming experience is significantly higher among the adopters of both technologies (dv =1, df = 1) and most of them are male and have farming as their main occupation. Also they have significantly smaller farm size and are located at a shorter distance from the extension centre. On the other hand, the adoption of HYV only (dv = 1, df = 0) has the lowest number of years of farming experience among those that adopt any technology. Their main occupation is mostly not farming. Of the rank variables, only high profit rank and ready market rank are significant across the adoption categories.

6.2. Determinants of Agricultural Technology Adoption by Cassava Farmers

The results of the determinants of adoption of agricultural technology as an estimation of the bivariate probit model for cassava farmers are presented in the tables below. The actual and predicted frequency of the adoption of HYV and/or fertilizer for cassava farmers is presented in Table 6.2. It shows that the predictabilities of both non-adoption and adoption of agricultural technologies is very strong but becomes weaker for only HYV adopters and non- existent for only fertilizer adopters. This reveals a very strong robust predictability for the two extremes and indicates the state of availability of agricultural inputs in Nigeria and the state of its agricultural extension services. The results of the bivariate probit analysis for cassava farmers are presented in Table 6.3. The key hypothesis that the correlation of the disturbance terms between the two equations dv and df is zero {푝(푑푣, 푑푓)} is rejected at the 10 percent level of significance; implying that the use of a bivariate model to determine agricultural technology adoption decisions among farmers is justified (Rahman, 2008). Five variables (Table 6.3) have a significant relationship with the decision to adopt HYV while four variables have a significant relationship with the decision to adopt fertilizer. This implies that farming experience, farm size, distance to extension centre, main occupation of household head and ready market (rank) decision to adopt HYV are all significantly related to the decision to adopt HYV; while family size, farm size, distance to extension centre and gender are significantly related to the decision to adopt fertilizer.

Complimentary Contributor Copy

Table 6.1. Summary statistics of the variables

Variables Unit of Measurement All sample Non- Only Only HYV Adopters of F-test Adopters fertilizer Adopters Both (dv=0, Adopters (dv=1, df=0) (dv=1, df=1) df=0) (dv=0, df=1) Mean Std. D Mean Family size Person per household 3.94 1.94 3.97 3.82 3.95 3.82 3.92*** Farming experience Years 19.21 13.50 17.07 23.77 20.06 24.36 22.19*** Educational level of farmer Completed years of schooling 8.10 4.78 8.40 6.69 8.16 6.30 13.98*** Farm size Hectare 0.58 0.34 0.62 0.47 0.57 0.49 13.88*** Distance to extension Kilometre (Km) 5.11 3.07 5.06 5.12 5.17 5.06 centre 26.88*** Gender Dummy (1 if male and 0 if female) 0.78 0.41 0.72 0.89 0.82 0.90 5.19*** Main occupation Dummy (1 if farmer and 0 if 0.51 0.50 0.44 0.63 0.55 0.69 otherwise) 14.41*** Farmers training Number (Dummy) 0.10 0.30 0.10 0.05 0.11 0.04 2.93** Proportion of rented-in 0.53 0.43 0.83 0.23 0.01 0.02 land 1.69 Extension contact Number 1.08 0.26 1.09 1.06 1.05 1.02 1.53 High yield rank Dummy (1 if yes and 0 if otherwise) 0.90 0.17 0.87 0.94 0.93 0.97 1.31 High profit rank Dummy (1 if yes and 0 if otherwise) 0.54 0.40 0.40 0.79 0.61 0.82 15.60*** High quality rank Dummy (1 if yes and 0 if otherwise) 0.86 0.20 0.81 0.92 0.89 0.94 0.30 Ready market rank Dummy (1 if yes and 0 if otherwise) 0.63 0.35 0.58 0.71 0.67 0.74 2.64** Number of respondents 344 175 30 69 70 Source: Field survey 2011. Note: *** significant at 1% (p<0.01), ** significant at 5% (p<0.05) and *significant at 10% (p<0.10) (One-way ANOVA using generalized linear model (GLM))

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 77

Table 6.2. Cassava farmer’s actual and predicted frequency of adopting HYV and/or adopting fertilizer

Adoption of fertilizer Total Non-adopter Adopter Adoption of HYV Non-adopter 175 (240) 30 (1) 205 (241) Adopter 69 (32) 70 (71) 139 (103) Total 244 (272) 100 (72) 344 (344) Accuracy of joint prediction (%) Non-adopter of any (dv=0 and df=0) 91 Only fertilizer adopter (dv=0 and df=1) 0 Only HYV adopter (dv=1 and df=0) 7 Adopter of both HYV and fertilizer (dv=1 and df=1) 70 Source: Field Survey 2011 (Chima, 2015) Note: (Figures in parentheses are the predicted frequencies). The marginal means in the model are the univariate probabilities that the two variables equal one. NLOGIT-4 analyses the condition mean: 퐸[푑푣\푑푓 = 1, 푍_1 푍_2] = 푃푟표푏[푑푣 = 1\푑푓 = 1, 푍_1 푍_2 𝜌]|푃푟표푏[푑푓 = 1|푍_1] (퐸푆퐼, 2007).

Therefore, the table shows that farming experience significantly and positively influences the decision to adopt HYV; which means that more experienced farmers are more likely to adopt HYV because they have the means and could afford the risk, as shown in the literature review (Chima, 2015). Also farm size, distance to extension centre and main occupation of household heads significantly and negatively influences the decision to adopt both HYV and fertilizer; this is consistent with the literature review. Ready market (rank) reason for adopting HYV has a significant but negative coefficient; meaning that even though it influences the decision to adopt HYV, it is not the most important factor. On the other hand, gender has a significant and positive relationship to the decision to adopt fertilizer, meaning that male farm heads of household are more likely to adopt fertilizer. Likewise, family size, farm size and distance to extension centre have significant but negative relationships with the decision to adopt fertilizer; as mentioned before this is consistent with literature review and similar studies like Ajibefun et al (2002), Chirwa, (2005), Rahman, (2008). On the other hand, the total marginal effect of the decision to adopt HYV and/or fertilizer is presented in Table 6.4; this is the combined effects of direct and indirect effects of the explanatory variables on the probability of joint adoption of HYV and fertilizer. The predicted joint probability of adopting HYV conditional on fertilizer adoption is estimated at 0.58. Seven variables have a significant predicted joint probability of adopting HYV conditional on fertilizer adoption. This means that a one percent increase in one year of farming experience will increase the probability of adopting HYV by +0.008, given that the farmer has already adopted fertilizer. Wiboonpongse et al. (2012); Rahman, (2008) and Shiyani, et al. (2002) all noted the positive impact of farming experience in modern technology adoption in their respective studies. Likewise, a one percent increase in either farm size, distance to extension centre or number of extension contacts will negatively increase the probability of adopting HYV by (- 0.191, -0.036 or 0.318) respectively, conditional on fertilizer adoption.

Complimentary Contributor Copy 78 Chidiebere Daniel Chima and Sanzidur Rahman

Table 6.3. Cassava farmer’s bivariate probit analysis of the decision to adopt HYV and/or fertilizer

Variables Adoption of HYV Adoption of fertilizer Coefficient t-ratio Coefficient t-ratio Constant 0.838 1.24 0.114 0.303 Family size -0.076 -1.49 -0.110** -2.279 Farming experience 0.025** 2.331 0.015 1.575 Education of farmer -0.023 -0.952 -0.030 -1.422 Farm size -0.567** -2.18 -0.367* -1.675 Extension distance -0.119*** -4.701 -0.111*** -5.911 Gender 0.289 1.282 0.425* 1.883 Main occupation -0.744*** -2.812 0.084 0.378 Farmers training 0.311 1.209 -0.365 -1.051 Proportion of rented-in land 0.005 0.017 -0.425 -1.318 Extension contact -0.479 -1.813 0.155 0.898 High yield rank 0.366 0.809 High profit rank 0.934 3.018 High quality rank -0.381 -0.758 Ready market rank -1.008*** -3.569 Model diagnostic Correlation between the error 0.376*** 3.319 terms: p(HYV, Fert) Log likelihood -340.7368 Number of respondents 344 Source: Field Survey 2011(Chima, 2015) Note: ***=significant at 1 percent level (p<0.01); **=significant at 5 percent level (p<0.05); *=significant at 10 percent level (p<0.10)

This underlines the scale of what it will take to make extension services and agricultural inputs readily available and effective in Nigeria as reviewed in the literature. The table also shows that an increase of one percent in the adoption of HYV because of high profit rank in one year will increase the probability of adoption of HYV by +0.39, conditional on fertilizer adoption. Likewise, an increase of one percent in the adoption of HYV due to ready market rank will reduce the probability of adopting HYV by -0.417, given that the farmer has already adopted fertilizer. This means that high profitability of the HYV is the key factor that influences the decision to adopt HYV and even though ready market for HYV influences the decision to adopt HYV, it is not one of the key factors that influence the decision.

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 79

Table 6.4. Cassava farmer’s bivariate probit total marginal effect of the variables on the decision to adopt HYV conditional on the adoption of fertilizer

Variables Total Marginal Effect Effect t-ratio Family size -0.019 -0.881 Farming experience 0.008** 1.959 Education of farmer -0.006 -0.592 Farm size -0.191* -1.787 Extension distance -0.036*** -3.073 Gender 0.069 0.729 Main occupation -0.318*** -2.893 Farmers training 0.172 1.587 Proportion of rented-in land 0.052 0.434 Extension contact -0.216** -2.081 High yield rank 0.151 0.81 High profit rank 0.387*** 3.02 High quality rank -0.158 -0.758 Ready market rank -0.417*** -3.543 Source: Field Survey 2011 (Chima, 2015) Note: The total marginal effect is decomposed into a direct effect produced by the presence of the variable in the first equation (i.e., dv) and an indirect effect produced by the presence of the same variable in the second equation (i.e., df) respectively. The total marginal effects are the partial derivatives of the explanatory variables on the probability of adopting HYV conditional on the adoption of fertilizer: i.e.;

퐸[푑푣|푑푓 = 1, 푍1,푍2] = 푃푟표푏[푑푣 = 1|푑푓 = 1, 푍1,푍2𝜌]| 푃푟표푏[푑푓 = 1|푍1]. The joint probability of adopting HYV conditional on the adoption of fertilizer is 0.58. The effects of the dummy variables are computed using 퐸 [푑푣\푑푓 = 1, 푣 = 1] − 퐸[푑푣|푑푓 = 1, 푣 = 0], where v is the dummy variable (ESI, 2007). ***=significant at 1 percent level (p<0.01); **=significant at 5 percent level (p<0.05); *=significant at 10 percent level (p<0.10).

7. CONSTRAINTS AFFECTING AGRICULTURAL TECHNOLOGY ADOPTION IN THE STUDY AREA

The result of the constraints affecting agricultural technology adoption in the study area is discussed in this section and presented in Table 7.1. It is a multi-section table showing respondents perception of constraints affecting the adoption of agricultural technology in the study area. Section I of the table discusses farmers’ perception of the constraints affecting agricultural technology adoption in the study area. It shows that in the study area, the farmers identified lack of extension agents and inadequate contacts of farmers with extension agents as the topmost constraints affecting the adoption of agricultural technology. Almost of equal importance are the lacks of credit facilities and farm inputs, and lack of basic infrastructures, while lack of well-articulated government policies and land tenure system are the least important constraint. These constraints are similar to those identified by ADP staffs in Chima, 2015, and consistent with those identified by Madukwe et al. (2002) and Anyasina, (2011) in their respective studies. This emphasises how important it is to address them and in the words of one of the farmers; “Even if I want to adopt a technology, I do not have the money; they Complimentary Contributor Copy 80 Chidiebere Daniel Chima and Sanzidur Rahman should teach us the technology and give us the credit (money) to enable us to adopt them” (Chima, 2015). Section II discusses the respondents’ perception of the constraints affecting agricultural technology training in the study area. Across all areas, the farmers identified high levels of illiteracy and inadequate contact of farmers with extension agents as the topmost constraints affecting agricultural technology training in the study area. Almost of equal importance is the lack of extension agents and of least importance are young people’s lack of interest in agriculture and government lack of interest in agriculture.

Table 7.1. Respondents’ perception of constraints affecting agricultural technology adoption in the study area

Study Area Constraints affecting the adoption of agricultural technology in percentage (Respectively) Section I Lack of extension Lack of credit Lack of basic Lack of good Land tenure officers/agents facilities and Infrastructure Governance system and inadequate farm inputs and policies extension contact with farmers Ebonyi 243 84.94 61.39 49.81 22.39 21.62 Anambra 100.0 53.90 24.82 17.02 5.67 101 All Areas 90.25 58.75 41.0 20.50 16.0 344 Section II Constraints affecting agricultural technology training in percentage (Respectively) Inadequate High level of Lack of Government Young people’s training/contact Illiteracy extension lack of lack of interest of farmers with officers/agents interest in in agriculture extension agriculture agents (ADP) Ebonyi 243 50.58 68.34 43.63 24.32 40.54 Anambra 60.99 36.17 56.03 22.70 9.93 101 All Areas 54.25 57.0 48.0 23.75 29.75 344 Section III Factors that will facilitate respondents adoption of agricultural technology in percentage (Respectively) Adequate Availability of Availability of Good Subsidization training and credit facilities basic Governance of cost of well-motivated and farm inputs Infrastructure and policies technology and extension agent the use of credit vouchers as subsidy Ebonyi 243 33.20 56.37 49.42 49.42 7.34 Anambra 52.48 33.33 28.37 25.53 32.62 101 All Areas 40.0 48.25 42.0 41.0 16.25 344 Source: Field Survey 2011 Note: (The underlined figures are the number of respondents in the states and all areas respectively. The percentages are against the number of respondents in the respective states and all areas).

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 81

Overall, some differences were noted between the two states, which may be due to the apparent differences between them as discussed in section 2.1. Inadequate contacts of farmers with extension agents, high level of illiteracy and lack of extension agents were identified as the topmost constraints affecting agricultural technology training in the study area. These constraints are similar to the ones identified by ADP staffs, and in line with similar studies (ibid) that noted the same findings in their respective studies. This underlines how important it is to address them if agricultural technology training is to improve in the study area. In the words of one of the farmers, “How could I be trained if I could not find an extension agent when needed; more needs to be done to increase the ratio of extension agents to farmers and motivate them to do their work”? (Chima, 2015). Section III discusses farmers’ perception of factors that will facilitate their adoption of agricultural technology in the study area. Table 7.1 shows that in the study area, the respondents suggested that the availability of credit facilities and farm inputs are the topmost factors that will facilitate their adoption of agricultural technology. Almost of equal importance are the availability of basic infrastructures, good governance and policies and adequate training and well-motivated extension agents respectively. While the use of credit vouchers as subsidy was identified as the least important factor that would facilitate their adoption of agricultural technology. Overall, these findings are in line with the suggestions of ADP staff in Chima, 2015 and consistent with the recommendations of FMARD, (2011) report on agricultural extension transformation in Nigeria and similar studies such as Madukwe, (2002) and Ayansina, (2011).

CONCLUSION AND POLICY IMPLICATIONS

The main aim of this study is to investigate the influence of socio-economic factors on the adoption of individual components of modern agricultural technology (i.e., HYV stem and inorganic fertilizers) in cassava and also to examine farm size–productivity and farm size– profitability relationships of cassava production at the farm-level in South-eastern Nigeria. The aim is also to discuss the constraints in the agricultural sector of Nigeria. The research is based on an in-depth farm-survey of 344 farmers from two states (243 from Ebonyi and 101 from Anambra states) of South-eastern Nigeria. The results show that sample respondents are dominated by small scale farmers (78.8% of total) owning land less than 1 ha. The average farm size is small estimated at 0.58 ha. The key hypotheses of the study are that farmers selectively adopt components of modern agricultural technology depending on their socio- economic circumstances and inverse farm size–technology adoption, size–productivity and size–profitability relationships exist in cassava production. The study clearly demonstrates that inverse farm size–technology adoption and farm size–productivity relationships exist in cassava production in this region of Nigeria but not inverse farm size–profitability. The level of modern technology adoption is low and mixed and farmers selectively adopt components of technologies as expected and use far less than recommended dose of fertilizers. Only 20.35% of farmers adopted both HYV stems and fertilizers as a package. The bivariate probit model diagnostic reveals that the decision to adopt modern technologies are significantly correlated, implying that univariate analysis of such decisions are biased, thereby, justifying use of the bivariate approach. Overall, the most

Complimentary Contributor Copy 82 Chidiebere Daniel Chima and Sanzidur Rahman dominant determinants of modern technology adoption is farming experience and remoteness of extension services influence negatively on modern technology adoption in cassava. A host of constraints are affecting Nigerian agricultural sector, which includes lack of extension agents and poor ratio of extension agent to farmers. Other constraints are lack of credit facilities, farm inputs, irrigation, support for ADP staff; value addition opportunities; the problem of corruption and ineffective government policies. Based on the findings of this study, several policy suggestions can be proposed. Since the level of adoption of technology as a package is very low and mixed, therefore the government and NGOs that have an interest in agricultural development should promote the adoption of modern agricultural technologies as a package. The package should be comprehensive and must include provision of necessary farm inputs. Profit motive was a dominant incentive in the adoption of HYV stem technology. Therefore, price policies to keep output prices high will promote HYV stem technology adoption in cassava, as this would induce farmers to invest in HYV stem, which in turn will increase productivity. This can be achieved by improving market and marketing infrastructure so that it works effectively and benefits all relevant stakeholders including the farmers. The study showed that although small farmers are more productive but they are the least profitable. This is because most small scale farmers take farming as a way of life and are not familiar with treating farming as a business. Therefore, training should be targeted at the small farmers to train them in business skills. Provision of credit services can be achieved through effective dissemination of credit through formal banking institutions and/or facilitating non-governmental development organizations (NGOs) targeted at the farming population. The constraints identified need not just one solution but a comprehensive, well- co-ordinated inter/intra departmental designed policy package to address them. There is a need for the government and NGOs to continue to encourage and support policies and programmes like the Cassava Plus project and Cassava Flour inclusion policy. Also the area of value adding chain for most cassava produce needs to be addressed. Generally, since agriculture in Nigeria is still predominantly dominated by small scale farms, most of these policies and packages should be designed with small scale farmers in mind. In order to increase agricultural productivity and for Nigeria to meet her aspiration of food security; there is a need to support and strengthen ADP systems in Nigeria, especially in the area of providing transportation (mobility) and increase in the ratio of extension agents to farmers. Although implementation of all these policies are formidable, but boosting cassava production in Nigeria is crucial in order to ensure food security of its massive population base as it is already an established staple diet for its people.

REFERENCES

Abolagba, E.O., Onyekwere, N.C., Agbonkpolor, B.N. and Umar, H.Y. (2010). Determinants of Agricultural Exports. J Hum Ecol, 29(3): 181-184 (2010). Action Plan for a Cassava Transformation in Nigeria (APFCTN), (2014). Draft copy by Federal Ministry of Agriculture Abuja Nigeria.

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 83

Adesina, A. A and Baidu-Forson, J. (1995) ‘Farmers perception and adoption of new agricultural technology: Evidence from analysis in Burkina Faso and Guinea, West Africa,’ Agricultural Economics, 13: 1-9. Adesina, A. A and Zinnah, M. M. (1993) ‘Technology characteristics, farmer’s perceptions and adoption decisions: A Tobit model application in Sierra Leone,’ Agricultural Economics, 9: 297-311. Agwu, A. E. (2004) ‘Factors influencing adoption of improved cowpea production technologies in Nigeria,’ Journal of International Agricultural and Extension Education, Vol. 11 No. 1. Ahmed, R. and Hossain, M. (1990) ‘Development Impact of Rural Infrastructure Bangladesh,’ IFPRI Research Report No. 83, International Food Policy Research Institute, Washington DC, USA. Ajayi, A.R (1996) Evaluation of the Soico-Economic Impacts of the Ondo State Ekiti-Akoko Agricultural Development Projects On the Rural Farmers. PhD Thesis, University of Nigeria, Nsukka, Nigeria. Ajibefun, I. A., Battese, G. E. and Daramola, A. G. (2002) ‘Determinants of technical efficiency in smallholder food crop farming: Application of stochastic frontier production function,’ Quarterly Journal of International Agriculture, 41 (3) 225-240. Arkin, H. and Colton, R.R. (1963). Table for Statisticians; 2nd Ed. Harper and Row Publishers: New York, NY, USA, 1963. Ayansina, S.O. (2011) ‘Farmers’ Perception of Public and Private Extension Services in South Western Nigeria,’ PhD Thesis, University of Ilorin, Nigeria. Barrett, C.B. et al. (2004). Better technology, better plots, or better farmers? Identifying changes in productivity and risk among Malagasy rice farmers. Am J. Agric. Econ. 86(4), 869-888. Chima, C.D. (2015). Socio-Economic Determinants of modern agricultural Technology Adoption in Multiple Food Crop and Its Impacts on Productivity and Food Availability at the Farm Level: A Case Study of South-Eastern Nigeria. Ph.D. Thesis, University of Plymouth UK, Plymouth, UK, 2015 http://pearl.plymouth.ac.uk. Chirwa, E.W. (2005) ‘Adoption of fertilizer and hybrid seeds by smallholders maize farmers in southern Malawi,’ Development Southern Africa, Vol. 22, No.1 March 2005. Ebonyi State Agricultural Policy (2010) Ebonyi state government plan and policy thrust document. A policy document of Ebonyi state government Nigeria. Abakaliki, Ebonyi State Nigeria, 2010. Econometric Software, Inc. (2007) LIMDEP and NLOGIT. Plainview, New York. Fabusoro, E., A. M. Omotayo, S. O. Apantaku and Okuneye, P. A. (2010) ‘Forms and Determinants of Rural Livelihoods Diversification in Ogun State, Nigeria,’ Journal of Sustainable Agriculture, 34:4, 417 – 438. Food and Agricultural Organization Statistics FAOSTAT (2009). IITA Cassava Handbook; Reinhardt Howeler (CIAT), Bangkok), TTDI. Food and Agricultural Organization Statistics FAOSTAT (2015). Consumption and Trade in Cassava Products. Food and Agricultural Organization Statistics FAOSTAT (2015). Trend of Cassava Production in Nigeria, from 1980-2013.

Complimentary Contributor Copy 84 Chidiebere Daniel Chima and Sanzidur Rahman

Floyd, C., Harding, A.H, Paudel, K.C, Rasali, D.P, Subedi, K and Subedi, P.P (2003) ‘Household adoption and the associate impact of multiple agricultural technologies in the western hills of Nepal,’ Agricultural Systems, 76: 715-738. FMARD (2011) The Agricultural Extension Transformation Agenda Report, Published by federal ministry of agriculture and rural development, Abuja, Nigeria. Greene, W.H (2003) Econometrics Analysis, 5th edition, New York: Macmillan Publishing. Guodong, L., David S. and Gary, K. E. (2012) How to convert liquid fertilizer into dry fertilizer in fertigation for commercial vegetable and fruit crop production, University of Florida publication. Available at www.edis.ifas.ufl.edu accessed 10th January 2014. Hossain, M. (1989) Green Revolution in Bangladesh: Impact on Growth and Distribution of Income, University Press Ltd, Dhaka. Igwe, P. A. (2013) Rural Non-farm Livelihood Diversification and Poverty Reduction in Nigeria, University of Plymouth PhD Thesis. Rural Micro and Small Enterprises, Published June, 2013. http://pearl.plymouth.ac.uk. IITA (2013a) Root and tuber systems: cassava. IITA publication on cassave, Avalable at www.old.iita.org. Junankar, P.N (1989) ‘The response of peasant farmers to price incentives: The use and misuse of profit functions,’ Journal of Development Studies, 25:375-399. Madukwe, M.C., Okoli, E.C and Eze, S. (2002) ‘Analysis and Comparison of the Agricultural Development Programme and University Agricultural Technology Transfer Systems in Nigeria,’ African technology policy studies network, Nairobi Kenya. Nang’ayo, F., Odera, G., Muchiri, N., Ali, Z. and Were-hire, P. (2007). A Strategy for Industrialization of Cassava in Africa. Proceedings of a small group meeting, 14-28 November 2005, Ibadan, Nigeria. African Agricultural Technology Foundation, Kenya. National Agricultural Extension and Research Liaison services (ABU) and National Programme on Agricultural and Food Security (2010) Agricultural Performance Survey of 2010 wet season of Nigeria. National Population Commission (2006) Official population figure of Ebonyi state Nigeria, NPC Nigeria. Nigerian Institute of Social and Economics Research (2013). Reducing Crop Losses through Post-Harvest Management in Nigeria, NISER Unpublished Research Study, 2013. NISER, Ibadan Nigeria. Nkamleu, G. B and Adesina, A. A. (2000) Determinants of chemical input use in Peri-urban Lowland systems: Bivariate Probit Analysis in Cameroon. Agricultural Systems, 63: 111- 121. Nkematu, J.A. (2000) Anambra state ADP Extension Services Reports for 1999. 14th Annual Farming System Workshop of ADP. Nwa, E. U. (2003) History of irrigation, drainage and flood control in Nigeria from pre- colonial era to 1999. Spectrum Books Limited, Ibadan, Nigeria. Nwibo, S. U. (2012) ‘Effect of Agricultural Exports on Food security in Ebonyi State Nigeria,’ Journal of agricultural Research and Development, Vol. 2(3) Pp. 77-82. Obasi, P. C; Henri-Ukoha, Ukewuihe, I. S. and Chidiebere-Mark, N. M. (2013) ‘Factors affecting agricultural productivity among arable crop farmers in Imo state, Nigeria,’ American Journal of Experimental Agriculture, 3(2):443-454, 2013.

Complimentary Contributor Copy Socio-Economic Determinants of Modern Technology Adoption … 85

Olukunle, O.T. (2016). Socio-Economic Determinants and Profitability of Cassava Production in Nigeria. International Journal of Agricultural Economics and Extension ISSN 2329-9797 Vol. 4(4). Pp. 229-249. 2016. Rahman, S. (2003) ‘Environmental Impact of Modern Agricultural Technology Diffusion in Bangladesh: an analysis of farmer’s perceptions and their determinants,’ Journal of Environmental Management, 68: 183-191. Rahman, S. (2008) ‘Determinants of crop choices by Bangladeshi farmers: A Bivariate Probit Analysis,’ Asian Journal of Agriculture and Development, Vol. 5:1. Rahman, S. (2011) Production and efficiency of maize production in Bangladesh. Unpublished PhD thesis, Bangladesh Agricultural University Mymensingh. Ransom, J, K., Paudyal, K., and Adhikari, K. (2003) ‘Adoption of improved maize varieties in the hills of Nepal,’ Agricultural Economics, 29: 299-305. Sanusi LS (2012). Industrial Agriculture Raw Materials: Critical Issues in Processing, Marketing and Investment. In: Ibrahim HD, Olugbemi BO, Marinho OJ ed. Re- engineering Raw Materials Resources: A Panacea for Economic and Industrial Development. 3rd Raw Materials Research and Development Council Annual International Conference on Natural Resources Development and Utilization. Pp. 90-95. Raw Materials Research and Development Council (RMRDC), Federal Ministry of Science and Technology, Abuja Nigeria. Sevilla-siero, C. (1991) ‘A on the use and misuse of profit functions for measuring the price responsiveness of peasant farmers: A comment,’ Journal of Dev. Studies, 27:123-136. Shiyani, R. L, Joshi, P. K, Asokan, M. and Bantilan, M. C. S. (2002) ‘Adoption of improved chicken pea varieties: KRIBHCO Experience in tribal regional of Gujarat, India,’ Agricultural Economics, 27: 33-39. Sugino, T. and Mayrowani, H. (2009). The Determinants of Cassava Productivity and Price Under the Farmer’s Collaboration with the emerging Cassava Processors: A Case Study in Eas Lampung, Indonesia. Journal of Development and Agricultural Economics Vol. 1(5), pp. 114-120, 2009. Tsegai, D and Kormawa, P.C (2002) Determinants of Urban Household Demand for Cassava Products in Kaduna, Nigeria. In Conference of International Research for Development, Witzenhause. Westby A. (2008). Cassava Utilization, Storage and Small Scale Processing. In Hillock,R.; Thresh, J.; Bellotti, AC eds., Cassava Biology Production and Utilization. CABI Publishing. Wallingford, UK, pp. 67-90. Wiboonpongse, A, Sriboonchitta, S. Rahman, S., Calkins, P. Sriwichailumphun, T (2012) ‘Joint determination of the choice of planting seasons and technical efficiency of potato in northern Thailand: A comparison of Greene’s versus Heckman’s sample selection approach,’ African journal of business management, 6: 4504-4513.

Chapter 5

CASSAVA FLOUR AS AN ALTERNATIVE TO PRODUCE GLUTEN-FREE BAKED GOODS AND PASTAS

Elevina Pérez1,*, Lilliam Sívoli2, Davdmary Cueto1 and Liz Pérez 1 1Instituto de Ciencia y Tecnología de Alimentos, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela 2Centro de Bioquímica Nutricional, Facultad de Ciencias Veterinarias, Universidad Central de Venezuela, Caracas, Venezuela

ABSTRACT

Celiac disease is an immune disorder in which people cannot tolerate gluten because it damages the inner lining of their small intestine and prevents it from absorbing nutrients. Gluten is a protein found in wheat, rye, and barley and occasionally in some other minor products. A lot of foods such as, baked food and pastas are manufactured using flour from wheat, rye, barley and oats, in which the gluten defines its functional properties. People who want to manufacture products containing gluten, have been looking for alternatives to solve this problem and to insure gluten-free products for the celiac population. Because, the cassava flour does not have gluten; the foods made with this flour could be one of the solutions for the development of food for gluten-intolerant consumers. Some research has been done in regard to substitute the gluten totally in order to produce baked goods, and pastas, quite similar in its functional properties, to those produced by wheat flour. The research was initiated producing flour from the edible portion of the cassava roots. Native and modified flour from cassava roots were made using different treatments of heat, water concentration, as well as the use of salt, emulsifier, hydrocolloids or enzymes. All of the flour obtained were characterized in their chemical composition, physical, and functionality. The research suggests that is feasibility to use these types of flour in the production of numerous gluten-free baked goods, bread, and pastas, because they showed a wide spectrum of nutritional and functional properties which is causes by the effect of the additives and the treatments applied. Therefore, at a pilot phase, a second research experiment has started to produce

* Email: [email protected]. Complimentary Contributor Copy 88 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al.

pastas, and formulation of mix flour for cake, and pancakes, all of them gluten-free. The formulations and procedures that were implemented, as a function of the cassava flour, are discussed in this chapter.

Keywords: cassava, celiac, gluten free, baked products, pastas

INTRODUCTION

Health problems related to poor nutrition can be classified as: malnutrition or diseases resulting from poor nutrition. The health effects of these diseases may be irreversible, but in some cases it is reversible and controllable by use of a balanced diet (Pérez, 2010). Diet is the sum of food intake and ‘balanced diet’ is foods providing adequate nutritional needs, as well as, extra allowance for stress from different foods belonging to different food groups in specific quantities and proportions. Since all foods do not have similar nutritional values, the nutrients provided and thus the health of an individual depends on the choice and quantity of foods selected (Mohan Chutani, 2008). Well-balanced nutrition requires ingestion and absorption of vitamins, minerals, and food energy in the form of carbohydrates, proteins, and fats. Dietary habits and choices play a significant role in the quality of life, health and longevity. Therefore, both undernutrition and overnutrition can lead to the development of diseases, and a combination of both is even worse. Consequently, countries in the developing world requires special attention to balanced nutrition (WHO, 2003). There is a rapidly increase of chronic diseases in public health around the world. In developing countries, 79% of deaths are attributed to chronic diseases, especially among middle-aged men. There is evidence of risks of chronic disease beginning in fetal life and persisting into old age. Thus, chronic diseases in adult reflect different cumulative lifetime exposure; such as physical harmful, and social environments (WHO, 2003). The close relationship between health and food is widely recognized. Today there is an effort to develop healthy food products, and to change their composition in order to decrease, eliminate or add nutrients to prevent deficiencies and excesses harmful to health (Diplock, et al., 1999, NOM-086-SSA1, 1994). The term Food for Special Dietary Regime is now been defined to describe food produced or prepared to satisfy particular dietary requirements. These requirements are determined by the physical or physiological condition of the individuals and/or specific diseases and disorders, which are presented with these conditions. The composition of these foods must differ significantly from the composition of ordinary foods of comparable nature, if such foods exist (FAO, 2008). There are different diseases that could be controllable or preventable by a Special Dietary Regime such as celiac disease, diabetes, intolerances to lactose, allergies to some food proteins, obesity, and hypertension, among others. In the developing countries around the world, there is a limited production of food for special dietary uses. In most developing countries these products are imported. As a result, the value of these is increased. The availability in the country depends on factors, such as importing procedures, foreign exchange supply, availability of resources, nationalization, among others, generating technological dependence. Moreover, there is not a wide range of these products because the market is small, manufacturers are scarce and there is little

Complimentary Contributor Copy Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods … 89 competition, so their availability is limited. On other hands the gluten-free products currently available in the market are considered of low quality and poor nutritional value. Science and Food Technology is a multidisciplinary area that has an extensive field or scope, extending from the study of food selection to the effects of a specific health component or functional ingredient (Ferrariet al., 2010). Therefore, this discipline should be also focus on the production of food for Special Dietary Regimes, with its consequently development and innovation of food products that shall cover the needs of these consumers. The offers of these foods must be a low cost, and availability sure and constant.

GENERALITIES OF THE CELIAC DISEASE (CD)

Celiac disease (CD) is healthy condition which can be controlled or prevented with dietary changes. Celiac disease is an autoimmune enteropathy caused by the ingestion of gluten in genetically susceptible individuals. It is characterized by the presence of autoimmune antibodies, systemic clinical manifestations, small intestinal enteropathy and genetic predisposition (Castillo et al., 2015). This chronic (long-term) digestive disease is a permanent intolerance to one of the gluten proteins, more specifically the prolamin protein fraction that is found in cereals such as wheat (gliadin), rye (secalin), barley (hordein) and oats (avenin) (Lorenzo et al, 2007). Gliadin is the water-soluble component of gluten, while glutenin is insoluble. Gliadin has primarily monomeric proteins, which differs from glutenin, which has primarily polymers, there are three main types of gliadin (α, γ, and ω) (Nikulina e al., 2004), to which the body is intolerant in CD. Thus, the intake of these proteins by individuals genetically predisposed, causes atrophy of the villi, and will prevents the proper absorption of nutrients (Ali et al, 2006). The treatment of CD consists exclusively in maintain a gluten-free diet, for life, to ensure the patient the opportunity to enjoy good health and grow normally. The following it is the explanation of the way of the CD acts in the body. When food containing the gluten protein arrives in the small bowel, the immune system reacts against the gluten, causing an inflammatory reaction in the wall of the bowel. The small intestine lining is covered by millions of villi, finger-like projections, which act to increase the surface area of the intestine allowing increased absorption of nutrients. The villi are damaged by the inflammation caused by CD, which results in a decrease in the absorption of food. When gluten is removed from the diet inflammation is reduced and the intestine begins to heal. The time when a patient develops symptoms varies from patient to patient after their first contact with the gluten protein. In many cases is may be decades before symptoms and signs develop, often precipitated by a trigger. Approximately 1 out of every 100 people may have CD though only 1 out of 10 people with celiac disease may be actually diagnosed and are aware that they have this disease. Some of these patients have mild forms of the disease and may have no symptoms or only mild symptoms. CD affects many ethnicities, whites and highest prevalence in Caucasians. Infants and children may have celiac disease, but CD is more commonly diagnosed in adulthood, and people can be diagnosed even in their seventies or eighties. Females are more likely to be diagnosed with celiac disease than males. Individuals that have type 1 diabetes, thyroid disorders, or relatives with CD are at greater risk for developing CD (Conor and Murray, 2016).

Complimentary Contributor Copy 90 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al.

Consequently, it is imperative to create an option in the food industry as a brand of the expansion department to cover the area of development and innovation of food for celiac consumer.

CASSAVA NUTRITIONAL PROPERTIES AND ITS DERIVATIVES

In the tropical countries, there are many potential vegetable sources to produce gluten- free ingredients for the elaboration of these foods (Pérez, 2007). Cassava (Manihot esculenta Crantz) roots are among them. The cassava plant has its origin in South America. The Amazonian Indians used cassava, instead of, or in addition to rice, potato or maize. Portuguese explorers introduced cassava to Africa during the 16th and 17th centuries through their trade in the African coasts and nearby islands. Africans then spread cassava further, and it is now found in almost all parts of tropical Africa. Today Nigeria and Congo-Kinshasa are the biggest producers of cassava after Brazil and Thailand (Department of Agriculture, Forestry and Fisheries, South Africa, 2010). The cassava is a perennial plant that, under cultivation, grows to a height of about 2.4 m. Its leaves and roots are edible, but the roots are he starch storage organ and have the greatest economic value (Alarcon and Dufour, 1998). Cassava roots are tuberous, long and tapered, with firm, homogeneous flesh encased in a detachable rind, about 1 mm thick, which is rough and brown on the outside. Commercial varieties can be 5 cm to 10 cm in diameter at the top, and around 15 cm to 30 cm long. A woody cordon runs along the root’s axis. The flesh can be chalk-white or yellowish. Cassava tubers are very rich in starch, and contain significant quantities of calcium (50 mg/100 g), phosphorus (40 mg/100 g) and vitamin C (25 mg/100 g). However, they are low in protein and other nutrients. (Department of Agriculture, Forestry and Fisheries, South Africa, 2010). The roots can be harvested after seven months of planting and cultivation, and can remain in the soil for a long time. Once harvested, they deteriorate in 3 or 4 days, therefore, should be eaten or processed without delay (Alarcon and Dufour, 1998). Although rich in carbohydrates, the cassava root is often classified as an inferior food because of its low protein content, which is why products made with them are also deficient in protein, and also because the presence of an anti-nutritional factor: the cyanide released from cyanogenic glycosides naturally present in the roots (Padmaja, 1995). Usually, cassava cultivars have been classified as sweet (low cyanide content; <0.01% of HCN as DM basis), or bitter (high cyanide content; 0.02-0.03% of HCN as DM basis) (Cock, 1985, Luyuku et. al., 2014). However, the concentration of cyanide can vary from one location to another, because its production of cyanide is affected by soil, weather and other geographical conditions (Bokanga et al., 1994, Charles, et al., 2005). Moreover, the FAO, 1990, have pointed out that bitterness is not necessarily a reliable indicator of cyanide content. Consequently, it is necessary to assay for cyanide content of cassava tubers irrespective of the cultivars before used for human consumption (Terver et al., 2015). There is of consensus that sweet varieties with low hydrogen cyanide (HCN) concentration are appropriate for direct consumption, while “bitter varieties” are for industrial purposes (Cassava Handbook, 2015). Improper preparation of cassava can leave enough residual cyanide to cause acute cyanide intoxication and goiters, and may even cause ataxia or

Complimentary Contributor Copy Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods … 91 partial paralysis. Then, eating uncooked cassava is not recommended because of the potentially toxic concentrations of cyanogenic glucosides. However, these concentrations are reduced to innocuous levels, through cooking. Then, the processing of cassava roots for human or animal consumption, not only looking to improve their palatability and storage time, but also reduce its toxicity, because with adequate processing of cyanogenic substances are reduced and cassava becomes a safe food for consumer. For hence, the type of processing that is given to the roots depends on the variety of cassava, ie, whether it is “bitter” or “sweet.” The Codex Alimentarius (2008) states that the appropriate reference value for acute toxicity, is the reference dose (ATV), ie, the maximum amount that can be consumed innocuously with confidence in one meal or one day. This dose is established in linamarina 0.7 mg/kg body weight equivalent to a ATV cyanide to 0.08 mg/kg body weight. Besides being high carbohydrate content and caloric input, the cassava root is a good source of minerals such as calcium, phosphorus, manganese, iron and potassium, and high amounts of dietary fiber. Cassava contains 38 grams of carbohydrates per 100-gram serving. Absence of the allergenic protein gluten makes cassava flour a good substitute for rye, oats, barley and wheat. Besides, cassava is a good source of saponins. These phytochemicals may help lower unhealthy cholesterol levels in your bloodstream. They do so by binding to the bile acids and cholesterol, thus preventing them from being absorbed through the small intestines. The antioxidant effects of saponins may help protect your cells from damage by free radicals (Cassava Handbook 2015). Cassava is a large agricultural crop of importance for food security in the tropical and subtropical areas of Latin America, Africa and Asia. In these places, the cassava root is consumed as soon as it is harvested without processing. Therefore, by processing the root to produce flour will help to prolong its life and reduce post-harvest losses, encourage its cultivation, and offers various alternatives for the consumers. Because the cassava flour does not have gluten, foods elaborated with this flour could become one of the solutions for development of food for celiac consumers (Pérez et al., 2007). People diagnosed with celiac disease and other gluten-related allergies can find relief in consuming foods made using tapioca or cassava flour. Although baking cakes, bread and other foods requires gluten to enable them to swell in size, it can be substituted with guar and xanthan gum. Around the world, cassava roots are eaten fresh, cooked on hot coals, boiled or fried in oil; alone, like bread or accompanied with sauces or combined with other foods. It turned into flour is very popular in Brazil, she made two major types of food: the farofa (ground cassava flour, lard and bacon) and pirão (flour boiled cassava in water or broth and served as bread) (Cartay, 2004). Cassava flour is also used to make breads, soups, food for elderly and babies, sauces, desserts, alcoholic and non-alcoholic beverages, etc. The most traditional and widespread form of consumption is like “cassabe” or cassava bread, a dry biscuit, round and thin. Cassava bread has high energy value for its high carbohydrate content, but the shortage of protein is a limitation from the nutritional point of view. However, its low moisture, gives great stability in storage and can stay several months without any deterioration (Carrizales, 1991). Alternatively, the cassava starch is another ingredient for developing gluten-free food. It has a strong competitor in corn starch, whose prices are stable and of high and consistent quality, which affects the expanded of the use of cassava starch. Products using starches from corn and cassava are sold in the same markets (for examples: breads, cookies, ice cream,

Complimentary Contributor Copy 92 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al. chocolates, meat). These starches are also used to make paper and cardboard, textiles, pharmaceuticals, glues and adhesives and modified starches (Cereda et al, 1996).

CASSAVA DERIVATIVE PERSPECTIVES AS INGREDIENT FOR DEVELOPMENT AND INNOVATION OF FOOD FOR CELIAC CONSUMER

Some research has been done in regard to complete substitution of gluten by using cassava flour to produce baked goods and pastas, quite similar in its functional properties, to those produced by wheat flour. However, the baked goods functional properties are reached mainly by the gluten. Gluten refers to the proteins found in cereal grain’s endosperm (the tissue produced in seeds that are ground to make flour). Gluten both nourishes plant embryos during germination, and later affects the elasticity of dough, which in turn affects the chewiness of baked products. Since cassava flour has not gluten-proteins, its functional properties are not sufficient for elaboration of gluten-free baked goods. However, if this flour is thermally and enzymatically modified, its properties are adequate to develop gluten-free baked product. Sivoli et al., in 2014 reports farinographic profile and ultrastructure of cassava flour modified by using heat and enzymatic treatments; all of them, with and without addition of stearoyl-2-lactylate (SSL), and proposing them, as ingredient for gluten-free food. Cassava flour produced by dehydration (60 °C x 24 hours) was modified: by boiling and autoclaving, aqueous solutions of flour at 10% (with 2% NaCl), and 33%, respectively, and dextrinized with Termamyl®. The boiling and retorting treatment have produced a type IV farinogram-curve. The SSL addition changed drastically the curves to those of type I, and the enzymatic treatment to the type II. These flour could be used for baked goods manufacture, and could also be blended with a flour type IV for bread making. The NaCl addition has shown poor effects in the farinographic profile. When analyzing (SEM) the modified flour has shown total granular disaggregation, indicative of gelatinization. The authors concluded that this flour can be used to produce baked goods and gluten-free bread. On the other hand, to improve the functional quality of the bakery products made with cassava flour, structuring agents that confer greater stability to the products during fermentation are used. Among the products used as substitutes for gluten are the xanthan gum and carboxymethyl cellulose and cellulose derivative, hydroxypropylmethylcellulose, guar gum, carrageenan and agar. These additives are permitted by the Food and Drug Administration (FDA) and its application needs to follow good manufacturing practices. The hydrocolloids create a cellular network that is strong enough to retain the carbon dioxide formed during fermentation, improve the cohesiveness of the dough, by increasing the attraction between the starch granules, however this may cause a decrease in binding mobility. These agents bind water to gelatinize the starch, this makes the crumb structure and bread volume increase and decrease the crumb firmness (Onyango et al, 2009. Shittu et al. 2009). Emulsifiers commonly used in the baking industry have the ability to improve aeration and stability of the gas bubbles during the process and until the cake structure is stable (Turabi et al. 2008).

Complimentary Contributor Copy Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods … 93

Native cassava flour or thermal treated have few protein content, so its derivative products must be enriched with a protein source. The protein content in the elaborated food can be reached by adding whey (liquid remaining after milk has been curdled and strained), legumes such as soy or others different beans, and pseudo cereals such as quinoa, amaranth, among others. Whey powder is a co-product of the manufacture of cheese or casein and has several commercial uses. Sweet whey is manufactured during the making of rennet types of hard cheese like cheddar or Swiss cheese. Acid whey (also known as “sour whey”) is a co-product produced during the making of acid types of dairy products such as cottage cheese or strained yogurt. Legumes are plants that bear their fruit (beans) in pods, which are casings with two halves, or hinges. Its dehydrated beans can be transformed into flour. The legume flour is a very healthy food, or ingredient in food production, because are low in fat and high in protein, with a high content of fiber and other nutrients. Pseudocereals are non-grasses that are used in much the same way as cereals (true cereals are grasses). Their seed can be ground into flour and otherwise used as cereals. Examples of pseudocereals are amaranth, quinoa, and buckwheat (University of Arkansas). Alvarez-Jubete et al., 2009 have shown results that suggest that the pseudocereals amaranth, quinoa and buckwheat can represent a healthy alternative to frequently used ingredients in gluten-free products.

CASSAVA FLOUR AS AN INGREDIENT FOR FOOD DEVELOPMENT: STUDIED CASE

Cueto and Pérez 2011and 2013, have used cassava flour in order to substitute totally the wheat flour to prepare a dry mix to make cakes and pancakes.

The authors have prepared flour, from two clones (experimental and commercial) from the Germplasm bank of the Agronomic Faculty Central University of Venezuela (FAGRO) to use as ingredient in the cake and pancakes mixes. The procedure for sweet cassava flour elaboration (with a yield of 34, efficiency index of 0.89, and less of 50 ppm of HCN) was as presented in Figure 1. Table 1 shown the proximate composition and dietary fiber, and amylose contents of the sweet cassava flour from the two clones. The experimental one is showing high crude protein, and available carbohydrates contents, but less amount of dietary fiber and crude fat, as compared to its commercial counterpart. These data could be interesting from a nutritional point of view, with the experimental clone showing more potential than the commercial one. Experimental cassava flour shows similar density (bulk and tapped) to commercial flour, however this flour is not as white and finer granulometry than commercial flour (Table 2). Even when both (experimental and commercial) flour have shown differences in their chemical composition, and physical properties, due to its inherent genetic differences, they have also shown a high performance and efficiency during the preparation process for food development. Furthermore, the results of the bulk, and tapped density in both flour are indicative that they can achieve good dosing during packaging industrial.

Complimentary Contributor Copy 94 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al.

Figure 1. Flow chart of cassava flour pilot level preparation.

Table 1. Proximate composition, dietary fiber and amylose contents of sweet cassava flour obtained from the two clones

Parameter Cassava flour experimental Cassava flour commercial Moisture (%) 5.72 ± 0.03 a 6.61 ± 0.04 b Crude Protein (%) 4.57 ± 0.01 b 1.50 ± 0.05 a Crude Fat (%) 0.15 ± 0.01 a 0.46 ± 0.01 b Ash (%) 2.43 ± 0.04 a 3.00 ± 0.07 b Dietary Fiber (%) 2.48 ± 0.01 a 3.25 ± 0.01 b Total Carbohydrates (%) 84.65 ± 0.04 a 85.18 ± 0.05 b Available Carbohydrates (%) 82.17 81.93 Amylose (%) 26.5 ± 0.36 b 23.3 ± 0.51 a Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

The size granular population, and physicochemical parameters (pH, WA, WAI and separation of phase), of the cassava flour are similar to those in other types of flour (Table 3). The amylographic profile (Table 4) shows significant differences between the flour. However, both are good for cooking, because its starch shows low tendency to retrograde, it is stable during the cooking, and its breakdown value is an index of an easily disintegration of

Complimentary Contributor Copy Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods … 95 the granules during baking. They can be used in the preparation of cakes, pancakes, breads, and as a substitute for wheat flour.

Table 2. Physical parameters of sweet cassava flour obtained from the two clones

Parameter Cassava flour experimental Cassava flour commercial Density Bulk 0.41 ± 0.02 a 0.45 ± 0.03 b Tapped 0.65 ± 0.03 a 0.69 ± 0.02 b Color L* 95.03 ± 0.02 a 95.49 ± 0.01 b a* -0.78 ± 0.01 a -0.60 ± 0.01 b b* 10.32 ± 0.02 b 7.87 ± 0.01 a ΔE 8.87 ± 0.02 b 6.59 ± 0.01 a WI (White index) 88.51 ± 0.01a 90.91 ± 0.01 b Granulometry (%) 60 mesh (0.2425 mm) 40 80 > 60 mesh (< 0.2425 mm) 55 17 Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

Table 3. Physicochemical parameters of sweet cassava flour obtained from the two clones

Parameter Cassava flour experimental Cassava flour commercial Water Solubility (WS) (%) 8.17 ± 0.60 a 8.53 ± 0.63 a Water Absorption Index (WAI) (g gel/g) 2.66 ± 0.02 a 2.76 ± 0.01 b Phase Separation (%) 17 ± 0.58 a 18 ± 0.58 a pH 6.61 ± 0.01 b 6.29 ± 0.01 a Titratable Acidity as H2SO4 (%) 0.016 ± 0.00 b 0.013 ± 0.00 a Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

Table 4. Gelatinization profile of sweet cassava flour obtained from the two clones

Parameter Cassava flour experimental Cassava flour commercial Initial Gelatinization Temperature (ºC) 68.6 a 70.7 b Initial Viscosity (BU) 31a 76 b Viscosity Peak (BU) 753 a 859 b Breakdown 544 a 628 b Set back - 329 a - 437 b Consistency 215 a 191 b Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

Complimentary Contributor Copy 96 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al.

CAKE FORMULATION AND PREPARATION USING BOTH CASSAVA FLOUR

For the formulation of the cake mix, raw cassava flour obtained from cassava roots was used, as well as whey (hydrolysate), an edible emulsifier, corn maltodextrin, refined sugar and baking powder. Additionally, other ingredients such as water, margarine, were required for the preparation. Fresh eggs were optional (Figure 2).

Water and margarine

Eggs or lecithin

Figure 2. Flow chart of cake preparation by using the cake optimal recipe, and elaboration parameters.

Preliminary different tests and formulations were performed in order to establish the optimal balance of ingredients. Once defined the optima formulation, the cakes were prepared by adding water and margarine. Eggs were optional. The use of the additives was done following the maximum level permitted by Norm COVENIN 910: 2000. In addition, formulation of cake parameters such as; time and stirring speed, baking time, cooking temperature, cooling time in the mold, were also previously established. All the ingredients were mixed and homogenized in a KitchenAid mixer. Model: K5SS (Michigan USA). Figures 2, and 3 show the sequence of cake preparation with the optimal recipe, and elaboration parameters.

Complimentary Contributor Copy Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods … 97

Cake mix (made with experimental and commercial cassava flour) has low moisture, fat, and micronutrient contents with a high carbohydrate content, and a protein contribution, which covers 7 and 4% respectively, of the Venezuelan protein daily requirement (Table 5). Cakes made with cassava flour and whey show a batter specific gravity, volume and acceptable symmetry (Table 6), with a crumb structure having more or less 1 mm2 alveoli, alveolus average size of 0.925 mm2 and a uniform crumb (Figure 4).

Figure 3. Sequence of pictures for cake preparation by using the cake optimal recipe, and elaboration parameters.

Table 5. Proximate composition of mixes for cassava cake

Parameter Cassava flour experimental Cassava flour commercial Moisture (%) 5.09 ± 0.05 5.14 ± 0.05 Crude Protein (%) 4.59 ± 0.01 2.63 ± 0.00 Crude Fat (%) 0.51 ± 0.01 0.34 ± 0.07 Ash 3.04 ± 0.06 3.21 ± 0.09 Total Carbohydrate 88.76 ± 0.03 89.83 ± 0.06 Niacin (mg/100g) 0.36 ± 0.06 0.33 ± 0.03 Riboflavin (mg/100g) 0.01 ± 0.00 0.01 ± 0.00 Gluten (ppm) < 5 < 5 Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

Complimentary Contributor Copy 98 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al.

Table 6. Cake batter parameters to make cassava flour cake

Parameter Cassava flour experimental Cassava flour commercial Batter Specific Gravity (g / cm3) 1.06 ± 0.01 1.05 ± 0.01 Weight (g) 198.43 ± 1.37 195.74 ± 3.75 Specific Volume (cm3/g) 2.71 ± 0.07 2.67 ± 0.09 Volume Index (cm) 13.62 ± 0.49 14.3 ± 0.13 Symmetry Index 0.48 ± 0.10 0.43 ± 0.04 Uniformity Index 0.08 ± 0.03 0.08 ± 0.04 Crust color: L* 53.90 ± 0.05 b 45.93 ± 0.01 a a* 12.29 ± 0.09 a 16.98 ± 0.04 b b* 42.24 ± 0.16 b 38.44 ± 0.07 a ΔE (compared to a wheat cake control) 7.38 ± 0.36a 6.75 ± 1.94a Brown Index 54.07 ± 0.01 46.13 ± 0.05 Crumb color L* 60.62 ± 0.06 a 64.13 ± 0.05 b a* 9.04 ± 0.01 b 7.19 ± 0.02 a b* 38.01 ± 0.09 b 31.95 ± 0.07 a ΔE (compared to a wheat cake control) 21.32 ± 2.22 16.21 ± 1.82 Average ± standard deviation, n = 3. Values with the same letter in rows (a, b) are not significantly different (p <0.05).

Figure 4. Pictures of cool cake. Left: cake made with experimental cassava flour. Right: cake made with commercial cassava flour.

Table 7. Textural profile of the cakes

Parameter Cassava flour experimental Cassava flour commercial Hardnesss (g.f) 2017.05 ± 154.17 b 1737.94 ± 277.99 a Cohesivity 0.42 ± 0.04 a 0.48 ± 0.10 a Elasticity 7.86 ± 0.87 a 8.39 ± 0.33 a Chewinesss (g.f) 7041.63 ± 443.90 a 7425.56 ± 185.07 a Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

Results of the sensorial evaluation of the cakes were above the average value of the hedonic scale, which was used for evaluation. Other than the hardness values, there was not found significant differences, between cakes when looking at the texture profile (Table 7). In fact, the sensorial evaluation judges did not detect any textural differences.

Complimentary Contributor Copy Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods … 99

PANCAKE FORMULATION AND PREPARATION USING BOTH CASSAVA FLOUR

In the formulation of the raw cassava pancake flour mix, both raw cassava flour obtained from roots of two kind of cultivars; one experimental, and one commercial were used, as well as, whey, refined sugar, baking soda, and cream of tartar. Water and margarine were used in the preparation of the pancake, adding eggs as ingredient optional. Preliminary different tests and formulations were performed in order to establish the optimal balance of ingredients. Once the best formulation was defined, the pancakes were prepared by incorporating water, margarine, and whole egg (optional) (Figure 5). In addition, preparation of pancake parameters such as; time and stirring speed, cooking time and temperature were previously established. All the ingredients were mixed and homogenized using a KitchenAid mixer. Model: K5SS (Michigan, USA). Pancake mix (made with experimental and commercial cassava flour) has low moisture, fat, and micronutrient contents with a high carbohydrate content, and a protein contribution, which covers 6 and 3% respectively, of the Venezuelan protein daily requirement (Table 8).

Figure 5. Flow chart and pictures sequence of pancake preparation by using the cake optimal recipe, and elaboration parameters.

Complimentary Contributor Copy 100 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al.

Table 8. Proximate composition of mixes for pancake from cassava flour

Parameter Cassava flour experimental Cassava flour commercial Moisture (%) 5.72 ± 0.16 a 5.81 ± 0.11 b Crude Protein (%) 3.86 ± 0.01 b 1.91 ± 0.01 a Crude Fat (%) 0.44 ± 0.01 a 0.68 ± 0.07 b Ash (%) 2.61 ± 0.09 a 2.73 ± 0.08 b Total Carbohydrates (%) 87.36 ± 0.06 a 88.87 ± 0.11 b Gluten (ppm) < 5 < 5 Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

The pancake batter parameters and color of the pancakes made with cassava flour and whey show similar properties to those presented in a fat cake batter. (Table 9).

Table 9. Pancake batter parameters to performs the cakes cassava flour

Parameter Cassava flour experimental Cassava flour commercial Batter Specific Gravity (g/cm3) 1.13 ± 0.01 1.14 ± 0.01 Weight (g) 6.87 ± 0.92 7.05 ± 0.87 Specific Volume (cm3/g) 1.35± 0.08 1.43 ± 0.05 Volume Index (cm) 14.3 ± 0.13 13.62 ± 0.49 Crust color: L* 56.41 ± 0.03 a 56.91 ± 0.05 b a* 10.38 ± 0.21 b 11.06 ± 0.06 a b* 28.37 ± 0.13 a 31.47 ± 0.03 b ΔE (compared to a wheat cake control) 7.10 ± 1.99a 4.47 ± 2.09a Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

There were no significant statistical differences observed in the objective test of the pancake texture profile, but in the subjective test, the judges detected statistically significant differences in texture (Table 10).

Table 10. Textural profile of the pancakes

Parameter Cassava flour experimental Cassava flour commercial Hardness (g.f) 4926.83 ± 115.77 a 4915.54 ± 99.94 a Cohesivity 0.75 ± 0.04 a 0.72 ± 0.03 a Gumminess (g.f) 3673.23 ± 114.77 a 3548.03 ± 83.54 a Elasticity 0.98 ± 0.23 a 0.99 ± 0.14 a Chewiness (g.f) 3630.37 ± 182.94 a 3513.21 ± 120.78 a Average ± standard deviation, n = 3. Values with the same letter in each rows (a, b) are not significantly different (p <0.05).

Complimentary Contributor Copy Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods … 101

PASTA FORMULATION AND PREPARATION USING CASSAVA FLOUR

A series of assays using two food sources (cassava and beet juice) were performed in order to substitute all wheat flour for cassava flour to make pasta (Pérez and Pérez, 2009). The assays were begun using three composite flours where the wheat semolina was replaced in proportions of 10, 15 and 20% cassava flour, adding beet juice instead of pure water. Fettucine pasta was elaborated, and its cooking parameters were assayed (Table 11).

Table 11. Cooking parameters

Pasta Cooking time Solid loss Gain of weigh (minutes) (g/10g) (g/8.0g sample) Control 100% semolina 15 a 1.25 a 22.4 a 80:20 (semolina: cassava flour) 15 a 1.28 a 28.0 b 85:15 (semolina: cassava flour) 15 a 1.27 a 24.5 cd 90:10 (semolina: cassava flour) 15 a 1.25 a 25.5 dc Average ± standard deviation, n = 3. Values with the same letter in each column (a,b,c,d) are not significantly different (p <0.05).

After this assay, a total substitution was performed mixing raw and thermally treated cassava flour, to prepare fettuccine pasta made with 100% cassava flour. The result was a fettuccine with similar cooking parameters to the control fettucine made with 100% semolina. However, further research must be done in order to establish the optimal pasta recipe.

CONCLUSION

The products (cakes, pancakes and fettuccini) were well accepted which indicates the feasibility of replacing wheat flour with cassava flour, and shows the potential for replacing conventional wheat flour with 100% cassava flour as an alternative in the diet for the celiac population.

REFERENCES

Alarcón, F. and Dufour, D. (1998). Almidón agrio de yuca en Colombia. Tomo 1: Producción y recomendaciones. Cali: CIAT. [Cassava starch in Colombia sour. Volume 1: Production and recommendations. Cali: CIAT.] Ali, I.M, Mariasch, N.C., Maurel, S.L, and Deschutter, S. (2006). Enfermedad celíaca: formas de presentación clínica en la población pediátrica. Revista de Postgrado de la VIa Cátedra de Medicina, 157, 3-6. [Celiac disease: clinical presentation in the pediatric population. Journal of Postgraduate Medicine Chair VIa, 157, 3-6.] Alvarez-Jubete, L., Arendt, E. K., and Gallagher, E. (2009). Nutritive value and chemical composition of pseudocereals as gluten-free ingredients. International Journal of Food Science Nutrition, 60(4), 240-257.

Complimentary Contributor Copy 102 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al.

Bokanga, M., Ekanayake, I.J., Dixon, A.G., and Porto, M.C. (1994). Genotype-Environment Interactions for Potential in Cassava. Acta Horticulturae, 375, 131-139. Cassava Handbook. (2015). Supported by: China-Cambodia-UNDP Trilateral Cooperation Cassava Project Phase II. Phnom Penh, Cambodia. Carrizales, V. (1991). Cassava bread technology and its future. Rome: FAO. Cartay, R. (2004). Difusión y comercio de la yuca (Manihot esculenta) en Venezuela y en el mundo. Agroalimentaria, 10(18), 13-22. [Dissemination and trade of cassava (Manihot esculenta) in Venezuela and the world, Agroalimentaria, 10(18), 13-22.] Castillo, N.E., Theethira, T.G., and Leffler, D.A. (2015). The present and the future in the diagnosis and management of celiac disease. Gastroenterology Rep, 3, 3–11. Cereda, M., Takitane, I., Chuzel G, and Vilpoux O. (1996). Starch potencial in Brazil. In Dufour, O`Brien y Best (Eds.), Cassava flour and starch: progress in research and development. Cali: CIAT. Charles, A., Chang, Y., Ko, W., Sriroth, K., and Huang, T. (2005). Influence of Amylopectin Structure and Amylose Content on the Gelling Properties of Five Cultivars of Cassava Starches. Journal of Agriculture and Food Chemistry, 53 (7), 2717-2725. Chutani, A.M. (2008). Nutritional Biochemistry. Nutrition and Dietary Habits. In: http://nsdl.niscair.res.in/jspui/bitstream/123456789/586/1/Nutrition Dietary.pdf. Cock, J.H. (1985) Cassava: New Potential for a Neglected Crop. Westview Press, Boulder Co., London, 191. CODEX (2008). Programa Conjunto FAO/OMS sobre normas alimentarias. Comité del CODEX sobre contaminantes de los alimentos. Documento de debate sobre los glucósidos cianogénicos. CX/CF 09/3/11. [Joint FAO / WHO Food Standards Programme. Codex Committee on contaminants in food. Discussion Paper on cyanogenic glucosides. CX / CF 09/03/11.] CODEX STAN (2008). 146-1985. http://www.fao.org/DOCREP/005/Y2770S/ y2770s04.htm. Accesed: in april 27/2016. Conor, G., Loftus, C.G., and Murray, J.A. (2016). Celiac Disease. Division of Gastroenterology and Hepatology. Mayo Clinic, Rochester, MN. The American College of Gastroenterology Recuperado de: www.acg.gi.org. COVENIN, 910: (2000). Norma general para aditivos alimentarios 2da Rev. FondoNorma. Caracas Venezuela. [General standard for food additives on the 2nd Rev. FONDONORMA. Caracas, Venezuela.] Cueto, D.E., Pérez, E.E., and Dufour, D. (2011). Formulación de mezcla para elaborar torta para regímenes especiales. Memorias del VIII Congreso Iberoamericano de Ingeniería de Alimentos CIBIA 8, Lima Perú. [Mix formulation for making cake for special dietary uses. Proceeding of the VIII Congreso Iberoamericano de Ingeniería de Alimentos CIBIA 8, Lima Perú.] Cueto, D.E., and Pérez, E., (2013). Formulación de mezcla para elaboración de panquecas a base de yuca (Manihot esculenta C.) y suero de leche bajo en fenilalanina para regímenes especiales. Memorias del V Congreso Venezolano en Ciencia y Tecnología de Alimentos “Dra. Mercedes Baragaño de Mosqueda.” Seguridad, Calidad e Inocuidad Alimentaria. (pp. 141-146). Caracas: Instituto de Ciencia y Tecnología de los Alimentos. [Formulation of pancake mix with cassava (Manihot esculenta C.) flour and milk serum low in phenylalanine for special dietary uses. Proceeding of the V Venezuelan Congress on

Complimentary Contributor Copy Cassava Flour as an Alternative to Produce Gluten-Free Baked Goods … 103

Food Science and Technology "Dra. Mercedes Baragaño Mosqueda. "Safety, Quality and Food Safety. (pp. 141-146). Caracas: Institute of Science and Food Technology.] Department of Agriculture, Forestry and Fisheries. (2010). Cassava Production Guidelines. Agriculture, Forestry and Fisheries Republic of South Africa. Diplock, A.T., Agget, P.J., Ashwell, M., Bornet, F., Fern, E.B., and Roberfroid, M.B. (1999). Scientific concepts of functional foods in Europe: consensus document. British Journal of Nutrition, 81(S1), 127. FAO (2008). Norma general para el etiquetado declaración de propiedades de alimentos preenvasados para regímenes especiales. [General Standard for the Labelling Claims for pre-packaged foods for special dietary uses.] FAO (1990). Toxic substances and antinutritional factors. Roots, tubers, plantains and bananas in human nutrition. Agriculture and consumer protection. Rome, Italy. Ferrari, G., Maresca, P., and Ciccarone, R. (2010). The application of high hydrostatic pressure for the stabilization of functional foods: Pomegranate juice. Journal of Food Engineering, 100(2), 245–253. Lorenzo, G., Zaritzky, N., and Califano, A. (2007). Optimization of non-fermented gluten free dough composition based on rheological behavior for industrial production of “empanadas” and pie crusts. Journal of Cereal Science, 48(1), 224-231. Luyuku, B., Okike, I., Dunca, A., Beveridge, M., and Blummel, M. (2014). ILRI 25. Discussion paper Cassava in livestock feed and aquaculture feeding program. Cassava and it residues as livestock feed: Limitations, processing, and nutritional composition. Nikulina, M., Habich, C., Flohé, S.B., Scott, F.W., and Kolb, H. (2004). Wheat gluten causes dendritic cell maturation and chemokine secretion. The Journal of Immunology, 173 (3), 1925–1933. Norma Oficial Mexicana NOM-086-SSA1- (1994). Bienes y servicios. Alimentos y bebidas no alcohólicas con modificaciones en su composición. Especificaciones nutrimentales. [Goods and services. Food and non-alcoholic beverages with changes in its composition. nutritional specifications.] Onyango, C., Unbehend, G., and Lindhaues, M. (2009). Effect of cellulose-derivatives and emulsifiers on creep-recovery and crumb properties of gluten-free bread prepared from sorghum and gelatinised cassava starch. Food Research International, 42 (8), 949-955. Padmaja, G. (1995). Cyanide detoxification in cassava for food and feed uses. Critical Reviews in Food Science and Nutrition, 35 (4), 299-339. Pérez, E.E. (2007). Raíces y Tubérculos. En: Edel, A. y Rosell, C. De tales harinas, tales panes: granos, harinas y productos de panificación en Iberoamérica. 1era edición, Córdoba, Argentina. [Such flours, such breads: grains, flour and bakery products in Latin America. 1st ed. Córdoba, Argentina.] Pérez, E.E, Lares, M., González, Z., and Tovar, J. (2007). Production and characterization of cassava (Manihot esculenta Crantz) flours using different thermal treatments. Interciencia, 32 (9), 615-619. Pérez, E.E, and Pérez, L. (2009). Effect of the addition of cassava flour and beetroot juice on quality of fettuccine. African Journal of Food Science, 3(11), 352-360. Pérez E. (2010). ¿Tecnología de alimentos en la Medicina? Perspectivas en Venezuela en la elaboración de productos de regímenes especiales. Tribuna de Investigador, 11(1-2):6-10. [Food technology in medicine? Perspectives in Venezuela in the manufacture of special dietary regimen. Researcher Tribune, 11(1-2):6-10.] Complimentary Contributor Copy 104 Elevina Pérez, Lilliam Sívoli, Davdmary Cueto et al.

Shittu, T.A., Aminu, R.A., and Abulude, E.O. (2009). Functional effects of xanthan gum on composite cassava-wheat dough and bread. Food Hydrocolloids, 23(8), 2254-2260. Sívoli, L.J., Ciarfella, A.T., and Pérez, E.E. (2014). Functional and Nutritional Characterization of Cassava Flours for Industrial Applications. En: Cassava: Production, Nutritional Properties and Health Effects. Ed Molinari FP. Nova Publishers. Turabi, E., Sumnu, G., and Sahin, S. (2008). Rheological properties and quality of rice cakes formulated with different gums and emulsifier blend. Food Hydrocolloids, 22(2), 305- 312. Ubwa, S.T., Otache, M.A., Igbum, G.O., and Shambe, T. (2015). Determination of Cyanide Content in Three Sweet Cassava Cultivars in Three Local Government Areas of Benue State, Nigeria. Food and Nutrition Sciences, 6(12), 1078-1085. University of Arkansa “Glossary of Agricultural Production, Programs and Policy.” (2006). University of Arkansas Division of Agriculture. Retrieved -12-31. WHO (2003). Dieta, nutrición y prevención de enfermedades crónicas. Informe de una Consulta Mixta de Expertos OMS/FAO/OMS, Serie de Informes Técnicos 916. [Diet, nutrition and prevention of chronic diseases. Report of a Joint WHO Expert / FAO / WHO Series 916 Technical Reports.]

Chapter 6

TECHNOLOGICAL ASPECTS OF PROCESSING OF CASSAVA DERIVATIVES

Elisa Cristina Andrade Neves1,3,*, Daniela Andrade Neves2, Kleidson Brito de Sousa Lobato2, Gustavo Costa do Nascimento3 and Maria Teresa Pedrosa Silva Clerici3 1Food Engineering Faculty, Institute of Technology, Federal University of Pará (UFPA), Belém, Brazil 2Department of Food Science, Food Engineering Faculty, State University of Campinas (UNICAMP), Campinas, Brazil 3Department of Food Technology, Food Engineering Faculty, State University of Campinas (UNICAMP), Campinas, Brazil

ABSTRACT

Cassava (Manihot esculenta Crantz) is a tuberous root grown in all regions of Brazil, mainly in the North region, and the state of Pará Pará is one of the largest producers. It is considered a high-energy food, rich in starch and fiber, but highly perishable, with moisture content of around 67.5%, used for direct human consumption or as raw material to produce cassava-derived products, by using the water activity principle for food conservation. Various products can be produced by artisanal or industrial processes, such as different types of cassava flour, cassava gums, fermented and native starch, tapioca flour, tucupi, among others. Flour is one of the main cassava products, and its use is widespread throughout the country as part of Brazilian eating habits, especially in the North and Northeast regions, consumed by rural, riverine, and urban populations of all social classes. However, the quality of cassava-derived products is very heterogeneous, often out of the standards established by Brazilian law, once they are produced by small producers following their decision-making processes. This chapter describes the technological differences in the manufacture of cassava-derived products, considering cassava varieties and processing stages, such as cassava fermentation before drying and drying process, as well as their effects on the physicochemical characteristics of the

* [email protected]. Complimentary Contributor Copy 106 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

products, including moisture, pH, acidity, particle size, color of the products and gel, helping to spread the potential of cassava and enhancement of regional products.

INTRODUCTION

Cassava (Manihot esculenta Crantz) is a tropical crop, widely grown around the world and one of the main food for about 800 million people. It was produced in 100 countries, with a world production of cassava of 276.7 million tons in 2015, according to the FAO in 2016. Nigeria is the largest producer with 19.5% of production, followed by Thailand with 10.9%, and Indonesia with 8.6% of production. Although Brazil was the largest cassava producer, currently is in 4th place with 7.7% of world production (EMBRAPA, 2016). In Brazil, cassava consumption was inherited from the indigenous eating habits, as well as many other delicacies typical of the Amazon. Cassava can grow in all regions of Brazil, as it adapts to adverse climatic conditions and different types of soils, due to its low demand for nutrients. However, it is most cultivated in the North and Northeast, and the state of Pará is one of the largest cassava producer. Despite generating jobs and income for the local and regional economy, with production mainly of family farming, cassava is considered an important agribusiness in the Brazilian economy, and has become staple food for many rural and low-income populations, being an essential for food safety of traditional populations (Cardoso, Souza & Gameiro, 2006). Its consumption has increased by patients of celiac disease, a public that needs a gluten-free diet (Rosa Neto, 2009).

COMPOSITION OF CASSAVA

Cassava is predominant in the daily diet of the low-income population. It is rich in starch (Cereda & Vilpoux, 2003), a good energy source, and contains fiber, thus being used in the preparation of different food products. Cassava roots have on average 67.5% moisture, 25.5% starch, 1.2% ash, 1.3% protein, 0.3% fat, 30.5% carbohydrates, and 0.3% other fibers. Starch is the predominant compound in cassava, which contains low protein and lipid levels (Albuquerque et al., 1993; El-Dash & Germani, 1994). In fresh roots are also found several vitamins, like vitamin C (14.9-50.0 mg/100 g), vitamin A (5.0-35.0 μg)/100 mg), and vitamin B such as thiamine (0.03-0.28 mg/100 g), riboflavin (0.03-0.06 mg/100 g) and niacin (0.60-1.09 mg/100 g). Cassava also presents calcium (19.0-176.0 mg/100 g), phosphorus (6.0-152.0 mg/100 g), iron (0.3-14.0 mg/100g), potassium (0.25-0.72 g/100g, magnesium (0.03-0.08 g/100g), copper (2.0-6.0 ppm), zinc (14.0-41.0 ppm), sodium (76.0-213.0 ppm) and manganese (3.0-10.0 ppm) (Montagnac, Davis & Tanumihardjo, 2009). The plant has a variety of uses, from the leaves to tuberous roots, which is the most important part of the plant, despite being highly perishable, with moisture content of around 67.5% (Albuquerque et al., 1993). Therefore, lowering the water activity of the product contributes for obtaining different products by artisanal or industrial way, such as various types of flour, tucupi, cassava gums, and starches (native or fermented).

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 107

CLASSIFICATION OF CASSAVA

There are various types of cassava that can be classified in relation to their color parameters, hydrocyanic acid content, applications, and biofortification.

Classification of Cassava According to Color Parameters

Cassava can be classified according to its coloring in white, cream, or yellow, which is defined by the carotenoids content in the roots. Ortega-Flores (1991) assessed the levels of pro-vitamin A carotenoids in cassava varieties of São Paulo and found that the main carotenoids were trans-β-carotene and cis isomers, 9-cis-β-carotene and 13-cis- β-carotene. This author also reported that cooking process promoted a reduction in the pro-vitamin A activity.

Classification of Cassava According to Hydrocyanic Acid Content

Cassava belongs to the group of cyanogenic plants, containing cyanogenic glycosides, known as linamarin and lotaustralin present in the roots in the ratio of 93: 7. After rupture of the cell root structure during peeling, cutting, grating, or grinding, the cyanogenic glycosides come in contact with the linamarase enzymes (class of β-glucosidase). These enzymes promote the hydrolysis of these compounds, resulting in glucose and α-hidroxinitriles. The latter, when catalyzed by a hydroxy nitrile lyase, are transformed into the corresponding ketones and hydrocyanic acid or hydrogen cyanide (HCN), highly toxic to humans, with a lethal dose of HCN from 50 to 60 mg / kg weight (Fennema, 1996). Cassava cultivars are classified according to the concentration of cyanogenic glycosides into sweet cassava (low) and bitter cassava (high). Although the concentration of these compounds is influenced by the environment, the main factors that affect its content are the age of the root at harvest time (Borges, Fukuda & Rossetti, 2002).

Sweet Cassava

Sweet cassava, also known as macaxeira or aipim, has low HCN levels (less than 50 mg/kg of pulp of fresh roots), and the highest linamarin concentrations are present in the bast, which is removed during peeling. It has a pleasant flavor and is suitable for cooking or table use, and can be eaten boiled, fried or used as raw materials in the preparation of cakes, snacks, and other foods (Mezette et al., 2009). Concerning its sensory characteristics, it is mainly evaluated for the parameters palatability, plasticity, and stickiness, which can vary according to the cooking time. The shorter cooking time can result in the better sensory quality of the cooked mass. Thus, the cooking time is used as an indirect measure of mass quality (Borges, Fukuda & Rossetti, 2002; Lorenzi, 1994).

Complimentary Contributor Copy 108 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

Sweet cassava is marketed by several ways, including in natura (without processing) either unpeeled or peeled chilled, or pre-treated in the minimally processed form, frozen or refrigerated, or pre-cooked and frozen, or fried (Rosa Neto, 2009).

Bitter Cassava

Bitter cassava contains high HCN levels, which imparts an unpleasant, bitter taste noticeable at high cyanogenic glycosides concentrations (greater than 100-150 mg eq. HCN kg-1 fresh weight), with poisoning risks to humans and animals (Schwengber, Smiderle & Mattioni, 2005). This class is intended for industrial use, however the reduction of the HCN contents is required before consumption, which is easily eliminated by volatilization after heating (Bokanga, 1994), during the drying stage (natural or artificial dryers) in the manufacture of flour and starch (produced both from sweet as bitter cassava). Bitter cassava is used as raw material in the preparation of several Brazilian products, such as white and yellow cassava flour, farinha d`água, starch, tapioca starch, sweet or sour starch, tapioca flour, tucupi, etc (Rosa Neto, 2009).

Classification of Cassava According to Its Application

According to its use, cassava can be classified into two broad categories: table cassava and industrial cassava. Table cassava usually belongs to the sweet variety, and most of them are marketed in natura. The roots used in the industry must provide pulp, cortex, and white colored film, no straps, large diameter, good conformation and thin film, which facilitates peeling, and ensures the quality of the final product. They should also contain high starch content, especially when used in the production of cassava starch (Cardoso, Souza & Gameiro, 2006; Cereda & Vilpoux, 2010).

Other Classifications

Cassava can also be classified according to its concentration of micronutrients, which may be conventional cassava and biofortified cassava, with the possibility of adding nutritional value to the roots for human consumption, by selecting yellow or pinkish cassava, with higher contents of carotenoids (Chávez et al., 2000), iron, and zinc. The production of biofortified cassava aims to contribute to reducing micronutrient deficiency, which is a serious problem of nutrition and public health throughout the world, especially in developing countries like Brazil.

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 109

CASSAVA DERIVATIVES

Different products can be made using cassava as raw material, including cassava flour, tapioca flour, and starches (fermented and non-fermented), cassava gum, carimã, etc.

Cassava Flour

In Brazil, there are a great number of cassava flour mills (flour`s house), most of them small, basically family-related enterprises. Small cassava processing units are usually installed near the plantations, with handmade character. In general, both harvesting and processing of cassava are performed by the same community group, often by members of the same family. Facilities are rustic buildings, covered, but usually open with the ground floor, using wooden equipment and utensils, without application of Good Manufacturing Practices. To increase the supply of quality cassava derivatives, the modernization of small rural mills, improvements in equipment efficiency and adaptation of the products to consumers' needs are required. However, this adaptation is a complex process, once the intervention in the traditional processes of small rural communities is essential, which are characterized by low level of education and high resistance to changes in the production chain (Cereda & Vilpoux, 2010). Medium-sized plants are often integrated into cooperatives or associations of family farming, with more structured facilities with complete equipment, such as mechanical peeler, strainers, grater, presses, and hand furnaces or mechanical oven, consisting of a cylindrical iron plate and planetary agitation system. The cassava-based agribusinesses in the state of Pará- Brazil, are usually local or regional, selling the products in other nearby towns, or in the metropolitan area of the capital, Belém, usually sold in bulk at trade markets (Figure 1A and B) or supermarkets (Figure 1C and D). According to Brazilian legislation (Brasil, 1995), flour is the product obtained by milling of the edible part of the plant, subjected to appropriate technological processes, declared by the word flour followed by the name of vegetable origin. Cassava flour is the product obtained by slight roasting of the grated mass of cassava roots previously peeled and washed, and containing low cyanide content. The product subjected to a new roasting step is called toasted cassava flour. Flour is one of the main cassava derivatives, considered a typically Brazilian product, despite equivalent products are found in some regions of Africa. Cassava flour is the staple food in many Brazilian states, mainly North and Northeast, where it is consumed by rural populations, riverine, and urban of all social classes, according to the customs of the various regions, as side dishes based on meat and fish. Although the great importance of cassava flour as a food product, there is no sufficient quality standards to classify all types of flour, mainly due to the artisanal manufacturing process (Cereda & Vilpoux, 2010; Dósea et al., 2010; Oluwamukomi, Oluwalana & Akinbowale, 2011). Thus, the main problems of currently produced cassava flour are the lack of uniformity and wide variation of composition, influenced by the variety, plant age, and time interval between harvest and processing. Other factors, such as farming, climate, soil, crop, among others, are also responsible for the

Complimentary Contributor Copy 110 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al. variability in the quality of cassava flour (Fiorda et al., 2003). However, the processing method is seen as the main factor responsible for the quality of flour (Cereda & Vilpoux, 2010; Dias & Leonel, 2006).

a b

c d

Figure 1. Marketing of tapioca flour and cassava flour: A and B: Ver-o-Peso street Market, C and D: supermarkets located in Belém (PA) - Brazil.

To increase the yield of cassava derivatives, including flour and starch, the roots are harvested from April to August, after 16 to 20 months of growth. Because cassava roots are highly perishable and undergo fermentation soon after being harvested, its processing should start no later than 36 hours after harvesting. The roots must be handled and transported carefully to avoid damage, which may favor fermentation and quality loss.

Characteristics of Cassava Flour

According to Brazilian legislation for cassava flour (Brasil, 2011), the standards include maximum 13% moisture, 1.4% ash, and 2.3% fiber. Cassava flour is rich in carbohydrates (± 91.4%), which are rapidly converted to glucose, essential to all processes in the human body, such as muscle contraction and recovery. Whereas it is a source of quick energy, cassava flour causes abrupt changes in glucose levels, which should be avoided by diabetics and obese patients. Although it is not considered the major fiber-rich flour, with 1.8% fiber, cassava flour is still a good source of these nutrients. Fibers are important to keep satiety for long periods and stimulate the intestinal functions. As defined by Brazilian law (Brasil, 2011), cassava flour can be classified into three groups, according to the manufacturing process, as follows: 1) dry flour that comes from the

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 111 washed roots that are peeled, grated, pressed, sieved, and dried in oven; 2) farinha d' água, obtained from water immersion of the roots, peeled or unpeeled, and fermented for varying time and peeled, grated, sieved, and oven dried to yield a product of coarse particle size; and 3) flour mix, which is the mixture of dry flour and farinha d' água. With respect to the color parameters, flours are classified mainly in white, which is the most common, yellow, common in North and Northeast, coming from yellow roots (natural color), or artificial dyed. Table 1 shows the color parameters of seven types of cassava flour and 2 types of tapioca flour purchased at fair in the Ver-o-Peso complex, located in Belém (PA) -Brazil, which was determined using spectrophotometer Mini Scan XE 45 / 0-L under the test conditions illuminant D65 and 10 ° viewing angle.

Table 1. Color parameters of different types of cassava flour purchased in Belém (PA) –Brazil

Color parameters* Cassava Flour Name in Brazil L a* b* White fine Branca fina 86.78±0.22 2.28±0.17 20.18±0.32 White medium Branca média 86.16±0.68 2.29±0.19 20.51±0.40 White coarse Branca grossa 83.44±0.97 2.43±0.07 21.05±0.45 Yellow fine Amarela fina 81.94±0.20 2.94±0.14 60.47±0.99 Yellow medium Amarela média 82.08±0.64 3.07±0.44 61.89±0.71 Yellow coarse Amarela grossa 77.55±1.03 4.53±0.41 61.05±0.45 Coarse farinha d' água D’água grossa 77.73±1.72 3.43±0.23 45.94±5.59 Coarse tapioca Tapioca fina 85.90±1.54 1.24±0.35 10.44±3.81 Coarse tapioca Tapioca Grossa 85.18±0.43 0.85±4.72 5.71±7.17

The yellow cassava flours showed high yellow and light red (Table 1), and farinha d`'água showed intermediate yellow intensity to yellow and white cassava flours. The tapioca flour exhibited a* and b* values close to zero, indicating white tone. The use of artificial coloring is due to potential consumers who have a preference for yellow dominant color with slight reddish hue. Thus, many manufacturers add dyes such as tartrazine yellow or turmeric after the grating step. The yellow dye tartrazine is a synthetic dye permitted for use in confectionery, but not regulated by Brazilian law for cassava flour (Souza et al., 2005), and especially without controlling of the concentrations used, which can cause allergy and poisoning in high concentrations, especially in children. Cassava flour can also be classified according to their particle size in coarse, medium, or fine, with variations mainly in artisanal processes, once it depends on the temperature and ability of the oven operator. The smaller the quantity of mass per furnace area, the higher the temperature is, and the finer the flour. Higher amounts of flour and higher moisture contents in hot furnace can lead to graininess characteristics in coarser flour due to starch gelatinization, as observed in farinha d' água. Table 2 shows the particle size distribution of 7 types of cassava flour purchased at the fair in the complex Ver-o-Peso located in Belém (PA) - Brazil, which was determined in Granutest apparatus using 14,16, 20, 35, 60 and mesh-sieves.

Complimentary Contributor Copy 112 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

The coarse flour showed over 97% of materials retained by a 14-mesh sieve, and the fine flour particles were distributed across the 20-, 35-, and 60-mesh sieves. It was observed that the apparent density (Table 3) was associated with the particle size. Table 3 presents the results of moisture, pH, and bulk density (mass/volume) of the cassava derivatives purchased at the Ver-o-Peso market complex, determined according to the methodolgies 31.1.02 (AOAC, 1995), 981.12 (AOAC, 1997), and AOAC (1995), respectively. The moisture content was less than 14%, thus it is in accordance with Brazilian legislation (Brasil, 2011), which contributes to its preservation. Another important parameter to be considered in cassava flour is acidity or pH (Table 3), once these parameters provide information about the fermentation process during manufacturing. The higher the acidity, the higher the intensity of fermentation or process time (cassava retting). Farinha d' água has low acidity due to the shorter fermentation time of 1-2 days, with no need to wait cassava soften as in the traditional cassava retting, with no negative effects on product's quality. The bulk density of a powder is determined by measuring the volume of a known mass of powder sample.

Table 2. Particle size distribution of cassava flour purchased in Belém (PA) – Brazil

Sieves (mesh) Cassava Flour < 60 60 35 20 16 14 White fine 4.92 23.31 27.08 18.25 7.67 18.77 White medium 2.77 15.94 18.67 12.77 5.06 44.89 White coarse 0.02 0.10 0.28 0.92 1.00 97.93 Yellow fine 6.71 24.16 23.07 17.67 7.77 21.02 Yellow medium 4.85 16.96 15.48 11.78 5.96 45.63 Yellow coarse 0.06 0.15 0.42 0.39 0.38 99.05 Coarse farinha d' água 0.19 3.62 10.15 12.38 7.93 66.36

Table 3. Moisture, bulk density, and pH of different types of cassava flour purchased in Belém (PA) –Brazil

Moisture Bulk density Cassava Flour pH (%) (g/mL) White fine 6.88±0.11 0.67 4.29±0.00 White medium 6.65±0.08 0.60 4.57±0.02 White coarse 8.07±0.10 0.54 4.42±0.00 Yellow fine 6.13±0.10 0.68 4.68±0.02 Yellow medium 7.65±0.26 0.68 4.27±0.01 Yellow coarse 7.33±0.06 0.56 4.15±0.02 Coarse farinha d' água 3.70±0.02 0.74 4.68±0.02 Coarse tapioca 3.22±0.08 0.21 4.06±0.02 Coarse tapioca 3.78±0.09 0.18 3.89±0.04

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 113

The total cyanide content in cassava flour is another parameter to be considered and depends on cultivars and processing conditions. Chisté et al. (2010) found a decrease in total cyanide concentration in cassava roots from 160 ± 11.8 mg HCN / kg to 149 ± 12.3 mg HCN / kg after grinding, 68 ± 2.5 mg HCN / kg after pressing, and 5 ± 0.2 mg HCN / kg in the final product after roasting. Lower total cyanide contents were observed in farinha d' água due to the additional fermentation process, which is a typical step that sets it apart from dried cassava flour. During fermentation, the cyanide content of the roots ranges from 297 ± 2.7 to 64 ± 2.3 mg HCN / kg after 96 hours of fermentation. Cyanide in cassava flour is not enough to cause poisoning (Chisté et al., 2010).

Dried Cassava Flour Processing

Dried cassava flour, table flour, or toasted flour are obtained by processing the edible parts of cassava roots. This type of flour is produced in all regions of Brazil, especially in the North and Northeast, and is considered the most consumed in the country, and one of the main energy sources for low-income populations.

Figure 2. Flowchart of the dried cassava flour processing (Cereda & Vilpoux, 2003).

Complimentary Contributor Copy 114 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

To obtain dried cassava flour (Figure 2), roots are received in the industrial flour mills, and the yield is determined by weighing, together with the determination of density in hydrostatic balance, once the payment is done according to the dry matter content (Cereda & Vilpoux, 2003). Then, the roots are washed and peeled, which can be done manually or mechanically. In manual peeling (Figure 3A), knives are used, removing the bark and the inner bark, rich in linamarin. The bark removal leads to a decrease in toxicity of the final product, and eliminates the phenolic compounds responsible for enzymatic browning, enhancing the quality of flour. This process is considered exhaustive, requiring a lot of workers, with a higher risk of accidents, longer processing time, and greater loss of raw material, which reduces production yield (Cereda & Vilpoux, 2003; Chisté et al., 2006). In the mechanical peeling (Figure 3B), bark is removed during cleaning in a washer- peeler, which can be rotating wooden drums, removing the bark (periderm) by abrasion, or semi-cylindrical, consisting of a shaft containing rods in movement and a perforated tube for passing washing water (Leite, 2003). This equipment removes only the outer bark, and in the case of residues on its surface, it is removed by manual peeling.

a b

Figure 3. Cassava peeling: (A) Manual, (B) Mechanical, in Moju (PA)-Brazil.

After peeling and washing, cassava is subjected to grating. This step is necessary to cut the roots until the formation of a wet mass which enables pressing. Grating leads to the disruption of cell tissues, causing various biochemical reactions, such as hydrolysis of cyanogenic compounds, contributing to its reduction. The grated mass is then subjected to the pressing process to remove excess water (20 to 30% water) before roasting, to facilitate the drying process, and to prevent gelatinization of starch. The operation is carried out using screw presses or weights (Figure 4), by the hydraulic or mechanical system. The process is conducted in timber rotating cylinders, provided with serrated blades fixed parallel to each Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 115 other, and in the longitudinal axis direction (Souza et al., 2005). As the cylinder rotates, the roots are pushed against them and turned into a wet grated mass, which is wrapped in bags and transferred to the press. Thus, a starch-rich mass is obtained, which can be recovered, and a pollutant liquid known as manipueira, containing cyanogenic compounds, is produced. Thus, pressing contributes to reducing the toxicity of the mass (Chisté & Cohen, 2008). In northern Brazil, artisanal pressing is performed using the tipiti, an instrument made of palm leaves, which provides an excellent pressing, however, it is limited by the small amount of mass per batch. The process produces manipueira, which can be used for the preparation of another cassava derivative, named tucupi. Traditionally, in the North, this liquid by-product is used in the manufacture of sauce for the preparation of traditional dishes, such as duck with tucupi and tacacá.

a b

Figure 4. Pressing the mass of grated cassava in Terra Alta (PA)-Brazil.

a b

Figure 5. (A) Pressed cassava mass, (B) Grating of cassava mass, in Patauateua (PA)-Brazil.

Complimentary Contributor Copy 116 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

a b

Figure 6. Cassava flour ovens: (A) Manual agitation, (B) Mechanical agitation, in Santa Isabel do Pará (PA)-Brazil.

After pressing, the mass in the form of compact blocks (Figure 5A) is subjected to flaking, which can be performed through a set of graters (Figure 5B), and / or sieves to disaggregate mass, retaining fibers and bark waste. The crumbly mass is subjected to roasting, which is held in metal furnaces, with manual or mechanical agitation. In general, wooden stirrers (Figure 6) and masonry under direct fire furnace are used. Stirring and temperature affects the sensory characteristics (flavor, color, and texture) of cassava flour, and contributes to the elimination of hydrocyanic acid, decreasing its toxicity (Souza et al., 2005). After roasting, cassava flour should present 14% moisture content to be packaged and stored.

Farinha D' Água Processing

Farinha d' água comes from the Amazon region and is consumed from the Amazonas to Maranhão state in Brazil. It is obtained from yellow cassava roots, being fermented or retted, and exhibiting greater particle size when compared to other flours, which provides a hard and grainy texture. For the production of farinha d' água (Figure 7), after reception and washing, maceration of roots in water followed by fermentation is required, known popularly as retting (Cereda & Vilpoux, 2003), which is a natural anaerobic fermentation process, with a predominance of the group of Lactobacillus bacteria. During fermentation, hydrolytic enzymes are released, including amylase and pectinase, which favors the softening of roots, the increase in acidity due to the formation of lactic acid and other acids, and reduction of cyanogenic compounds (Souza et al., 2005). Traditionally, cassava retting is carried out on the banks of rivers and streams (Figure 8A), with cold and clean water, with a minimum duration of 5 to 6 days, varying according to climate conditions. The installation of masonry tanks or water tanks (Figure 8B) allows the communities to control water volume and pollution, and reduces the fermentation time for 1 to 3 days. The fermentation time has an influence on acidity, flavor, aroma, and texture, because the longer the fermentation period, the greater formation of acid and the softer the texture, due to higher water absorption and action of enzymes.

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 117

Figure 7. Flowchart of the farinha d' água processing (Cereda & Vilpoux, 2003).

a b

Figure 8. Traditional cassava retting on the bank of stream in Terra Alta (PA)-Brazil (A) and a masonry tank in Patauateua (PA)-Brazil (B). Complimentary Contributor Copy 118 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

During retting, starch and most soluble compounds, including cassava sugars, are metabolized and leached, and can be drawn into the fermenting water. Fermentation softens the roots, thus separating bark, sapwood, and cassava pieces more easily, followed by manual peeling, contributing to its transformation into a mass. Subsequently, the mass is pressed, similar to dried cassava flour, flaked, and toasted. Roasting of farinha d' água is a specific operation for this type of product, as it includes cooking and some modification of flour characteristics, different from dried cassava flour, where the crumbly mass is roasted in the oven (Cereda & Vilpoux, 2003). Heating is performed in the temperature range between 150 and 250°C, which causes starch gelatinization and formation of granules typical of farinha d' água. Baking and drying are responsible for the final particle size and product's quality. This product must contain a minimum of fine dust and large particles. To obtain a more homogeneous product, as desired by consumers, flour should be sieved by passing through a 0.5 mm-sieve to remove the finer powder, and a 5 mm-sieve to separate the coarser grain (Vilela & Juste Junior, 1987).

Cassava Starch

Cassava starch is a fine, white powder, odorless, tasteless, which produces slight crackle when squeezed between fingers. In an aqueous medium at room temperature, it forms a milky suspension which when is allowed to stand, the starch slowly precipitates to the bottom. When the suspension is subjected to heating at starch gelatinization temperature (between 65 and 70°C), there is the formation of clear translucent unstable paste, with high viscosity (Arias, 2000). Starch is considered one of the cassava derivatives with high added value for having high starch levels. Cassava starch comprises about 18% amylose and 82% amylopectin. Amylose is an essentially linear molecule composed of glucose units connected by α-1,4 glycoside bonds, while amylopectin is also composed of glucose units connected by α-1,4 glycoside bonds, being a highly branched molecule, with 5 to 6% bonds α-1,6 glycoside bonds, at the branch points. Most existing cassava starch manufacturers in Brazil is located in the northwest and western regions of Parana, in the south of Mato Grosso do Sul, west of São Paulo, and Santa Catarina and Pará (ABAM, 2016), which increased its production in recent years (Figure 9). The production of cassava starch is performed by artisanal scale, varying only the type of equipment, or by industrial scale. In industrial scale, root starch content directly influences yield and production costs. According to the acidity of cassava starch, it can be classified into sweet cassava starch or sour cassava starch.

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 119

Figure 9. Evolution of the Brazilian production of cassava starch (1990-2015) * (1000 t) (CEPEA, 2016).

Cassava Starch Processing

To obtain sweet cassava starch (Figure 10) by the industrial process, cassava roots are subjected to peeling and washing, which is carried out simultaneously, removing only the bark. Grinding and disintegration is performed to form a mass and release the starch granules, followed by extraction in rotary screens (Figure 11A), and washing with water to separate starch fiber. The resulting liquid is separated by centrifugation (Figure 11B), and the retentate (starch) is sieved using vibrating screens. The mass is concentrated and vacuum filtered (Figure 11C), resulting in starch with 40% moisture, which is subsequently subjected to drying in a rotary dryer or flash dryer, obtaining cassava starch with 12-14% moisture, which is then cooled and packaged (CEPEA, 2016). Starch can be used in the paper, textile, and food industries, for example in the manufacturing of bakery and meat products. The manufacturing process of sour cassava starch (Figure 10) is similar to sweet cassava starch. However, starch should be fermented by natural fermentation in an aqueous medium, in open or closed tanks, preferably coated masonry or stainless steel, at room temperature. A thin layer of starch is coated with 10 to 20 cm of water. After several days (15-40 days), the liquid portion has become turbid with bubbles on the surface. The production of acids can be detected by the characteristic aroma, and fermentation is terminated when the acidity reaches 5%. At the end of fermentation, the supernatant liquid is drained off, and the surface material is removed to eliminate impurities. The product is crumbly, subjected to drying to achieve 14% moisture, packed and stored (Ferreira Filho et al., 2013).

Complimentary Contributor Copy 120 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

Figure 10. Flowchart of the Cassava starch (Cereda & Vilpoux, 2003).

The fermentation process leads to changes in the starch characteristics, favoring the expansion of the processed products, without the need of yeasts or extrusion, for example in the preparation of pão de queijo, biscuits, and gluten-free products. Various other products are produced from sweet starch, including sago, creams, puddings, infant food, sauces, soups, broths, tapioquinha, tapioca flour, among others.

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 121

a b

Figure 11. Cassava starch separation: (A) Rotary screeners, (B) Centrifuge, (C) Vacuum filter in Moju (PA)-Brazil.

Tapioca Flour Processing

Tapioca flour is produced using cassava starch as raw material, and can be defined as a product with irregular polyhedral or spherical granules. The production of tapioca flour may be performed by artisanal or industrial processes. In artisanal production, cassava starch is hydrated and sieved in 3.0 mm-sieve. The fine particles are then pressed with slight rounding movements on a cotton fabric to form granules, which are sieved again in the same mesh. Then, scalding on the oven plate at approximately 180°C with constant tumbling for 5 minutes is performed. The granules are maintained at rest for 24 hours at room temperature, followed by heating at 240°C with constant tumbling, which causes expansion of the particles, similar to the popping expansion (Chisté et al., 2012; Silva et al., 2013; Souza et al., 2005). In the industrial production (Figure 12), cassava starch is hydrated to 40% moisture in a rotating cylinder, forming large clumps which are then milled. The resulting hydrated mass is transferred to a second rotating cylinder, leading to the formation of granules of different diameters, which are classified to standardize the particle size of tapioca flour. Scalding is then performed, in which the granules are transferred to an

Complimentary Contributor Copy 122 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al. oven with mechanical and manual stirring (Figure 13A) for starch gelatinization. The resulting granules are sorted by diameter. Thereafter, the blanched granules are transferred to another furnace, under constant stirring, for expansion (Figure 13B), due to the temperature rise, thus forming tapioca flour, which is sorted once more and packed.

Figure 12. Flowchart of the tapioca flour processing (Cereda & Vilpoux, 2003).

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 123

Figure 13. Tapioca flour processing (A) Scalding, (B) Expansion in Santa Isabel do Pará (PA)-Brazil.

Cassava Gum Processing

Cassava gum is widely used in the northern of the country in regional cuisine, to make tacacá and tapioca dish, with great consumption in Brazil due to the increased demand for gluten-free products. By hand, it is processed on a smaller scale, and unlike cassava starch, which is extracted directly from the roots, it is obtained from manipueira, a liquid by-product extracted from the pressed cassava. Manipueira is allowed to stand, and the starch precipitates to the bottom, which is then separated, without being subjected to drying. The cassava gum is also marketed in the form of hydrated and packaged starch, which should be stored under refrigeration, due to its high water activity.

Carimã Processing

Carimã or puba flour is a cassava derivative obtained by spontaneous fermentation of fresh cassava roots, whole or broken. The roots are dipped in water for about five days until softening and loosening of the bark. Then, they are crushed, washed, and sieved to remove fiber. The fiber-free mass is washed several times, subjected to sun drying or mechanical dryers, until reaching 50% moisture in the case of wet puba, or 13% for dry puba. Carimã is used to prepare cakes, porridges, cuscuz, etc. (Ferreira Filho et al., 2013).

Complimentary Contributor Copy 124 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

Maniva Processing

Although cassava roots are the basis for consumption and production of derivatives, the aerial parts, especially the leaves, are also used in animal feed and human food, due to their high contents of protein, vitamin, iron, calcium, vitamin C, and phosphorus, despite containing linamarin and ethyl methyl ketone cyanohydrin, which release HCN after hydrolysis. According to Bokanga (1994), the cyanogenic potential of cassava leaves is 5 to 20 times larger than the roots, due to the greater activity of linamarase. For consumption, the leaves are dried and used in the form of flour in multimixtures, or cooked for a long time for the preparation typical dishes, including maniçoba, which contains feijoada ingredients, except beans that are replaced by the crushed cassava leaves, also known as maniva (Rosa Neto, 2009).

FUTURE PERSPECTIVES

 Increasing the supply of quality cassava derivatives requires the monitoring of producers, modernization of small rural cassava flour mills, improving equipment efficiency, and adaptation of products to consumers' expectations.  Increasing the demand for new cassava varieties with a nutritional and functional appeal, with the expansion of new agricultural frontiers and greater incentive for the development of new alternatives to the use of derivatives.  Changes in consumers' habits and the possibility of cassava participate in other markets, such as partial wheat flour replacement in bakery products can contribute to increasing the cassava chain with reduced wheat imports.  Adding value to raw materials through differentiated products, such as pre-cooked and frozen cassava, and an increase in industrial starch production, making cassava as a source of income, especially for small and medium producers. There is a reduction of its use in food and feed in traditional ways and an increase in industrial use.

REFERENCES

ABAM. (2016). Associação brasileira dos produtores de amido de mandioca http://www.abam.com.br/. [ABAM. (2016). The Brazilian Association of Cassava Starch Producers. http://www.abam.com.br/] Albuquerque, T. T. O., Miranda, L. C. G., Salim, J., Teles, F. F. F. and Quirino, J. G. (1993). Composição centesimal da raiz de 10 variedades de mandioca (Manihot esculenta Crantz) cultivadas em minas gerais. Revista Brasileira de Mandioca, 12(1): 7-12. [Albuquerque, T. T. O., Miranda, L. C. G., Salim, J., Teles, F. F. F., Quirino, J. G. (1993). Chemical composition of the root of 10 cassava varieties (Manihot esculenta Crantz) grown in Minas Gerais. Brazilian Journal of Cassava 12(1): 7-12.]. AOAC. (1995). Association of official analytical chemists. Official methods of analysis of the aoac international. Arlington: AOAC.

Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 125

AOAC. (1997). Association of official analytical chemistry. Official methods of analysis of aoac 16 ed. Gaithersburg: AOAC. 1141p. Arias, L. V. B. (2000). Fécula de mandioca e polvilho azedo para fabricação de pão de queijo. In Pizzinato, A. O., R.de C.S.S (Ed.), Seminário pão de queijo: Ingredientes, formulação e processo, Campinas: Instituto de Tecnologia de Alimentos. (pp. 1-4). [Arias, L. V. B. (2000). Cassava starch and sour starch for the manufacture of pão de queijo. In: Pizzinato, A, Ormese, R.de C.S.S. Pão de Queijo Workshop: ingredients, formulation and process. Campinas: Institute of Food Technology, p.1-14]. Bokanga, M. (1994). Processing of cassava leaves for human consumption. In 375 ed., (pp. 203-208): International Society for Horticultural Science (ISHS), Leuven, Belgium. Borges, M. F., Fukuda, W. M. G. and Rossetti, A. G. (2002). Avaliação de variedades de mandioca para consumo humano. Pesquisa Agropecuária Brasileira, 37(11): 1559-1565. [Borges, M. F., Fukuda, W. M. G, Rossetti, A. G. (2002). Evaluation of cassava varieties for human consumption. Brazilian Agricultural Research, 37(11): 1559-1565]. Brasil. (1995). Ministério da Agricultura, Pecuária e Abastecimento. Portaria n. 554, de 30 de agosto de 1995, Norma de identidade, qualidade, apresentação, embalagem, armazenamento e transporte da farinha de mandioca. Diário Oficial da República Federativa do Brasil. Brasília. [Brazil (1995). Ministry of Agriculture, Livestock and Supply. Ordinance n. 554, august 30, 1995. Identity Standards, Quality, Presentation, Packaging, Storage and Transportation of Cassava Flour. Official diary, Brasília]. Brasil. (2011). Ministério da Agricultura, Pecuária e Abastecimento. Instrução Normativa nº 52, de 07 de novembro de 2011, Regulamento técnico da farinha de mandioca. Diário Oficial da República Federativa do Brasil. Brasilia. [Brazil (2011). Ministry of Agriculture, Livestock and Supply. Normative Instruction nº 52, November 07, 2011. Technical Regulation of cassava flour. Official diary, Brasilia]. Cardoso, C. E. L., Souza, J. S. and Gameiro, A. H. (2006). Aspectos econômicos e mercado. In: Embrapa. Aspectos socioeconômicos e agronômicos da mandioca. In, Cruz das Almas, BA: Embrapa. (pp. 41-70). [CARDOSO, C. E. L.; SOUZA, J. S.; GAMEIRO, A. H. (2006). Economics and market aspects. In: Embrapa. Socio-economic and agronomic aspects of cassava. Cruz das Almas, BA: Embrapa p 41-70]. CEPEA. (2016). Centro de estudos avançados em economia aplicada- esalq/usp http://cepea.esalq.usp.br/mandioca/. [CEPEA. (2016). Center for Advanced Studies in Applied Economics- ESALQ/USP http://cepea.esalq.usp.br/mandioca/] Cereda, M. P. and Vilpoux, O. (2010). Metodologia para divulgação de tecnologia para agroindústrias rurais: Exemplo do processamento de farinha de mandioca no maranhão. Journal of Management and Regional Development, 6: 219-250. [Cereda M. P., Vilpoux, O. F. (2010). Methodology for dissemination of technology for rural agribusinesses: example of cassava flour processing in Maranhão. Journal of Management and Regional Development, 6:219-250]. Cereda, M. P. and Vilpoux, O. F. (2003). Tecnologia, usos e potencialidades de tuberosas amiláceas latino americanas v. 3. São Paulo: Fundação Cargill. [Cereda M. P., Vilpoux, O. F. (2003). Technology, uses and potential of Latin American starchy tuberous. São Paulo, Cargill Foundation, v.3, 2003]. Chávez, A. L., Bedoya, J. M., Sánchez, T., Iglesias, C., Ceballos, H. W. and Roca, W. (2000). Iron, carotene, and ascorbic acid in cassava roots and leaves. Food and Nutrition Bulletin, 21: 410-413. Complimentary Contributor Copy 126 Elisa Cristina Andrade Neves, Daniela Andrade Neves et al.

Chisté, R. C. and Cohen, K. O. (2008). Determinação de cianeto total nas farinhas de mandioca do grupo seca e d’água comercializadas na cidade de belém-pa. Revista Brasileira de Tecnologia Agroindustrial, 2: 96-102. [Chisté, R. C., Cohen, K. O. (2008). Total cyanide determination from cassava flour of dry and water groups traded in the city of Belém-PA. Journal of Agroindustrial Technology, 2:96-102]. Chisté, R. C., Cohen, K. O., Mathias, E. A. and Oliveira, S. S. (2010). Quantificação de cianeto total nas etapas de processamento das farinhas de mandioca dos grupos seca e d'água. Acta Amazonica, 40(1): 221-226. [Chisté, R. C., Cohen, K. O., Mathias, E. A., Oliveira. S. S. (2010). Total cyanide quantification in processing stages of cassava flour from dry and water groups. Acta Amazonica, 40(1): 221-226]. Chisté, R. C., Cohen, K. O., Mathias, E. A. and Ramoa Júnior, A. G. A. (2006). Qualidade da farinha de mandioca do grupo seca. Food Science and Technology, 26: 861-864. [Chisté, R. C., Cohen, K. O., Mathias, E. A., Ramoa Júnior, A. G. A. (2006). Quality of cassava flour from a dry group. Food SCience and Technology, 26(4): 861-864]. Chisté, R. C., Silva, P. A., Lopes, A. S. and da Silva Pena, R. (2012). Sorption isotherms of tapioca flour. International Journal of Food Science & Technology, 47(4): 870-874. Dias, L. T. and Leonel, M. (2006). Caracterização físico-química de farinhas de mandioca de diferentes localidades do brasil. Ciência e Agrotecnologia, 30(4): 692-700. [Dias, L. T., Leonel, M. (2006). Phisico-chemical characteristics of cassava flours from different regions of Brazil. Science and Agrotechnology, 30(4): 692-700]. Dósea, R. R., Marcellini, P. S., Santos, A. A., Ramos, A. L. D. and Lima, A. S. (2010). Qualidade microbiológica na obtenção de farinha e fécula de mandioca em unidades tradicionais e modelo. Ciência Rural, 40: 411-416. [Dósea, R. R., Marcellini, P. S., Santos, A. A., Ramos, A. L. D., Lima, A. S. (2010). Microbiological quality in the flour and starch cassava processing in traditional and model unit. Rural Science, 40:441-446]. El-Dash, A. and Germani, R. (1994). Tecnologia de farinhas mistas: Uso de farinhas mistas de trigo e mandioca na produção de pães. Brasília, DF: Embrapa. [El-Dash, A., Germani, R. (1994). Technology of mixed flours: use of mixed flour, wheat and cassava, in bread production. Brasília, DF: Embrapa]. EMBRAPA. (2016). Empresa brasileira de pesquisa agropecuária https://www.embrapa.br/mandioca-e-fruticultura. [EMBRAPA. Brazilian Agricultural Research Corporation (2016). https://www.embrapa.br/ mandioca-e-fruticultura] Fennema, O. R. (1996). Food chemistry 3 ed. New York: Marcel Dekker. 1069 p. Ferreira Filho, J. R., Silveira, H. F., Macedo, J. J. G., Lima, M. B. and Cardoso, C. E. L. C. (2013). Cultivo, processamento e uso da mandioca: Instruções práticas. Brasília-DF: Embrapa. [Ferreira Filho, J. R., Silveira, H. F., Macedo, J. J. G., Lima, M. B., Cardoso, C.E.L.C. (2013). Cultivation, processing and using of cassava: Practice directions. Brasília-DF: Embrapa]. Fiorda, F. A., Soares Junior, M. S., Silva, F. A., Souto, L. R. F. and Grossmann, M. V. E. (2003). Farinha de bagaço de mandioca: Aproveitamento de subproduto e comparação com fécula de mandioca. Pesquisa Agropecuária Tropical, 43: 408-416. [Fiorda, F. A., Soares Junior M. S., Silva, F. A., Souto, L. R. F., Grossmann, M. V. E. (2003). Cassava bagasse flour: utilization of by-product and comparison with cassava starch. Tropical Agricultural Research, 43:408-416]. Leite, E. J. (2003). Iniciando um pequeno grande negócio agroindustrial: Processamento da mandioca. Brasília: Embrapa Informação Tecnológica. 115. [Leite, E. J. (ed.) (2003). Complimentary Contributor Copy Technological Aspects of Processing of Cassava Derivatives 127

Starting a big small agroindustrial business: cassava processing. Brasília: Embrapa .115p.]. Lorenzi, J. O. (1994). Variação na qualidade culinária das raízes de mandioca. Bragantia, 53(2): 237-245. [Lorenzi, J. O. Culinary quality variation in cassava roots. Bragantia, 53(2): 237-245]. Mezette, T. F., Carvalho, C. R. L., Morgano, M. A., Silva, M. G., Parra, E. S. B., Galera, J. M. S. V. and Valle, T. L. (2009). Seleção de clones-elite de mandioca de mesa visando a características agronômicas, tecnológicas e químicas. Bragantia, 68: 601-609. [Mezette, T. F., Carvalho, C. R. L., Morgano, M. A., Silva, M. G. da, Parra, E. S. B., Galera, J. M. S. V., Valle, T. L. (2009). Selection of cassava elit clones for agronomic, technological, and chemical characteristics. Bragantia, 68: 601-609]. Montagnac, J. A., Davis, C. R. and Tanumihardjo, S. A. (2009). Nutritional value of cassava for use as a staple food and recent advances for improvement. Comprehensive reviews in food science and food safety, 8: 181-194. Oluwamukomi, M. O., Oluwalana, I. B. and Akinbowale, O. F. (2011). Physicochemical and sensory properties of wheat-cassava composite biscuit enriched with soy flour. African Journal of Food Science, 5: 5:50-56. Ortega-Flores, C. I. (1991). Carotenoides com atividade pró vitamínica a e teores de cianeto em diferentes cultivadores de mandioca (Manihot esculenta Crantz) do estado de são paulo. USP, São Paulo. [Ortega-Flores, C. I. (1991). Carotenoids with pro vitamin A activity and levels of cyanide in different cassava (Manihot esculenta Crantz) growers in the state of São Paulo. Dissertation (Master in Food Sciences) - Pharmaceutical Sciences Faculty, USP- São Paulo]. Rosa Neto, C. (2009). A cadeia agroindustrial da mandioca em rondônia: Situação atual, desafios e perspectivas. Porto Velho - RO: Embrapa Rondônia: SEBRAE. [Rosa Neto, C. (Coord.) (2009). The agro-industrial supply chain of cassava in Rondônia: current situation, challenges, perspectives, and recommendations for planting in Rondônia. Porto Velho, RO: Embrapa Rondônia: SEBRAE]. Schwengber, D. R., Smiderle, O. J. and Mattioni, J. A. M. (2005). Mandioca: Recomendações para plantio em roraima (circular técnica, 5). Boa Vista: Embrapa Roraima. [Schwengber, D. R., Smiderle, O. J., Mattioni, J. A. M. (2005). Cassava: recommendations for planting in Roraima, Boa Vista: Embrapa Roraima. 30 pp. (Technical Circular, 5)]. Silva, P. A., Cunha, R. L., Lopes, A. S. and Pena, R. S. (2013). Obtenção da farinha de tapioca: Parte 1- avaliação do processo. Boletim do Centro de Pesquisa de Processamento de Alimentos, 31(1): 13-24. [Silva, P. A., Cunha, R. L., Lopes, A. S., Pena, R. S. (2013). Tapioca flour obtaining: Part 1- Process evaluation. Bulletin Research Center for Food Processing, 31(1): 13-24]. Souza, L. S., Farias, A. R. N., Mattos, P. L. P. and Fukuda, W. M. G. (2005). Processamento e utilização da mandioca. Cruz das Almas: EMPRAPA Mandioca e Fruticultura Tropical. [Souza, L. S., Farias, A. R. N., Mattos, P. L. P., Fukuda, W. M. G. (2005). Cassava processing and use. Cruz das Almas: EMPRAPA. 547p]. Vilela, E. R. and Juste Junior, E. S. G. (1987). Tecnologia da farinha de mandioca. Informe Agropecuário, 145(13): 60-62. [Vilela, E. R., Juste Junior, E. S. G. (1987). Cassava flour technology. Agriculture Information, Belo Horizonte, 145(13): 60-62].

Chapter 7

SUSTAINABLE MANAGEMENT OF CASSAVA PROCESSING WASTE FOR PROMOTING RURAL DEVELOPMENT

Anselm P. Moshi1, and Ivo Achu Nges2 1Department of Industrial Research, Tanzania Industrial Research and Development Organization (TIRDO), Dar es Salaam, Tanzania 2Departmentof Biotechnology, Lund University, Sweden

ABSTRACT

Cassava is the third-most important food source in the tropics after rice and maize. Cassava is the staple food for about half a billion people in the World. It is a tropical crop grown mainly in Africa, Asia, and South America. It can be cultivated on arid and semi- arid land where other crops do not thrive. During the processing of cassava into chips, flour or starch, enormous amount of wastes are generated ca. 0.47 tons for each ton of fresh tubers processed. This waste consists of peels, wastewater and pulp that contain between 36 to 45% (w/w) of starch and from 55 to 64% (w/w) of lignocellulosic biomass. An innovative processing system is therefore essential to take into account the transformation of this waste into value added products. This will address both the environmental pollution and inefficient utilization of these resources. The starch and lignocellulosic cassava processing waste can be converted into renewable energy carriers such as biogas through anaerobic digestion (AD), bio-ethanol through fermentation and bio-hydrogen through dark fermentation. In the case of AD, the waste can be used directly as substrate while for fermentation; the waste must be pre-treated to release monomeric sugars, which are substrates for bioethanol and bio-hydrogen production. There is possibility of sequential fermentation for either bio-ethanol or bio-hydrogen and AD for biogas production thereby making use of all the fractions of the cassava waste. Generation of renewable energy from cassava waste could benefit rural populations where access to electricity is very poor. This would also reduce the dependence on firewood and charcoal that are known to provide almost 90 percent of domestic energy

Corresponding Author: Anselm P. Moshi, email: [email protected], telephone +255756547634. Complimentary Contributor Copy 130 Anselm P. Moshi and Ivo Achu Nges

requirements. Such a development could help save trees, lower emissions that cause climate change and reduce the fumes from millions of tons of firewood that threaten human health, especially the health of women and children. Although deforestation and land degradation are well-known, the charcoal and firewood consumption that causes them is still on the rise. This chapter, therefore, explores the use of cassava waste for production of fuel energy with a focus for use as domestic cooking fuel. It also proposes an efficient approach to cassava processing to ensure efficient resource utilization in which every part of the tuber is converted to value added products mitigating environmental pollution and improving human health.

1. INTRODUCTION

1.1. Cassava Production and Growing Conditions

Cassava (Manihot esculenta Crantz) is a high-yielding crop that grows well in tropical and subtropical climate. It is widely cultivated in Africa, Asia and Latin and South America. Cassava is the third most important food source in the tropics after rice and maize and is the staple food for about 500 million people [1]. Its starch content is more than 30% (w/w of fresh tuber), which is higher than any other stem tuber plants. Moreover, cassava has many desirable growing characteristics such as drought and flood-tolerance and is suitable to grow on light sandy soils and medium texture soils. It can be cultivated on arid and semi-arid land where other crops do not thrive [2]. Global production of cassava in 2014 according to data derived from FAO biannual report 2015 Outlook [3] was estimated at 2.91 ×108 tones with Africa contributing 1.70×108tonnes (57%), Asia 0.87 ×108 (30%) Latin America 0.33 ×108 tones (11.4%), other regions contribute only 0.05 ×108 tonnes (1.6%). Cassava is a high yield crop with low requirement of agricultural input [4]. According to IITA improved varieties yield up to 20 to 40 tonnes per hectare at minimum cost of $75 to 125 [5, 6]. Therefore, cassava stands a competitive crop for food security as well as industrial raw material.

1.2. Wastes Generated During Processing and Conventional Handling

During processing of each unit of cassava tubers into chips, flour, starch or other products at both small and large-scale colossal amount of wastes are generated. For every tone of tubers processed ca.0.47 tones of wastes are produced [7]. This means, for example in 2014 according to FAO biannual report [3] a total of ca. 1.4×108 tonnes of waste was generated which comprised of peels, pulps, starch rich liquid waste, gaseous (vapour and cyanide). In Nigeria, processing of a given unit of raw cassava into gari, yield is about 34% while generating 30%, 19.8% and 16.2% of solid, gaseous and liquid wastes, respectively [8]. Although various studies have suggested a variety of ways of handling cassava waste [9- 11], still, the waste generated causes serious environmental pollution [7]. The cassava waste (e.g., peels and pulp) consists of vital macromolecules which can save as raw material for various value added products including biofuels. For example, cassava peels consist of cellulose (25%), hemicelluloses (7%) and crude protein (5%) [12], whereas cassava pulp from Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 131 starch processing factories consists of residual starch (60%), fibres (20%) and lignin (3%) [13, 14]. These wastes can be used for production of value added products such as bioethanol, biogas, biohydrogen and platform chemicals. The result of this is an additional production line in the cassava value chain or industry, which will make it more profitable, more competitive and sustainable.

1.3. Limited Energy Access in Rural Cassava Growing Regions

Considering that energy demand in the areas where cassava is cultivated (e.g., Sub- Sahara Africa), has reached crisis level causing serious deforestation (through haphazard harvesting of trees for firewood and charcoal), an innovative way of converting these bioresource to address specific energy challenges is imminent. Fire or fuel wood and charcoal provides more than 90% of domestic energy requirements for people living in Sub-Sahara Africa and South East Asia. These are the areas where cassava is cultivated in large amounts (more than 80% of global production) [15]. According to the International Energy Agency World Outlook worldwide [16], globally 1.2 billion people lack access to electricity. More than 95% of the people with no access to electricity live in developing countries and in rural areas, mainly in South Asia and sub- Saharan Africa. Also ca. 3 billion people lack modern fuels for cooking and heating relying on solid fuels (mostly in Sub-Sahara Africa and South East Asia) [17] and suffer the health consequences such as respiratory infections, including pneumonia, tuberculosis and chronic obstructive pulmonary disease, low birth weight, cataracts, cardiovascular diseases[18]. These effects can be reduced by shifting from solid fuels to cleaner energy. This article offers innovative solutions of using biogas and bioethanol which can be produced from agrowastes such as cassava waste as clean domestic cooking fuel.

1.4. Proposed Solution: Efficient Processing System Which Generate Energy for Domestic Use

From previous studies, a sequential bioethanol-biogas process can be employed to produce very high ethanol concentration [19] (and biogas) and therefore achieving maximum energy yield from cassava processing waste [20]. The system has been improved further in an ongoing pilot study where the very high concentration ca. 200g/L i.e., (20%) is distilled to 50- 60% and bottled to be used as cooking fuel in specifically designed cooking stoves. The fermentation residue which contains cellulose, hemicellulose, fermentation metabolites (e.g., volatile acids) and large number of yeast cells saves as substrate for biogas production. The biogas produced is divided into 3 portions using control valves in which 20% is recycled to heat the AD process so that temperature in the AD is maintained at 50ºC for higher reaction rates and hence higher yield and productivity (AD is discussed in section 2.2). About 20% fuels the ethanol distiller and 60% goes to low pressure cylinders to be sold at a price lower than natural gas for domestic cooking. The biogas manure (digestate or bio-fertilizer) is dried and packed in bags and sold at very low price to improve soil fertility. This will also save farmers from having to purchase the rather expensive artificial fertilizers. Individual bioenergy (biogas, bio-ethanol and bio-hydrogen) generating processes and a combination of the processes are discussed in sections 2.2-2.5.

Complimentary Contributor Copy 132 Anselm P. Moshi and Ivo Achu Nges

Figure 1. Current cooking conditions and improved cooking situation using bioethanol from cassava processing waste.

2. CHARACTERISTICS OF CASSAVA WASTE

Cassava waste has been reported to be rich in starch, cellulose and hemicelluloses (fiber), with small amounts of lignin, fats and proteins [21, 22]. Cyanide content have been reported to be as high as 390 mg/kg TS [23, 24]. The major criteria for the selection of organic matter for bio-energy production are availability, cost, carbohydrate content, and biodegradability [25]. Because of its high biomass yield and its ability to grow well in arid areas, cassava has been promoted as an ideal energy crop for bio-energy production [26]. Cassava processing waste type and composition depends on the industrial process and can be divided into four categories (a) pulp is the fibrous solid waste produced as a consequence of starch production which contains high starch content (50–60% w/w [21], (b) peelings from initial processing contain up to 45% w/w starch and lignocellulose up to 20% w/w [27], starch residues after starch settling and waste water [9]. Whereas cassava peels is the skin which is peeled from the tuber while the chaff that results from processing the root into cassava product termed “foofoo” in the West Africa is called root sieviate. Table 1 summarises composition of different types of cassava waste.

Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 133

Table 1. Composition of Cassava Waste (%w/w)

Composition Type of waste Amount References Ash Pulp 2.0 [28] Cellulose Pulp 24.99 [28] Hemi-cellulose Pulp 6.67 [28] Starch Pulp 65 -69 [21, 28] Glucan Pulp 19.1 [13] Xylan Pulp 42.0 [13] Arabicanan Pulp 1.4 [13] Galactan Pulp 0.5 [13] Mamnan Pulp 0.7 [13] lignin Pulp 3.0 [14] Protein Pulp 1.6 [21] Fat Pulp 0.12 [21] Starch peels 45 [12] Cellulose peel 5.4-8.4 [4, 12] Hemicelluloses peel 2.6 -22 [4, 12] Protein peel 5.5 -8.1 [4, 12] Lignin peel 15 -16 [4, 12] Cellulose root sivieate 9.0 [12] Hemicellulose root sivieate 9.0 [12] Protein root sivieate 2.1.0 [12] Lignin root sivieate 23.0 [12]

2.1. Pre-Treatment of Cassava Waste

Cassava pretreatment methods are often employed to aid the conversion of starch into fermentable sugars or reduced particle sizes. The goal of any pre-treatment is the modification of the (chemical) structure of the feedstock thereby removing the hurdles or hindrances to hydrolysis and enhances the yields of fermentable sugars [29]. There exist many pretreatment methods wherein enzymatic and acid hydrolysis are commonly reported in literature especially in case of bio-ethanol or bio-hydrogen production from cassava waste [30]. Biomass pre-treatment can be grouped under three main categories; (1) mechanical/physical, (2) chemical and (3) biological pretreatments wherein a combination of these methods is often performed [31, 32]. For biogas production, mechanical pre-treatment is often sufficient [33] while enhanced pre-treatments involving a combination of mechanical and chemical, mechanical and biological or a combination of all three methods are often necessary in case of bio-ethanol or bio-hydrogen production [34]. Mechanical pre-treatment often entail the increase in the accessible surface area or pore size as in milling, chopping, irradiation etc.[31]. Other more specialized forms of mechanical pre-treatment include amongst others steam explosion, liquid hot water treatment [29] and thermal or heat pre-treatments [35]. Chemical pre-treatments with the use of acids and base are known to remove or dissolve lignin and hemicelluloses from the feedstock thereby improving the recovery of glucose from cellulose. The acids and base commonly used for such pre-treatments are H2SO4, HCl and

Complimentary Contributor Copy 134 Anselm P. Moshi and Ivo Achu Nges

NaOH [29, 36]. In recent years, other acids and bases such H3PO4, Ca (OH)2 and NH3 have also been used in chemical pre-treatments. Biological pre-treatment involves the use of microorganisms and or parts thereof such as enzymes to enhance the hydrolysis of the feedstock. Enzymatic hydrolysis is particularly common in bio-ethanol and bio-hydrogen production wherein reduced sugars such as glucose and xylose are precursors [31, 37]. White-rot-fungi are microorganisms specially endowed with the ability to degrade lignin and polysaccharides via the secretion of enzymes such as lignin peroxidase, manganese peroxidase and laccase [38]. Ensiling is common biological pre-treatment method which is also a means of biomass storage wherein the feedstock is fermented (at low pH) via addition of lactic acid bacteria under anaerobic conditions [39, 40]. Biological pre-treatments are safer, present low energy requirements and have a rather low environmental footprint [31].

2.2. Anaerobic Digestion, AD

There has been a sharp increase of interest in AD over the recent years due to the fact via AD a renewable energy can be produced, waste can be treated and the effluent from the process can be used as a bio-fertilizer [41]. Renewable energy production via AD may also lead to a reduction in greenhouse emissions and promote a sustainable development of energy supply [42]. As compared to other renewable energy production technologies, AD has been reported as the most cost-effective and environmentally benign technology [42, 43]. The AD is widely recognized as a promising, robust, low-cost technique for treatment of various organic waste streams with or without pre-treatment. Amongst the different bio-fuels, currently biogas presents the most preferred alternative to the rural populations as a major low-carbon fuel source [44]. The AD process comprises of four major biological steps i.e., hydrolysis, acidogenesis, acetogenesis, and methanogenesis which are performed in tandem, whereby bacteria and Archaea break down biodegradable matter in the absence of oxygen to produce a mixture of gases (biogas), amongst them energy-rich methane. In general terms, methanogensis is the rate limiting step in AD process due to the fastidious, slow-growing nature of Archaea [45]. However, hydrolysis is often the rate-limiting step in the AD of particulate, solid or lignocellulosic material [46, 47]. Therefore, for an efficient and effective AD of solid waste biomass such as cassava waste to biogas, a suitable pre-treatment (as discussed above) is necessary. The AD process can be classified as either liquid or dry depending on the nature or water content of the feedstock. Processes run on feedstock with total solids (TS) values higher than 15% of wet weight are often termed dry AD while processes run on feedstock with TS values less than 15% are usually termed wet AD [42]. The biogas or AD processes can be operated either in batch or continuous modes under mesophilic or thermophilic conditions [48]. In general, the biogas process or AD is influenced by ambient factors, which might slow or stall the process if not within a certain range [49]. These factors are amongst others temperature, pH, inhibitors/nutrients, water content, organic loading residence time, mixing etc. [50, 51].

Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 135

The produced biogas can be use directly as fuel in combined heat and power gas engines [42], for heating, cooking (as has been highlighted above), and lighting or be upgraded to biomethane which can be a substitute for natural gas [52, 53]. Cassava waste is rich in carbohydrates (starch), see section 2 and Table 1, which are easily converted to biogas via AD. However, cassava waste may also contain cyanides which are potent inhibitors in the AD process [54, 55]. The waste is also poor in micronutrients such as iron, zinc and manganese [56] which are integral parts of enzymes or co-factors unswervingly liaised in the biochemistry of methane production [57]. Co-digestion of cassava and pig manure which is rich in nutrients has been reported as a way forward in the AD of cassava waste [58]. Though cassava waste is rich in readily convertible starch to biogas, its use as feedstock for biogas (bio-energy) production is still in its infancy. There are however a few studies on biogas production or combined bioethanol and biogas production from cassava waste [54, 58, 59]. AD is also easy to perform and the residue or effluent from the process called digestate can be used as a bio-fertilizer. The bio-fertilizer may become a future fertilizer of choice due to the easy availability of plant nutrients such as nitrogen, phosphorus and potassium and its short-term fertilizing effect [42]. The AD process also minimizes the proliferation of pathogens which is vital when the digestate is used as a bio-fertilizer [42]. Cassava wastes may, therefore, become a feedstock or co-feedstock of choice in the – (Co-) AD process because of its high biodegradability [54, 59].

2.3. Bio-Ethanol Fermentation

Bio-ethanol production as a bio-energy carrier has gained heightened attention as an alternative solution to energy security and environmental pollution among nations [60]. Bio- ethanol is a renewable energy source produced through fermentation of sugars and used as a partial gasoline replacement in a few countries in the world [61]. Amongst others, the cost of feedstock has been reported to hamper the full development of the bio-ethanol industry [62]. Cassava waste with its high starch content could be a cheap source of biomass that can be used for bio-ethanol fermentation. Processing of cassava waste to bio-ethanol will entail four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/purification (distillation) [63]. Pre-treatment is a major unit operation without which bio-ethanol fermentation cannot proceed. Various pre-treatments options have been discussed under section 2.1. Enzymatic hydrolysis performed separately from the fermentation step is known as separate hydrolysis and fermentation (SHF). Hydrolysis of biomolecules carried out in the presence of the fermentative microorganism is referred to as simultaneous saccharification and fermentation (SSF). Simultaneous saccharification of both cellulose (to glucose) and hemicellulose (to xylose and arabinose) and co-fermentation of both glucose and xylose (SSCF) could be carried out by genetically engineered microbes that ferment xylose and glucose in the same broth as the enzymatic hydrolysis of cellulose and hemicellulose. SSF and SSCF are preferred since both unit operations can be performed in the same tank, resulting in lower costs [63]. There is a body of knowledge about bio-ethanol production from cassava waste [13, 14, 28, 64].

Complimentary Contributor Copy 136 Anselm P. Moshi and Ivo Achu Nges

The second cost item after raw material in bioethanol production is energy expended mainly in the pretreatment and recovery stages. This has been a bottleneck to lower ethanol price for decades. Therefore, design of ethanol production process to lower energy in these steps is crucial for the competitiveness of the bioethanol industry. With substrate that is readily degradable like cassava waste the best approach is to employ minimum pretreatment such as partial liquefaction with minimum amount of enzymes followed by pulse fed-batch simultaneous saccharification. This has an advantage in that the liquefying and the saccharifying enzymes in the SSF act synergistically to achieve a more effective hydrolysis at a pace that does not impair glycolytic enzymes in the yeast cell. In this way very high ethanol titre up to 20% w/v can be obtained which significantly reduces energy demand in the downstream recovery stage [65]. Further, synergy of the liquefying and saccharifying enzymes can also be explored through the use of co-culture of saccharifying microorganisms and fermenting microorganisms. This has proved to be possible at the fermentation temperature thus downsizing the cost of both enzyme and energy [66].

2.4. Bio-Hydrogen Fermentation

Bio-hydrogen production from renewable sources, often termed ‘green energy’ has been in the lamplight most recently as an approach towards sustainable development and energy security [22]. Bio-hydrogen is non-polluting, high energy yielding and alternative to fossil- derived fuel [67]. In fact, combustion of hydrogen has only water as a by-product. Hydrogen can be produced by a number of processes, including electrolysis of water, thermocatalytic reformation of hydrogen-rich organic compounds, and biological processes. Biological production of hydrogen (biohydrogen), using microorganisms, is an exciting new area of technology development that offers the potential production of usable hydrogen from a variety of renewable resources [68] such as cassava waste. On a sustainability perspective, it is environment-friendly and less energy-intensive as compared to thermo-chemical and electrochemical processes [69]. Thus, this chapter will consider only biological hydrogen production. Biological hydrogen production can be categorized into four groups based on the biological route of its generation, which are photo-biological fermentation, anaerobic formation, enzymatic and microbial electrolysis or a combination of this processes [68, 69]. Fermentative bio-hydrogen can be operated in batch, continuous and fed-batch modes [70]. It has also been reported that fermentative hydrogen production processes have some edge over the other biological processes [69]. More so, dark fermentation has been shown to be highly efficient as compared to photo-fermentation [70]. Studies on sequential dark fermentation and photo-fermentation have reported particularly in bio-hydrogen production from cassava waste [22]. Environmental samples from a variety of sources including the sewage sludge from waste-water treatment plant can serve as a microbial consortium for hydrogen fermentation under anaerobic conditions after the hydrogen-consuming methanogens are inactivated (or not) via heat or load-shock treatment, chloroform, acid, base and sodium-2 bromoethanesulfonate[71-74]. These treatments are often effectuated to suppress the proliferation of hydrogen consuming methanogens and therefore promote the growth of hydrogen production bacteria. Such mixed cultures are often preferred because they provide Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 137 stability, diversity of biochemical functions, and possibility to use a variety of (unsterilized) substrates [74-76]. Bio-hydrogen production from cassava waste using treated and un-treated anaerobic sludge have proved successful in both batch and continuous processes wherein the processes with untreated sludge showed higher bio-hydrogen yields [74]. However, increasing attention is being paid on bio-hydrogen (dark) fermentation via the use of specialized thermophilic pure cultures such as Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana wherein the microorganism are reported to utilize pentose, hexoses, and oligosaccharides [77]. These microorganisms can produce hydrogen at yields near to the theoretical maximum of 4 mol/hexose consumed [78]. C. saccharolyticus can also ferment a wide range of poly-, oligo- and mono-saccharides including sugars present in lignocellulosic hydrolysate [79] and starchy feedstock such as that from cassava waste [22]. Bio-hydrogen production from various fractions of cassava waste has been reported in literature [80-82] and also enhanced bio-hydrogen production during co-digestion of cassava waste with food waste [25].

2.5. Combined Bio-Ethanol/Bio-Hydrogen and Biogas Production

A sustainable solution for the complete utilization of the residual organic matter in the effluents from bio-ethanol and bio-hydrogen processes is to convert them to biogas and use the residual effluents as a bio-fertilizer on agricultural soil [83, 84]. Such a development will lead to efficient biomass utilization and a sustainable system. An important part in this is to create a system that allows the recycling of the process effluent to farmland as a bio-fertilizer. This gives a sustainable recycling of nutrients, improves soil carbon content, and an application that is also generally most cost efficient [85]. In fact, bio-fertilizers have emerged as vital component in integrated nutrient supply system and can improve crop yield through environmentally sound nutrient supplies [86]. Apart from the ills of burning fossil fuel such as global warming, it is also very important that nutrients be returned to the farmland else they leak into watersheds and cause environmental hazards such as eutrophication. Recycling of plant nutrient such as phosphorous (P) is vital since peak P is eminent and quality of the remaining phosphate rock is decreasing [87]. Practically, the highest maximum ethanol or hydrogen production is unachievable during (dark) fermentation [78, 88]. The effluent or residue from the dark fermentative process can be transferred to an anaerobic digester, wherein acetate and other unutilized fractions can be converted to biogas via AD, which is a reliable and an industrially established process [88]. Bio-ethanol fermentation followed by biogas production is well established with many studies reported in the scientific literature and even applied in a bio-refinery concept [88, 89]. Bio-refinery entails the total conversion of all components of a feedstock to usable end- products. Such a development is both economically and environmentally important paying cognizance to the high cost of some feedstock and potential environmental degradation liaised to the dumping of organic matter. Bio-hydrogen production followed by the second stage of AD, forming a combined system have proved feasible in harvesting the residual energy (biogas), and a high overall energy recovery of the combined system [90]. There exist a few studies detailing the combined bio-ethanol and biogas production from cassava waste [54, 59, 91] and combined bio-hydrogen and biogas production [25].There are also studies on sequential bio-ethanol and bio-hydrogen production from cassava waste (stillage) [92]. Complimentary Contributor Copy 138 Anselm P. Moshi and Ivo Achu Nges

3. BIOGAS AND BIOETHANOL FOR DOMESTIC COOKING

3.1. Purification and Packing of Biogas in Pressurized Cylinder

Application of biogas is restricted to the place where it is produced. Commonly the gas produced in the AD is transported to the desired places e.g., kitchen by pipe line, facilitated by the pressure developed in the biogas digester dome itself. However, this is not sufficient to transport gas to farther distances from the generation site. Consequently, biogas use especially in Africa until now is not produced at a persuasive amount as large-scale biogas production lack economic incentive. For it to have wide use in domestic cooking it has to be bottled in cylinders. Biogas is composed of mainly CH4 and CO2. In most biogas processes, the CH4 content is usually in the range of 50-70% and at this CH4 concentration, biogas burns very well and can be used as a alternate to kerosene, charcoal and fire wood for cooking and lighting. This conserves forest, and also saves health of especially women and children who suffer chronic respiratory illness as a result of using solid biomass in cooking (as outlined above). Typical composition of biogas is given in Table 2 [93].

Table 2. Biogas Composition

Substances Symbol Percentage (%) Methane CH4 50-70 Carbon dioxide CO2 30-40 Hydrogen H2 5-10 Nitrogen N2 1-2 Water vapor H2O 0.3% Hydrogen Sulphide H2S Traces

The utilization of biogas as an efficient energy source depends strongly on its CH4 concentration. However, biogas which is not upgraded has low energy per unit volume almost twice less the energy value for natural gas. Furthermore, it is technically difficult to compress biogas which also contains high amount of CO2, traces of H2S and water vapour. Therefore, biogas purification is essential in order to have more energy per unit volume of compressed biogas and to get rid of the corrosive effect of H2S. Biogas can easily be upgraded up to 97% by introducing a scrubber containing KOH (3M) and silica gel between the digester and the collecting bag to remove CO2, H2S and water vapor [94, 93], thus also eliminating the greenhouse gas. Then after upgraded biogas can be packed in compressed cylinders and used in transportation or domestic cooking in the same way as compressed natural gas. The use of biogas can significantly reduce health consequences ascribed to the use of solid biomass in domestic cooking. In rural households, wood, charcoal and other solid fuels (mainly agricultural residues and coal) are often burned in open fires or poorly functioning stoves. Incomplete combustion leads to the release of small particles and other constituents such as carbon monoxide, benzene, butadiene, formaldehyde, polyaromatic hydrocarbons and many other compounds that are hazardous to human health in the household environment [95]. The consequence is acute infections of the lower respiratory tract (pneumonia) in young children, the chief killer of children worldwide and the disease responsible for the most lost life in earlier years in the world; and chronic obstructive pulmonary disease, such as chronic

Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 139 bronchitis and emphysema, in adult women who have cooked over unvented solid fuel stoves for many years [95]. It is estimated that solid biomass indoor cooking claims 800,000 to 2,400,000 premature lives each year [96]. Total or partial replacement of wood fuel with renewable energy sources such as biogas and bioethanol will significantly mitigate these ills.

3.2. Bioethanol as Efficient Domestic Cooking Fuel

In utilization of cassava processing waste, the most efficient approach is combining bioethanol and biogas production. This avoids expensive pretreatments, taking advantage of the readily degradable starch component of up to 40% w/w of cassava waste for bioethanol, the lignocellulosic component of up to 37% w/w, protein of about 8% w/w plus biomass (yeast cells) and fermentation metabolites for biogas production [7]. By employing a pose fed-batch simultaneous saccharification and fermentation of partially liquefied starch in the ethanol fermentation step it is possible to achieve very high ethanol concentration up to 20% w/v [19]. Consequently, a very small amount of energy is used to distill to 50-60%. A Stove running on 50% ethanol-water mixture has proved to be as effective as Liquefied Petroleum Gas and kerosene stoves. Ethanol produced in this way is cheaper than kerosene. Due to its high energy density, easy transportability, easy storage and local availability of its substrate (e.g., cassava processing waste), bioethanol is considered a fuel of choice in rural settings. Ethanol can be produced locally from a variety of materials that can be classified as sugar-containing (e.g., sugar cane and sweet sorghum), starch-containing (e.g., cassava, maize and grain sorghum). For example in India ethanol can be produced inexpensively from sugar cane and distributed at retail prices of less than 35 cents (USD) [97]. Thus, combining innovative fermentation technology and the low cost and abundantly available raw materials for the production of ethanol, make it very competitive compared with the other fuels used for cooking. Nigeria, the world leader in cassava production, has recently embarked on a gigantic project to replace wood and kerosene with cassava based bioethanol in domestic cooking [98]. Although this initiative has been credited as being beneficial in terms of job creation, boost rural agriculture, conserve forest from fuel wood exploitation, alleviate poverty and prevent indoor pollution, it has however been blamed to release large volume of waste stream. This waste stream (as discussed above) can serve as substrate for biogas production while creating a valuable bio-fertilizer from the effluent. This chapter therefore offers a comprehensive and more efficient solution in that the peeled cassava tuber ca. 50% of total biomass w/w is used for more valuable products and the waste also ca. 50% w/w of total tuber weight goes to bioethanol, biogas and bio-fertilizer. Furthermore, ethanol fuel especially ethanol gel fuel is suitable for domestic cooking and has very low indoor emissions of particulate matter and carbon monoxide [99].

CONCLUSION AND RECOMMENDATIONS

The conversion of cassava waste into cooking energy in Africa, Eastern Asia and Latin America where cassava is cultivated in abundance, constitutes not only a sustainable cassava

Complimentary Contributor Copy 140 Anselm P. Moshi and Ivo Achu Nges waste management scheme but also a solution to the energy crisis these regions are facing. This should stimulate both industrial and agricultural development as well as mitigating climate change. Furthermore, this approach in which agro-wastes are wholly converted to energy and bio-fertiliser resulting into zero waste is recommended for all agro-based subsectors in the agricultural based economies.

REFERENCES

[1] A.P. Cardoso, E. Mirione, M. Ernesto, F. Massaza, J. Cliff, M. Rezaul Haque, J.H. Bradbury, Processing of cassava roots to remove cyanogens, J. Food. Comp. Anal. 18 (2005) 451-460. [2] D. Dai, Z. Hu, G. Pu, H. Li, C. Wang, Energy efficiency and potentials of cassava fuel ethanol in Guangxi region of China, Energ. Conv. Mgt. 47 (2006) 1686-1699. [3] F. Outlook, Biannual Report on Global Food Markets, FAO, Rome, Italy 2015, pp. 133. www.fao.org/3/a-I5003E.pdf. Accessed on 26 June 2016. [4] A.P. Moshi, C.F. Crespo, M. Badshah, K.M. Hosea, A.M. Mshandete, E. Elisante, B. Mattiasson, Characterisation and evaluation of a novel feedstock, Manihot glaziovii, Muell. Arg, for production of bioenergy carriers: Bioethanol and biogas, Bioresour.Technol. 172 (2014) 58-67. [5] V.M. Manyong, A.G.O. Dixon, K.O. Makinde, M. Bokanga, and J. Whyte, The Contribution of IITA-improved Cassava to Food Security in Sub-Saharan Africa, IITA 2000. www.fao.org/docs/eims. Accessed on 26 June 2016. [6] N. Nassar, R. Ortiz, Breeding cassava to feed the poor, Sci. Am. 302 (2010) 78-84. [7] A.P. Moshi, S.G. Temu, I.A. Nges, G. Malmo, K.M.M. Hosea, E. Elisante, B. Mattiasson, Combined production of bioethanol and biogas from peels of wild cassava Manihot glaziovii, Chem. Eng. J. 279 (2015) 297-306. [8] E.I. Ohimain, D.I. Silas-Olu, J.T. Zipamoh, Biowastes generation by small scale cassava processing centres in Wilberforce Island, Bayelsa State, Nigeria, Green. J.Environ. Mgt Pub.Saf. 2 (2013) 51-59. [9] Ubalua, Cassava wastes: treatment options and value addition alternatives, Afr. J.Biotech.6 (2007) 2065-2073. [10] S. Sivakumar, P. Senthilkumar, V. Subburam, Carbon from cassava peel, an agricultural waste, as an adsorbent in the removal of dyes and metal ions from aqueous solution, Bioresour. Technol. 80 (2001) 233-235. [11] F.C. Ogbo, Conversion of cassava wastes for biofertilizer production using phosphate solubilizing fungi, Bioresour. Technol. 101 (2010) 4120-4124. [12] F. Aderemi, F. Nworgu, Nutritional status of cassava peels and root sieviate biodegraded with Aspergillus niger, American-Eurasian J.Agric. Environ. Sci. 2 (2007) 308-311. [13] Kosugi, A. Kondo, M. Ueda, Y. Murata, P. Vaithanomsat, W. Thanapase, T. Arai, Y. Mori, Production of ethanol from cassava pulp via fermentation with a surface- engineered yeast strain displaying glucoamylase, Renew. Energ. 34 (2009) 1354-1358.

Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 141

[14] U. Rattanachomsri, S. Tanapongpipat, L. Eurwilaichitr, V. Champreda, Simultaneous non-thermal saccharification of cassava pulp by multi-enzyme activity and ethanol fermentation by Candida tropicalis, J.Biosci. Bioeng. 107 (2009) 488-493. [15] A.D. Uchechukwu-Agua, O.J. Caleb, U.L. Opara, Postharvest handling and storage of fresh cassava root and products: A review, Food.Bioproc. Technol.8 (2015) 729-748. [16] I.E.,A. IEA, World Energy Outlook, http://www.worldenergyoutlook.org, 2013. Accessed on 15th June 2016. [17] W. World Bank, Annual report www.worldbank.org/en/topic/energy/x/energyaccess, 2016. Accessed on 15th June 2016. [18] D.G. Fullerton, N. Bruce, S.B. Gordon, Indoor air pollution from biomass fuel smoke is a major health concern in the developing world, Trans. Roy. Soc. Trop. Med. Hyg. 102 (2008) 843-851. [19] A.P. Moshi, C.F. Crespo, M. Badshah, K.M. Hosea, A.M. Mshandete, B. Mattiasson, High bioethanol titre from Manihot glaziovii through fed-batch simultaneous saccharification and fermentation in Automatic Gas Potential Test System, Bioresour. Technol.156 (2014a) 348-356. [20] A.P. Moshi, S.G. Temu, I.A. Nges, G. Malmo, K.M.M. Hosea, E. Elisante, B. Mattiasson, Combined production of bioethanol and biogas from peels of wild cassava Manihot glaziovii, Chem.Eng.J.279 (2015) 297-306. [21] K. Sriroth, R. Chollakup, S. Chotineeranat, K. Piyachomkwan, C.G. Oates, Processing of cassava waste for improved biomass utilization, Bioresour. Technol. 71 (2000) 63- 69. [22] W. Zong, R. Yu, P. Zhang, M. Fan, Z. Zhou, Efficient hydrogen gas production from cassava and food waste by a two-step process of dark fermentation and photo- fermentation, Biom. Bioeng. 33 (2009) 1458-1463. [23] V.I.E., Ajiwe, I.E., Anyadiegwu, Recovery of silver from industrial wastes, cassava solution effects, Sep. Pur. Technol. 18 (2000) 89-92. [24] T249 cm-85 Carbohydrate composition of extractive-free wood and wood pulp by gas- liquid chromatography, Test Methods 1998–1999, TAPPI Press, Atlanta, 1999. [25] W. Wang, L. Xie, G. Luo, Q. Zhou, Enhanced fermentative hydrogen production from cassava stillage by co-digestion: The effects of different co-substrates, Int. J.Hydrogen Energ. 38 (2013) 6980-6988. [26] Z. Hu, P. Tan, G. Pu, Multi-objective optimization of cassava-based fuel ethanol used as an alternative automotive fuel in Guangxi, China, Appl. Energ. 83 (2006) 819-840. [27] A.P. Moshi, C.F. Crespo, M. Badshah, K.M. Hosea, A.M. Mshandete, E. Elisante, B. Mattiasson, Characterisation and evaluation of a novel feedstock, Manihot glaziovii, Muell. Arg, for production of bioenergy carriers: Bioethanol and biogas, Bioresour. Technol. 172 (2014b) 58-67. [28] T. Srinorakutara, L. Kaewvimol, L.-a. Saengow, Approach of cassava waste pretreatments for fuel ethanol production in Thailand, J. Sci. Res.Chula. Univ 31 (2006) 77-84. [29] N. Mosier, C.E. Wyman, B. Dale, R. Elander, Y.Y. Lee, M. Holtzapple, M. Ladisch, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresour. Technol. 96 (2005). [30] A.P. Moshi, C.F. Crespo, M. Badshah, K.M.M. Hosea, A.M. Mshandete, B. Mattiasson, High bioethanol titre from Manihot glaziovii through fed-batch simultaneous Complimentary Contributor Copy 142 Anselm P. Moshi and Ivo Achu Nges

saccharification and fermentation in Automatic Gas Potential Test System, Bioresour. Technol. 156 (2014) 348-356. [31] M. Taherzadeh, K. Karimi, Pretreatment of Lignocellulosic Wastes to Improve Ethanol and Biogas Production: A Review, Int. J. Mol. Sci. 9 (2008) 1621-1651. [32] Berlin, M. Balakshin, N. Gilkes, J. Kadla, V. Maximenko, S. Kubo, J. Saddler, Inhibition of cellulase, xylanase and β-glucosidase activities by softwood lignin preparations, J.Biotech.125 (2006) 198-209. [33] Q. Zhang, J. He, M. Tian, Z. Mao, L. Tang, J. Zhang, H. Zhang, Enhancement of methane production from cassava residues by biological pretreatment using a constructed microbial consortium, Bioresour.Technol. 102 (2011) 8899-8906. [34] M.P. Divya Nair, G. Padmaja, S.N. Moorthy, Biodegradation of cassava starch factory residue using a combination of cellulases, xylanases and hemicellulases, Biom. Bioenerg. 35 (2011) 1211-1218. [35] T.V. Fernandes, G.J. Klaasse Bos, G. Zeeman, J.P.M. Sanders, J.B. van Lier, Effects of thermo-chemical pre-treatment on anaerobic biodegradability and hydrolysis of lignocellulosic biomass, Bioresour. Technol. 100 (2009) 2575-2579. [36] I.A. Nges, C. Li, B. Wang, L. Xiao, Z. Yi, J. Liu, Physio-chemical pretreatments for improved methane potential of Miscanthus lutarioriparius, Fuel 166 (2016) 29-35. [37] B.C. Saha, Hemicellulose bioconversion, J. Indust.Microbiol. Biotech. 30 (2003) 279- 291. [38] P. Nigam, N. Gupta, A. Anthwal, Pre-treatment of Agro-Industrial Residues, in: P. Nigam, A. Pandey (Eds.) Biotechnology for Agro-Industrial Residues Utilisation: Utilisation of Agro-Residues, Springer Netherlands, Dordrecht, 2009, pp. 13-33. [39] E. Kreuger, I.A. Nges, L. Björnsson, Ensiling of crops for biogas production: effects on methane yield and total solids determination, Biotech. Biof. 4 (2011) 44. [40] O. Pakarinen, A. Lehtomäki, S. Rissanen, J. Rintala, Storing energy crops for methane production: Effects of solids content and biological additive, Bioresour. Technol. 99 (2008) 7074-7082. [41] I.A. Nges, A. Björn, L. Björnsson, Stable operation during pilot-scale anaerobic digestion of nutrient-supplemented maize/sugar beet silage, Bioresour. Technol. 118 (2012) 445-454. [42] P. Weiland, Biogas production: current state and perspectives, Appl.Microbiol. Biotech. 85 (2010) 849-860. [43] S.A. Gebrezgabher, M.P.M. Meuwissen, B.A.M. Prins, A.G.J.M.O. Lansink, Economic analysis of anaerobic digestion—A case of Green power biogas plant in The Netherlands, NJAS - Wageningen J.Life Sci. 57 (2010) 109-115. [44] V. Okudoh, C. Trois, T. Workneh, S. Schmidt, The potential of cassava biomass and applicable technologies for sustainable biogas production in South Africa: A review, Renew. Sust. Energ. Rev. 39 (2014) 1035-1052. [45] M.H. Gerardi, The microbiology of anaerobic digesters., Wiley-Interscience. John Wiley & Sons, Inc. (2003) 94 -129. [46] S. Shen, I.A. Nges, J. Yun, J. Liu, Pre-treatments for enhanced biochemical methane potential of bamboo waste, Chem. Eng. J. 240 (2014) 253-259. [47] I.A. Nges, J. Liu, Effects of anaerobic pre-treatment on the degradation of dewatered- sewage sludge, Renew.Energ. 34 (2009) 1795-1800.

Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 143

[48] P. Weiland, Biomass Digestion in Agriculture: A Successful Pathway for the Energy Production and Waste Treatment in Germany, Eng. Life. Sci. 6 (2006) 302-309. [49] Angelidaki, L. Ellegaard, B. Ahring, Applications of the Anaerobic Digestion Process: Biomethanation II, in: B. Ahring, B. Ahring, I. Angelidaki, J. Dolfing, L. Euegaard, H. Gavala, F. Haagensen, A. Mogensen, G. Lyberatos, P. Pind, J. Schmidt, I. Skiadas, K. Stamatelatou (Eds.), Springer Berlin / Heidelberg2003, pp. 1-33. [50] Y. Chen, J.J. Cheng, K.S. Creamer, Inhibition of anaerobic digestion process: A review, Bioresour. Technol. 99 (2008) 4044-4064. [51] K.V. Rajeshwari, M. Balakrishnan, A. Kansal, K. Lata, V.V.N. Kishore, State-of-the-art of anaerobic digestion technology for industrial wastewater treatment, Renew. Sust. Energ. Rev.4 (2000) 135-156. [52] P. Weiland, Production and energetic use of biogas from energy crops and wastes in germany, Appl Biochem Biotechnol 109 (2003) 263-274. [53] E. Kreuger, B. Sipos, G. Zacchi, S.E. Svensson, L. Bjornsson, Bioconversion of industrial hemp to ethanol and methane: The benefits of steam pretreatment and coproduction, Bioresour. Technol. 102 (2011) 3457-3465. [54] A.P. Moshi, S.G. Temu, I.A. Nges, G. Malmo, K.M.M. Hosea, E. Elisante, B. Mattiasson, Combined production of bioethanol and biogas from peels of wild cassava Manihot glaziovii, Chem.Eng. J.279 (2015) 297-306. [55] N. Cuzin, J.L. Farinet, C. Segretain, M. Labat, Methanogenic fermentation of cassava peel using a pilot plug flow digester, Bioresour. Technol. 41 (1992) 259-264. [56] W.P. Clarke, P. Radnidge, T.E. Lai, P.D. Jensen, M.T. Hardin, Digestion of waste bananas to generate energy in Australia, Wast. Mgt. 28 (2008) 527-533. [57] M. Takashima, K. Shimada, R.E. Speece, Minimum requirements for trace metals (iron, nickel, cobalt, and zinc) in thermophilic and mesophilic methane fermentation from glucose, Water Environ. Res. 83 (2011) 339-346. [58] P. Panichnumsin, A. Nopharatana, B. Ahring, P. Chaiprasert, Production of methane by co-digestion of cassava pulp with various concentrations of pig manure, Biom. Bioenerg. 34 (2010) 1117-1124. [59] A.P. Moshi, C.F. Crespo, M. Badshah, K.M.M. Hosea, A.M. Mshandete, E. Elisante, B. Mattiasson, Characterisation and evaluation of a novel feedstock, Manihot glaziovii, Muell. Arg, for production of bioenergy carriers: Bioethanol and biogas, Bioresour. Technol. 172 (2014) 58-67. [60] REN21, Renewables 2011: Global Status Report, (2011). http://www.ren21.net/Portals. Accessed on 26th June 2016. [61] H. von Blottnitz, M.A. Curran, A review of assessments conducted on bio-ethanol as a transportation fuel from a net energy, greenhouse gas, and environmental life cycle perspective, J. Clean.Prod. 15 (2007) 607-619. [62] P. Sassner, M. Galbe, G. Zacchi, Techno-economic evaluation of bioethanol production from three different lignocellulosic materials, Biom.Bioenerg. 32 (2008) 422-430. [63] N. Mosier, C. Wyman, B. Dale, R. Elander, Y.Y. Lee, M. Holtzapple, M. Ladisch, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresour. Technol. 96 (2005) 673-686. [64] A.P. Moshi, S.G. Temu, I.A. Nges, G. Malmo, K.M. Hosea, E. Elisante, B. Mattiasson, Combined production of bioethanol and biogas from peels of wild cassava Manihot glaziovii, Chem. Eng. J. (2015). Complimentary Contributor Copy 144 Anselm P. Moshi and Ivo Achu Nges

[65] A.P. Moshi, C.F. Crespo, M. Badshah, K.M. Hosea, A.M. Mshandete, B. Mattiasson, High bioethanol titre from Manihot glaziovii through fed-batch simultaneous saccharification and fermentation in Automatic Gas Potential Test System, Bioresour. Technol. 156 (2014) 348-356. [66] A.P. Moshi, K.M. Hosea, E. Elisante, G. Mamo, L. Önnby, I.A. Nges, Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of inedible wild cassava flour to bioethanol, J.Biosci. bioeng. (2015). [67] S.K.S. Patel, H.J. Purohit, V.C. Kalia, Dark fermentative hydrogen production by defined mixed microbial cultures immobilized on ligno-cellulosic waste materials, Int.J. Hydrog. Energ. 35 (2010) 10674-10681. [68] D.B. Levin, L. Pitt, M. Love, Biohydrogen production: prospects and limitations to practical application, Int.J. Hydrog. Energ. 29 (2004) 173-185. [69] D. Das, T.N. Veziroǧlu, Hydrogen production by biological processes: a survey of literature, Int.J.Hydrog.Energ.26 (2001) 13-28. [70] H. Argun, F. Kargi, Bio-hydrogen production by different operational modes of dark and photo-fermentation: An overview, Int. J.Hydrog. Energ. 36 (2011) 7443-7459. [71] R. Datar, J. Huang, P.-C. Maness, A. Mohagheghi, S. Czernik, E. Chornet, Hydrogen production from the fermentation of corn stover biomass pretreated with a steam- explosion process, Int.J.Hydrog. Energ. 32 (2007) 932-939. [72] S.V. Ginkel, S. Sung, J.-J. Lay, Biohydrogen Production as a Function of pH and Substrate Concentration, Environ.Sci.Technol.35 (2001) 4726-4730. [73] B.E. Logan, S.-E. Oh, I.S. Kim, S. Van Ginkel, Biological Hydrogen Production Measured in Batch Anaerobic Respirometers, Environ.Sci.Technol. 36 (2002) 2530- 2535. [74] G. Luo, L. Xie, Z. Zou, W. Wang, Q. Zhou, Evaluation of pretreatment methods on mixed inoculum for both batch and continuous thermophilic biohydrogen production from cassava stillage, Bioresour. Technol. 101 (2010) 959-964. [75] L.T. Angenent, K. Karim, M.H. Al-Dahhan, B.A. Wrenn, R. Domíguez-Espinosa, Production of bioenergy and biochemicals from industrial and agricultural wastewater, Trend. Biotech. 22 (2004) 477-485. [76] Wang, W. Wan, Factors influencing fermentative hydrogen production: A review, Int. J.Hydrog. Energ. 34 (2009) 799-811. [77] T. de Vrije, R.R. Bakker, M.A. Budde, M.H. Lai, A.E. Mars, P.A. Claassen, Efficient hydrogen production from the lignocellulosic energy crop Miscanthus by the extreme thermophilic bacteria Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana, Biotech.r Biof.2 (2009) 1-15. [78] S.S. Pawar, V.N. Nkemka, A.A. Zeidan, M. Murto, E.W.J. van Niel, Biohydrogen production from wheat straw hydrolysate using Caldicellulosiruptor saccharolyticus followed by biogas production in a two-step uncoupled process, Int. J.Hydrog.Energ. 38 (2013) 9121-9130. [79] F.A. Rainey, A.M. Donnison, P.H. Janssen, D. Saul, A. Rodrigo, P.L. Bergquist, R.M. Daniel, E. Stackebrandt, H.W. Morgan, Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: An obligately anaerobic, extremely thermophilic, cellulolytic bacterium, FEMS Microbiol.Lett. 120 (1994) 263-266. [80] T. Sreethawong, S. Chatsiriwatana, P. Rangsunvigit, S. Chavadej, Hydrogen production from cassava wastewater using an anaerobic sequencing batch reactor: Effects of Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 145

operational parameters, COD:N ratio, and organic acid composition, Int. J.Hydrog. Energ. 35 (2010) 4092-4102. [81] G. Luo, L. Xie, Z. Zou, W. Wang, Q. Zhou, Exploring optimal conditions for thermophilic fermentative hydrogen production from cassava stillage, Int.J. Hydrog. Energ. 35 (2010) 6161-6169. [82] B.M. Cappelletti, V. Reginatto, E.R. Amante, R.V. Antônio, Fermentative production of hydrogen from cassava processing wastewater by Clostridium acetobutylicum, Renew.Energ. 36 (2011) 3367-3372. [83] M. Torry-Smith, P. Sommer, B.K. Ahring, Purification of bioethanol effluent in an UASB reactor system with simultaneous biogas formation., Biotechnol. Bioeng. 84 (2003) 7-12. [84] D. Liu, D. Liu, R.J. Zeng, I. Angelidaki, Hydrogen and methane production from household solid waste in the two-stage fermentation process, Wat. Res. 40 (2006) 2230- 2236. [85] M. Lantz, A. Ekman, P. Börjesson, Systemoptimerad produktion av fordonsgas. Rapport 69, Miljö- och Energisystem, LTH, Lunds Universitet., (2009). [86] S.C. Wu, Z.H. Cao, Z.G. Li, K.C. Cheung, M.H. Wong, Effects of biofertilizer containing N-fixer, P and K solubilizers and AM fungi on maize growth: a greenhouse trial, Geoderma 125 (2005) 155-166. [87] D. Cordell, J.-O. Drangert, S. White, The story of phosphorus: Global food security and food for thought, Glob. Environ. Chang. 19 (2009) 292-305. [88] P. Kaparaju, M. Serrano, A.B. Thomsen, P. Kongjan, I. Angelidaki, Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept, Bioresour. Technol. 100 (2009) 2562-2568. [89] A.P. Moshi, S.G. Temu, I.A. Nges, G. Malmo, K.M.M. Hosea, E. Elisante, B. Mattiasson, Combined production of bioethanol and biogas from peels of wild cassava Manihot glaziovii, Chem. Eng. J. [90] E. Corneli, F. Dragoni, A. Adessi, R. De Philippis, E. Bonari, G. Ragaglini, Energy conversion of biomass crops and agroindustrial residues by combined biohydrogen/biomethane system and anaerobic digestion, Bioresour. Technol. 211 (2016) 509-518. [91] S. Yu, J. Tao, Energy efficiency assessment by life cycle simulation of cassava-based fuel ethanol for automotive use in Chinese Guangxi context, Energy 34 (2009) 22-31. [92] G. Luo, L. Xie, Z. Zou, Q. Zhou, J.-Y. Wang, Fermentative hydrogen production from cassava stillage by mixed anaerobic microflora: Effects of temperature and pH, Appl. Energ. 87 (2010) 3710-3717. [93] R.B. Nallamothu, A. Teferra, B.A. Rao, Biogas Purification, Compression and Bottling, Glob. J.Eng.Desig. Technol. 2 (2013). [94] M. Badshah, D.M. Lam, J. Liu, B. Mattiasson, Use of an automatic methane potential test system for evaluating the biomethane potential of sugarcane bagasse after different treatments, Bioresour. Technol. 114 (2012) 262-269. [95] K.R. Smith, Health impacts of household fuelwood use in developing countries, UNASYLVA-FAO- 57 (2006) 41. [96] M.A. Desai, S. Mehta, K. Smith, Indoor smoke from solid fuels, Assessing the environmental burden of disease at national and local levels. Environ.Burden.Dis. Ser.(2004) 675-740. Complimentary Contributor Copy 146 Anselm P. Moshi and Ivo Achu Nges

[97] A.K. Rajvanshi, S. Patil, B. Mendonca, Low-concentration ethanol stove for rural areas in India, Energ.Sust. Develop. 11 (2007) 94-99. [98] E.I. Ohimain, The benefits and potential impacts of household cooking fuel substitution with bio-ethanol produced from cassava feedstock in Nigeria, Energ. Sust. Develop.16 (2012) 352-362. [99] N. Schlag, F. Zuzarte, Market barriers to clean cooking fuels in sub-Saharan Africa: a review of literature. Working Paper. Stockholm Environment, Stockholm (2008). http://sei-international.org/ publications?pid=80 (last accessed 26th June 2016).

BIOGRAPHICAL SKETCH

Anselm Patrick Moshi

Affiliation: Tanzania Industrial Research and Development Organization Education: BSc Food Technology (SUA), MSc Food Technology (Massey), PhD Biotechnology (Lund/UDSM) Research and Professional Experience: Researcher scientists since 2005 to date Professional Appointments: Principal Research Scientist and acting director of Industrial Research, Board member, Tanzania Journal of Science Honors: MSc Tech, Massey University New Zealand

Publications Last Three Years: 1. Moshi, A. P.,Crespo, C. F., Badshah, M., Hosea, K. M., Mshandete, A. M., &Mattiasson, B. (2014a). High bioethanol titre from Manihot glaziovii through fed batch simultaneous saccharification and fermentation in Automatic Gas Potential Test System. Bioresource technology, 156, (1) 348-356. 2. Anselm P. Moshi, Carla F. Crespo, Malik Badshah, Kenneth. M. M. Hosea, Anthony Manoni Mshandete, Bo Mattiasson, 2014b. Evaluation of a Novel Feedstock, Manihot glaziovii, for production of Bioenergy Carriers: Bioethanol and Biogas. Bioresource technology. 172 (1) 58–67. 3. Carla F. Crespo, Anselm Moshi, Kenneth. M. M. Hosea, Bo Mattiasson 20013. Metabolic response of Caloramator boliviensis to fed-batch fermentation (In Carla F. Crespo, Caloramator boliviensis, a New Thermoanaerobe with Interesting Metabolic Properties, ISSN: 978-91-89627-85-7LTH). 4. Anselm P. Moshi, Ken. M. M. Hosea, Emrode Elisante, Anthony Manoni Mshandete, and Ivo Achu Nges. Production of bioethanol from wild cassava Manihot glaziovii through various combinations of hydrolysis and fermentation in stirred tank bioreactors. British Biotechnology Journal, 5(3): 123-139, 2015. 5. Anselm P. Moshi, Ken M. M. Hosea, Emrode Elisante, G. Mamo, Bo Mattiasson. Simultaneous saccharification and fermentation of inedible wild cassava (Manihot glaziovii) flour at high temperature using the thermoanaerobe Caloramator boliviensis. Bioresource technology: Bioresource technology 180 (2015) 128–136.

Complimentary Contributor Copy Sustainable Management of Cassava Processing Waste … 147

6. Anselm P. Moshi, Stella G. Temu, Ivo Achu Nges, Gashaw Malmo, Ken M. M. Hosea, Emrode Elisante, Bo Mattiasson, (2025). Combined production of bioethanol and biogas from peels of wild cassava Manihot glaziovii. Chemical Engineering Journal 279 (2015) 297–306. 7. Anselm P. Moshi, Jane P. Nyandele, Humphrey P. Ndossi, Sosovele M. Eva, Ken M. Hosea. Feasibility of bioethanol production from tubers of Dioscorea sansibarensis and Pyrenacantha kaurabassana. Bioresource Technology 196 (2015) 613–620. 8. Anselm P. Moshi, Ken M.M. Hosea, Emrode Elisante, Gashaw Mamo, Linda Önnby, and Ivo Achu Nges. Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of inedible wild cassava flour to bioethanol. Journal of Bioscience and Bioengineering. 121 (4), 457-463, 2016. 9. Stella Gilbert Temu, Anselm P. Moshi, Ivo Achu Nges, Anthony Manoni Mshandete, Amelia Kajumulo Kivaisi and Bo Mattiasson Mixed Palm Oil Waste Utilization through Integrated Mushroom and Biogas Production. British Biotechnology Journal 11(4): 1-12, 2016, Article no.BBJ.23385.

Chapter 8

WASTEWATER FROM CASSAVA PROCESSING AS A PLATFORM FOR MICROALGAE-MEDIATED PROCESSES

Tatiele C. do Nascimento1, Erika C. Francisco2, Leila Queiroz Zepka1 and Eduardo Jacob-Lopes1,* 1Department of Food Science and Technology, Federal University of Santa Maria (UFSM), Santa Maria, RS, Brazil 2Departament of Environmental Engineering, University of Passo Fundo (UPF), Passo Fundo, RS, Brazil

ABSTRACT

Cassava is widely produced worldwide, and it is a suitable source of carbohydrates (roots), proteins and minerals (leaves). Because of perishability in fresh form, it is widely marketed in the form of gums and flour. Often, its roots have high amounts of cyanohydrin that emanates cyanide, which is highly toxic to human health. This toxic molecule is significantly present in the wastewater from the cassava processing. For this reason, the resulting wastewater, also known as manipueira, when dumped in the environment, causes huge damage to soil and to water sources. The environmental problem can be avoided by advances in industrial biotechnology, which offer potential opportunities for economic utilization of agro-industrial residues. Manipueira has high levels of organic matter and nutrients, which can serve as an ideal platform for bioprocesses mediated by microorganisms, especially microalgae, to obtain products with a high value, such as, carotenoids, phycobilins, polysaccharides, vitamins, fatty acids, and several natural bioactive compounds, which are applicable to foods, pharmaceutical products and bioenergy. This chapter describes the use of the wastewater from cassava processing as a platform for microalgae-mediated processes aiming to obtain bioproducts of commercial value. Divided into five parts, the chapter covers topics on cassava processing, the characteristics of waste from cassava, the impact of cassava waste on the environment, the potential industrial processes for wastewater conversion and the

* Corresponding Author’s address: Email: [email protected]. Complimentary Contributor Copy 150 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al.

bioproducts from microalgae, summarizing a range of useful techno-economic opportunities to be applied on cassava processing plants.

Keywords: cassava, manipueira, microalgae, bioprocess, bioproducts

INTRODUCTION

Cassava provides the staple food of an estimated 800 million people worldwide, and is a tropical root crop, originally from Amazonia. Almost 70 percent of the world production is concentrated in five countries, namely Nigeria, Brazil, Thailand, Indonesia and the Democratic Republic of the Congo [1]. Since 2000, the world’s annual production has increased by an estimated 100 million tonnes, driven in Asia by demand for dried cassava and starch for use in livestock feed and industrial applications, and in Africa by expanding urban markets for cassava food products [2]. The quick postharvest deterioration of cassava causes quantitative and qualitative losses; thus, in order to enable distribution, the initial characteristics of this raw material have to be modified through processing, and, this is why it is marketed widely in the form of gums and flour [3]. However, most forms of cassava processing produce large amounts of wastewater, whose type and composition are governed by the processing method and sophistication of the technology in use. The prodution of flour and gums, in particular, requires large volumes of water [4]. Wastewater is the result of various unit operations of the industrial process; by definition, it is the set of fractions of raw materials, which were not incorporated into the final product [5]. In the cassava processing industry, a large part of the required water is not aggregated to the final product; according to [2], on average about 300 liters of manipueira are generated per ton of processed cassava. In Brazil, one of the world’s largest producers of cassava, about 250-300L of wastewater is generated during processing of flour and starch per ton of processed cassava; therefore, it significantly contributes to environmental pollution, given the presence of elevated levels of cyanide [4]. In addition to organic material, the cyanide released during cassava processing poses a problem, especially in the processes that generate large amounts of “squeezed juice.” Care should be taken for waste containing cyanide, either diluted or stored in such a manner that cyanide concentration can be reduced over time. This is usually the case, even if waste is stored for a short period of time. The current models of exploration of wastewater provided from starch industries support recycling, where the wasted starch could still be sedimented and collected before discharge into the environment [4]. An economically and ecologically attractive option is the search of alternatives based on biotechnological processes involving microorganisms [6]. The use of these microscopic beings for the detoxification of waste has many advantages compared to the treatments usually employed. It is a promising alternative because simultaneity, high removal rate and large amount of valuable biomass are achieved [7]. In this context, microalgae, including cyanobacteria and green algae, are largely explored nowadays, since they have high biotechnological potential associated with the possibility to obtain numerous metabolites of commercial interest that can be widely applied as intermediate inputs and final products of processes relative to bioenergy, food and Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 151 pharmaceuticals products [8, 9, 10, 11, 12, 13, 14]. Thus, types of wastewater such as manipueira are considered ideal platforms for microalgae-mediated processes [7]. Thus, the objective of this chapter is to evaluate the impact of cassava processing, as well as the characteristics of the generated waste, the impact on the environment, the potential industrial processes for wastewater conversion and the bioproducts from microalgae that represent a range of useful techno-economic opportunities to be applied on cassava processing plants.

CASSAVA PROCESSING

Although cassava is one of the crops that can be grown under adverse growth conditions, the production advantages of cassava are, however, partly offset by the rapid deterioration of the roots, which can begin as quickly as 24 hours after harvest. This physiological deterioration of cassava roots can lead to substantial quantitative and qualitative post-harvest losses, thus causing high losses and market risks [1]. As a result of short shelf-life, the industrial transformation process of cassava has played a major role in ensuring better use, accessibility and distribution of this feedstock. Cassava is the basis of a wide range of products; in terms of food, it mainly includes flour and gums. These may be considered as convenience foods, since they are easy to buy, store and prepare [4]. Cassava flour is defined as the product obtained from healthy roots from Euforbiácea family plants, Manihot genus, subject to an appropriate technological manufacturing process that includes cleaning, peeling, grinding, pressing, drying, sieving and processing [15]. By comparison, the processing of starch can therefore be divided into preparation and extraction, purification, removal of water by centrifuging/drying and finishing. The separation of starch granules from the tuber in a way as pure as possible is essential in the manufacture of cassava flour. The granules are locked in cells together with proteins, soluble carbohydrates, fats and other constituents of the protoplasm, which can only be removed by a purification process in the watery phase [16]. During cassava manufacturing processes, the dough pressing step used for the production of flour and the process of extraction and purification to obtain the starch are essential to ensure the quality of the final product. The pressing step must occur after crushing to avoid fermentation and to prevent the flour from darkening. It is held in screw presses or hydraulic presses and aims to reduce the moisture in the ground mass to a minimum to prevent the appearance of unwanted fermentation, save time and fuel in the roasting, and enable uniform roasting [17]. In the manufacture of starch, the purpose of the extraction stage is to separate it from the yucca fiber by means of rotating conical sieves, with the water inlet with countercurrent flow o increase separation efficiency. The remaining liquid also known as milk starch, proceeds to the step of purification, by the addition of water and centrifugation to remove soluble starches and foreign particles [18]. During these stages, large amounts of manipueira, a highly toxic and polluting liquid waste, are generated [19]. In general, it is estimated that 1 ton of cassava produces, on average, about 300 liters of this liquid extracted from pressing manioc [2]. This waste has

Complimentary Contributor Copy 152 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al. high organic load, in addition to other nutrients, such as nitrogenated and phosphorated compounds, which can be reused as culture medium in bioprocesses.

CHARACTERISTICS OF WASTE FROM CASSAVA

The cassava industry can be considered as an inexhaustible source of raw material in the case of solid and liquid waste. Such waste results from the processing of cassava roots in general, and specifically of sectors such as cassava flour manufacturing, starch extraction and production of sour cassava starch. The processing of one ton of cassava generates about 3 m3 of wastewater and 150 kg of cassava bagasse [20]. The production of cassava in Brazil only amounts to about 25-30 million tonnes, and this commodity is the thirteenth largest crop production in the world [21]. Taking into account the Brazilian cassava production in 2011, it is estimated that about 3-5 million tons of pulp and 75-90 × 106 m3 of waste were generated by the cassava industry [22]. In the structure of cassava, 16% of its dry weight is waste such as peel and fiber, composed of about 70% of water and 30% of dry weight. The dry weight fraction is usually composed of 3.5% protein, 10% crude fiber, 11% lignin, 14% cellulose and 27% hemicelluloses [23, 24]. In general, the liquid waste, cassava wastewater, has high organic load, nitrogenated and phosphorated compounds, cyanide, among others nutrients, which could be reused as a culture medium in bioprocesses. Currently, it is desirable that the industrial and agro-industrial wastewater be incorporated into production processes in order to provide the reuse of nutrients that have high added value [25]. The productive chain of products from cassava in general produce two types of liquid waste. The first arises from washing and stripping roots and is performed in a rotary drum; it generally contains a large amount of inert material such as chemical oxygen demand (COD). The second type is due to the drainage of the starch sedimentation tank, and has a higher contaminant load of COD and biochemical oxygen demand (BOD) [26]. According to Damasceno [27], cassava wastewater has a variable composition : approximately 62 gL-1 of total solids, 60 gL-1 of COD, 58 gL-1 of total sugars, 1.6 gL-1 total nitrogen, 83 mgL-1 phosphorus, 895 mgL-1 of potassium, 184 mgL-1 of calcium, 173 mgL-1 of magnesium, 38 mgL-1 sulfur, 8 mgL-1 iron, 0.8 mgL-1 copper and pH of 5.5. Table 1 shows the composition of the effluent arising from cassava flour processing in a company located in Brazil. Furthermore, the physicochemical characteristics of this wastewater are directly related to the variety of processed cassava, the level of technology of the plants and the water retention time in the settling tanks [28]. One of the varieties derived cassava products is the sour cassava starch, also called tapioca starch; this product is processed by extraction of cassava starch from processes such as cleaning, peeling, chopping, pressing and straining of cassava roots. The fiber is separated from the starchy water (‘starch milk’), then the starch is separated from the water and then dehydrated [29], or subjected to natural fermentation for the production of cassava starch [30]. During fermentation, yeasts and bacteria produce organic acids, aromatic compounds and vitamins, among various other substances [31, 32, 33]. Organic acids commonly found in fermentation are lactic, butyric, acetic and propionic acids. Lactic acid is present in a higher amount than the others, about 60-80% of the total composition [30]. The wastewater resulting

Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 153 from sour cassava starch production, generated during the fermentation of starch, is not only composed of a variety of compounds as mentioned above, but also has high concentrations of COD and BOD, with a highly polluting character [34]. In addition to the wastewater originating from fermentation process, solid waste is produced. It generally consists of molecules of amylose and amylopectin hydrolyzate, organic acids, and residual microorganism cells. Thus, this waste represents a potential source of products, such as organic acids, which could be retrieved and applied in different industrial processes [35].

Table 1. Composition of cassava processing wastewater

Parameter Value pH 5.47 -1 BOD (mgL ) 20880 COD (mgL-1) 24000 C/N 96 N/P 1.50 Total Solid (mgL-1) 35410 Suspended Solid (mgL-1) 25315 Volatile Solid (mgL-1) 10095 Turbidity (FTU) 2100 Inorganic compounds Calcium (mgL-1) 26.61 Copper (mgL-1) 0.18 Hardness (mgL-1) 365.21 Magnesium (mgL-1) 63.13 N-TKN (mgL-1) 250.00 4- -1 P-PO3 (mgL ) 166.48 Potassium (mgL-1) 1095.65 Sodium (mgL-1) 3.12 Sulfur (mgL-1) 0.63 Total iron (mg/L) 338.76 Zinc (mg/L) 0.27 Organic compounds Fructose (mgL-1) 1005 Glucose (mgL-1) 10 Starch (mgL-1) 1320 Adapted from [106].

In his studies, Maieves [37] evaluated ten Brazilian cassava cultivars and the composition of bagasse originated from processing roots. The results showed a starch fraction which ranged from 72.92 to 74.51% and a fiber fraction between 4.70 and 7.12% on a dry basis. Factors relative to environmental conditions, genetic and agricultural practices are seen as responsible for the variation in chemical composition of cassava roots [38, 39]. Cassava bagasse is a fibrous product containing about 50% of starch on a dry basis; for each ton of fresh roots when processed, another ton of cassava bagasse (85% moisture) is produced [40, 41]. Since the amount of moisture is extremely high, storage and transport practices are

Complimentary Contributor Copy 154 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al. difficult to implement; therefore, the material becomes highly perishable. The dry fraction of the bagasse is composed by equivalent quantities of starch and fiber compounds, and low levels of minerals, proteins and lipids which, together, compose less than 5% of it on a dry basis [38]. Farias [42] analyzed the chemical composition of bagasse cassava in mass percentage on a moist basis in two different regions of Brazil; the results confirm the major fraction of starch (42.7-46.9% m/m) and fiber (42.2-42.9% m/m), the fractions that were less representative were 5.57-7.15; 1.93-2.43; 1.89-1.92; 0.11-0.17, and 1.31-2.66% m/m for moisture, ash, protein, lipids, and carbohydrates, respectively. Also, they stated that the cassava stem, as solid waste, is not frequently studied and taught. After the cassava root is directed to the production of flour and starches, biofuel, or for allocation for human consumption, which are of paramount importance to billions of people in tropical and subtropical areas, the rod is wasted [43]. Some studies indicate that the composition of cassava includes a high starch content, with up to 30% of dry matter. It is estimated that around 32-38 Tg dry mass is generated worldwide based on a stem/root mass ratio of 42-50% and considering the average cassava root production in 2009-2013 [44, 45]. The vast majority of cassava stem of small and large industries are abandoned or burned, while only 10 to 20% is used for soil fertilization, culture propagation or as substrates for mushrooms. The lack of use of this source of nutrients is mainly due to the lack of knowledge about the constitution of this starch in the stems [46, 47]. Therefore, the productive chain that employs the culture of cassava results in a considerable variety and quantity of waste that consists of substances that could be reused, but in many regions, they are released into the environment, mainly affecting rural areas and population in its surroundings.

IMPACT OF CASSAVA WASTES ON THE ENVIRONMENT

Agricultural development has brought about significant environmental impact and, in this context, the originating products of cassava industry, such as starch and flour, are examples of this global problem. Impacts are initially reported in the agricultural phase because of the intensive use of land, depletion of nutrients needed for cultivation and soil erosion [48, 49, 50]. During processing, the use of fossil energy that results in greenhouse gas emissions and the large volume of freshwater are pointed out as the main environmental impacts [51]. The volume of freshwater for the production of starch ranges from 10 to 60 m3/t, according to reports from countries with high production of this comodity, e.g., Brazil, Colombia, Thailand and Vietnam [52, 53, 55]. Consequently, the major use of water in the production process results in significant volumes of waste that should necessarily be referred for treatment processes such as facultative aerobic lagoons before being thrown into the water body [51]. The effluents produced in different sectors of the cassava industry, have a varying composition, for example, chemical agents such as sulfur dioxide (SO2), used to prevent microbial growth during processing or cyanide contained in the cassava roots, substances that can cause significant impacts. In most industries, these substances are used in small concentrations and are highly diluted (<250 mgL-1); thus they have rapid degradation, and do

Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 155 not cause significant impact [56, 57, 58, 54]. However, intensive and uncontrolled use of wastewater may pose threats to the environment. The manipueira generated when the cassava is pressed to produce flour or starch has a yellowish color and milky consistency, has a varied constitution, and its constituents include macro and micronutrients, and cyanogenic glycosides [59]. Among glycosides, linamarin is the most abundant and, when hydrolyzed, it releases cyanide gas via cyanohydrin intermediates, and it is toxic to many organisms [60]. This feature can be extremely aggressive and cause the death of fish and other aquatic organisms, when released untreated directly into the water body. An example of this problem was reported by Santos [61], whose studies showed that certain stretches of the river Santa Rita in the state of Bahia, Brazil, had a decline in water quality as a result of the disposal of manipueira in its course, without any treatment. Cyanide, when disposed in soil, can be biodegraded by microorganisms such as bacteria, fungi and yeasts, which can convert this substance into other compounds of cyanide through alternative pathways in metabolism, e.g., hydrolytic, oxidative, reductive or substitution reactions [62]. The biochemical properties of this wastewater allow its application in agriculture for different purposes, such as plant nutrition and control of organisms such as insects, parasites, nematodes and fungi that can harm plants [63, 64, 65, 66, 67]. However, there are reports of wastewater, such as vinasse, whose direct application to the soil can compromise their characteristics because of its low pH, high concentrations of sulfate and organic matter, which could compromise the productivity of cultures such as sugarcane [68]. These potential problems can be extrapolated to the cassava wastewater, since they have similar characteristics. Still, disposal of wastewater composed of cyanogenic substances has been a concern in recent years in areas such as the Brazilian northeastern, because this practice can lead to environmental imbalances. According to Souza [69], natural waters and soils have high interaction with cyanide ions (CN-) are the major sources of these ions to the environment. In freshwater, 50% of organic carbon is associated with aquatic humic substances (AHS), and humic substances (HS), which are the main natural organic complexing agents in different systems. HS are heterogeneous mixtures produced by biological degradation of animal and vegetable waste, and are made up of different functional groups, which influence the complexation of ions and, consequently, their bioavailability [70, 71, 72]. Some authors suggest that HS behave like macromolecules capable of polymerizing, of aggregating to form micelles and supramolecular assemblies. Moreover, such micelles promote Van der Waals interaction with anions such as Cl- and CN- [73]. However, studies show a greater complexation of cyanide by SHS (2.98-3.22 mmol CN- g-1 HS) when compared with AHS (1.86-2.07 mmol CN- g-1 HS). These values show the importance of HS for cyanide retention in the environment. Thus, the concentrations of macro and micronutrients in manipueira suggest a promising use of this wastewater as an agricultural fertilizer, since the cyanide present in its constitution is eliminated before the wastewater is disposed [69]. One of the major environmental damage caused by potentially toxic effluents, such as manipueira, refers to reduced availability of oxygen in aquatic environments, resulting in damage to aquatic life [74]. This problem is related to the high biochemical oxygen demand (BOD) associated with a considerable amount of hydrocyanic acid and large volumes dropped in the water body. BOD is the amount of oxygen required to oxidize the organic matter during Complimentary Contributor Copy 156 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al. its microbial decomposition. Thus, the wastewater has high organic loads, requires large amounts of oxygen for water degradation and, consequently, results in reduction of this gas in the aquatic environment. According to Correa [75], the manipueira BOD ranges from 14000- 34000 mgL-1, a concentration equivalent to the load generated in residential wastewater for about 200 people/day. DBO is also related to the eutrophication process, where effluents with high concentrations of nitrate and nitrite, such as cassava wastewater, lead to increased BOD and deterioration of water bodies; nitrate is less harmful in water bodies than nitrite, given its higher oxidation [76, 69, 77]. Previous studies also indicate a predominance of potassium between mineral constituents of manipueira, whose significant levels of phosphors are larger than those found in other agro-industrial effluents, such as vinasse, known for its polluting load [69]. One factor that needs to be highlighted is that the concentrations of some minerals can provide a large difference, due to the kind of technology employed in the production process. For example, it is higher in industrial processes when compared with home processes [75]. Still, the composition of this wastewater shows variations according to the different seasons, can the macro and micronutrient, and potentially toxic metals, have lower concentrations in dry seasons, and high ones in rainy periods [69]. Finally, the solid fraction of the waste generated by the production of flour and starch is of vegetable origin, such as stems and stal, but also waste of the pressing process, such as bagasse. In general, these types of waste are not rationally valued and often abandoned in the open air, on the ground and close to water bodies. Therefore, they may cause contamination of different compartments of the environment, since they have high moisture and are putrecible. Case studies have reported examples of neglect of this portion of waste in municipal waste, disposal in rivers or gutter water in the city of Kinshasa, capital of the Democratic Republic of the Congo [79]. This practice probably involves the proliferation of microbes and various disease vectors. However, when solid waste is properly managed, it could go on to have a biomass nature for the production of methane by anaerobic biodegradation [80]. Environmental problems in general, and especially those posed by agricultural waste, such as cassava processing industry, can not be solved by one single technology. The way suggested as more reasonable and efficient, addresses interdisciplinary applications, since the composition of waste has a wide variety, and so does its physical state, which can be liquid, solid and pasty. Still, the vast composition of waste, as well as its relatively low sales value, makes it attractive for different industrial sectors; it is reused, for example, as organic substrates or for generation of energy.

POTENTIAL INDUSTRIAL PROCESSES FOR WASTEWATER CONVERSION

Worldwide, millions of tons of hazardous and non-hazardous waste are generated each year, demonstrating the need for better management of waste through the reduce, reuse and recycle concepts [81]. So far, several renewable substrates including wastewater, waste and by-products, agricultural and industrial origin, have been intensively studied for the cultivation of microorganisms, production of bioproducts and power generation [82, 83, 84,

Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 157

85]. The concept of waste reuse generally provides new directions to the production chain by applying the so-called Cleaner Production practices based on the concept of sustainability. Still, practices and waste reuse technologies enable the evaluation of the waste and, in some cases, direct the production process toward the concept of biorefineries. Recent research indicates that one of the oldest methods of reuse of effluents, irrigation of crops, widespread throughout the world, is a valuable technology both for improvement in soil fertility, and for the reduction of large amounts of frashwater that are directed to agriculture [86]. However, the practice of inserting treated wastewater or not, needs to be managed carefully, so that problems such as changes in soil properties, as well as the decrease in crop quality, do not result in economic losses and environmental damage. In 2007, at least one tenth of the world's population was consuming food produced by wastewater, and approximately 200 million hectares in 50 countries applied raw sewage irrigation or partially treated wastewater [87]. Pakistan is an example of the immense dissemination of this practice: 80% of its inhabitants use sewage for irrigation [88]. One of the factors behind the spread of wastewater reuse in agriculture is a valuable composition with inorganic and organic substances required for the nutrition of crops [89]. Currently, the practice of wastewater conversion in the industrial sector has been gaining momentum throughout the world, mainly because of the search for sustainability in the supply chain and products with lower final cost, targeting the consumer market. Use of wastewater is being developed in different sectors for production of bio-energy, high value- added products, food and feed, fine chemicals, and application as substrates for microorganisms in obtaining bioproducts. The citric acid production sector has examples of watewater reuse in the production process. Citric acid is an important organic acid used in the food, pharmaceutical, beverage, chemical and metallurgy industry [90, 91]. Several materials can be used to obtain this acid, including cassava, which is an attractive option because of its low cost and high productivity [92]. In the search for conversion of the wastewater generated by fermentation of cassava starch, Zhang [93] there are studies that have applied recycling processes. The process consists in treating the wastewater by ion exchange and subsequent recycling as culture media for the next fermentations. Thus, the effluent is not disposed and becomes a raw material in the production process. Over the decades, the high demand for energy, associated with increased economic activity, resulted in higher consumption of fossil fuels and hence environmental degradation. For this reason, renewable energies, such as biofuels, had to be searched. One of the alternatives to fossil fuels is hydrogen, which can also be applied to a raw material for various branches of the chemical industry [94]. The higher H2 production rates, coupled with the capacity of agroindustrial waste to be rich in carbohydrates, led a significant amount of research to develop methodologies for fermentations in the dark [95, 96]. This technology encompasses processes for obtaining hydrogen and other products such as methane, and the reduction of the organic load, such as cassava wastewater [97]. In the face of broad access to more diversified products and a competitive market, choice of cheap raw materials is of great importance for the global economy of industries, especially the sectors of biotechnological processes, since they account for 50% of the final cost of the product. In this context, the best way to reduce cost substrate for biotechnology is the use of wastewater composed of essential nutrients for growth of micro-organisms to obtain bioproducts, which also results in environmental benefits. Complimentary Contributor Copy 158 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al.

Among the wide variety of bioproducts obtained by micro-organisms, biopolymers such as chitin and chitosan are readily available and show promising features such as biodegradability, biocompatibility, non-toxicity and several industrial applications [98]. Berger [99] have shown that cassava wastewater employment for obtaining chitin and chitosan from a fungal species, such as Cunninghamella elegans, effectively reduced production costs. Thus, the biotechnological application of these biopolymers is economically viable, and the use of effluents refers to the production chain, and has an environmentally friendly character. Another byproduct obtained from micro-organisms is biosurfactants, which can be used in industrial processes such as oil recovery, lubricants, bioremediation of pollutants, food processing and health promotion [100]. Among the various classes of biosurfactants, the lipopeptides obtained from Bacillus subtilis are relevant because of their therapeutic potential [101, 102]. Despite its great diversity of application, one disadvantage is high production costs, and the strategy referred to circumvent this problem is the use of manipueira as a means of cultivation [103]. A sector that has a representative increase year after year in the development of technologies, encompasses the industries that apply wastewater conversion processes using microalgae and cyanobacteria [104]. These microrganisms are considered to be sources of a diverse range of bioproducts, for example, pigments, proteins, carbohydrates and lipids. Still, they are seen as raw materials for the production of biofuels, since under certain growing conditions, they result in the accumulation of high lipid concentrations. According to Queiroz [14], the main technological routes identified as having potential for escalation are photoautotrophic and heterotrophic. Moreover, closed bioreactors are limited by engineering factors, and open tank bioreactors, by biological factors. Heterotrophic cultivation on a large scale is generally advantageous in terms of costs compared with photosynthetic systems. However, the superior performance of biomass productivity is the driving force behind these systems. Controlled growth in fermenters under aseptic conditions reduces the loss of nutrients and increases product quality. The final cost of biomass productivity is an essential feature for cultivation systems and essentially depends on the conditions and the culture medium [105]. Given the use of an exogenous source of organic carbon in heterotrophic cultures, it is estimated that 80% of the total of the culture medium comes from this substrate. For this reason, the investigation of organic carbon sources is necessary to run the microalgal organic processes. Several studies indicate the use of agroindustrial wastewater, rich in organic and inorganic nutrients, as a viable source as replacement for synthetic culture media [106, 107, 14]. In their studies, Francisco [106] demonstrated the conversion efficiency of manipueira employing the cyanobacterium Phormidium sp. under heterotrophic cultures through different operation systems. Conversion substrates are essential for heterotrophic metabolism of cyanobacteria, in the range of 63-66% (organic carbon), 19-50% (nitrogen) and 17-52% (phosphorus), for the systems in batch, fed-batch and continuous modes. Among the operating systems being studied, the one on continuous mode is seen as a promising for the production of biomass and lipids. Still, the representative productivity of lipids, combined with a fatty acid profile suitable for biodiesel synthesis, enables the production of bulk oil from microrganisms such as cyanobacteria and microalgae.

Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 159

BIOPRODUCTS FROM MICROALGAE AS TECHNO-ECONOMIC OPPORTUNITIES APPLICABLE ON CASSAVA PROCESSING PLANTS

Microalgae have shown great biotechnological potential associated with the production of numerous high value-added metabolites [108]. The biodiversity and consequent variability in their biochemical composition enable these microorganisms to be used in various applications [109], as shown in Figure 1. This metabolic versatility allows high productivity of biomass from agroindustrial residues, such as those generated from cassava processing, which may be potentially exploited as raw material in biorefinery systems. Through the production of various bioproducts of commercial interest, a biorefinery can use all of the biomass components and intermediates, thereby maximizing the value derived from the biomass feedstock [13]. Varying species and growth conditions can produce a wide variety and abundance of bioproducts such as lipids, proteins, carbohydrates, and feedstocks that are important for production of biofuel and nutraceuticals [110]. Their cellular content shows considerable composition of pigments such as carotenoids and chlorophyll [111]. Table 2 shows the range of bioproducts obtained from different microalgal species.

Figure 1. Biodiversity and consequent variability in microalgae biochemical composition enable these microorganisms to be used in various applications.

Complimentary Contributor Copy 160 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al.

Table 2. Bioproducts obtained from microalgal biomass

Microalgae Bioproduct Application Ref. Nitzschia laevis Eicosapentaenoic acid Food additive; [112] Pharmaceutics Chlorella protothecoides Carotenoids Pharmaceutics [113] Galdieria sulphuraria Phycocyanin Pharmaceutics [114] Aphanothece microscopica Amino acid Functional foods [10] nageli Chlorella protothecoides Fatty acid Biofuels [115] Aphanothece microscopica Lipids, carbohydrates, Functional foods; [116] nageli minerals, proteins Pharmaceutics Phaeodactylum tricornutum Docosahexaenoic acid and Functional foods; [117] eicosapentaenoic acid Pharmaceutics Phormidium autumnale Fatty acid Biofuels [106] Phormidium autumnale Carotenoids Food additive; [118] Medicine; Pharmaceutics Phormidium autumnale Carotenoids, Chlorophyll a Pharmaceutics [119] and b, Phycocyanin Phormidium autumnale Natural aromas Food additive [120] Chlorella vulgaris Mono-oligo- and Pharmaceutics [121] polysaccharides

The production of single cell proteins by residual biomass provides an economically viable source of protein, known as meal for use in animal feed, since they often meet the nutritional requirements [122]. Furthermore, because of their amino acid profiles, some microalgae can be used as nutraceuticals or functional foods that can help treat some diseases and prevent damage to cells or tissue [123]. The protein content associated with the amino acid balance indicates the high biological value of these proteins [9]. Algal lipids are typically composed of glycerol, sugars or bases esterified to fatty acids with carbon number ranging from 12 to 22, and make up 1 to 40% of dry weight biomass [124]. The wastewater from cassava processing was considered as an ideal platform for obtaining biofuels by means of cyanobacteria Phormidium sp.; it has reached parallel high productivity of biomass and oil, 320.1 and 43.8 mg.L-1.h-1 respectively, moreover, the properties of the biodiesel are shown according to acceptable standards [106]. Currently, another appeal for the exploitation of microalgal oil is the manipulation of metabolic pathways for safe and sustainable production of polyunsaturated fatty acids (PUFAs), i.e., docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which are of high commercial value [125]. Some types of microalgae, especially Chlorella, Dunaliella, Chlamydomonas, Scenedesmus, and Spirulina species, have abundant carbohydrates in their metabolites [126]. Depending on the biological task that they fulfill, they can be found in different forms, such as starch, cellulose, glycolipids or glycoproteins. Starch is the major part of total carbohydrates, at least 46.5%, followed by mono- and oligosaccharides in solution released after mechanical cell disruption (26.4%) [121]. These waste carbohydrates after oil extraction can be used to produce bioethanol, biohydrogen and antibiotics. These are economically

Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 161 valuable applications, considering only the branch of antibiotics (penicillin, cephalosporin, monobactam, carbapenem, and beta-lactamase), which represent 57% of the global antibacterial drug market, valued at 43.55 $ billion in 2012 [127]. Carotenoids are a major operation field of biotechnology microalgae with a wide range of applications [83]. They have important properties both for food quality as well as for human health. Considering the structural specificity of these pigments, is possible to obtain interesting pharmacological properties, such as antioxidant activity [119]. Through economic feasibility analysis, [128] showed that the use of agro-industrial waste for the production of oleoresins rich in carotenoids microlagais can reach a profit margin of 70.6%. For the production of single-cell carotenoids in agroindustrial wastewater, an all-trans-zeaxanthin production of up to 15,061.8 kg/year can be estimated on an industrial scale [118]. In this chapter, we discussed cassava processing, as well as the characteristics of generated waste, its impact on the environment, the potential industrial processes for wastewater conversion and the bioproducts from microalgae that summarize a range of useful techno-economic opportunities. In short, these techno-economic opportunities demonstrate the exploration potential of microalgae in cassava processing plants based on the use of their waste as platform for bioprocesses.

REFERENCES

[1] FAO. (2000). The world cassava economy. World Food and Agriculture, Food and Agriculture Organization of the United Nations, Rome. [2] FAO. (2013). State of Food Insecurity in the World – The multiple dimensions of food security, Food and Agriculture Organization of United Nations, Rome. [3] FAO. (1995). Post-harvest deterioration of cassava. World Food and Agriculture, Food and Agriculture Organization of the United Nations, Rome. [4] FAO. (2001). The global cassava strategy. World Food and Agriculture, Food and Agriculture Organization of the United Nations, Rome. [5] Garcia-Serna, J.; Perez-Barrigon, L.; Cocero, M.J. (2007). New trends for design towards sustainability in chemical engineering: Green engineering. Chemical Engineering Journal 133, 7-30. [6] Gargouri, B.; Karray, F.; Mhiri, N,.; Aloui, F.; Sayadi, S. (2011). Application of a continuously stirred tank bioreactor (CSTR) for bioremediation of hydrocarbon-rich industrial wastewater effluents. Journal of Hazardous Materials 189, 427-434. [7] Liang, Y. (2013). Producing liquid transportation fuels from heterotrophic microalgae. Applied Energy 104, 860-868. [8] Queiroz, M. I.; Jacob-Lopes, E.; Zepka, L. Q.; Bastos, R. G.; Goldbeck, R. (2007). The kinetics of the removal of nitrogen and organic matter from parboiled rice effluent by cyanobacteria in a stirred batch reactor. Bioresource Technology 98, 2163-2169. [9] Jacob-Lopes, E.; Queiroz, M. I.; Zepka, L. Q.; Netto, F. M. (2006). Caracterização da fração proteica da cianobactéria Aphanothece microscopica Nägeli cultivada no efluente da parboilização do arroz. Food Science and Technology 26, 482-488.

Complimentary Contributor Copy 162 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al.

[10] Jacob-Lopes, E.; Zepka, L. Q.; Queiroz, M. I.; Pinto, L. A. A. (2007). Characteristics of thin-layer drying of the cyanobacterium Aphanothece microscopica Nägeli. Chemical Engineering and Processing 46, 63-69. [11] Jacob-Lopes, E.; Lacerda, L. M. C. F.; Franco, T. T. (2008). Biomass production and carbon dioxide fixation by Aphanothece microscopica Nägeli in a bubble column photobioreactor. Biochemical Engineering Journal 40, 27-34. [12] Jacob-Lopes, E.; Revah, S.; Shirai, K.; Hernandez, S.; Franco, T. T. (2009). Development of operational strategies to remove carbon dioxide in photobioreactors. Chemical Engineering Journal 153, 120-126. [13] Jacob-Lopes, E.; Scoparo, C. H. G.; Queiroz, M. I.; Franco, T. T. (2010). Biotransformations of carbon dioxide in photobioreactors. Energy Conversion and Management, 51 894-900. [14] Queiroz, M. I.; Hornes, M. O.; Silva-Manetti, A. G.; Jacob-Lopes, E. (2011). Single- cell oil production by cyanobacterium Aphanothece microscopica Nägeli cultivated heterotrophically in fish processing wastewater. Applied Energy 88, 3438-3443. [15] BRAZIL (1995). Ordinance nº 554 of 30 Augusth 1995. Official Federal Gazette, Ministry of Environment, Brasilia. [16] FAO. (1977). Cassava processing. World Food and Agriculture, Food and Agriculture Organization of the United Nations, Rome. [17] Embrapa. (2006). Manufacturing process effect of cassava flour. Brazilian Agricultural Research Corporation, Embrapa Eastern Amazon, Belem. [18] Leonel, M.; Cereda, M.P. (2000). Starch extraction from cassava fibrous residue. Food Science and Technology 20, 122-127. [19] Campos, A.T.; Daga, J.; Rodrigues. E.E.; Franzener, G.; Mary M. T. Suguiy, M.M.T.; Syperreck, V.L.G. (2006). Residuary water treatment of fecularia by means of stabilization lagoons. Engenharia Agrícola 26, 235-242. [20] Hien, P.G.; Oanh, L.T.K.; Viet, N.T.; Lettinga, G. (1999). Closed wastewater system in the tapioca industry in Vietnam. Water Science and Technology 39, 89-96. [21] Food and Agricultural Organization of United Nations (FAO). Available: http://www.fao.org/index_en.htm. [22] Cardona, C.A.; Sanchez, O.J.; Gutierrez, L.F. (2010). Feedstocks for fuel etanol production. In: Process Synthesis for Fuel Ethanol Production. CRC Press, USA, 43-75. [23] Babayemi, O.J.; Ifut, O.J.; Inyang, U.A.; Isaac, L.J. (2010). Quality and chemical composition of cassava wastes ensiled with Albizia saman Pods. Agricultural Journal 5(3), 225-228. [24] Ratnadewi, A.A.I.; Santoso, A.B.; Sulistyaningsih, B.; Handayani, W. (2016). Application of Cassava Peel and Waste as Raw Materials for Xylooligosaccharide Production using Endoxylanase from Bacillus subtilis of Soil Termite Abdomen. Procedia Chemistry 18, 31-38. [25] Subhadra, B.G.; Edwards, M. (2011). Coproduct market analysis and water footprint of simulated commercial algal biorefineries. Applied Energy 88, 3515-3523. [26] Gijzen, H.J.; Bernal, E.; Ferrer, H. (2000). Cyanide toxicity and cyanide degradation in anaerobic wastewater treatment. Water Research 34 (9), 2447-2454. [27] Damasceno, S.; Cereda, M.P.; Pastore, G.M.; Oliveira, J.G. (2003). Production of volatile compounds by Geotrichum fragrans using cassava wastewater as substrate. Process Biochemistry 39, 411-414. Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 163

[28] Colin, X.; Farinet, J.-L.; Rojas, O.; Alazard, D. (2007). Anaerobic treatment of cassava starch extraction wastewater using a horizontal flow filter with bamboo as support. Bioresource Technology 98, 1602-1607. [29] Vilela, E.R.; Ferreira, M.G. (1987). Tecnologia de produção e industrialização do amido da mandioca. Informe Agropecuário 13, 69-73, Belo Horizonte. [30] Demiate, I.M.; Barana, A.C.; Cereda, M.P.; Wosiacki, G. (1999). Organic acid profile of commercial sour cassava starch. Food Science and Technology 19, 131-135. [31] Morlon-Guyot, J.; Guyot, J.P.; Pot, B.; Jacobe De Haut, I. (1998). Lactobacillus manihotvorans sp. a new starch-hydrolysing lactic acid bacterium isolated during cassava sour starch fermentation. International Journal of Systematic Bacteriology 48, 1101-1109. [32] Lacerda, I.C.A.; Miranda, R.L.; Borelli, B.M.; Nunes, A.C.; Nardi, R.M.D.; Lachance, M.-A.; Rosa, C.A. (2005). Lactic acid bacteria and yeasts associated with spontaneous fermentations during the production of sour cassava starch in Brazil. International Journal of Food Microbiology 105, 213-219. [33] Silveira, I.A.; Carvalho, E.P.; Pádua, I.P.M.; Dinízio, F.L.; Marques, S.C. (2003). Isolamento e caracterização da microbiota ácido lática envolvida no processo fermentativo para produção de polvilho azedo. Pró Homine: Revista Científica da Unilavras 2(2), 7-14. [34] Avancini, S.R.P.; Faccin, G.L.; Vieira, M.A.; Rovaris, A.A.; Podestá, R.; Tramonte, R.; de Souza, N.M.A.; Amante, E.R. (2007). Cassava starch fermentation wastewater: Characterization and preliminary toxicological studies. Food and Chemical Toxicology 45, 2273-2278. [35] Aquino, A.C.M.S. et al. (2015) Validation of HPLC and CE methods for determination of organic acids in sour cassava starch wastewater. Food Chemistry 172, 725-730. [36] Pereira, J.M.; Aquino, A.C.M.S.; de Oliveira, D.C.; Rocha, G.; de Francisco, A.; Barreto, P.L.M.; Amant, E.R. (2016). Characteristics of cassava starch fermentation wastewater based on structural degradation of starch granules. Ciência Rural 46(4), 732-738. [37] Maieves HA, Oliveira DC, Bernardo C, Müller CMO, Amante ER. (2012). Microscopy and texture of raw and cooked cassava (Manihot esculenta Crantz) roots. Journal of Texture Study 43, 164-173. [38] Teixeira, E.M.; Pasquini, D.; Curvelo, A.A.; Corradini, E.; Belgacem, M.N.; Dufresne, A. (2009). Cassava bagasse cellulose nanofibrils reinforced thermoplastic cassava starch. Carbohydr Polymers 78(3), 422-431. [39] Teixeira, E.M.; Curvelo, A.A.S.; Corrêa, A,C.; Marconcini, J.M.; Glenn, G.M.; Mattoso, L.H.C. (2012). Properties of thermoplastic starch from cassava bagasse and cassava starch and their blends with poly (lactic acid). Industrial Crops and Products 37(1), 61-68. [40] Maieves, H.A.; Oliveira, D.C.; Frescura, J.R.; Amante, E.R. (2011). Selection of cultivars for minimization of waste and of water consumption in cassava starch production. Industrial Crops and Products 33, 224-228. [41] Pandey, A.; Soccol, C.R.; Nigam, P.; Soccol, V.T.; Vandenberghe, L.P.S.; Mohan, R. (2000). Biotechnological potential of agro-industrial residue II: cassava bagasse. Bioresource Technology 74, 81-87.

Complimentary Contributor Copy 164 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al.

[42] Farias, F.O.; Jasko, A.C.; Colman, T.AD.; Pinheiro, L.A.; Schnitzler, E.; Barana, A.C.; Demiate, I.M. (2014) Characterisation of Cassava Bagasse and Composites Prepared by Blending with Low-Density Polyethylene. Brazilian Archives og Biology and Technology 57(6), 821-830. [43] FAOSTAT. http://faostat.fao.org/. [44] Howeler, R.H. (2012). The Cassava Handbook e a Reference Manual Based on the Asian Regional Cassava Training Course. International Centre for Tropical Agriculture, Cali, Colombia. [45] Zhu, W.B.; Lestander, T.A.; Orberg, H.; Wei, M.G.; Hedman, B.; Ren, J.W.; Xie, G.H.; Xiong, S.J. (2013). Cassava stems: A new resource to increase food and fuel production. GCB Bioenergy. [46] Howeler, R.H.; Lutaladio, N.; Thomas, G. (2013). Save and Grow: Cassava. http:// www.fao.org/ag/save-and-grow/cassava/ [47] Wei, M.; Zhu, W.; Xie, G.; Lestander, T.A.; Xiong, S. (2015). Cassava stem wastes as potential feedstock for fuel etanol production: A basic parameter study. Renewable Energy 83, 970-978. [48] Howeler, R.H. Cassava mineral nutrition and fertilization. (2002). In: Hillocks, R.J.; Thresh, J.M.; Bellotti, A.C. (Ed.). Cassava: biology, production and utilization. Oxon, United Kingdom: CABI Publishing, 115-47. [49] Valentin, C.; Agus, F.; Alamban, R.; Boosaner, A.; Bricquet, J.P.; Chaplot, V.; de Guzman, T.; de Rouw, A.; Janeau, J.L.; Orange, D.; Phachomphonh, K.; Do Duy Phai; Podwojew-ski, P.; Ribolzi, O.; Silvera, N.; Subagyono, K.; Thiebaux, J.P.; Tran Duc Toan; Vadari, T. (2008). Runoff and sediment losses from 27 upland catchments in Southeast Asia: impact of rapid land use changes and conservation practices. Agriculture, Ecosystems and Environment 128, 225-38. [50] Phan Ha Hai An; Huon, S.; Henry des Tureaux, T.; Orange, D.; Jouquet, P.; Valentin, C.; de Rouw, A.; Tran Duc Toan. (2012). Impact of fodder cover on runoff and soil erosionat plot scale in a cultivated catchment of North Vietnam. Geoderma 177-178, 8- 17. [51] Trana, T.; Da, G.; Moreno-Santander, M.A.; Vélez-Hernández, G.A.; Giraldo-Toro, A.; Piyachomkwan, K.; Sriroth, K.; Dufour, D. (2015). A comparison of energy use, water use and carbon footprint ofcassava starch production in Thailand, Vietnam and Colombia. Resources, Conservation and Recycling 100, 31-40. [52] Marder, R.C.; Cruz, R.A.; Moreno, M.A.; Curran, A.; Trim, D.S. (1996). Investigating sour starch pro-duction in Brazil. In: Dufour, D.; O’Brien, G.M.; Best, R. (Ed.). Cassava flour andstarch: progress in research and development. CIAT publication n. 271, Cali, Colombia, 247-58. [53] Rivier, M.; Moreno, M.; Alarcon, F.; Ruiz, R.; Dufour, D. (2001). Cassava sour starch in Colombia. Processing plant: description, plans, and layout. CIAT publication, n. 323, Cali, Colombia, v. 2. [54] Sriroth, K.; Piyachomkwan, K.; Wanlapatit, S.; Oates, C. (2000). Cassava starch technology: the Thai experience. Starch-Stärke 52, 439-49. [55] Zakhia, N.; Dufour, D.; Chuzel, G.; Griffon, D. (1996). Review of sour cassava starch productionin rural Colombia areas. Tropical Science 36, 247-55.

Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 165

[56] Piyachomkwan, K.; Wanlapatit, S.; Chotineeranat, S.; Sriroth, K. (2005). Transformation and bal-ance of cyanogenic compounds in the cassava starch manufacturing process. Starch-Stärke 57, 71-8. [57] Rojas, O.C.; Alazard, D.; Aponte, L.R.; Hidrobo, L.F. (1999). Influence of flow regime on the concen-tration of cyanide producing anaerobic process inhibition. Water Science and Technology 40(8), 177-85. [58] Somboonchai, W.; Songkasiri, W.; Nopharatana, M. (2008). Kinetics of cyanide oxidationby ozone in cassava starch production process. J. Food Eng. 84(4), 563-8. [59] Chitwood, D.J. Phytochemical based strategies for nematode control. (2002). Annual Review of Phytopathology 40, 221-249. [60] Magalhães, C.P.; Xavier-Filho, J.; Campos, F.A.P. (2000). Biochemical basis of the toity of manipueira (liquid extract of cassava roots) to nematodes and insects. Phytochemistry 11, 57-60. [61] Santos, G.P.; Rego, N.A.C.; Santos, J.W.B.; Delano Júnior, F.; Silva Júnior, M.F. (2012). Avaliação espaço-temporal dos parâmetros de qualidade da água do rio Santa Rita (BA) em função do lançamento de manipueira. Ambiente e Água 7, 261-278. [62] Nasu, E.G.,C.; Formentini, H.M.; Furlanetto, C. (2015). Effect of manipueira on tomato plants infected by the nematode. Crop Protection 78, 193-197. [63] Baker, K.R.; Koenning, S.R. (1998). Developing sustainable systems for nematode management. Annual Review of Phytopathology 36, 165-205. [64] Duarte, A.S.; Silva, E.F.F.; Rolim, M.M.; Ferreira, R.F.A.L.; Malheiros, S.M.M.; Albuquerque, F.S. (2012). Uso de diferentes doses de manipueira na cultura da alface em substituição à adubação mineral. Revista Brasileira de Engenharia Agrícola e Ambiental 16, 262-267. [65] Freire, F.C.O. Uso da manipueira no controle do oídio da cerigueleira: resultados preliminaries. (2001). Embrapa Agroindústria Tropical, Com. Téc. 70. [66] Gonzaga, A.D.; Ribeiro, J.D.A.; Vieira, M.F.; Alécio, M.R. (2007). Toxidez de três concentrações de Erva-de-rato (Palicourea marcgravii A. St.-Hill) e manipueira (Manihot esculenta Crantz) em Pulgão Verde dos Citros (Aphis spiraecola Patch) em Casa de Vegetação. Rev. Bras. Bioc. 5, 55-56. [67] Jesus, S.C.P.; Mendonça, F.A.C. (2012). Atividade do extrto aquoso da madioca sobre a mortalidade e reprodução do pulgão da couve. Rev. Bras. Ciênc. Agr. 7, 826-830. [68] Fuess, L.T.; Garcia, M.L. (2015). Bioenergy from stillage anaerobic digestion to enhance the energy balance ratio of ethanol production. Journal of Environmental Management 162, 102-114. [69] Souza, S.O.; Oliveira, L.C.; Cavagis, A.D.M.; Botero, W.G. (2014). Cyanogenic Residues: Environmental Impacts, Complexation with Humic Substances, and Possible Application as Biofertilizer. Water Air and Soil Pollution 225-2223. [70] Botero, W.G.; Oliveira, L.C.; Rocha, J.C.; Rosa, A.H.; Santos, A. (2010). Peat humic substances enriched with nutrients for agricultural applications: competition between nutrients and non-essential metals present in tropical soils. Journal of Hazardous Materials 177(4), 307-311. [71] Sloboda, E.; Vieira, E.M.; Dantas, A.D.B.; Berando, L.D. (2009). A Influência das substâncias húmicas aquáticas características na eficiência da coagulação com cloreto férrico. Quimica Nova, 32(4), 976-982.

Complimentary Contributor Copy 166 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al.

[72] Tadini, A.M.; Moreira, A.B.; Bisinoti, M.C. (2014). Fractionation of aquatic humic substances and dynamic of chromium species in an aquatic body influenced by sugarcane cultivation. Journal of the Brazilian Chemical Society 25(1), 119-125. [73] Pacheco, M.L.; Pena-Méndes, E.M.; Havel, J. (2003). Supramolecular interactions of humic acids with organic and inorganic xenobiotics studied by capillary electrophoresis. Chemosphere 51(1), 95-108. [74] Wosiacki, G.; Cereda, M.P. (2002). Recovery of waste processing cassava. The Sciences and Engineering 8(1), 27-43. [75] Correa, A.D.; Santos, C.D.; Natividade, M.A.E.; Abreu, C.M.P.; Xisto, A.L.R.P.; Carvalho, V.D. (2002). Farinha de mandioca deixa: I. efeito de secagem sobre a atividade de linamarase. Ciência Agrotecnica 26(4), 368-374. [76] Sodre, F.F.; Grassi, M.T. (2006). Changes in copper speciation and geochemical fate in freshwaters following sewage discharges. Water, Air, and Soil Pollution 178(3), 103- 112. [77] Thebaldi, M.S.; Sandri, D.; Felisberto, A.B.; Rocha, M. S.; Neto, S.A. (2011). A qualidade da água de um córrego sob influência de abate de bovinos efluente tratado. Revista Brasileira de Engenharia Agricola e Ambiental 15(4), 302-309. [78] Nasu, E.G.,C.; Pires, E.; Formentini, H.M.; Furlanetto, C. (2010). Effect of manipueira on Meloidogyne incógnita through in vitro and in vivo essays on tomatoes in greenhouse. Tropical Plant Pathology 35(1), 32-36. [79] Nzuzi, L. (1999) La gestion des déchets domestiques: bilan annuel d’une expérience pilote de l’Hôtel de Ville de Kinshasa. Med Fac Landbouww Univ Gent 64(1), 107- 114. [80] Ngoma, P.M.; Hiligsmann, S.; Zola, E.S.; Culot, M.; Fievez, T.; Thonart, P. (2015). Comparative study of the methane production based on the chemical compositions of Mangifera Indica and Manihot Utilissima leaves. SpringerPlus 4(75), 1-12. [81] Makkar, R.S.; Cameotra, S.S. (2002). Na update on the use of unconventional substrates for biosurfactant production and their new applications. Applied of Microbiology and Biotechnology 58(4), 435. [82] Mendes, A.; Guerra, P.; Madeira, V.; Ruano, F.; da Silva, T.L.; Reia, A. (2007). Study of docosahexaenoic acid production by the heterotrophic microalga Crypthecodinium cohnii CCMP 316 using carob pulp as a promising carbon source. World Journal of Microbiology and Biotechnology 23(9), 1209-1215. [83] Perez-Garcia, O.; Escalante, F.M.; De-Bashan, L.E.; Bashan, Y. (2011). Heterotrophic cultures of microalgae: metabolism and potential products. Water Research 45(1), 11- 36. [84] Plaza, G.A.; Pacwa-Plociniczac, M.; Piotrowska-Seget, Z.; Jangid, K.; Wilk, K.A.; (2011). Agroindustrial wastes as unconventional substrates for growing of Bacillus strains and production of biosurfactante. Environment Protection Engineering 37(3), 63-71. [85] Schmidt, R.A.; Wiebe, M.G.; Eriksen, N.T. (2005). Heterotrophic high celldensity fed- batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria. Biotechnology and Bioengineering 90(1), 77-84. [86] Abegunrin, T.P.; Awe, G.O.; Idowu, D.O.; Adejumobi, M.A. (2016). Impact of wastewater irrigation on soil physic-chemical properties, growth and water use pattern of two indigenous vegetaables in southwest Nigeria. Catena 139, 167-178. Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 167

[87] Kauser, S. (2007). Water use in vegetable production in district Faisalabad (MSc Thesis) Department of Environmental and Resource Economics, University of Agriculture, Faisalabad, Pakistan. [88] Khalil, S.; Kakar, M.K. (2011). Agricultural use of untreated wastewater in Pakistan. Asian J. Agric. Rural Develop. 1, 21-26. [89] Al-Hamaiedeh, H.; Bino, M. (2010). Effect of treated grey water reuse in irrigation on soil and plants. Desalination 256, 115-119. [90] Ali, S.; Haq, I.; Qadeer, M.A. (2001). Effect of mineral nutrient on the production of citric acid by Aspergillus niger. International Journal of Biological Sciences 32, 31-35. [91] Dhillon, G.S.; Kaur, S.; Brar, S.K.; Verma, M. (2013). Green synthesis approach: extraction of chitosan from fungus mycelium. Critical Reviews in Biotechnology 33, 379-403. [92] Chiumarelli, M.; Ferrari, C.C.; Sarantopoulos, C.I.G.L.; hubinger, M.D. (2011). Fresh cut “Tommy Atkins” mango pre-treated with citric acid and coated with cassava (Manihot esculenta Crantz) starch of sodium alginate. Innovative Food Science and Emerging Technologies 12, 381-387. [93] Zhang, H-J.; Zhang, J-H.; Xu, J.; Tang, L.; Mao, Z-G. (2014). A novel recycling process using the treated citric acid wastewater as ingredients water for citric acid production. Biochemical Engineering journal 90, 206-213. [94] Sikora, A.; Blaszczyk, M.; Jurkowski, M.; Zielenkiewics, U. (2013). Lactic acid bacteria in hydrogen-producing consortia: on purpose or by coincidence? In: Kongo, M. (Ed.), Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes. InTech. [95] Ntaikou, I.; Antonopoulou, G.; Lyberatos, G. (2010). Biohydrogen production from biomass and wastes via dark fermentation: a review. Waste Biomass 1, 21-39. [96] Rosa, P.R.F.; Gomes, B.C.; Varesche, M.B.A.; Silva, E.L. Characterization and antimicrobial activity of latic acid bacteria from fermentative bioreactors during hydrogen production using cassava processing wastewater. Chemical Engineering Journal, 284, 1-9, 2016. [97] Khongkliang, P.; Kongjan, P.; O-Thong, S. Hydrogen and methane from starch processing wastewater by thermophilictwo-stage anaerobic digestion. Energy Procedia, 79, 827-832, 2015. [98] Dhillon, G.S.; Kaurc, S.; Sarma, S.J.; Brar, S.K. (2013). Integrated process for fungal citric acid fermentation using apple processing wastes and sequential extraction of chitosan from waste stream. Industrial Crops and Products 50, 346–351. [99] Berger, L.R.R.; Stamford, T.C.M.; Stamford-Arnaud, T.M.; Franco, L.O.; do Nascimento, A.E.; Cavalcante, H.M.M.; Macedo, R.O.; de Campos-Takaki, G.M. (2014). Effect of Corn Steep Liquor (CSL) and Cassava Wastewater (CW) on Chitin and Chitosan Production by Cunninghamella elegans and Their Physicochemical Characteristics and Cytotoxicity. Molecules 19, 2771-2792. [100] Banat, I.M.; Makkas, R.S.; Cameotra, S.S.; (2000). Potential comercial applications of microbial surfactants. Applied Microbiology and Biotechnology 53, 495-508. [101] Besson, F.; Michel, G. Biosynthesis of iturin and surfactin by Bacillu subtilis. (1992). Evidence for amino acid activating enzymes. Biotechnology Letters 14, 1013-1018.

Complimentary Contributor Copy 168 Tatiele C. do Nascimento, Erika C. Francisco, Leila Q. Zepka et al.

[102] Sandrin, C.; Peypoux, F.; Michel, G. (1990). Coproduction of surfactin and iturin A lipopeptides with surfactante and antifungal properties by Bacillus subtilis. Applied Biochemistry and Biotechnology 12, 370-375. [103] Nitschke, M.; Pastore, G.M. (2003). Cassava flour wastewater as a substrate for biosurfactant production. Applied Biochemistry and Biotechnology 106, 295-302. [104] Jacob-Lopes, E.; Franco, T.T. (2013). From oil refinery to microalgal biorefinery. Journal of CO2 Utilization 2, 1-7. [105] Borowitzka, M.A. (1992). Algal biotechnology products and processes: Matching science and economics. Journal of Applied Phycology 4, 267-279. [106] Francisco, E.; Franco, T.T.; Zepka, L.Q.; Jacob-Lope, E. (2015). From waste-to-energy: the process integration and intensification for bulk oil and biodiesel production by microalgae. Journal of Environmental Chemical Engineering 3, 482-487. [107] Lu, Y.; Zhai, Y.; Liu, M.; Wu, Q. (2010). Biodiesel production from algal oil using cassava (Manihot esculenta Crantz) as feedstock. Journal of Applied Phycology 22(5), 573-578. [108] Vidotti, E.C.; Rollemberg, M.C.E. (2004). Algas: da economia nos ambientes aquáticos à biorremediação e à química analítica. Química Nova 27, 139-145. [109] Borowitzka, M.A. (1999). Commercial production of microalgae: ponds, tanks, tubes and fermenters. Journal of Biotechnology, 70, 313-321. [110] Ramírez-Mérida, L., Zepka, Q.L., Jacob-Lopes, E. (2014). Microalgae and cyanobacteria: application in medicine. Revista Electrónica PortalesMedicos 9, 149. [111] Schwender, J.; Seemann, M.; Lichtenthaler, H.K.; Rohmer, M. (1996). Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehyde 3-phosphate non-mevalonate pathway in the green alga Scenedesmus obliquus. Biochemical Journal 316, 73-80. [112] Wen, Z.Y.; Chen, F. (2000). Heterotrophic production of eicosapentaenoic acid by the diatom Nitzschia laevis: effects of silicate and glucose. Journal of Industrial Microbiology and Biotechnology 25, 218-224. [113] Shi, X. M. et al. (2000). Heterotrophic production of biomass and lutein by Chlorella protothecoides on varios nitrogen sources. Enzyme and Microbial Technology 27, p.312-318. [114] Schmidt, R.A. et al. (2005), Heterotrophic high cell-density fed-batch cultures of the phycocyaninproducing red alga Galdieria sulphuraria. Biotechnology and Bioengineering 90, 77-84. [115] Xiong, W. et al. (2008). High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production. Applied Microbiology and Biotechnology 78, 29-36. [116] Queiroz, M. I.; Hornes, M. O.; Silva-Manetti, A. G.; Zepka, L. Q.; Jacob-Lopes, E. (2013). Fish processing wastewater as a platform of the microalgal biorefineries. Biosystems Engineering 115(2), 195-202. [117] Ceron-Garcia, M.C.; Fernandez-Sevilla, J.M.; Sanchez-Miron, A.; Garcia-Camacho, F.; Contreras-Gomez, A.; Molina-Grima, E. (2013). Mixotrophic growth of Phaeodactylum tricornutum on fructose and glycerol in fed-batch and semi-continuous modes. Bioresource Technology 147, 569.

Complimentary Contributor Copy Wastewater from Cassava Processing as a Platform … 169

[118] Rodrigues, D.B.; Flores, E.M.M.; Barin, J.S.; Mercadante, A.Z.; Zepka, L.Q.; Jacob- Lopes, E. (2014). Production of carotenoids from microalgae cultivated using agroindustrial wastes. Food Research International 65, 144-148. [119] Rodrigues, D. B.; Menezes, C.R.; Mercadante, A. Z.; Jacob-Lopes, E.; Zepka, L. Q. Bioactive pigments from microalgae Phormidium autumnale. Food Research International 77, 273-279. [120] Santos, A.B.; Fernandes, A.S.; Wagner, R.; Jacob-Lopes, E. Zepka, L.Q. (2016). Biogeneration of volatile organic compounds produced by Phormidium autumnale in heterotrophic bioreactor. Journal of Applied Phycology 28, 1561-1570. [121] Ortiz-Tena, J. G.; Rühmann, B.; Schieder, D.; Sieber, V. (2016). Revealing the diversity of algal monosaccharides: Fast carbohydrate fingerprinting of microalgae using crude biomass and showcasing sugar distribution in Chlorella vulgaris by biomass fractionation. Agae Resarch 17, 227-235. [122] Voltolina, D.; Gómez-Villa, H.; Correa, G. (2005). Nitrogen removal and recycling by Scenedesmus obliquus in semicontinuous cultures using artificial wastewater and a simulated light and temperature cycle. Bioresource technology 96, 359-362. [123] Raposo, M. F. D. J. et al. (2013). Bioactivity and applications of sulphated polysaccharides from marine microalgae. Marine drugs 11, 233-252. [124] Becker, W. (2004). Microalgae in human and animal nutrition. In: RICHMOND, A. (Ed). Handbook of microalgal culture: biotechnology and applied phycology. London: Blackwell Science, 312-351. [125] Chautona, M. S.; Kjell Inge Reitana, K. I.; Norskerc, N. H.; Tveteråsd, R.; Kleivdal, H.T. (2015). A techno-economic analysis of industrial production of marine microalgae as a source of EPA and DHA-rich raw material for aquafeed: Research challenges and possibilities. Aquaculture 436, 95-103. [126] Kim, K.H.; Choi, I.S.; Kim, H.M; Wi, S.G.; Bae, H.J. (2014). Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation. Bioresourch Technology 153, 47-54. [127] Bloomberg, J. (2014). Antibacterial Drugs Market Expected to Reach USD 45.09 Billion Globally in 2019: Transparency Market Research. Pharmaceutical news on ulitzer. [Accessed:26.06.2016] Available from: URL: http://pharmaceuticals.ulitzer. com/node/3023186. [128] Roso, G.R.; Queiroz, M.I.; Streit, N.; Menezes, C.R.; Zepka, L.Q.; Jacob-Lopes, E. (2015). The bioeconomy of microalgal carotenoid-rich oleoresins produced in agroindustrial biorefineries. Journal of Chemical Engineering and Process Technology 6, 1-7.

Chapter 9

CASSAVA WASTEWATER AS SUBSTRATE IN BIOTECHNOLOGICAL PROCESSES

Cristiano José de Andrade1, Ana Paula Resende Simiqueli2, Fabiola Aliaga de Lima1, Juliana Bueno da Silva3, Lidiane Maria de Andrade1 and Ana Elizabeth Cavalcante Fai4 1Department of Chemical Engineering, Polytechnic School, University of São Paulo (USP), São Paulo, SP, Brazil 2National Agricultural Laboratory - Brazilian Ministry of Agriculture, Livestock and Food Supply (LANAGRO-SP/MAPA), Campinas, SP, Brazil 3Department of Food Science, College of Food Engineering, University of Campinas (UNICAMP), Campinas, SP, Brazil 4Department of Basic and Experimental Nutrition, Institute of Nutrition, Rio de Janeiro State University (UERJ), Rio de Janeiro, RJ, Brazil

ABSTRACT

Progresses in biotechnological processes offer a vast array of possibilities for economic use of agro-industrial residues, such as cassava wastewater. Due to its chemical composition, cassava wastewater is an interesting substrate for microbial processes for the production of value-added bioproducts. Cassava wastewater comes from the manufacture of cassava (Manihot esculenta spp. esculenta) flour which has up to 90% of starch in its root (w/w) and is easily cultivable. The main producers of cassava in 2014 - Nigeria, Thailand, Indonesia and Brazil - were responsible for 48.61% of the total world production of 27.03 × 107 metric tons of the raw crop, which is mainly used as food and feed, but also as feedstock for biofuels and biochemicals. However, the industrial manufacturing of cassava roots generates a large amount of liquid (cassava wastewater – 2.5 liters/10 kg of cassava) and solid (bagasse) residues, in which are usually burned or disposed incorrectly. Cassava wastewater has a high content of nutrients including carbohydrates (9.6-37 g/L), protein (2.3 g/L), nitrogen (0.1-1.3 g/L) and minerals as phosphorous, potassium, calcium, magnesium, sulphur, iron, zinc, cooper, etc in pH value 5.5. Therefore, due to the plenty availability, non-market value, high content of nutrient and the continuous supply throughout the year (perennial crop), there is an

Complimentary Contributor Copy 172 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

interesting potential for the utilization of cassava wastewater as an alternative substrate in biotechnological processes, which would be in consonance with biorefinery approach. In this sense, during the past years, several biotechnological processes using cassava wastewater as substrate have been described, which are an alternative to reduce the production costs and the environmental impact. The various products which have been obtained from cassava waste water include biofuels (hydrogen, ethanol, butanol, methane), biosurfactants; organic acids (citric acid, lactic acid and succinic acid), volatile fatty acids (acetic, propionic, butyric and valeric acids), aromatic compounds, enzymes and prebiotics.

1. INTRODUCTION

The valorization and utilization of organic residues for the biosynthesis of value-added products has been encouraged and studied from multiple viewpoints, due to environmental issues and the potential of cost-efficient technological development (Pleissner et al., 2015; Bentsen et al., 2014). Amount of 3.7 × 109 t of agricultural residues are yearly generated as residues from agricultural industries around the world. In addition, approximately one-third of the edible parts of food produced globally are discarded, which amounts to 1.3 billion tons per year (Pleissner et al., 2015; Gustavsson et al., 2011). Food is lost or wasted across the supply chain, from agricultural production through to final household consumption. Regarding medium to high-income countries, food is mostly wasted at consumption level, suggesting that it is thrown out even if it is still edible. Substantial losses also take place early in the food supply chains in the predominantly industrialized country regions. On the contrary, in low-income countries, food is lost frequently through the early (post-harvest) and middle (processing) levels of the food supply chain and a smaller amount of food is wasted at the retail and consumer stage (Gustavsson et al., 2011). In any case, the food supply chain generates a high volume of residues and food industrial processes, which are among the major contributors of environmental waste; mainly organic, high biological oxygen demand (BOD) and chemical oxygen demand (COD). Their chemical and physical composition may vary according to seasonal fluctuations and handling procedures. These residues additionally present some other characteristics of severe concern, such as: susceptibility to bacterial contamination due to the high water activity content and high accumulation amounts; disposal management complications (environmental impact) and the costs related to them. Waste management, hence, involves careful technological and financial investments, mainly for procedures on an industrial scale. In this sense, novel technological approaches to develop value-added bio-based products must be considered a positive strategy to minimize this problem and to maintain industrial interest in the utilization of these residues (Ravindran and Jaiswal, 2016; Agyei et al., 2015). A rising global population generates a growing need for food production and the industry related to it, and thus the generation of high volumes of food waste. This problem is increased as a result of insufficient development strategies for waste management and valorization (Ravindran and Jaiswal, 2016). Materials defined as waste from food processing comprise of dairy waste, oil waste, fruit and vegetable residues, and meat, poultry, and seafood by products, which may be expressed as a pool of diversified fibers and non-fiber carbohydrates, proteins, lipids, and other micronutrients and bioactive compounds (Alibardi and Cossu, Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 173

2016; Agyei et al., 2015). Thus, as the total amount of residues generated by the food industry is increasing, the challenge for its management and valorization is rising as well. In this context, biorefinery is an emerging conception, proposing that all types of biomass-derived materials might be transformed into high value-added products, providing economic and environmental sustainability to the process (Zhang et al., 2016; Gustavsson et al., 2011). Cassava wastewater (Andrade et al., 2016a-b; Zhang et al., 2016; Fai et al., 2015); olive oil mill effluent (Ahmad et al., 2016); lactic whey (Pescuma et al., 2015); soybean curd residue (Vong and Liu, 2016); potato process effluent (Li et al., 2015); fruits and vegetables residues, such as stems, stalks and seeds (Panda et al., 2016), among others, have been reported in literature as interesting residues for biotechnological processes due to their chemical composition and high accumulation‐rate. Different processes and products have been developed from those materials in recent years, including electricity generation, sports supplements, nutraceuticals and functional food ingredients, pulp and paper production, edible coatings and films and products based on fermentation. Important technological advances have emerged from this last option and present really interesting opportunities to build powerful biotechnological industries (Panda et al., 2016; Zhang et al., 2016; Panesar and Kaur, 2015; Soo et al., 2015). Among various food processing residues, cassava wastewater has been highlighted due to its nutrient content and abundant availability as an ideal alternative substrate for biosynthesis and biotransformation of a wide range of products from different bacteria, mold and yeasts. Cassava wastewater is a liquid waste obtained in large volumes, around 300 L/t of processed roots from cassava-based industries. The discard of this effluent leads to environmental issues due to its high organic load; this residue is composed of carbohydrates, nitrogen, minerals, and trace elements and therefore has potential to be biologically converted into various high- value products (Zhang et al., 2016). Based on the above reasons, cassava wastewater has become an important topic to review and discuss, in order to contribute to its industrial large-scale bioapplications. This chapter aims to provide an overview of both current and potential innovative applications, using cassava wastewater as an alternative substrate for the production of biosurfactants (surfactin, rhamnolipids and mannosylerytritol lipids), organic acids (citric, lactic, butyric, propionic and valeric acids), alternative fuels (bioacetone, butanol and bioethanol, biomethane and biohydrogen), bioflavours, chemicals, food ingredients and other high value-added products.

2. VALORIZATION OF AGRO-INDUSTRIAL RESIDUES - CASSAVA WASTEWATER UTILIZATION AS AN ALTERANTIVE SUBSTRATE FOR BIOTECHNOLOGICAL PROCESSES

According to Maiti et al., (2016) agro-based industries are facing an increasing surge around the world. It has been estimated that about 60-70% of the agro-industrial waste generated, especially from the food and beverage processing industries, is discharged into the environment with no treatment or management, which could lead to seriously damaging effects. On the other hand, unstable and expensive nonrenewable resources have encouraged research concerning the utilization of biomass resources for the production of energy, fuels and other goods (Sindhu et al., 2016; Pleissner et al., 2015). In general, the food residues have

Complimentary Contributor Copy 174 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al. a chemical composition comprised approximately of 30-60% starch, 5-10% proteins and 10- 40% (w/w) lipids, which provides good potential to be used as a suitable feedstock in biotechnological processes (Pleissner et al., 2015; Bentsen et al., 2014). The biotechnological utilization of residues, derived from food supply chains, exploits many metabolic pathways, which are associated to their chemical composition, structure and amount (Agyei et al., 2015). Lin et al., (2013) has defined the conventional techniques, such as manufacturing feed and composites, as first generation production; and the producing of high value-added products using wastes from the conventional techniques, such as biofuels, food ingredients and other chemical compounds, as second-generation techniques. The 2nd generation bioprocesses have proved to be effective in the production of a wide range of interesting biocompounds, thereby it is aligned to green chemistry concept and also shows economic advantages (Sindhu et al., 2016; Agyei et al., 2015). Although biotechnology products (enzymes, biomass, and other metabolites) exhibit a large number of interesting applications, it is still essential to reduce manufacturing costs. The use of agro-industrial residues as substrates, which are abundant, inexpensive and rich of nutrients as low-cost substrates, may be a feasible strategy in achieving the reduction of manufacturing costs (Maiti et al., 2016; Fai et al., 2015). For instance, for biosurfactants microbial production, culture media may represent about 30% of production costs (Andrade et al., 2016a-b; Fai et al., 2015). In this sense, practical studies on upstream and downstream processing have advanced, however they present some important challenges. For instance, bio-butanol which costs the feedstock is the main economic limitation, representing 60% of the total cost of the process. Thus, bio-butanol production from lignocellulose and/or starch based residues, such as cassava wastewater, might be a promising renewable energy source for countries with high amounts of agro-industrial waste biomass (Maiti et al., 2016). In addition, the integration of fermentative processes for agro-industrial residues should reduce the cost of production (e.g., capital savings in the plant, energy-saving process, cell recycling, multiple bioproducts), (Andrade et al., 2016a-b; Fai et al., 2015; Soo et al., 2015; Barros et al., 2013). Fai et al. (2015) demonstrated the technical viability of an integrated process: biosurfactant and galactooligosaccharides production from Pseudozyma tsukubaensis using cassava wastewater as a low-cost culture medium to produce biosurfactant and to provide biomass for galactooligosaccharides synthesis. Andrade et al., (2016a-b) evaluated different concentrations of alterative substrates for the simultaneous production of surfactin and 2.3-butanediol by Bacillus subtilis LB5a and observed that the best culture composition for both bioproducts was whey (27.7-34 g/L), activated carbon (25 g/L) and cassava wastewater (74 g/L), in which the bench experiments resulted in 27.07 mg/L of surfactin and 330 mg/L of 2.3-butanediol. Barros et al., (2013) demonstrated that the integrated production of amylases, proteases and biosurfactants by Bacillus subtilis in cassava wastewater was not only promising, but also that it was better than that in the synthetic medium, showing the feasibility of this agro-industrial residue as an alternative substrate. Therefore, cassava wastewater offers one of the most remarkable biotechnological potentials for cost-efficient production of chemicals (Zhang et al., 2016), enzymes (Barros et al., 2013), biofuels and bioenergy (Intanoo et al., 2014; Nuwamanya et al., 2012; Sreethawong et al., 2010), biosurfactants (Andrade et al., 2016a-b; Kummer et al., 2016), food ingredients (Fai et al., 2015; Tosungnoen et al., 2014).

Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 175

3. BIOPRODUCTS DERIVED FROM CASSAVA WASTEWATER

As stated above, various biotechnological processes face the key challenge of, simultaneously, reducing the costs of culture media and producing very selective products with a high yield. In this sense, inexpensive and renewable substrates have been studied in recent years as an appropriate strategy for the economic transformation of fermentation technology (Panda et al., 2016; Panesar and Kaur, 2015; Panesar et al., 2015; Soo et al., 2015; Banat et al., 2014). It is interesting to note that distinct residues applied as a substrate might modify microbial metabolic expression and result in bioproducts with different characteristics with multiple potential routes in the biotechnological industry (Novelli et al., 2016). The multivariate association of biochemical pathways of different microorganisms, combined with composition, structure and pretreatment of matrix substrates, permits the biotechnological synthesis of a wide variety of industrially important bio-based compounds (Pleissner et al., 2015). Thus, factors such as the concentration of micronutrients; carbon and nitrogen sources in agro-based residues; C/N ratio; type of microorganism and its metabolic engineering variations; inoculum age and concentration; dissolved oxygen; temperature; incubation time; moisture content; among others, are important keys to explore novel agro-industrial residues applications for biotechnological industrial purposes (Alibardi and Cossu, 2016; Andrade et al., 2016a-b; Novelli et al., 2016; Bution et al., 2015; Fai et al., 2015). The following discussion will focus on (i - item 4) the chemical composition of cassava effluent residue, the 2nd generation food waste valorization for biosynthesis and biotransformation processes, especially using cassava wastewater as a low-cost substrate for the production of biosurfactants (ii - item 5), organic acids (iii - item 6), alternative fuels (biogas, bioacetone, butanol and bioethanol) (iv - item 6) and bioflavours (v - item 7).

4. CHEMICAL COMPOSITION OF CASSAVA EFFLUENT RESIDUE

The roots of cassava have high moisture content and their major macronutrient is carbohydrate, which is mainly composed of starch (Table 1). However, different varieties of cassava have different concentrations of cyanogenic glycoside compounds, which allows the classification of cassava into two groups, (i) sweet cassava and (ii) bitter cassava. Sweet cassava roots can contain approximately 50 ppm of cyanide on a fresh weight basis, although the bitter ones may contain around 400 ppm (Okudoh et al., 2014). Thus, cassava is consumed as food and feed (whole root, flour, grated, chips, among others) only after appropriate treatment (Okudoh et al., 2014). Sweet cassava roots are consumed after cooking (the process includes peeling and boiling in water). Bitter cassava roots, on the other hand, require the removal of cyanide. First of all, bitter cassava roots are peeled and grated, and then soaked for enough time that allows leaching and fermentation (thus releasing the volatile cyanide gas) (Okudoh et al., 2014; Ponte, 2006). Cassava wastes are obtained during the manufacturing process, such as water washing (roots), selection of roots, flour production, among others. Cassava wastewater is the main residue of cassava flour production. Others significant residual products from the

Complimentary Contributor Copy 176 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al. manufacturing of cassava are roots, the skins and fibers, that are mainly composed of cellulose (Nitschke and Pastore, 2004a-c). The extraction process to obtain starch from cassava roots produces bagasse residues, that may have 40 to 75% of starch (dry weight) and 15-50% of fibers, such as cellulose, and others polysaccharides. Despite this, the concentration of protein in bagasse is very low, as shown in Table 2.

Table 1. Chemical composition of cassava (fresh and dry weight)

Composition Fresh weight (%) Dry weight (%) Moisture 62 12 Protein 1 1.5 Lipids 0.2 0.5 Carbohydrates 35 85 Starch 30 80 Fiber 1 1.5 Nitrogen 0.22 0.45 Phosphorus 0.03 0.08 Potassium 0.4 0.8 Calcium 0.027 0.096 Sodium 0.002 0.01 Iron 0.001 0.008 Niacin 0.0006 0.0008 Cyanide 0.8 2 Adaptation from Okudoh et al., (2014).

Table 2. Chemical composition of cassava wastewater

Composition Concentration (ppm) Carbohydrates 41.000 Nitrogen 425 Phosphorus 259 Potassium 1853 Calcium 227 Magnesium 405 Sulfur 195 Iron 15 Zinc 4 Copper 11 Manganese 3 Boron 5 Cyanide 42 Adaptation from Ponte, (2006); Nitschke and Pastore, (2004a-b).

As Tables 1 and 2 show, cassava residues such as bagasse and wastewater have minerals, carbohydrates and nitrogen. Therefore, these residues can be used as substrates in

Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 177 biotechnology processes that involve the growth of microorganisms for the production of chemicals and high value-added products. It is important to emphasize the presence and concentration of cyanide (which affects the microbial growth). In this sense, cassava wastewater needs to be pretreated (detoxiﬁcation) (Nitschke and Pastore, 2004a-c).

5. THE PRODUCTION OF BIOSURFACTANTS USING CASSAVA WASTEWATER AS A SUBSTRATE

Surfactants are amphipathic compounds, meaning that their structures comprise of both hydrophobic and hydrophilic groups. This characteristic causes them to accumulate in an oriented regular manner in the interfaces, such as liquid-air or liquid-liquid interfaces, resulting in a reduction in the interfacial tension. There is a wide range of applications for surfactants, such as detergent, emulsifier, de-emulsifier and foaming, wetting, dispersant and solubilizing agents (Herman and Maier, 2002; Harshada, 2014). Surfactants can be produced by living beings, and are thus called biosurfactants, with several different structures and intracellular and extracellular functions to the organisms (Bognolo, 1999). They are produced by the human body, such as bile acids (Bognolo, 1999) and pulmonary surfactant (Harshada, 2014), and plants are known for producing saponins (Bognolo, 1999). However, biosurfactants with industrial potential are those produced by microorganisms, more specifically by bacteria (Healy et al., 1996; Maier, 2003). There is a wide range of different structures of biosurfactants that can be first classified by high molecular weight, e.g polimerical surfactantants, or low molecular weight, and these are subsequently divided according to the nature of their hydrophilic moiety, such as lipopeptides, glicopeptides and phospholipides (Bognolo, 1999; Desai and Banat, 1997; Healy et al., 1996; Herman and Maier, 2002; Rebello et al., 2014; Marchant and Banat, 2012; Singh and Saini, 2014). The type of microbial or even the isoform of the surfactant produced depends not only on the source and species of microorganism, but also on the carbon source used, which also represents an opportunity to “design” surfactants better suited to each commercial application (Herman and Maier, 2002; Singh and Saini, 2014). Biosurfactant synthesis by bacteria is related to some microbial needs, such as the production of substances with activity against competing microorganisms (Maier, 2003), causing bacterial adhesion or de-adhesion to hydrophobic surfaces (Neu, 1996; Cameotra and et al., 2010; Herman and Maier, 2002), nutrients emulsification, the transport of material through cell membrane (Bognolo, 1999; Lin, 1996; Maier, 2003) and cell recognition (Costa, 2005). It is possible that biosurfactants are an evolutionary defense strategy, since their production has an effect on microbial survival (Cameotra et al., 2010). Nowadays, some authors consider biosurfactants as the answer to the demand of “greener,” milder and more efficient surfactants, since they are less toxic and more easily biodegradable than their chemical counterparts (Herman and Maier, 2002; Rebello et al., 2014). Besides this, they also have as advantages their structural diversity, specificity, stability under extreme conditions (Cameotra and Makkar, 2004; Kosaric, 1992; Marchant and Banat, 2012), biological activities, such as antimicrobial, antiviral, anti-tumorigenic and anti adhesive (Barros, 2007; Gomes and Nitschke, 2012; Cameotra and Makkar, 2004) and application for bioremediation purposes (Harshada, 2014). This plurality of different

Complimentary Contributor Copy 178 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al. applications represents the potential use of biosurfactants as multipurpose ingredients or additives (Nitschke and Costa, 2007). Even though there has been an increase in the development of biosurfactants, 70 to 75% of all surfactants used in industrialized countries are still synthetized by the petrochemical industry. In 2012, the total market value of surfactants was approximately 12 million tons against 3.5 million tons of bio-based surfactants (Campos et al., 2013). This can be explained by the 3 to 10 times higher production costs of biosurfactants compared to synthetic surfactants, what hinder their competition on a commercial scale (Amaral et al., 2010; Barros, 2007; Makkar and Cameotra, 1997; Singh and Saini, 2014). One of the strategies that has been studied along the years to reduce production costs in order to turn the use of these compounds into a feasible option on an industrial scale is the replacement of traditional synthetic media by agro-industrial wastes, reducing, thus, the cost of carbon source. Besides the economical aspect of the use of inexpensive alternative substrates, this could reduce the ecological damage due to the incorrect discard of these residues (Amaral et al., 2010; Costa et al., 2008; Makkar et al., 2011; Oliveira et al., 2013; Johny, 2013; Singh and Saini, 2014). The use of residues from cassava processing has been extensively studied for lipopeptides production; nevertheless their use for the production of other kinds of microbial surfactants is still sparse. Bacteria from the genus Bacillus are known for the production of lipopeptides, being the most studied surfactin. Several papers describe surfactin production by Bacillus subtilis using cassava wastewater as a substrate either in laboratorial (Nitschke et al., 2004a-c; Nitschke and Pastore, 2006), bench (Costa, 2005) or pilot scale (Barros et al., 2008a-b; Quadros et al., 2011). Surfactin obtained when B. subtilis was grown in this medium was stable to freezing and heating, as well as changes in ionic strength and pH from 6 to 10 (Barros et al., 2008a-b; Nitschke and Pastore, 2006), presented antimicrobial (Araújo et al., 2016; Nitschke et al., 2004b; Quadros et al., 2011), antiviral (Nitschke et al., 2004b), anti-adhesion and antibiofilm activities (Araújo et al., 2016; Gomes and Nitschke, 2012) and had potential to be used for remediation of soils and treatment of effluents contaminated by heavy metals (Kummer et al., 2016). Even though cassava wastewater is considered an appropriate medium for surfactin production, it was not suitable for the production of the lipopeptide lichenysin by Bacillus licheniformis. However, the authors considered that cassava starch could be an interesting alternative to be explored in the future (Coronel-León et al., 2016). Monteiro-Rodríguez et al., (2015) assessed the use of an alternative culture medium containing both cassava wastewater and corn waste oil for biosurfactant production by Serratia marcescens UCP/WFCC 1549 and the resulting crude biosurfactant was considered a sustainable option for application in the bioremediation processes of hydrophobic pollutants derived from petroleum. Similarly, Andrade et al., (2015) evaluated glycolipid production by Candida glabrata UCP/WFCC 1556 in media containing cassava wastewater, whey and corn steep liquor using central composite designs. However, the best results, with lower surface tension, were achieved when using only whey and corn steep liquor. Pseudomonas aeruginosa rhamnolipids were also obtained in a medium containing cassava wastewater and mixed salts solution. This study showed that the medium could be diluted in water up to 50% with no statistical difference in the surface tension obtained (Bezerra et al., 2012). Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 179

Costa et al., (2009) and Fai et al., (2015) studied integrated processes for the production of microbial surfactants and other commercially interesting products by fermentation in media containing cassava wastewater. Costa et al., (2009) developed a medium containing cassava wastewater and waste cooking oil for the simultaneous production of rhamnolipids and poly-hydroxyalkanoates, a potential biodegradable thermoplastic, by several strains of P. aeruginosa. While Fai et al., (2015) assessed the bench-scale production of mannosylerytritol lipids by Pseudozyma tsukubaensis on cassava wastewater and, subsequently, the reutilization of the biomass for galactooligosaccharides synthesis.

6. ORGANIC ACIDS

The production of organic acids using agro-industrial residues by microorganisms is very interesting and feasible for food and pharmaceutical industrial applications, as well as their common use in the chemical industry. Many studies report the biotechnological production of organic acids, using as a substrate cassava wastewater and other cassava by-products, as shown in Table 3. The main organic acids produced by microorganisms are i) short-chain fatty acids (SCFA) as main representatives, acetic (HAc), butyric (HBu), propionic (HPr) and valeric (HVa) acids (Aquino et al., 2015, Streehawong et al., 2010), and, ii) other acids such as citric (HCi) and lactic (HLa) acids (Chookietwattana, 2014, Tosungnoen et al., 2014, Lima et al., 2010, Avancini et al., 2007, Colin et al., 2007). SCFA and other organic acids as well may be obtained by direct production (Studies 1-7, Table 3), in which these acids are the product of cassava wastewater fermentation, or also through an indirect production of biotechnological processes, such as the production of biogas (organic acids are an intermediary metabolites of biogas production) (8-13 studies, Table 3). In the next sections (6.1 and 6.2) the direct and indirect production of organic acids using cassava wastewater as a substrate will be discussed.

6.1. Direct Production of Organic Acids through Cassava Wastewater Fermentation and Other Cassava By-Products

Lactic Acid Bacteria (LAB) play an important role in the production of organic acids through the natural fermentation of cassava wastewater caused by the LAB present in this residue, or by the addition of other microorganisms in the fermentative process, such as Lactobacillus plantarum or Lactobacillus rhamnosus. Cassava wastewater natural fermentation can be used to make beverages with probiotic properties. Avancini et al., (2007) observed low toxicity in these beverages for rats. Moreover, the authors evaluated the lactic acid content produced by the biotechnological process by LAB as 1.9 g/L. From cassava wastewater natural fermentation at 31°C for up to 75 days, Aquino et al., (2015) identified and quantified four organic acids by HPLC: HLa (0.06 g/L, 48 d), HAc (1.25 g/L, 62 d), HBu (1.98 g/L, 75 d) and HPr (0.20 g/L, 75 d).

Complimentary Contributor Copy 180 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

Inoculation of Lactobacillus plantarum 702 MSUL (amylases producer) to natural fermentation of cassava wastewater promotes the saccharification of starch and favors the production of lactic acid. The saccharification starch resulted in the production of lactic acid 28.71 g / L in 48 h (Tosungnoen et al., 2014). Chookietwattana (2014), obtained 39.70 g/L of lactic acid from the LAB selection from cassava with amylolytic activity followed by a fermentation process supplemented with Lactobacillus plantarum MSUL 903 for 96 h at 45°C. We noted that the cassava wastewater fermentation showed an advantage in the production time (48 h) of HAc (Tosungnoen et al., 2014) compared with the starch fermentation from cassava, which took 96 h to produce an equivalent amount of this acid (Chookietwattana, 2014). Cassava wastewater fermentation using other LAB, as Lactobacillus rhamnosus LR-1, also favored the production of lactic acid (35.54 g/L) in 48 h at 37ºC, with a productivity of 1.31 g/Lh (Lima et al., 2010). Besides the LAB, others microorganisms, such as filamentous fungi, may also be used for the production of these organic acids. Citric acid production can be obtained by the biotechnological process of fermentation by Aspergillus niger using carbon sources such as glucose, molasses, and/or cassava wastewater (Adeoye et al., 2015, Zhang et al., 2014). According to Zhang et al., (2014), 108.8 g of HCi/L using Aspergillus niger WML-016 that was inoculated in cassava wastewater and incubated at 60°C could be reached. In another study with Aspergillus niger, Adeoye et al., (2015) compared the citric acid production efficiency of a wild strain of Aspergillus niger (Aspergillus niger FUO 2) with another one of the same fungus modified by UV irradiation (Aspergillus niger FUO 110). They observed an increase in HCi of 4.87 times in the mutant strain compared to the wild- type. According to the authors, it was possible to optimize the citric acid production (88.43 g/L) with the mutant strain of A. niger (FUO 110) using a mixture of substrates, such as cassava peel and sorghum malt in a proportion of 80:20, with 84 h of fermentation.

6.2. Indirect Production of Organic Acids by Cassava Wastewater Fermentation and Other Cassava by-Products

Biogas production, such as biohydrogen and biomethane, is an interesting technology for further discussion (item 7.2). In addition, during the carbohydrate conversion to biogas the formation of organic acids occurs: intermediary metabolites of biogas production (Gomes et al., 2016, Intanoo et al., 2014; Rosa et al., 2014, Lu et al., 2012, Colin et al., 2007). Studies 8-13 mentioned in Table 3 show varying contents of organic acids (HAc, HPr, HBu, HLa, HVa) formed during biogas production from cassava wastewater fermentation as well using the cassava sub-products. Among these, the levels of organic acids are, in decreasing order: HBu (up to 5.6 g/L), HVa (up to 4.8 g/L), HAc (up to 3.68 g/L), HPr (up to 1.6 g/L) and HLa (up to 1.54 g/L), The majority of studies have highlighted the production of lactic acid as the main organic acid obtained from the cassava wastewater natural fermentation (Rosa et al., 2014, Tosungnoen et al., 2014, Colin et al., 2002), although the lactic acid can be further converted into other organic acids as HBu, HVa and HAc (biogas production).

Complimentary Contributor Copy

Table 3. Organic acid production from the fermentation cassava wastewater and other by-products of cassava (Direct and Indirect)

*Studies **Organic Acid Content (g/L), Substrate Microorganism Biotechnology process Time (hours or days), Temperature (ºC) Direct production 1 HLa (28.71 g/L, 48 h) Cassava Wastewater Lactobacillus plantarum MSUL 702 Lactic acid production 2 HLa (34.54 g/L, 48 h, 37 °C) Cassava Wastewater Lactobacillus rhamnosus RL-1 Fermentative production of HLa 3 HLa (1.9 g/L) Cassava Wastewater Lactobacillus Toxicological studies 4 HLa (39.70 g/L, 96 h, 45ºC) Starch Cassava Lactobacillus plantarum MSUL 903 Lactic acid production 5 HCi (88.73 g/L, 84 h, 30ºC) Agro Waste: Cassava peels: Aspergillus niger mutant (FUO 110) Citric acid production malted sorghum (80:20) 6 HCi (108.8 g/L, 60ºC) Cassava Wastewater Aspergillus niger WML-016 Citric acid and methane production 7 HLa (0.06 g/L, 48 d) Cassava Wastewater Natural fermentation Determination organic acids HAc (1.25 g/L, 62 d) HBu (1.98 g/L, 75 d) HPr (0.20 g/L, 75 d, 31°C) Indirect production 8 HBu (~0.5-2 g/L) HPr (~0.3-1.2g/L) Cassava Wastewater Lactic acid bacteria Hydrogen production 9 HAc (~0.3-4 g/L), HBu (~0.27-5.6 g/L) Cassava Wastewater Hydrogen-producing bacteria Production of H2 and CH4 HPr (~0.22-1.6 g/L) HVa (~0.16-4.8 g/L) 10 HLa (0.04-1.2 g/L) HAc (0.17-0.75 g/L) Cassava Wastewater Lactobacillus/Methanobacterium Hydrogen production HBu (0.2-0.4 g/L) HPr (<0.120 g/L, 10 h) 11 HAc (0.38-0.87 g/L) HBu (0.53-3.7 g/L) Cassava Wastewater Anaerobic sequencing batch reactors Hydrogen production HPr (0.07-0.36 g/L) HVa (0.18-1.28 g/L) 12 HLa (1.54 g/L) HAc (0.38 g/L) Cassava Wastewater Methanogenic consortia Biogas production 13 HAc (3.68 g/L) HBu (2.68 g/L) Cassava Bagasse Hydrolysate Clostridium acetobutylicum JB200 n-butanol production *References: 1. Tosungnoen et al., 2014. 2. Lima et al., 2010. 3. Avancini et al., 2007. 4. Chookietwattana, 2014. 5. Adeoye, et al., 2015. 6. Zhang et al., 2014. 7. Aquino et al., 2015. 8. Gomes et al., 2016. 9. Intanoo et al., 2014. 10. Rosa et al., 2014. 11. Streethawong, et al., 2010. 12. Colin et al., 2007. 13. Lu et al., 2012. ** Abbreviation of organic acids. HLa: Lactic acid. HCi: Citric acid. HAc: Acetic Acid. HBu: Butyric acid. HPr: Propionic Acid. HVa: Valeric acid.

Complimentary Contributor Copy 182 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

Gomes et al., (2016) (study 8 in Table 3) reported that during cassava wastewater natural fermentation by LAB the organic acids HBu and HPr reached 50% and 30% out total organic acids, respectively. Intanoo et al., (2014) (study 9, Table 3) also quantified high levels of HBu and HAv among the organic acids involved in the production of biohydrogen. Both studies (8 and 9, Table 3) discussed that the prevalence of these three acids can be justified by the concomitant production of organic acids by microbial metabolic pathways during the production of hydrogen. Furthermore, according to Antonopoulou et al., (2014), during the hydrogen production, microorganisms can convert organic acids into other acids, justifying the increase of some acids. As already discussed, organic acids have awakened industrial interest. They are intermediate metabolites inherently produced during the biogas production using cassava wastewater fermentation. Thus, the biogas production generates two large types of compounds i) biohydrogen / biomethanol (final product) and ii) organic acids (intermediate), which leads to remarkable industrial flexibilty. In this sense, Zhang et al. (2014) combined the production of biogas (CH4) and HCi from the fermentation of a mixture of cassava and corn (4:1, w/w) by Aspergillus niger WNL-016 with further simultaneous purification of HCi and CH4. In their results, the authors described the high content of citric acid 108 g/L and 16 consecutive runs of stable fermentation. Therefore, further studies should investigate the concomitant production of organic acids and biogas in further detail.

7. ALTERNATIVE FUELS

7.1. Production of Bioethanol and Acetone-Butanol-Ethanol Fermentation Using Cassava Wastewater and Other Constituents of Cassava Manufacturing

On the one hand, the use of fossil fuels as primary energy (oil, natural gas and coal) is already set worldwide (structure, knowledge, etc.) and represents approximately 86.7% of total primary energy (Chen et al., 2016). On the other hand, the use of fossil oils has been a rising concern regarding global climate change, with a volatile market (price) and oil depletion. In addition, there is an increase in energy demand by population and industries, which leads to searches for alternatives to the dependence on fossil fuels. The United States and Brazil are the most important producers of first generation fuels (alternative primary energies) using starch based (food crop) as substrate (Chen et al., 2016, Ghosh, 2016; Li et al., 2016). Regarding biofuels that are sugar-based produced, biobutanol and bioethanol are the most promising ones. In addition, biobutanol and bioethanol are very aligned to green chemistry concept (Akponah and Akponie, 2012; Ghosh, 2016). Many microorganisms such as Saccharomyces cerevisiae, Escherichia coli and Zymomonas mobilis can convert sugar into ethanol using anaerobic metabolic pathways(bioethanol is, directly, metabolized from fermentable sugars (glycolysis → pyruvate → acetaldehyde → ethanol) (Ostergaard et al., 2000; Dien et al., 2003; Elemike et al., 2015). However, S. cerevisiae shows advantages on

Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 183 the production of ethanol such as aerobic respiration and good tolerance of high ethanol concentrations and of inhibitors. Biobutanol is also produced in anaerobic conditions by solventogenic microorganisms, emphasizing 4 species of Clostridium: C. beijerinckii, C. acetobutylicum, C. saccharoperaceto-butylicum and C. saccharobutylicum, in which particular strains that do not naturally produce acetone and also uses a wide range of carbohydrates as carbon source (e.g lactose, inulin, glycerol, pentoses and hexoses) (Patakova et al., 2013; Guo et al., 2015; Li et al., 2016). This bioprocess is known as acetone–butanol–ethanol (ABE) fermentation, since bioethanol and bioacetone are the main co-products (Guo et al., 2015; Li et al., 2016). However, during the production of biobutanol, two different phases are observed, (i) acidogenic and (ii) solventogenic. The first phase (acidogenic), occurs during logarithmic phase and produces mainly acetic and butyric acids, whereas solventogenic phase occurs from late logarithmic phase to early stationary phase, in which acetic and butyric acids are metabolized into ABE. The acidogenic phase is switched to the solventogenic phase by limitation of substrates but mainly by pH >5 and butyric acid content (2 g/L) (Li et al., 2016). Drop-in biofuels (mainly butanol) are an alternative as primary energy that have similar properties of oil derived fuels, which allow them, for instance, to be fully interchangeable or blended with conventional jet fuels resulting in little structural change (with regard to current transportation pipelines and the automobile engine design) (Ouephanit et al., 2011; Cantarella et al., 2015; Guo et al., 2015). As already mentioned, bioethanol is also a powerful energy source (primary energy) to replace fossil fuels. The energy density of bioethanol corresponds approximately to 67% of gasoline (Guo et al., 2015). Over the past few decades, the global production of bioethanol on an industrial scale increased, in particular between the years 2000 and 2010 there was an increase from4.5 to 23.3 billion gallons. In 2013, United States (56.8%) and Brazil (26.7%) represented more than 83.5% of the global production of bioethanol (Guo et al., 2015; Ghosh, 2016). In this sense, theoretically all vegetables (e.g., cassava) can be used to produce biofuels through various biomass converting technologies such as gasification, fast pyrolysis, liquefaction, enzymatic hydrolysis, fermentation, etc (Guo et al., 2015; Cantarella et al., 2015; Ghosh, 2016). However, as emphasized by Chen et al., (2016), biomass such as cassava and sweet sorghum have better environmental advantages (cassava low-added value and is non- competitive with foods) for bioethanol production rather than maize, wheat and rapeseed (Elemike et al., 2015; Li et al., 2016). Different substrates have been used to produce bioethanol, for instance in the United States (corn), Brazil (sugar cane), Europe (beet), and China (corn, wheat and cassava) (Elemike et al., 2015; Chen et al., 2016; Guo et al., 2015). Obviously, the main advantage of the bioethanol production using corn, beet, wheat or other food crops is their competition for animal feed or human food (Guo et al., 2015; Elemike et al., 2015). An interesting strategy for competitive pricing of bioethanol is to use low-valued biomasses as substrates including agricultural wastes, municipal wastes, food industrial wastewaters, etc., which makes the process even more environmentally friendly and does not compete with food demand (Ghosh, 2016). Thus, many low-valued biomasses can be used for the production of biofuels, and the choice of substrate should be taken into account using 4 basic parameters: environmental, social, energetic and economical. In this sense, compared to sweet sorghum, cassava usually Complimentary Contributor Copy 184 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

shows a higher production cost and energy input and lower absorption of CO2 (Chen et al., 2016). Biofuels production using cassava wastewater and their derivatives as substrate has some challenges. For example, important provinces in China (Guangxi and Hainan) do not grow cassava on an industrial scale because of the fluctuating market that makes it difficult for the factories, and which depend on importation to control the risk (Chen et al., 2016). Therefore, beyond the reduction of climate change, the possibility of using low- aggregated value substrates such as cassava wastewater as a precursor for biofuels production such as bioethanol and ABE has been a high potential.

7.1.1. Production of Bioethanol Using Cassava Wastewater and Other Constituents of Cassava Manufacturing As already mentioned, cassava is a starch-based material and S. cerevisiae is the main bioethanol microbial producer. However, S. cerevisiae does not produce amilolitic enzymes (e.g., α-amylases, β-amylases, pullulanase, isoamylase and glucoamylase). Thus, the production of bioethanol using cassava itself or its derivatives as substrates are summarized in 5 phases, (i) pretreatment, (ii) sacchariﬁcation (e.g α-amylase), (iii) fermentation, (iv) distillation and dehydration (Chen et al., 2016). In this sense, heterologous expression of glucoamylase from Aspergillus awamori in S. cerevisiae (host), or glucoamylase and α- amylases from Schwanniomyces occidentalis also in S. cerevisiae (host), among others, were successfully reported; that is, metabolic engineering of S. cerevisiae has led to the enhancement of bioethanol productivity (Ostergaard et al., 2000). Bioethanol production using cassava wastewater has not been much explored. Akponah and Akponie, (2012) studied the optimization of bioethanol production from cassava effluent using S. cerevisiae, by pH variations, culture age and nutrient supplementation. The improved results using pH of 5.5, 3-day-old culture and NPK fertilizer as a nutrient supplement showed 14.5 (% v/w) of ethanol. Similarly, Nuwamanya et al., (2012) explored the feasibility of using the non-edible parts of cassava for biofuel production. The inexpensive nature of cassava stems and peel biomass as well as their abundance, creates an opportunity for better cassava exploitation in terms of the green chemistry concept. Roots, peels and stems showed a similar bioethanol yield of (in 500 mL batch), 55.8, 43.5, and 52.4 mL, respectively; whereas, leaves presented a significantly lower ethanol yield of 11.3 mL. Wei et al., (2015) studied the production of bioethanol using the Cassava (Manihot esculenta Crantz.) stem, which is, relatively, a new approach. The author's estimation indicated an increase of approximately 26% on the first generation ethanol by the use of cassava stem. Elemike et al., (2015) studied a pretreatment of cassava cellulosic waste (Manihot esculenta) by using pH 6.9, at a temperature at 100oC for 1 h of enzymatic activity (α- amylase) which produced disaccharide, followed by the sacchariﬁcation using amyloglucosidase at pH 4.6 at temperature 55oC for 20 h. The pretreatment allowed S. cerevisiae to produce, approximately, 2.7 g ethanol/15 g cassava cellulosic waste (Elemike et al., 2015). A similar approach was described by Akaracharanya et al., (2010) in which cassava pulp was used. The starch in the cassava pulp was enzymatically treated by α- amylase (100°C, 10 min) and glucoamylase (60 °C, 2 h), whereas the by-product of the enzymatic treatment, the lignocellulosic fibers, were pretreated with sulfuric acid followed by saccharification (cellulase) at a temperature of 40°C for 72 h. The maxim production of Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 185 bioethanol using starch and lignocellulosic fiber hydrolysates by S. cerevisiae TISTR 5596, pH 4.5, at 30°C after 48 h, reached 9.9 and 11.9 g/L, respectively (Akaracharanya et al., (2010). In other study, Moshi et al., (2015) carried out the fermentation of starch hydrolysate from inedible wild cassava for ethanol production at high temperature. First of all, cassava flour was hydrolysed using α-amylase at 60°C and under a pressure of 590 Pa for 1 h, followed by saccharification using glucomylase in the same conditions used before (temperature, pressure and time). Subsequently the fed-batch fermentation process with Caloramator boliviensis was performed in the bioreactor (530 mL). The microorganism was able to produce an ethanol concentration of up to 33 g/L and productivity of 0.15-0.2 g/L, achieving 83-84% of conversion efficiency, related to the substrate used in this study.

7.1.2. Production of ABE Using Cassava Wastewater and Other Constituents of Cassava Manufacturing Currently, the bottlenecks of ABE fermentation on an industrial scale are related to cost of substrate, low productivity and high downstream process costs. In addition, approximately 0.23 g of ABE/(L/h), 0.15 g of butanol/(L/h), and 19 g of ABE/L could be reached, in which 50% is effectively recovery after purification (Li et al., 2016). The concentration of 6.7 g of 1-butanol/L reached by C. tetanomorphum using a culture medium composed of 60 g of glucose/L, whereas by the use of lactose as a carbon source by C. acetobutylicum NCIB 2951, 1.5 g of 1-butanol/L (1-butanol/acetone ratio of 10:1) was obtained. In addition, C. saccharobutylicum produced 12 g of ABE/L with a synthetic glucose medium (Patakova et al., 2013). Ouephanit et al., (2011) compared the solvent production (ethanol and butanol) by two species, C. butyricum and C. acetobutylicum, using glucose, starch, carboxymethylcellulose, solid cassava waste and tapioca starch washed wastewater. The highest production of solvent was obtained by C. butyricum using tapioca starch washed wastewater at pH 5.5, 0.144 g of ethanol/L and 1.810 g of butanol/L. The authors indicated the potential of tapioca starch wastewater (with its complex nutrients) as a substrate for solvent production. Li et al., (2016) tested two culture media as substrates for the production of ABE, cassava flour and synthetic mineral by C. acetobutylicum SE25. The bioprocess using cassava flour (80 g/L) showed a longer phase shift (acidogenesis → solventogenesis), 12 h instead of 6 h when a synthetic culture medium was used (Li et al., 2016). Theoretically, subtle phase shift (correlated to pH) favors the ABE production. After concluding the advantages of cassava flour medium over synthetic medium, the authors evaluated the pH regulation by the addition of CaCO3, resulting in an even more subtle phase shift and subsequent enhancement on butanol yield. Meesukanun and Satirapipathkul, (2014) explored the production of ABE by using cassava rhizome hydrolysate and Clostridium saccharobutylicym. In this study, the cassava rhizome was pretreated by NaOH at 100°C for 30 min followed by hydrolysis at 100 rpm for 48 h at 50°C. The maxim production of ABE (10.57 g/L) was reached after 96 h using 60 g/L of reducing sugar in batch fermentation. In conclusion, it is very likely that the production of bioethanol and biobutanol will become one of the most important primary energies worldwide. In this sense, alternatives to the first generation of biofuel have drawn attention. Oddly, although there is a huge potential in the use of cassava wastewater for the production of bioethanol and biobutanol, there is a Complimentary Contributor Copy 186 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al. lack of deep information about the bioprocesses. Thus, research on microbial fuel producers (screening, metabolic engineering, etc.), bioprocesses, scale-up, substrates, purification, etc., is still needed. The yields of bioethanol production using cassava wastewater supplemented with NPK fertilizer reached 14.5 (% v/w) of ethanol. The ABE fermentation can achieve 19 g of ABE/L, in which with the use of tapioca starch washed wastewater at pH 5.5, approximately 0.144 g of ethanol/L and 1.810 g of butanol/L were obtained by C. butyricum. Cassava wastewater has remarkable potential as a substrate for ABE and bioethanol bioprocesses. In particular, during ABE production, the use of pH control (phase shift) such as CaCO3 could enhance the productivity.

7.2. Biogas Production: Anaerobic Digestion of Cassava Wastewater

Biogas is one of the most important forms of renewable energy. Industrial residues and animal wastes are being widely applied to biogas production, which can be used for heating, electricity and combustion, among others. That is, biogas can be used to replace natural gas (Sreethawong et al., 2010; Cappelletti et al., 2011; O-Thong et al., 2011; Intanoo et al., 2014; Ren et al., 2015; Suwanasri et al., 2015). The main biogases produced on an industrial scale are methane and hydrogen. The substrate conversion into H2 or methane depends mainly on the microorganisms (species, genus, etc.) fermentative pathway used by microorganisms, the bioprocess conditions and substrates (Cappelletti et al., 2011). Biogas is usually produced by the anaerobic digestion of carbohydrates, proteins, and fats by microorganisms, in particular bacteria. Methane biogas is composed of CH4 (55-70%) and CO2 (30-45%) with traces of other gases (1-5%) as H2S, NH3, CO, H2, N2 and H2O vapour, etc. The hydrogen biogas production is similar to methane biogas production (without the methanogenesis stage) (Ofoefule and Uzodinma, 2009; Sreethawong et al., 2010; Budiyono and Kusworo, 2011; Okudoh et al., 2014). Most studies reporting on biogas production came from Germany (7,000 units of biogas in 2011 that produced ≥ 2.8 gigawatts) in Europe, Colombia in South America, Nigeria, Tanzania and Kenya in Africa, and Asian Countries, in particular Thailand and China (Suwanasri et al., 2015; Zhang et al., 2016). It is worth noting that Thailand reached biogas production on an industrial scale due to financial support, tax incentives, small power purchase tariffs and environmental law enforcement (Okudoh et al., 2014; Suwanasri et al., 2015). Biogas has a high calorific value ≈ 6,000 kcal/m3, which is equivalent to 0.7 m3 of natural gas, 0.7 kg of fuel oil, 3.5 kg of wood and 4 kWh of electrical energy (Sreethawong et al., 2010; Okudoh et al., 2014). Anaerobic digestion results in both biogas production and the treatment of industrial wastes (lower chemical oxygen demand, nitrogen content, etc). For instance, Kpata-Konan et al., (2015), reported that the chemical oxygen demand of culture medium and total Kjeldahl nitrogen were significantly reduced from 27.46 to 5.01 and 3.87 to 2.08, respectively. Therefore, biogas production using industrial wastes is, by itself, an interesting strategy, but also it reduces post-treatment costs (after the bioprocess), which is aligned with the principles of green chemistry (Intanoo et al., 2016).

Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 187

7.2.1. The Biological Processing of Biogas Production – Methane and Hydrogen Biogas production is a very complex process, which covers interactions between substrates and different species of microorganisms (syntrophic metabolism), both facultative and obligate anaerobes (Okudoh et al., 2014). Some patents detail the enhancement of biogas production by specific bacterial inoculums, for instance United States Patent 20.080.124.775, United States Patent 20.070.062.866 and United States Patent 7.560.026 (Budiyono and Kusworo, 2011). In this sense, microbial consortia or sole microbial biogas producers (such as Clostridium acetobutylicum) have already been investigated (Zhang et al., 2016). In addition, higher yields of biogas production were described under thermoplilic temperature (45-57oC) instead of mesophilic temperatures (35-37oC), very likely due to the thermophilic microorganisms (such as Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum, Anoxybacillus sp. and Geobacillus sp.) using a wide range of substrates and producing lower bioprocess end-products (compared to mesophilic microorganisms), for instance higher acetic acid production and lower butyric acid, ethanol and lactic acid. However, thermophilic operations have higher operating costs ≈ 10% (O- Thong et al., 2011; Leaño and Babel, 2012; Intanoo et al., 2014; Okudoh et al., 2014). Regarding the bioreactor and fermentative design, when substrates with high organic loading but low solid content are used (such as cassava wastewater), the continuous-stirred tank reactor is often used (I stage of anaerobic digestion) (Intanoo et al., 2004; Okudoh et al., 2014; Zhang et al., 2016), or an anaerobic fixed film reactor, during which the culture medium is fed at the bottom of bioreactor to achieve the biofilm, then, during the fermentative process, biogas is recovered at the top of bioreactor. In this sense, nylon is one of the best materials to use as a fixed film media (low cost and weight) (Suwanasri et al., 2015). The fermentative process of biogas production comprises I-III stages (hydrogen) or I-IV stages (methane) are described below (Sreethawong et al., 2010; Okudoh et al., 2014; Intanoo et al., 2014; Intanoo et al., 2016; Ren et al., 2015). (I) hydrolysis - complex organic compounds are hydrolyzed by microbial enzymes into simpler water soluble organic compounds, for instance proteins → amino acids. In this stage, the following species are particularly essential - Clostridium spp., Ruminococcus spp., and Bacteroides spp. (II) acidogenesis – from these products (I) acidogenic bacteria produce organic acids (e.g, carbonic and lactic acids), short-chain fatty acids (SCFA) (butanoic acid, valeric acid, acetic acid, propionic acid), NH3, H2S, CO2, H2 and alcohols. In this sense, the highest conversion of glucose to hydrogen (4 mols) occurs when acetic acid is produced as a by-product (see Equation 1). Ammonia that is produced from the microbial metabolism of amino acids and proteins acts as buffer (ammonium bicarbonate). Thus, a harsh lower pH indicates an excessive production of SCFA (the toxicity of SCFA in methane-producing bacteria in methanogenesis stage is approximately 350 - 400 mg/L, whereas in hydrogen producing bacteria it is 10,000 mg/L), which leads to low biogas production. When substrates such as cassava wastewater are used, Peptoccus spp., Clostridium spp., Lactobacillus spp. and Propionibacterium spp are usually found during this stage.

C6H12O6 + 2H2O → 2CH3COOH (acetic acid) + 2CO2 + 4H2 (1)

Complimentary Contributor Copy 188 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

C6H12O6 → CH3CH2CH2COOH (butanoic acid) + 2CO2 +2H2 (2)

(III) acetogenesis – mainly from SCFA (II) acetogenic bacteria produce acetic acid (CH3COOH), CO2 and H2 (Equations 3 and 4). During this stage H2 and CO2 (electron acceptor) are at a low titer. In addition, obligate acetogenic bacteria such as Syntrophobacter spp., Desulfovibrio spp. and Syntrophomonas spp., metabolize longer chain fatty acids.

CH3CH2CH2COOH (butanoic acid) + 2H2O → 2CH3COOH (acetic acid) + 2H (3)

CH3CH2COOH (propionic acid) + 2H2O → CH3COOH (acetic acid) + CO2 + 3H2 (4)

(IV) methanogenesis – All the acetic acid, H2 and CO2 are converted into methane and CO2 as per the equations 5 and 6.

CH3COOH (acetic acid) → CH4 + CO2 (acetotrophic methanogenesis) (5)

CO2 + 4H2 → CH4 + 2H2O (hydrogenotrophic methanogenesis) (6)

Methanogenic Euryarchaeota microorganisms such as Methanosarcina spp. and Methanosaeta spp. (acetotrophic); Methanobacterium spp. and Methanobrevibacter spp. (hydrogenotrophic methanogenesis) that require organic acids and hydrogen, are responsible for the production of methane biogas (acetotrophic and hydrogenotrophic). In this sense, there is a predominance of hydrogenotrophic methanogens when wastes are used as substrates (Okudoh et al., 2014). Ren et al., (2015), detailed the taxonomic of microorganisms during the hydrogen biogas production (single culture system; dark fermentation), in which genus Succinivibrio, Serratia, Sphingomonas, Anaerobacter and Sarcina were found, but genus Clostridium was predominant (> 99%). This predominance trend is aligned with information described by Cappelletti et al., (2011).

7.2.2. Biogas Productions Using Cassava Wastewater as Substrate and Their Yields The chemical oxygen demand of cassava wastewater ranges from 14,500 mg/L, (Intanoo et al., 2014), 20,000 mg/L (Sreethawong et al., 2010); 5,000-15,000 mg/L) (Cappelletti et al., 2011); 432.1 mg/L nitrogen (Intanoo et al., 2014), 294.35 mg/L phosphorous (Intanoo et al., 2014). It seems that pretreatments of cassava wastewater are significant for enhance the biogas production, however this topic has not been fully explored. Leaño and Babel, (2012), evaluated three strategies, sonication, cellulase and α-amylase, in which α-amylase showed the highest H2 (mL)/COD removed (g). As in most cases, pH value of culture medium is fundamental in biotechnological processes, for instance low pH will affect the activity of hydrogenase with consequent decrease of hydrogen biogas production (Leaño and Babel, 2012). Sreethawong et al., (2010) indicated that the pH should be controlled at 5 in order to optimize hydrogen production, whereas Ren et al., (2015), indicated optimal pH should be at pH 7 – 8. Cappelletti et al., (2011), described that the hydrogen biogas production stopped at pH 4, then pH was corrected to 7 by NaOH solution. Obviously, the optimal pH depends on the microorganism(s),

Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 189 substrate(s), etc., Zhang et al., (2016), suggested a co-digestion crops and their residues due to more subtle pH changes and reduction free ammonia/ammonium inhibition. The ideal culture medium for biogas production must have the relation of C/N between ≈20-30, mainly during acedogenic stage (Sreethawong et al., 2010; Budiyono and Kusworo, 2011). However, cassava wastewater has C/N ≈ 86. That is, on the one hand cassava wastewater has a high content of organic compounds. Whereas on the other hand, a low content of nitrogen (0.6 - 0.8 g/L. In addition, the phosphorus content in cassava wastewater allows for microbial growth (do not needing supplementation). Therefore, cassava wastewater has to be supplemented by a nitrogen source, in order to enhance the yields of biogas production (Budiyono and Kusworo, 2011; Kpata-Konan et al., 2015). However, an excess of nitrogen will increase the concentration of SCFA. As already mentioned an increasing of SCFA leads to lower hydrogen production Sreethawong et al., 2010). In this sense, studies have indicated a co-digestion of cassava wastewater with cow dung, chicken manure, food-processing and distillery wastewater in order to improve the C/N ratio (Okudoh et al., 2014). To overcome the C/N ratio (cassava wastewater) for biogas production, Kpata-Konan et al., (2015) mixed cassava wastewater with two residues with high nitrogen concentration, human urine and cow dung. Moreover, due to high concentration of fermentable sugars in cassava wastewater, fast growth of forming-bacteria can be observed, which obviously acidifies the medium. At a low pH, methane bacteria show a slow growth rate of cells (Budiyono and Kusworo, 2011). Boncz et al., (2008) described that excess acid can be avoid by 3 strategies (i) first digestion (acidification), buffering followed by a second digestion; (ii) applying low organic rates and process automation; (iii) adding an alkaline compounds such as bicarbonate. Besides this, cassava wastewater contains cyanide, which acts as growth inhibitor (Boncz et al., 2008).

Table 4. Experimental design for biogas production studied by Budiyono and Kusworo, (2011)

Independent variable Dependent variable Tank 1 CWW* (ungelled) and yeast Urea and ruminant bacteria Tank 2 CWW* (ungelled) Urea and ruminant bacteria Tank 3 CWW* (gelled) and yeast Urea and ruminant bacteria Tank 4 CWW* (ungelled) and microalgae Urea and ruminant bacteria Tank 5 CWW* (ungelled), yeast and microalgae Urea and ruminant bacteria *Cassava wastewater (CWW).

A significant enhancement was noted in the productivity of biogas production, when the gelled was used (thermal treatment) of cassava wastewater (Tank 3), very likely due to the higher solubility of cassava starch (Table 4). On the other hand the highest yield (biogas/gram total solid) was obtained in Tank 1 (Budiyono and Kusworo, 2011; Okudoh et al., 2014). Thus, the addition of microalgae was not a good nitrogen source for biogas production by ruminant bacteria or biostabilisator of pH. Hydrogen biogas production in a co-culture system (microalgae) using cassava wastewater as substrate showed a yield of 183.3 mL of H2/L of cassava wastewater (see Table 5 and total lipid (most from microalgae) 0.26 g/L of cassava wastewater (Ren et al., 2015).

Complimentary Contributor Copy 190 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

Table 5. Productivity of biogas using cassava wastewater

H2 (mL)/COD removed (g) CH4 (mL)/COD removed (g) References 186 Sreethawong et al., 2010 438 Sreethawong et al., 2010a 169 921 Intanoo et al., 2014b 39.83 115.23 Intanoo et al., 2016 5.5 Ren et al., 2015c 1.87 Ren et al., 2015d 101 Cappelletti et al., 2011e 3.41 Kpata-Konan et al., 2015f aTwo-stage process. b Nitrogen-supplemented. c Calculated using graphs of references – single culture system. dCalculated using graphs of references – co-culture system. e Cassava wastewater concentrations of 30 g/L. f Average concentration of methane 79.63%.

7.2.3. Perspectives for Biogas Production from Cassava Wastewater Even being a feasible bioprocess on an industrial scale (white biotechnology), the biogas production has bottlenecks. The main challenges in biogas production (dark fermentation) are related to low productivity (longstanding and low conversion rates) and metabolic inhibition (feedback) by volatile fatty acids and alcohols (Ren et al., 2015). In addition, the cost of biogas production needs to be comparable or lower than other energy source production, the substrate should be overly abundant, inexpensive and easily accessible throughout the year, in order to avoid seasonal fluctuation (O-Thong et al., 2011). There is now a natural trend to replace petrochemical derivatives. Thus, biogas production will be an interesting alternative, in particular for tropical countries such as Brazil, which has an abundance of agro-industrial substrates (wastes). Therefore, based on the above- mentioned information, researches are needed on cassava wastewater nitrogen supplementation, in particular wastes with high nitrogen concentration, metabolic engineering (biogas producers) and pretreatments of cassava wastewater.

8. BIOFLAVOURS USING CASSAVA WASTEWATER AS A SUBSTRATE

Natural foods have been widely promoted. An alternative to the production of natural food ingredients and food products is the application of microorganisms and enzymes. In particular, the synthesis of bioflavour compounds by biotransformation (Bicas et al., 2010; Damasceno et al., 2003). Inherently, microorganisms produce several bioflavours during any bioprocesses, in which substrates such as cassava wastewater and cassava bagasse have been investigated. Solid State Fermentation (SSF) has been used to increase the productivity of enzymes and other compounds by microorganisms (Pengthamkeerati et al., 2012).

Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 191

SSF is performed using non-soluble materials as nutrients (absence or near-absence of free water). This technique reproduces natural microbiological processes that occur in composting and ensiling. The reduced moisture restricts the microbial growth, in which yeasts, fungi and a few bacteria usually show a good performance (Longo et al., 2006). SSF has been described by several studies that were interested in the production of enzymes, flavours and other chemicals, in which the flavors were produced by Neurospora sp., Zygosaccharomyces sp. and Aspergillus sp., using a substrate with rice and cellulose fiber production (Pengthamkeerati et al., 2012; Longo et al., 2006). Longo et al., (2006) evaluated the fruit flavour produced by Ceratocystis fimbriata during the SSF process using several agro-industrial wastes, in which fruity flavours were produced using cassava bagasse, apple pomace and soybean. In another study, Kluyveromyces marxianus produced fruit flavour compounds in SSF using cassava bagasse or giant palm bran as a substrate. The analysis showed the production of various compounds from palm bran and cassava bagasse substrates, such as alcohols, esters and aldehydes. Although ethyl acetate, ethanol and acetaldehyde were the main compounds yielded. Similar flavour composition was obtained in solid-state system with cultures of K. marxianus in a packed bed column bioreactor and cassava bagasse (substrate). The compound 2-phenylethanol is an aromatic alcohol with flavour similar to essential oils present in flowers as hyacinth, jasmine, lilies and daffodils (Oliveira et al., 2015). This aroma has been used in a variety of applications in cosmetics and food. An alternative to obtain 2-phenylethanol is using the conversion of L-phenylalanine into 2-phenylethanol by yeast, through the Ehrlich pathway. In this way, the amino acid is converted into phenylpyruvate by enzymes action, then in phenyl acetaldehyde and finally in phenylethanol (Oliveira et al., 2015). The 2-phenylethanol may be synthesized by the Shikimate pathway, with the conversion of compounds pentose phosphate to phenylpyruvate, then phenyl acetaldehyde to phenylethanol. Although saying this, the process with phenylalanine showed a great performance to obtain 2-phenylethanol rather than the dextrorotatory process (Oliveira et al., 2015). Rhizopus oryzae was also evaluated using the residual cassava bagasse as raw material, but lower concentrations of aldehydes and esters were identified (Oliveira et al., 2015). Medeiros et al., (2000) evaluated the production of aroma compounds by the yeast Kluyveromyces marxianus using as substrate cassava bagasse. The experimental strategy was carried out by the supplementation of glucose to obtain ethyl acetate compound, which showed higher yields of fruit flavour. Fusarium oxysporum, Aspergillus sp. and Penicillium sp. were used to produce terpineol and other products through limonene biotransformation. Compared to other media, the uses of cassava wastewater significantly increased the yield of biotransformation (Damasceno et al., 2003; Bicas et al., 2010). Another example of a flavour production process using cassava wastewater was achieved by Bicas et al., (2010) using a strain of Penicillium sp. that synthesized a blend of cis- and trans-rose oxides from citronellol as the sole carbon source. These studies show that a wide range of microorganisms are able to use cassava wastes as an alternative carbon source for bioflavors compounds with a good fermentation performance.

Complimentary Contributor Copy 192 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

9. PERSPECTIVES

The sustainable manufacture of high-value bioproducts from the food supply chain - during the early and middle levels - is a key challenge for effective biorefinery accomplishment (Zhang et al., 2016). Utilization of these residues offers great opportunities and a number of industrial processes are going in this direction in order to develop the economically and ecofriendly sustainable production of bioenergy and other value added goods (Sindhu et al., 2016). In addition, for some high value products, a bioprocess operated with renewable feedstocks and alternative substrates might be the only route economically viable to compete with conventional industry (Panesar et al., 2015; Jiménez-González and Woodley, 2010). In this sense, several researches point out that cassava wastewater can be used as a low-cost substrate for biofuels, chemicals, biosurfactants, food ingredients and others value goods production, supporting environmental goals and costs reduction in biotechnological processes. Feasibility and interest in the use of cassava wastewater as an alternative substrate may be due to its chemical composition and high generation amounts, associated with the high costs of waste management systems (Andrade et al., 2016a-b); Ravindran and Jaiswal, 2016; Barros et al., 2013). Development of integrating fermentative processes with simultaneous production of more than one value added bioproduct from cassava wastewater is encouraging in order to make the process efficient and economically viable (Andrade et al., 2016a-b; Fai et al., 2015; Barros et al., 2013). Yet there are major challenges in this area for future wide utilization of cassava wastewater as an alternative substrate in industry. These include; development of integrated systems; improvement of property optimized statistical models in large-scale production; detection of microorganisms gene expression and regulation of key enzymes in order to increase the specificity and yield of specific products; the upgrading of economical techniques for separation, purification and the analysis of bioconverted products; among others strategies of green processes (Andrade et al., 2016a-b;; Zhang et al., 2016; Bution et al., 2015; Fai et al., 2015; Morais et al., 2014; Jiménez-González and Woodley, 2010).

REFERENCES

Adeoye, AO; Lateef, A; Gueguim-Kana, EB. Optimization of citric acid production using a mutant strain of Aspergillus niger on cassava peel substrate. Biocatalysis and Agricultural Biotechnology, 2015, 4, 568–574. Agyei, D; Shanbhag, BK; He, L. Enzymes for food waste remediation and valorization. In: Yada, R, editor. Improving and tailoring enzymes for food quality and functionality, Cambridge UK: Elsevier; 2015; 123-145. Ahmad, A; Buang, A; Bhat, AH. Renewable and sustainable bioenergy production from microalgal co-cultivation with palm oil mill effluent (POME): a review. Renewable and Sustainable Energy Reviews, 2016, 65, 214-234. Akaracharanya, A; Kesornsit J; Leepipatpiboon, N; Srinorakutara, T; Kitpreechavanich, V; Tolieng, V. Evaluation of the waste from cassava starch production as a substrate for ethanol fermentation by Saccharomyces cerevisiae, Annals of Microbiology, 2010, 61, 431-436.

Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 193

Akponah, E; Akpomie, OO. Optimization of bio-ethanol production from cassava effluent using Saccharomyces cerevisiae, African Journal of Biotechnology, 2012, 11, 8110-8116. Alibardi, L; Cossu, R. Effects of carbohydrate, protein and lipid content of organic waste on hydrogen production and fermentation products. Waste Management, 2016, 47, 69-77. Amaral, PFF; Coelho, MAZ; Marrucho, IMJ; Coutinho, JAP. Biosurfactants from yeasts: Characteristics, production and application. In: Sen, R, editor. Biosurfactants: Advances in experimental medicine and biology. New York: Springer New York; 2010; 236–249. Andrade, CJ; Andrade, LM; Bution, ML; Dolder, MAH; Barros, FFC; Pastore, GM. Optimizing alternative substrate for simultaneous production of surfactin and 2,3- butanediol by Bacillus subtilis LB5a.Biocatalysis and Agricultural Biotechnology, 2016a,6, 209-218. Andrade, CJ; Barros, FFC; Andrade, LM; Rocco, AS; Sforça, ML; Pastore, GM; Jauregi, P. Ultraﬁltration based puriﬁcation strategies for surfactin produced by Bacillus subtilis LB5A using cassava wastewater as substrate. Journal of Chemical Technology and Biotechnology, 2016b, DOI: 10.1002/jctb.4928. Andrade, RFS; Antunes, AA; Lima, RA; Araújo, HWC; Resende-Stoianoff, MA; Franco, LO; Campos-Takaki, GM. Enhanced production of an glycolipid biosurfactant produced by Candida glabrata UCP/WFCC1556 for application in dispersion and removal of petroderivatives. International Journal of Current Microbiology and Applied Sciences (IJCMAS), 2015, 4, 563–576. Antonopoulou, G; Gavala, HN; Skiadas, IV; Lyberatos, G. Effect of substrate concentration on fermentative hydrogen production from sweet sorghum extract. International Journal of Hydrogen Energy, 2011, 36, 4843-4851. Aquino, ACMS; Azevedo, MS; Ribeiro, DHB; Costa, ACO; Amante, ER. Validation of HPLC and CE methods for determination of organic acids in sour cassava starch wastewater. Food Chemistry, 2015, 172, 725-730. Araújo, LV; Guimarães, CR; Marquita, RLS; Santiago, VMJ; Souza, MP; Nitschke, M; Freire, DMG. Rhamnolipid and surfactin : Anti-adhesion/antibiofilm and antimicrobial effects. Food Control, 2016, 63, 171-178. Avancini, SRP; Faccin, GL; Vieira, MA; Rovaris, AA; Podestá, R; Tramonte, R; De Souza, N MA; Amante, ER. Cassava starch fermentation wastewater: Characterization and preliminary toxicological studies. Food and Chemical Toxicology, 2007, 45, 2273–2278. Banat, IM; Satpute, SK; Cameotra, SS; Patil, R; Nyayanit, NV. Cost effective technologies and renewable substrates for biosurfactants’ production. Frontiers in Microbiology, 2014, 5, 697. Barros, FFC. Study of process variables and scale-up of the production of biosurfactant by Bacillus subtilis in cassava wastewater. Dissertation, State University of Campinas, Campinas, Brazil, 2007. Barros, FFC; Pereira, C; Pastore, M. Studies of emulsifying properties and stability of the biosurfactant produced by Bacillus subtilis in cassava wastewater. Ciência e Tecnologia de Alimentos, 2008a, 28, 979–985. Barros, FFC; Ponezi, AN; Pastore, GM. Production of biosurfactant by Bacillus subtilis LB5a on a pilot scale using cassava wastewater as substrate. Journal of Industrial Microbiology and Biotechnology, 2008b, 35, 1071–8.

Complimentary Contributor Copy 194 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

Barros, FFC; Simiqueli, APR; Andrade, CJ; Pastore, GM. Production of enzymes from agroindustrial wastes by biosurfactant-producing strains of Bacillus subtilis.Biotechnology Research International, 2013, 2013: 1-9. Bentsen, NS; Felby, C; Thorsen, BJ. Agricultural residue production and potentials for energy and materials services. Progress in Energy and Combustion Science, 2014, 40, 59-73. Bezerra, MS; Holanda, VCD; Amorim, JÁ; Macedo, GR; Santos, ES. Biotensoative production using Pseudomonas aeruginosa (P.A.) and agroindustry waste (cassava wastewater) as substract. Holos, 2012, 28, 14–27. Bicas, JL; Silva, JC; Dionísio, AP; Pastore, GM. Biotechnological production of bioflavors and functional sugars. Ciência Tecnologia de Alimentos, 2010, 30, 7-18. Bognolo, G. Biossurfactants as emulsifying agents for hydrocarbons. Colloids and Surfactants, 1999, 12, 41-52. Boncz, MA; Bezerra, LP; Ide, CN; Paulo, PL. Optimisation of biogas production from anaerobic digestion of agro-industrial waste streams in Brazil. Water Science & Technology, 2008, 58, 1659-1664. Budiyono, B; Kusworo, TD. Biogas production from cassava starch effluent using microalgae as biostabilisator. International Journal of Science and Engineering, 2011, 2, 4-8. Bution, ML; Molina, G; Abrahão, MRE; Pastore, GM. Genetic and metabolic engineering of microorganisms for the development of new flavor compounds from terpenic substrates. Critical Reviews in Biotechnology, 2015, 35, 313-325. Cameotra, SS; Makkar, RS; Kaur, J; Mehta, SK. Synthesis of biosurfactants and their advantages to microorganisms and mankind. In: Sen, R, editor. Biosurfactants: Advances in experimental medicine and biology. New York: Springer New York; 2010; 261-280. Cameotra, SS; Makkar, RS. Recent applications of biosurfactants as biological and immunological molecules. Current Opinion on Microbiology, 2004, 7, 262-266. Campos, JM; Montenegro, TL; Sarubbo, LA; Luna, JM; Rufino, RD; Banat, IM. Microbial biosurfactants as additives for food industries. Biotechnology Progress, 2013, 29, 1097- 1108. Cantarella, H; Nassar, AM; Cortez, LAB; Junior, RB. Potential feedstock for renewable aviation fuel in Brazil, Environmental Development, 2015, 15, 52-63. Cappelletti, BM; Reginatto, V; Amante, ED; Antônio, RV. Fermentative production of hydrogen from cassava processing wastewater by Clostridium acetobutylicum. Renewable Energy, 2011, 36, 3367-3372. Chen, H; Xu, M-L; Guo, Q; Yang, L; Ma, Y. A review on present situation and development of biofuels in China, Journal of the Energy Institute, 2016, 89, 248-255. Chookietwattana, K. Lactic acid production from simultaneous saccharification and fermentation of cassava starch by Lactobacillus plantarum MSUL 903. APCBEE Procedia, 2014, 8, 156-160. Colin, X; Farinet, J-L; Rojas, O; Alazard, D. Anaerobic treatment of cassava starch extraction wastewater using a horizontal flow filter with bamboo as support.Bioresource Technology, 2007, 98, 1602-1607. Coronel-León, J; Marqués, AM; Bastida, J; Manresa, A. Optimizing the production of the biosurfactant lichenysin and its application in biofilm control. Journal of Applied Microbiology, 2016, 120, 99–111. Costa, SGVAO; Lépine, F; Milot, S; Déziel, E; Nitschke, M; Contiero, J. Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 195

polyhydroxyalkanoates by Pseudomonas aeruginosa. Journal of Industrial Microbiology and Biotechnology, 2009, 36, 1063–1072. Costa, GAN. Produção biotecnológica de surfactante de Bacillus subtilis em resíduo agroindustrial, caracterização e aplicações. Thesis, State University of Campinas, Campinas, Brazil, 2005. Costa, SGVAO; Lépine, F; Milot, S; Déziel, E; Nitschke, M; Contiero, J. Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by Pseudomonas aeruginosa. Journal of Industrial Microbiology and Biotechnology, 2009, 36, 1063–1072. Costa, SGVAO; Nitschke, M; Contiero, J. Fats and oils wastes as substrates for biosurfactant production. Ciência e Tecnologia de Alimentos, 2008, 28, 34-38. Damasceno, S; Cereda, MP; Pastore, GM; Oliveira, JG. Production of volatile compounds by Geotrichum fragrans using cassava wastewater as substrate. Process Biochemistry, 2003, 29, 411–414. 2003. Desai, JD; Banat, IM. Microbial production of surfactants and their commercial potential. Microbiology and Molecular Biology Reviews, 1997, 61, 47-64. Dien, BS; Cotta, MA; Jeffries, TW. Bacteria engineered for fuel ethanol production: Current status, Applied Microbiology and Biotechnology, 2003, 63, 258-266. Elemike, EE; Oseghale, OC; Okoye, AC. Utilization of cellulosic cassava waste for bioethanol production, Journal of environmental chemical engineering, 2015, 3, 2797-2800. Fai, AEC; Simiqueli, APR; Andrade, CJ; Ghiselli, G; Pastore, GM. Optimized production of biosurfactant from Pseudozyma tsukubaensis using cassava wastewater and consecutive production of galactooligosaccharides: an integrated process. Biocatalysis and Agricultural Biotechnology, 2015, 4, 535-542. Ghosh, SK. Biomass & bio-waste supply chain sustainability for bio-energy and bio-fuel production, Procedia Environmental Sciences, 2016, 31, 31-39. Gomes, SD; Fuess, LT; Mañunga, T; Clairmont, P; Gomes, FL; Zaiat, M. Bacteriocins of lactic acid bacteria as a hindering factor for biohydrogen production from cassava flour wastewater in a continuous multiple tube reactor. International Journal of Hydrogen Energy, 2016, 41, 8120-8131. Gomes, MZV; Nitschke, M. Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria. Food Control, 2012, 25, 441-447. Guo, M; Song, W; Buhain, J. Bioenergy and biofuels: History, status, and perspective, Renewable and Sustainable Energy Reviews, 2015, 42, 712-725. Gustavsson, J; Cederberg, C; Sonesson, U; Otterdijk, RV; Meybeck, A. Global food losses and food waste: Extent, causes and prevention. 2016, August 15th. Available from: http://www.fao.org/docrep/014/mb060e/mb060e.pdf. Harshada, K. Biosurfactant: A potent antimicrobial agent. Journal of Microbiology & Experimentation, 2014, 1, DOI: 10.15406/jmen.2014.01.0003. Healy, MG; Devine, CM; Murphy, R. Microbial production of biosurfactants. Resources, Conservation and Recycling, 1996, 18, 41-57. Herman, DC; Maier, RM. Biosynthesis and applications of glycolipid and lipopeptide biosurfactants. In: Gardner, HW; Kuo, TM, editors. Lipid Biotechnology. CRC Press; 2002; 629-655. DOI: 10.1201/9780203908198.ch32.

Complimentary Contributor Copy 196 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

Intanoo, P; Rangsanvigit, P; Malakul, P; Chavadej, S. Optimization of separate hydrogen and methane production from cassava wastewater using two-stage upflow anaerobic sludge blanket reactor (UASB) system under thermophilic operation. Bioresource Technology, 2014, 173, 256-265. Intanoo, P; Chaimongkol, P; Chavadej, S. Hydrogen and methane production from cassava wastewater using two-stage upﬂow anaerobic sludge blanket reactors (UASB) with an emphasis on maximum hydrogen production. International Journal of Hydrogen Energy, 2016, 41, 6107–6114. Jiménez-González, C; Woodley, JM. Bioprocesses: Modeling needs for process evaluation and sustainability assessment. Computers & Chemical Engineering, 2010, 34, 1009-1017. Johny, JM. Inhibitory effect of biosurfactant purified from probiotic yeast against biofilm producers. IOSR Journal of Environmental Science, Toxicology and Food Technology, 2013, 6, 51–55. Kosaric, N. Biosurfactants in industry. Pure and Applied Chemistry, 1992, 64, 1731-1737. Kpata-Konan, EN; Gnagne, T; Konan, FK; Kouamé, MK; Kouamé, FY; Tano, K. Biogas production from anaerobic co-digestion of cassava effluent and human urine, Pakistan Journal of Biotechnology, 2015, 12, 93-98. Kummer, L; Cosmann, NJ; Pastore, GM; Simiqueli, APR; Melo, VF; Gomes, SD. Adsorption of copper, zinc and lead on biosurfactant produced from cassava wastewater. African Journal of Biotechnology, 2016, 15, 110-117. Leaño, EP; Babel S. Effects of pretreatment methods on cassava wastewater for biohydrogen production optimization. Renewable Energy, 2012, 39, 339-346. Li, H; Zang, Q; Yu, X; Wei, L; Wang, Q. Enhancement of butanol production in Clostridium acetobutylicum SE25 though accelerating phase shift by different phases pH regulation from cassava flour, Bioresource Technology, 2016, 201, 148-155. Li, Y; Liu, B; Song, J; Jiang, C; Yang, Q. Utilization of potato starch processing wastes to produce animal feed with high lysine content. Journal of Microbiology and Biotechnology, 2015, 25, 178-184. Lima, CJB; Coelho, LF; Contiero, J. Fermentative production of L-lactic acid from cassava flour wastewater by Lactobacillus rhamnosus RL-1. Journal of Biotechnology, 2010, 150, 511. Lin, SC. Biosurfactants: recents advances. Journal of Chemical Technology and Biotechnology, 1996, 66, 109-120. Lin, CSK; Pfaltzgraff, LA; Herrero-Davila, L. Mubofu, EB; Abderrahim, S; Clark, JH; Koutinas, AA; Kopsahelis, N; Stamatelatou, K; Dickson, F; Thankappan, S; Mohamed, Z; Brocklesby, R; Luque, R. Food waste as a valuable resource for the production of chemicals, materials and fuels. Current situation and global perspective. Energy & Environmental Science, 2013, 6, 426-464. Longo, MA; Sanromán, MA. Production of food aroma compounds, Food Technology and Biotechnology, 2006, 44, 335–353. Lu, C; Zhao, J; Yang, S-T; Wei, D. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresource Technology, 2012, 104, 380-387. Maier, RM. Biosurfactants: evolution and diversity in bacteria. Advances in Applied Microbiology, 2003, 52, 101-121.

Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 197

Maiti, S; Sarma, SJ; Brar, SK; Bihan, YL; Drogui, P; Buelna, G; Verma, M. Agro-industrial wastes as feedstock for sustainable bio-production of butanol by Clostridium beijerinckii. Food and Bioproducts Processing, 2016, 98, 217-226. Makkar, RS; Cameotra, SS; Banat, IM. Advances in utilization of renewable substrates for biosurfactant production. AMB Express, 2011, 1, 1-19. Makkar, RS; Cameotra, SS. Biosurfactant production by a thermophilic Bacillus subtilis strain. Journal of Industrial Microbiology and Biotechnology, 1997, 18, 37-42. Marchant, R; Banat, IM. Biosurfactants: A sustainable replacement for chemical surfactants? Biotechnology Letters, 2012, 34, 1597–1605. Medeiros, ABP; Pandey, A; Freitas, RJ; Christen, P; Soccol, CR. Optimization of the production of aroma compounds by Kluyveromyces marxianus in solid-state fermentation using factorial design and response surface methodology. Biochemical Engineering Journal, 2000, 6, 33-39. Meesukanun, K; Satirapipathkul, C. Production of acetone-butanol-ethanol from cassava rhizome hydrolysate by Clostridium saccharobutylicum BAA 117. Chemical Engineering Transactions, 2014, 37, 421-426. Montero-Rodríguez, D; Andrade, RFS; Ribeiro, DLR; Rubio-Ribeaux, D; Lima, RA; Araújo, HWC; Campos-Takaki, GM. Bioremediation of petroleum derivative using biosurfactant produced by Serratia marcescens UCP/WFCC 1549 in Low-Cost Medium. International Journal of Current Microbiology and Applied Sciences, 2015, 4, 550-562. Morais, ARC; Mata, AC; Bogel-Lukasik, R. Integrated conversion of agroindustrial residue with high pressure CO2 within the biorefinery concept. Green Chemistry, 2014, 16, 4312- 4322. Moshi, AP; Hosea, KMM; Elisante, E; Mamo, G; Mattiasson, B. High temperature simultaneous saccharification and fermentation of starch from inedible wild cassava (Manihot glaziovii) to bioethanol using Caloramator boliviensis. Bioresource Techonology, 2015, 180, 128-136. Neu, TR. Significance of bacterial surface-active compounds in interactions of bacteria with interfaces. Microbiological Reviews, 1996, 60, 151-166. Nitschke, M; Costa, SGVAO. Biosurfactants in food industry. Trends in Food Science and Technology, 2007, 18, 252-259. Nitschke, M; Ferraz, C; Pastore, GM. Selection of microorganisms for biosurfactant production using agroindustrial wastes. Brazilian Journal of Microbiology, 2004a, 35, 81-85. Nitschke, M; Haddad, R; Costa, GAN; Gilioli, R; Meurer, EC; Gatti, MS; Eberlin, MN; Höehs, NF; Pastore, GM. Strutural characterization and biological properties of a lipopeptide surfactant produced by Bacillus subtilis on cassava wastewater medium. Food Science and Biotechnology, 2004b, 13, 591-596. Nitschke, M; Pastore, GM. Biosurfactant production by Bacillus subtilis using cassava- processing effluent. Applied Biochemistry and Biotechnology, 2004c, 112, 163-172. Novelli, PK; Barros, MM; Fleuri, LF. Novel inexpensive fungi proteases: production by solid state fermentation and characterization. Food Chemistry, 2016, 198, 119-124. Nuwamanya, E; Chiwona-Karltun, L; Kawuki, RS; Baguma, Y. Bio-ethanol production from non-food parts of cassava (Manihot esculenta Crantz), AMBIO, 2012, 41, 262–270.

Complimentary Contributor Copy 198 C. José de Andrade, A. P. Resende Simiqueli, F. Aliaga de Lima et al.

Ofoefule, AU; Uzodinma, EO. Biogas production from blends of cassava (Manihot utilissima) peels with some animal wastes. International Journal of Physical Sciences, 2009, 4, 398-402. Okudoh, V; Trois, C; Workneh, T; Schmidt, S. The potential of cassava biomass and applicable Technologies for sustainable biogas production in South Africa: A review. Renewable and Sustainable Energy Reviews, 2014, 39, 1035–1052. Oliveira, DWF; França, IWL; Félix, AKN; Martins, JJL; Giro, MEA; Melo, VMM; Gonçalves, LRB. Kinetic study of biosurfactant production by Bacillus subtilis LAMI005 grown in clarified cashew apple juice. Colloids and Surfaces B: Biointerfaces, 2013, 101, 34-43. Oliveira, SMM; Gomes, SD; Sene, L; Christ, D; Piechontcoski, J. Production of natural aroma by yeast in wastewater of cassava starch industry. Revista Engenharia Agrícola, 2015, 35, 721-732. Ostergaard, S; Olsson, L; Nielsen, J. Metabolic Engineering of Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews, 2000, 64, 34-50. O-Thong, S; Hniman, A; Prasertsan, P; Imai, T. Biohydrogen production from cassava starch processing wastewater by thermophilic mixed cultures. International Journal of Hydrogen Energy. 2011, 36, 3409-3416. Ouephanit, C; Virunanon, C; Burapatana, V; Chulalaksananukul, W. Butanol and ethanol production from tapioca starch wastewater by Clostridium spp. Water Science & Technology, 2011, 64, 1774-1780. Panda, SK; Mishra, SS; Kayitesi, E; Ray, RC. Microbial-processing of fruit and vegetable wastes for production of vital enzymes and organic acids: biotechnology and scopes. Environmental Research, 2016, 146, 161-172. Pandey, A; Soccol, CR; Nigam, P; Soccol, VT; Vandenberghe, LPS; Mohan, R. Biotechnological potential of agro-industrial residues. II: cassava bagasse. Bioresource Technology, 2000, 74, 81-87. Panesar, PS; Kaur, S. Bioutilisation of agro-industrial waste for lactic acid production. International Journal of Food Science & Technology, 2015, 50, 2143-2151. Panesar, R; Kaur, S; Panesar, PS. Production of microbial pigments utilizing agro-industrial waste: a review. Current Opinion in Food Science, 2015, 1, 70-76. Pengthamkeerati, P; Numsomboon, S; Satapanajaru, T; Chairattanamanokorn, P. Production of α-amylase by Aspergillus oryzae from cassava bagasse and wastewater sludge under solid state fermentation. Environmental Progress & Sustainable Energy, 2012, 31, 122- 129. Patakova, P; Linhova, M; Rychtera, M; Paulova, L; Melzoch, K. Novel and neglected issues of acetone–butanol–ethanol (ABE) fermentation by clostridia: Clostridium metabolic diversity, tools for process mapping and continuous fermentation systems. Biotechnology Advances, 2013, 31, 58-67. Pescuma, M; Valdez, GF; Mozzi, F. Whey-derived valuable products obtained by microbial fermentation. Applied Microbiology and Biotechnology, 2015, 99, 6183-6196. Pleissner, D; Qi, Q; Gao, C; Rivero, CP; Webb, C; Lin, CSK; Venus, J. Valorization of organic residues for the production of added value chemicals: a contribution to the bio- based economy. Biochemical Engineering Journal, DOI: 10.1016/j.bej.2015.12.016. Ponte, JJ. Cartilha da manipueira: Uso do composto como insumo agrícola. 3 edition. Fortaleza: Banco do Nordeste do Brasil; 2006. Complimentary Contributor Copy Cassava Wastewater as Substrate in Biotechnological Processes 199

Quadros, CP; Duarte, MCT; Pastore, GM. Biological activities of a mixture of biosurfactant from Bacillus subtilis and alkaline lipase from Fusarium oxysporum. Brazilian Journal of Microbiology, 2011,42, 354-361. Ravindran, R; Jaiswal; AK. Exploitation of food industry waste for high-value products. Trends in Biotechnology, 2016, 34, 58-69. Rebello, S; Asok, AK; Mundayoor, S; Jisha, MS. Surfactants: toxicity, remediation and green surfactants. Environmental Chemistry Letters, 2014, 12, 275–287. Ren, H; Liu, B; Kong F; Zhao, L; Ren, N. Hydrogen and lipid production from starch wastewater by co-culture of anaerobic sludge and oleaginous microalgae with simultaneous COD, nitrogen and phosphorus removal. Water Research, 2015, 85, 404– 412. Rosa, PRF; Santos, SC; Sakamoto, IK; Varesche, MBA; Silva, EL. The effects of seed sludge and hydraulic retention time on the production of hydrogen from a cassava processing wastewater and glucose mixture in an anaerobic fluidized bed reactor. International Journal of Hydrogen Energy, 2014, 39, 13118-13127. Sindhu, R; Gnansounou, E; Binod, P; Pandey, A. Bioconversion of sugarcane crop residue for value added products: an overview. Renewable Energy, 2016, 98, 203-215. Singh, PB; Saini, HS. Exploitation of agro-industrial wastes to produce low-cost microbial surfactants. In: Brar, KS; Dhillon, SG; Soccol, RC, editors. Biotransformation of waste biomass into high value biochemicals. New York: Springer New York; 2014; 445–471. Soo, CS; Yap, WS; Hon, WM; Phang, LY. Mini review: hydrogen and ethanol co-production from waste materials via microbial fermentation. World Journal of Microbiology and Biotechnology, 2015, 31, 1475-1488. Sreethawong, T; Chatsiriwatana, S; Rangsunvigit, P; Chavadej, S. Hydrogen production from cassava wastewater using an anaerobic sequencing batch reactor: effects of operational parameters, COD:N ratio, and organic acid composition. International Journal of Hydrogen Energy, 2010, 35, 4092-4102. Suwanasri, R; Trakulvichean, S; Grudloyma, U; Songkasiri, W; Commins, T; Chaiprasert, P; Tanticharoen, M. Biogas – key success factors for promotion in Thailand, Journal of Sustainable Energy & Environment, Special Issue, 2015, 25-30. Tosungnoen, S; Chookietwattana, K; Dararat, S. Lactic acid production from repeated-batch and simultaneous saccharification and fermentation of cassava starch wastewater by amylolytic Lactobacillus plantarum MSUL 702. Procedia APCBEE, 2014, 8, 204-209. Vong, WC; Liu, SQ. Biovalorisation of okara (soybean residue) for food and nutrition. Trends in Food Science & Technology, 2016, 52, 139-147. Wei, M; Zhu, W; Xie, G; Lestander, TA; Xiong, S. Cassava stem wastes as potential feedstock for fuel ethanol production: A basic parameter study, Renewable Energy, 2015, 83, 970-978. Zhang, M; Xie, L; Yin, Z; Khanal, SK; Zhou, Q. Biorefinery approach for cassava-based industrial wastes: Current status and opportunities. Bioresource Technology, 2016, 215, 50-62. Zhang, H. -J., Zhang, J. -H., Xu, J., Tang, L., Mao, Z. -G. (2014). A novel recycling process using the treated citric acid wastewater as ingredients water for citric acid production. Biochemical Engineering Journal, 90:206-213.

Chapter 10

TECHNICAL, COST AND ALLOCATIVE EFFICIENCY OF PROCESSING CASSAVA INTO GARI IN DELTA STATE, NIGERIA

Brodrick O. Awerije† and Sanzidur Rahman‡ 1Tree Crops Unit, Ministry of Agriculture and Natural Resources, Asaba, Delta State, Nigeria 2School of Geography, Earth and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth, UK

ABSTRACT

The present chapter examines productivity, technical, cost and allocative efficiencies of processing cassava into gari by applying Data Envelopment Analysis (DEA) of 278 farmers/processors from three regions of Delta State, Nigeria. Results revealed that the mean levels of technical, cost and allocative efficiencies of gari processing is low estimated at 0.55, 0.35 and 0.64, respectively, implying that gari production can be increased substantially by reallocation of resources to optimal levels, given input and output prices. Inverse size–productivity and size–efficiency relationships exist in gari processing. In other words, marginal and small processors are significantly more productive and efficient relative to large processors. Availability of credit significantly improves technical and cost efficiencies. Extension contact significantly reduces efficiencies which is counterintuitive. Female processors are technically efficient relative to male processors while both perform equally well with respect to allocative and cost efficiencies in processing gari. Significant differences in efficiencies exist across regions as well. Processors located in Delta North and Delta South is relatively more efficient than processors located in Delta Central. A host of constraints affect gari processing

 The chapter was developed from the first author’s PhD thesis submitted at the School of Geography, Earth and Environmental Sciences, University of Plymouth, UK in 2014. The data required for this project was generously funded by the Seale-Hayne Educational Trust, UK. † E-mail: [email protected] ‡ Address for correspondence: Sanzidur Rahman, PhD, Associate Professor (Reader) in International Development, School of Geography, Earth and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth, United Kingdom.Phone: +44-1752-585911, Fax: +44-1752-584710, E-mail: [email protected]. Complimentary Contributor Copy 202 Brodrick O. Awerije and Sanzidur Rahman

which include lack of transportation, information, processing equipment and infrastructure and high cost of raw materials. Policy implications include investment in education targeted at small farmers/processors, improving agricultural credit services, processing equipments, infrastructure and transportation facilities and reforming extension services in order to make it effective in disseminating information regarding cassava processing.

Keywords: technical, cost and allocative efficiency; DEA; cassava processing; Delta state; Nigeria

1. INTRODUCTION

Cassava is an important crop that has great potential to support agricultural growth in Nigeria because of its wide range of use spanning from consumption to industries. Africa produces 40%–50% of the world cassava output (Awerije, 2014). According to Ohimain (2014), cassava (Manihot esculenta Crantz) is the chief source of dietary food energy for majority of the people living in low land tropics and much of the sub-humid tropics of the West and Central Africa. An estimated 600-700 million people derive more than 500 kcal/day of dietary energy from cassava (Ohimain, 2014). Nigeria is the largest producer of cassava in the world, producing 36.8 million mt with an average yield of 11.7 mt/ha (Awerije, 2014; Babatunde, 2012; Knipscheer et al., 2007). It is the basic staple food for more than 70% of the Nigerian population who consume cassava at least once every day (Eke-Okoro and Njuko, 2012). Eke-Okoro and Njuko (2012) further noted that about 90% of the cassava produced in Nigeria is used locally for food, animal feed, industrial and pharmaceutical uses and an unspecified quantity of the remaining 10% are exported. As a human food, cassava is processed into over 50 food forms – mainly gari, lafun, bread, flakes, flour etc. (Denton et al., 2004). The utility attributes of cassava is inexhaustible especially with increase in capacity and sophistication of the processing technologies. Furthermore, recent studies have shown cassava to be a promising crop for international trade. The demand for cassava derivatives such as starch, gari, tapioca, etc. doubled over the last two decades (Awerije and Rahman 2014). However, the current cassava yield is only 12.3 mt/ha, whereas the potential yield is 28.0 mt/ha (Nkonya et al., 2010), and the yield of improved varieties at research stations range from 13–40 mt/ha (Eke-Okoro and Njoku, 2012). Asogwa et al., (2012) also noted that although Nigeria is producing 40 million mt of cassava roots, the average yield is about 10 mt/ha whereas the yield level could be easily increased three folds to 30 mt/ha by removing a range of constraints affecting the cassava sector. Gari is the most commonly used form of cassava products in Nigeria and it accounts for 70% of the entire cassava production in Nigeria (IITA, 1990). It has been estimated that between 4 and 5 million mt of cassava roots are used each year for this purpose. Other products such as fufu, lafun, starch, pellets, chips and cassava flour and the raw root constitute the remaining 10–11 million mt. In the production of gari, fresh cassava roots are peeled, washed and grated. The grated pulp is packed in sacks (Jute or polypropylene) and placed under heavy stones or pressed with a hydraulic jack between wooden platforms for 3–

Complimentary Contributor Copy Technical, Cost and Allocative Efficiency of Processing Cassava … 203

4 days to express excess liquid from the pulp while it is fermenting (Olasoro et al., 2013; Adejumo and Raji, 2009). One of the most obvious means to improve production of cassava is to improve production efficiency by allocating resources to its optimal level given existing level of input and output prices. A number of studies looked into production efficiency of cassava in different states of Nigeria using either parametric or non-parametric approaches (e.g., Oladeebo and Oluwaranti, 2012; Raphael, 2008; Udoh and Etim, 2007; Ogundari and Ojo, 2007; Awerije and Rahman, 2014). But the focus of all these studies was on the estimation of efficiency measures of cassava root tubers only. Only Rahman and Awerije (2015) estimated overall pure technical and scale efficiencies of cassava production as a system where the efficiency measures of cassava processed into gari entered as a sub-system in the production process. In other words, to our knowledge no study was conducted to estimate the technical, cost and allocative efficiencies of gari processing for Nigeria. Given this backdrop, the specific objectives of this study are: (a) to determine productivity of processing cassava into gari by farm size categories, (b) to estimate technical, allocative and cost efficiency of gari production by farm size categories, and (c) to analyse the socio-economic determinants of technical, allocative and cost efficiency of gari production. The contribution of this research to the existing literature are three folds: (a) the study specifically estimated a range of efficiency measures of processing cassava into gari using a fairly large sample of 278 farmers/processors; (b) the study also specifically tested the role of farm operation size on the aforementioned objectives in order to test the existence of inverse size-productivity and size-efficiency relationships with respect to gari processing, which was also not addressed in the previous studies; and (c) use of the non-parametric DEA approach to estimate all three measures of efficiency simultaneously which then provides information on the potential to improve productivity of gari without resorting to additional use of resources given existing levels of input and output prices.

2. METHODOLOGY

In order to estimate technical, allocative and cost efficiency of gari production, DEA method is applied. And to identify the determinants of DEA efficiency scores, a tobit model is estimated in the second stage. The details of the methods used are presented below preceded by a description of the study area, sampling procedure and the data.

2.1. Study Area, Sampling Procedure and the Data

Data used for the study were drawn from the three geopolitical zones of the Delta state of Nigeria which is situated at the South-southern (Niger Delta) part of Nigeria. These are, North, Central and South Delta. The major food crops grown in Delta state are cassava (leading producer), yam, plantain, maize, and vegetables (MANR, 2006). Farm sampling was based on the cell structure developed by the Agricultural Developmental Programme. First, nine local government areas (LGAs) of the total 25 LGAs in the state were selected randomly. Then three cells per LGA were chosen randomly. Next,

Complimentary Contributor Copy 204 Brodrick O. Awerije and Sanzidur Rahman

105 cassava growers from each LGA were selected using a stratified random sampling procedure with cassava farm operation size as the strata. The cut-off points for farm size followed the nationally defined categories (Apata et al., 2011). These are: marginal farms – upto 1.00 ha; small farms – 1.01 to 2.00 ha; medium farms – 2.01 to 10.00 ha and large farms – >10.01 ha. This provided a total of 315 cassava farmers of which 278 farmers also processed cassava into gari and thus form the final sample for the study. For primary data collection, a structured questionnaire was administered containing both open and closed type questions. A team of two research assistants were trained by one of the authors and all three members were involved in collecting primary data using face to face interview method. Demographic and socio-economic information from each of the farm households included information such as age of the farmer, years of farming experience, number of household members, number of working adult household members, level of education (completed year of schooling) of the head of household, cassava farm operation size, contact with extension services and training received over the past one year, and gender of the household head. Input-output data included information on the quantities of cassava output, family and hired labour, fertilizers, pesticides, and seeds used. Also, information on all input and output prices were collected from each farm household based on memory recall of the farmers. The survey was conducted during September to December, 2008.

2.2. DEA Approach to Analyse Technical, Cost and Allocative Efficiency

Data Envelopment Analysis (DEA), a non-parametric approach, has been widely applied to measure relative efficiency of decision making units (DMUs) engaged in the production of goods and services (Kao and Hwang, 2008; Charnes et al., 1978). An advantage of DEA is its capacity to analyse multiple output–multiple input production technologies without assuming any functional form or behaviour of the DMUs or markets. The analysis provides DMU specific relative efficiency measures in comparison to its most efficient peers so that one can identify what factors are responsible for inefficient performance of DMUs. Technical efficiency relates to the degree to which a farmer produces the maximum feasible output from a given bundle of inputs, or uses the minimum feasible amount of inputs to produce a given level of output. These two definitions of technical efficiency lead to what are known as output-oriented and input-oriented efficiency measures, respectively (Coelli et al., 2002). These two measures of technical efficiency will coincide when the technology exhibits constant returns to scale, but are likely to differ otherwise. In this study, the input-oriented efficiency measures were used because these lead to a natural decomposition of cost efficiency into its technical and allocative components (Coelli et al., 2002). Since most of the sampled farmers have very small areas of land, the technology is unlikely to be significantly affected by non-constant returns to scale. Allocative efficiency refers to a producer’s ability to maximise profit given technical efficiency. It refers to a producer’s ability to utilise the inputs in optimal proportions, given observed input prices, in order to produce at minimum possible cost. A producer may be technically efficient but allocatively inefficient (Hazarika and Alwang, 2003). Cost efficiency, also known as economic efficiency, results from both technical efficiency and allocative efficiency. Therefore, cost efficiency refers to a producer’s ability to produce the maximum possible output from given quantity of inputs at the lowest possible cost. Complimentary Contributor Copy Technical, Cost and Allocative Efficiency of Processing Cassava … 205

The DEA production frontier is constructed using linear programming technique, which gives a piece-wise linear frontier that ‘envelopes’ the observed input and output data. Technologies produced in this way possess the standard properties of convexity and strong disposability, which are discussed in Färe et al., (1994). The DEA model is used to simultaneously construct the production frontier and obtain the technical efficiency measures. Following Coelli et al. (2002) the general model for data on K inputs and M outputs for each of the N farms is presented. For the ith farm, input and output data are represented by the column vectors xi and yi, respectively. The KN input matrix, X, and the MN output matrix, Y, represent the data for all N farms in the sample. The DEA model used for the calculation of technical efficiency (TE) is:

푀푖푛휃,휆 휃, 푆푢푏푗푒푐푡 푡표: − 푦𝑖 + 푌휆 ≥ 0 휃푥𝑖 − 푋휆 ≥ 0 푁1′휆 = 1 휆 ≥ 0 (1) where  is a scalar, N1 is an N1 vector of ones, and  is an N1 vector of constants. The value of  obtained is the technical efficiency score for the ith farm. It will satisfy:   1, with a value of 1 indicating a point on the frontier and hence a technically efficient farm, according to the Farrell (1957) definition. Note that the linear programming problem must be solved N times, to obtain a value of  for each farm in the sample. The cost and allocative efficiencies are obtained by solving the following additional cost minimisation DEA problem:

′ ∗ 푀푖푛휆,푥𝑖∗ 푤𝑖 푥𝑖 , 푆푢푏푗푒푐푡 푡표: − 푦𝑖 + 푌휆 ≥ 0 ∗ 푥𝑖 − 푋휆 ≥ 0 푁1′휆 = 1 휆 ≥ 0 (2)

th where wi is a vector of input prices for the i farm and xi* (which is calculated by the model) th is the cost-minimising vector of input quantities for the i farm, given input prices wi and the th output levels yi. The total cost efficiency (CE) of the i farm is calculated as

CE = (wixi*)/(wixi).

That is, CE is the ratio of minimum cost to observed cost for the ith farm. The allocative efficiency (AE) is then calculated residually by

AE = CE/TE.

Complimentary Contributor Copy 206 Brodrick O. Awerije and Sanzidur Rahman

2.3. Determinants of Efficiency: A Tobit Model

Tobit model is the most suitable when the dependent variable is truncated because it uses all observations, both those at the limit, usually zero (e.g., inefficient), and those above the limit (e.g., across the efficiency spectrum upto fully efficient score), to estimate a regression line as opposed to other techniques that use observations which are only above the limit value (McDonald and Moffit, 1980). Let the efficiency function for a particular efficiency category be:

* i ' XYii (3) where Xi is the vector of regressors, γ is the vector of parameters to be estimated, and µi is the * error term. For processors’ producing gari, Yi equals actual level of efficiency (Yi). For those * who are not producing gari, Yi is an index reflecting potential efficiency such that:

YY* if X  0' i i ii (4)  if X ii 0'0

In practice, all processors actually processed gari but their efficiency scores vary within a range of 0 (fully inefficient) to 1 (fully efficient). The following farm-specific socio-economic characteristics were used as regressors to identify the determinants of technical, cost and allocative efficiencies. These are farmers’ experience in years (V1), subsistence pressure (V2), educational level of the head of the household (V3), a set of dummy variables to identify the following: marginal farms (V4), small farms (V5), main occupation is farming (V6), extension contact (V7), training received (V8), credit received (V9), gender (V10), Delta North (V11), and Delta South (V12). Choice of these variables are based on existing literature and justification thereof (e.g., Rahman and Awerije, 2015; Awerije and Rahman 2014; Gelan and Murithi, 2012; Aye and Mungatana, 2011; and Coelli et al., 2002).

3. RESULTS

The summary statistics of the sample farms are presented in Table 1. The average value of gari produced is N 354,153.7; the average level of education is 6.84 years of schooling; family size is 5.86 persons; farming experience is 16.2 years; 10% are marginal farms; 67% are small farms, and 22% are medium and/or large farms; 41% of the processors are female; 31% of the farmers received credit; 36% of the farmers had extension contacts and only 10% received any training.

Complimentary Contributor Copy Technical, Cost and Allocative Efficiency of Processing Cassava … 207

Table 1. Definition, measurement and summary statistics of the variables

Variables Definition Mean Standard deviation Output Value of gari produced Naira 354153.710 447959.80 Inputs Cassava root tuber kg 11435.13 11015.28 Labour person days 123.21 161.36 Other material costs Naira 27269.49 38351.20 Prices Cassava price Naira per kg 16.84 0.79 Wage rate Naira per day 544.24 21.98 Other material price Naira per kg of gari produced 5.08 0.96 Socio-economic factors Education Completed years of schooling 6.84 4.84 Subsistence pressure Number of family members/working adult 5.86 3.31 Experience Years engaged in farming 16.24 11.71 Delta North Dummy (1 if Central, 0 otherwise) 0.30 -- Delta South Dummy (1 if South, 0 otherwise) 0.35 -- Delta North Dummy (1 if South, 0 otherwise) 0.35 -- Main occupation Dummy (1 if farmer, 0 otherwise) 0.82 -- Extension contact Dummy (1 if had extension contact in the past one year, 0.36 -- 0 otherwise) Credit received Dummy (1 if had received credit, 0 otherwise) 0.31 -- Training received Dummy (1 if had received training, 0 otherwise) 0.10 -- Marginal farms Dummy (1 if cultivated area upto 1.00 ha, 0 otherwise) 0.11 Small farms Dummy (1 if cultivated area between 1.01 – 2.00 ha, 0.67 -- 0 otherwise) Medium/large farms Dummy (1 if cultivated area >2.01 ha, 0 otherwise) 0.22 -- Gender Dummy (1 if female, 0 otherwise) 0.41 -- Note: Exchange Rate: GBP1.00 = Naira 200.00.

3.1. Productivity of Gari Produced from Per Ha of Cassava Root Tuber

Table 2 presents the results of the productivity analysis of cassava production classified by farm size categories as well as regions. It is clear from Table 2 that productivity of gari processed from per ha of cassava root tuber production varies significantly by farm size categories as well as by regions. The average productivity of gari is highest for marginal farms (2948 kg/ha) and lowest for small farms (2293.2 kg/ha) with a very high standard deviation. This finding points toward the existence of inverse size-productivity relationship in gari processing. Awerije and Rahman (2014) noted presence of inverse size-productivity relationship in cassava production in Nigeria. Amongst the regions, gari productivity is highest in Delta North (3098.6 kg/ha) and lowest in Delta South (1993.0 kg/ha).

Complimentary Contributor Copy 208 Brodrick O. Awerije and Sanzidur Rahman

Table 2. Productivity of gari processed from one hectare of cassava root tuber production by farm size and region.

Variables Region Farm Sizes Category Overall Delta Delta Delta North Marginal Small Medium/Large Central South Mean 2443.80 1993.01 3098.57 2948.69 2293.16 2840.53 2483.62 Std Deviation 1156.53 733.62 1672.08 1510.00 1011.63 1769.37 1297.21 Minimum 750.00 1200.00 750.00 833.00 776.99 750.00 750.00 Maximum 8333.00 3920.00 8000.00 7333.00 8000.00 8333.00 8333.00 F-value 18.26*** 6.47*** for regional differences (ANOVA) Note:*** = significant at 1 percent level (p < 0.01). Source: Computed from Field Survey, 2008.

Table 3. Technical, cost and allocative efficiency of gari processing by region and by farm size

Regions Delta Central Delta South Delta North Overall TE AE CE TE AE CE TE AE CE TE AE CE Mean 0.55 0.64 0.33 0.49 0.31 0.65 0.65 0.43 0.65 0.55 0.64 0.35 Std Deviation 0.16 0.11 0.08 0.11 0.08 0.09 0.19 0.20 0.21 0.17 0.14 0.14 Minimum 0.34 0.33 0.15 0.35 0.18 0.35 0.38 0.05 0.08 0.34 0.08 0.05 Maximum 1.00 0.87 0.61 1.00 0.79 0.90 1.00 1.00 1.00 1.00 1.00 1.00 F-value 26.89*** 0.18 20.05*** for regional differences (ANOVA) Farm size Marginal Small (1.01 – Medium/Large Overall categories (upto 1.00 ha) 2.00 ha) >2.01) Mean 0.73 0.66 0.48 0.52 0.66 0.34 0.57 0.57 0.33 0.55 0.64 0.35 Std Deviation 0.18 0.16 0.17 0.14 0.11 0.11 0.19 0.18 0.15 0.17 0.14 0.14 Minimum 0.41 0.34 0.29 0.34 0.32 0.15 0.36 0.08 0.05 0.34 0.08 0.05 Maximum 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 F-value 23.55*** 10.17*** 15.65*** for farm size differences (ANOVA) Note:*** = significant at 1 percent level (p < 0.01). Source: Computed from Field Survey, 2008.

3.2. Technical, Cost and Allocative Efficiency of Cassava Production

Results of efficiency estimates using DEA are presented in Table 3 classified by farm size categories and by regions. The overall mean levels of TE, AE and CE are 55%, 64% and 35% respectively, with significant difference across regions as well as farm size categories. The implication is that there is substantial scope to boost gari productivity by reallocating Complimentary Contributor Copy Technical, Cost and Allocative Efficiency of Processing Cassava … 209 resources to optimal levels, given input and output prices. The TE and CE estimates are higher than cassava production but the AE is lower (Awerije and Rahman, 2014). As with the case of productivity, a clear inverse size-efficiency relationship is observed with marginal farms scoring highest levels of TE, AE and CE. Therefore, based on the results from Table 2 and Table 3, it can be safely concluded that the classic inverse size-productivity as well as size-efficiency relationship exist in gari production in these sample farms of Delta State, Nigeria. Awerije and Rahman (2014) also noted inverse size-efficiency relationship in cassava production in Nigeria. Among the regions, farms located in Delta North performed better than the other two regions. It should be noted that there is no significant difference across regions with respect to AE implying that farmers/processors of all three regions are able to maximize profits given technical efficiency levels.

4. DETERMINANTS OF TECHNICAL, COST AND ALLOCATIVE EFFICIENCY OF CASSAVA PRODUCTION

Table 4 presents the parameter estimates of the Tobit model presented in Eq. (4). A total of 12 variables representing farm-specific socio-economic factors were used to identify the determinants of observed technical, cost and allocative efficiencies of cassava production. The model diagnostics revealed that these variables jointly explain variation in farm-specific efficiency levels quite satisfactorily. A total of 19 coefficients out of 36 in three models (excluding the intercept) were significant at the 10% level at least, implying that these factors exert differential effect on the observed measures of technical, cost and allocative efficiencies. Since the Tobit model cannot reveal the magnitude of influence directly, we present the elasticities of the influences of individual socio-economic factors on our three measures of efficiency in Table 5. Table 5 clearly shows that marginal farms are more efficient relative to small and medium/large farms. In fact, small farms are also significantly more allocative and economically efficient as compared to the medium/large farms. The magnitude of the influence is much larger for small farms as compared with the marginal farms. Therefore, taken all these information together, the results econometrically confirm the existence of inverse size-efficiency relationship in gari processing observed in Table 3. Technical efficiency is significantly higher for female processors, which is encouraging. Extension contact, however, is negatively associated with all efficiency measures. The implication is that farmers who have extension advice are using too much of inputs and not achieving the expected yield of gari (hence technical efficiency is lower). And because the farmers are using too much of the inputs, their cost efficiency is low. Also, they are not being able to respond to price information. Awerije and Rahman (2014) noted negative influence of extension contact on TE and CE in cassava production. Aye and Mungatana (2011) also reported negative significant influence of extension contact on technical efficiency in maize production in Nigeria. They concluded that extension services in Nigeria in general have not been effective, especially after the withdrawal of the World Bank funding from the Agricultural Development Project, which is the main agency responsible for extension services (Aye and Mungatana, 2011).

Complimentary Contributor Copy 210 Brodrick O. Awerije and Sanzidur Rahman

Table 4. Determinants of technical, cost and allocative efficiencies in gari processing

Variables Technical efficiency Cost efficiency Allocative efficiency Constant 0.4730*** 0.2472*** 0.5515*** Delta North§ 0.1146*** 0.1126*** 0.0373* Delta South§ 0.0232 0.0495** 0.0502* Education 0.0013 0.0020 0.0030 Main occupation§ 0.0034 -0.0154 -0.0318 Subsistence pressure -0.0002 0.0014 0.0026 Experience 0.0004 -0.0004 -0.0004 Extension contact§ -0.0777*** -0.0788*** -0.0599** Training received§ -0.0062 0.0087 0.0155 Credit received§ 0.0422** 0.0279* -0.0007 Marginal farms§ 0.1941*** 0.1964*** 0.1099*** Small farms§ 0.0011 0.0699*** 0.1127*** Gender§ 0.0365** 0.0098 -0.0174 Model diagnostic Log likelihood 152.54 210.47 163.71 Chi-squared(12 df) 101.65*** 96.34*** 34.87*** Number of observations 278 278 278 Note:*** = significant at 1 percent level (p < 0.01). ** = significant at 5 percent level (p < 0.05). * = significant at10 percent level (p < 0.10). § = dummy variables.

Table 5. Elasticities of technical, cost and allocative efficiencies in gari processing

Variables Technical efficiency Cost efficiency Allocative efficiency Delta North§ 0.0620*** 0.0953*** 0.0173*** Delta South§ 0.0145*** 0.0485*** 0.0270* Education 0.0156 0.0379** 0.0316* Main occupation§ 0.0051 -0.0360 -0.0408 Subsistence pressure -0.0015 0.0227 0.0237 Experience 0.0106 -0.0186 -0.0094 Extension contact§ -0.0506*** -0.0803*** -0.0335** Training received§ -0.0011 0.0024 0.0023 Credit received§ 0.0237** 0.0245* -0.0003 Marginal farms§ 0.0367*** 0.0581*** 0.0178*** Small farms§ 0.0014 0.1332*** 0.1178*** Gender§ 0.0271** 0.0113 -0.0111 Number of observations 278 278 278 Note:*** = significant at 1 percent level (p < 0.01). ** = significant at 5 percent level (p < 0.05). * = significant at10 percent level (p < 0.10). § = dummy variables.

Education significantly influences cost and allocative efficiencies. This is expected because educated farmers are more able to derive correct information regarding prices of inputs and outputs. This enables them to maximize profits and/or able to produce at a lowest cost given the level of technical efficiency and input and output prices. Access of agricultural credit significantly increases technical and cost efficiencies, which is encouraging. Awerije and Rahman (2014) did not find any influence of education and credit on cassava production.

Complimentary Contributor Copy Technical, Cost and Allocative Efficiency of Processing Cassava … 211

Location of farmers has an important effect on efficiency scores of gari processing. Farmers located in the Delta North and Delta South are technically, allocatively and economically efficient as compared with farmers in Delta Central. The reasons for such differences may lie with respect to differences in the regional features (e.g., soil conditions, topography, weather, and other unknown factors) and market conditions (e.g., input prices, timely availability, market infrastructure, market competition, etc.). However, Awerije and Rahman (2014) noted differential influence of location on the TE and AE of cassava production.

5. CONSTRAINTS IN PROCESSING GARI INTO CASSAVA

Farmers/processors were also asked about the constraints to adding value to cassava through processing. The respondents identified a lack of transportation and adequate information as the top two constraints (Table 6). Approximately 91.5% of the processors agreed that transportation of cassava root tubers from the farm/market to the processing site is costly as the average distance from the farmers/processors to the nearest marketplace is estimated to be 2.93 km (±3.13 km) with a maximum distance of 15 km. Akinnagbe (2010) and Tonukari (2004) also noted distance as a major factor adversely affecting the cost and efficiency of processing.

Table 6. Constraints to adding value in cassava through processing

Constraints % of processors responding Rank Transportation Difficulties 91.5 1 Lack of Adequate Information 91.4 2 Too Many Buyers for Limited Raw Materials 76.6 3 Lack of Processing Equipment 76.2 4 High Cost of Raw Materials/Processing Equipment 72.4 5 Lack of Adequate Infrastructure 70.5 6 Others 23.8 7 Source: Adapted from Rahman and Awerije (2016).

CONCLUSION AND POLICY IMPLICATIONS

The present study examined the productivity level as well as technical, cost and allocative efficiency of processing cassava into gari as well as determinants of efficiency using a sample of 278 farmers/processors from three regions of Delta State, Nigeria. The study also tested whether the hypothesis of inverse size-productivity and size-efficiency relationships exists in gari processing. Productivity of gari processed from per ha of cassava root tuber varies significantly by farm size categories as well as regions. The average levels of TE, AE, and CE are 55%, 64%, and 35%, respectively, implying that gari production can be boosted substantially by reallocation of resources to optimal levels, given input and output prices. The results also confirmed that gari production in the Delta State, Nigeria demonstrated inverse size-

Complimentary Contributor Copy 212 Brodrick O. Awerije and Sanzidur Rahman productivity as well as size-efficiency relationships. The smallest scale farms, i.e., the marginal farms are the most productive and efficient followed by small farms. Education level significantly influences cost and allocative efficiency of gari processing. Female processors are more technically efficient. Extension contact significantly reduces all efficiency measures which is highly counterintuitive. Farmers located in Delta North and Delta South regions are more efficient relative to Delta Central (the effect of which is subsumed in the constant term). The following policy implications can be drawn for the results of this study. First, investment in education targeted at the farmers/processors will result in significant improvement in cost and allocative efficiencies of gari processing which in turn will increase revenue of the farmers because price of gari is significantly higher than cassava root tuber (Rahman and Awerije, 2014). Second, there is the need for investment in improving cassava processing facilities and utilities. Third, measures to enhance involvement of female processors will increase gari productivity. Fourth, measures to improve access and provision of agricultural credit services through financial institutions as credit significantly influences efficiencies. Fifth, the agricultural extension services in Nigeria needs to be revitalized so that it contributes to improving production efficiency of gari production for all categories of farmers because mean efficiency levels are still very low across the board. This would require investment in developing capacity of the extension workers on new and improved technologies as well as dissemination strategies so that they can effectively serve to benefit the farmers. And sixth, measures are needed to target farmers located in Delta Central to support them to overcome low level of inefficiency relative to Delta North and Delta South. This may take the form of providing infrastructural and marketing support to bring them at par with the facilities and opportunities available for farmers in Delta North and Delta South. Although the policy options are challenging, effective implementation of these measures will increase processing of cassava into gari that could contribute positively to farmers’ revenue in Delta State, Nigeria.

REFERENCES

Adejumo, B. A. and Raji, A. O. (2009). An appraisal Gari packaging in Ogbomoso, Southwestern Nigeria. Journal of Agricultural and Veterinary Sciences, 2:120– 126. Akinnagbe, O. (2010). Constraints and strategies towards improving cassava production and processing in Enugu north agricultural zone of Enugu State, Nigeria. Bangladesh Journal of Agricultural Research, 35: 387–394. Amos, O.A., Imam, R.S., Idowu, A. B. and Mubarak, A. A. (2013). Analysis of Cassava product (Gari) marketing in Ekiti Local Government Area, Kwara State, Nigeria. Asian Journal of Agriculture and rural Development, 3: 736–745, 2013. Apata, T.G., Folayan, A., Apata, O.M. and Akinlua, J. (2011). The economic role of Nigeria’s subsistence agriculture in the transition process: implications for rural development. Paper presented at the 85th Agricultural Economics, UK conference at Warwick University during 18-20 April, 2011.

Complimentary Contributor Copy Technical, Cost and Allocative Efficiency of Processing Cassava … 213

Asogwa, B.C., Umeli, J.C. and Okwoche, V.A. (2012). Agricultural Policy in Cassava Sub- Sector: Implication for welfare of cassava farmers in Nigeria. British Journal of Science, 6: 81–98. Awerije, B.O. (2014). Exploring the potential of cassava for agricultural growth and economic development in Nigeria. Unpublished PhD thesis. University of Plymouth, UK. Awerije, B.O. and Rahman, S. (2014). Profitability and efficiency of cassava production at the farm-level in Delta State, Nigeria. International Journal of Agricultural Management. 3: 210–218. Aye, G.C. and Mungatana, E.D. (2011). Technological innovation and efficiency in the Nigerian maize sector: Parametric stochastic and non-parametric distance function approaches. Agrekon: Agricultural Economics Research, Policy and Practice in Southern Africa, 50: 1–24. Babatunde, J. (2012). 40% cassava inclusion in flour: Are the miller fighting back? Nigeria Vanguard, June, 2012. Charnes, A., Cooper, W. W. and Rhodes, E. (1978). Measuring the Efficiency of Decision Making Units. European Journal of Operational Research, 2: 429–444. Coelli, T.J., Rahman, S. and Thirtle, C. (2002). Technical, allocative, cost and scale efficiencies in Bangladesh rice cultivation: a non-parametric approach. Journal of Agricultural Economics, 53: 607–626. Denton, F.T., Azogu, I. I. and Ukoll, M.K. (2004). Cassava based recipes for house hold utilization and home generation. AIDU, Federal Department of Agriculture, Abuja, Nigeria. Eke-Okoro, O.N. and Njoku, D.N. (2012). A review of cassava development in Nigeria from 1940-2010. ARPN Journal of Agricultural and Biological Science, 7: 59–65. Färe, R., Grosskopf, S. and Lovell, C.A.K. (1994). Production Frontiers. Cambridge: Cambridge University Press. Farrell, M.J. (1957). The Measurement of Productive Efficiency. Journal of the Royal Statistical Society Series A, CXX (Part 3): 253–290. Gelan, A. and Muriithi, B.W. (2012). Measuring and explaining technical efficiency of dairy farms: a case study of smallholder farms in east Africa. Agrekon: Agricultural Economics Research, Policy and Practice in Southern Africa, 51: 53–74. Hazarika, G. and Alwang, J. (2003). Access to credit, plot size, and cost inefficiency among smallholder tobacco cultivators in Malawi. Agricultural Economics, 29: 99–109. IITA (1990). Cassava in Tropical Africa. A Reference Manual. International Institute for Tropical Agriculture, Ibadan, Nigeria. Kao, C. and Hwang, S.N. (2008). Efficiency decomposition in two-stage data envelopment analysis: An application to non-life insurance companies in Taiwan. European Journal of Operational Research, 185: 418–429. Knipscheer, H., Ezedinma, C., Kormawa, P., Asumugha, G., Mankinde, K., Okechukwu. R. and Dixon, A. (2007). Opportunities in the Industrial Cassava Market in Nigeria. International Institute for Tropical Agriculture (IITA). MANR (2006). Ministry of Agriculture and Natural Resources, Delta State, Nigeria. Agricultural Development Policy Report. McDonald, J.F. and Moffit, R.A. (1980). The uses of Tobit analysis. Review of Economics and Statistics, 61: 318–321.

Complimentary Contributor Copy 214 Brodrick O. Awerije and Sanzidur Rahman

Nkonya, E., Pender, J., Kato, E., Omobowale, O., Phillip, D. and Ehui, S. (2010). Enhancing Agricultural Productivity and Profitability in Nigeria. Nigeria Strategy Support Program, Brief # 19. International Food Policy Research Institute, Washington, D.C. Ogundari, K. and Ojo, S.O. (2007). An Examination of Technical, Economic and Allocative Efficiency of Small Farms: The Case Study of Cassava Farmers in Osun State of Nigeria. Bulgarian Journal of Agriculture, 13: 185–195. Ohimain, E.I. (2014).The prospects and challenges of cassava inclusion in wheat bread policy in Nigeria. International Journal of sciences, Technology and Society. 2: 6 –17. Oladeebo, J.O. and Oluwaranti, A.S. (2012). Profit Efficiency among Cassava Producers: Empirical Evidence from South Western Nigeria. Journal of Agricultural Economics and Development, 1: 46–52. Olasore, A.A., Imam, R.S., Idowu, A.B. and Mubarak, A.A. (2013). Analysis of Cassava Product (Garri) Marketing in Ekiti Local Government Area, Kwara State, Nigeria. Asian Journal of Agriculture and Rural Development. 3: 736–745 Rahman, S. and Awerije, B.O. (2014). Marketing efficiency of cassava products in Delta State, Nigeria: A stochastic profit frontier approach”. International Journal of Agricultural Management, 4: 28–37. Rahman, S. and Awerije, B.O. (2015). Technical and scale efficiency of cassava production system in Delta State, Nigeria: an application of Two-Stage DEA approach. Journal of Agriculture and Rural Development in the Tropics and Subtropics, 116: 59–69. Rahman, S. and Awerije, B.O. (2016). Exploring the potential of cassava in promoting agricultural growth in Nigeria. Journal of Agriculture and Rural Development in the Tropics and Subtropics, 117: 149–163. Raphael, I.O. (2008). Technical Efficiency of Cassava Farmers in South Eastern Nigeria: Stochastic Approach. Agricultural Journal, 3: 152–156. Tonukari, N.J. (2004). Cassava and the future of starch. Electronic Journal of Biotechnology, 7: 5–8. Udoh, E.I. and Etim, N–A. (2007). Application of Stochastic Production Frontier in the Estimation of Technical Efficiency of Cassava Based Farms in Akwa Ibom State, Nigeria. Agricultural Journal, 2: 731–735.

BIOGRAPHICAL SKETCHES

Dr. Brodrick O. Awerije is an Assistant Chief Agriculture Officer at the Tree Crops Unit, Ministry of Agriculture and Natural Resources, Asaba, Delta State, Nigeria. He is involved in planning, evaluation and implementation of agricultural policies in Delta State. He holds a Master’s Degree in Sustainable Crop Production and a PhD Degree from the University of Plymouth, UK completed in 2004 and 2014, respectively. His main research interest is in the economics of agricultural production and marketing as well as agricultural policies. He has published around the topics in international journals.

Dr. Sanzidur Rahman is Associate Professor (Reader) in International Development with the School of Geography, Earth and Environmental Sciences, University of Plymouth, UK. The core area of his research is to improve understanding of the range of factors

Complimentary Contributor Copy Technical, Cost and Allocative Efficiency of Processing Cassava … 215 affecting agricultural and rural development in developing economies and to promote their integration into policy and practice. His specialization is in agricultural economics, specifically, on efficiency and productivity measurements, and underlying determinants of technological change, innovation, and diffusion in agriculture. He has published widely on the topic in leading international journals.

Chapter 11

STATUS OF CASSAVA PROCESSING AND CHALLENGES IN THE COASTAL, EASTERN AND WESTERN REGIONS OF KENYA

1, 2 2 C. M. Githunguri *, M. Gatheru and S. M. Ragwa 1Kenya Agricultural and Livestock Research Organization (KALRO) Food Crops Research Centre Kabete, Nairobi, Kenya 2KALRO Katumani, Machakos, Kenya

ABSTRACT

Whether cassava can be relied upon as a low cost staple food in urban centres and a source of steady real income for rural households will to a larger extent depend on how well it can be processed and presented to urban consumers in safe and attractive forms at competitive prices to those of cereals. A study was conducted in the coastal, eastern, central, and western regions of Kenya where only the major processors were visited and interviewed randomly using a structured questionnaire. At the coast, 62.5% of the processors were sole proprietors while 37.5% were in partnership. In the eastern region, 66.7% of the processors were sole proprietors while 33.3% were in partnership. In the western region, the only processor interviewed was a company based in Busia. At the coast, 75% of respondents had their own initial capital while in eastern 33% of respondents reported the same. Only 25% and 33% of respondents at the coast and eastern regions, respectively, had acquired their initial capital on credit. In western, the respondent had acquired initial capital through own resources and credit. In the study regions, all processors (100%) met their operating costs. In the coastal region (Mombasa), among the respondents interviewed, 50% made cassava crisps, 17% chapatti and 8% bhajia. In eastern region (Kibwezi), 50% made Nimix (composite flour) and 50% boiled cassava. In western region (Busia), 100% of respondents made composite flour (cassava mixed with other cereals). The major products reported were crisps, fried chips, composite flours (cassava mixed with cereals, legumes, leaves etc). Golden coloured crisps, fiber free cassava and sweet taste were preferred by consumers. Even though processors maintained high standards, none of the processors had their products patented.

* Email: [email protected], Cellphone: +254 726959592. Complimentary Contributor Copy 218 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Processing of cassava products showed a rising trend which were marketed in supermarkets, direct consumers, retailers and wholesalers. Except for the eastern region, most processors could access raw materials throughout the year. Only a few processors in the coastal region had contractual arrangements with suppliers, whereas there was none in the other regions. Processing equipment were locally fabricated except in the eastern region where they were imported. The processors had reliable sources of power and water. The major constraints included market fluctuations, inadequate supply of cassava, city council regulations, competition from other related products like maize and sweetpotatoes, lack of credit facilities, market and capital, and processing equipment.

INTRODUCTION

African farmers grow cassava under field conditions where one or more of the resources are limiting while due to the nature of measurements, most of the research work carried out is under optimum management conditions (Githunguri et al. 2006). Cassava is gaining importance as an industrial crop in several countries within the tropics and especially sub- Saharan Africa (Ayinde et al., 2004; Azogu et al., 2004; EFDI-Technoserve, 2005; Ezedinma et al., 2005; Githunguri, 1995; Onyango et al., 2006). Safety of cassava products is important and could be affected by agro-ecological zones and genotypes (Githunguri, 2002; Odongo G. O., 2008; Tivana. and Bvochora, 2005). Cassava is the most perishable root crop and deteriorates at ambient temperatures in 2-4 days. Cassava’s twin problems of rapid post-harvest deterioration and cyanide toxicity have been solved through the development of processing methods that increase its shelf life and detoxifies it in various countries but this technology has not taken root in Kenya. Traditional methods like heap fermentation in western Kenya could be mechanized to make them more commercially competitive. In six households in Uganda, it was found that heap fermentation followed by sun-drying of cassava roots reduced the cyanogenic potential from 436 to 20ppm on dry weight basis (Essers et al., 1995). Heap fermentation for four days in three households in Mozambique followed by sun-drying reduced the cyanogenic potential of cassava roots from 660 to 19ppm on dry weight basis (Tivana, 2005). Although heap fermentation is important in reducing total cyanogens in cassava roots, the above levels were still above the World Health Organization (WHO) safe level of 10ppm (FAO/WHO, 1991). The removal of cyanogens by heap fermentation has been found to be less effective than those reported above and that an initial cyanogenic potential of less than 32ppm is required for cassava roots, if the flour is to reach the WHO safe level of 10ppm (Tivana and Bvochoro, 2005). Perhaps the WHO safe level of 10ppm should be revised upwards. The human body, even with very low protein intake, is able to detoxify 12.5mg of cyanide every 24 hours. In a well-nourished adult, the body can detoxify about 50 to 100mg of cyanide every 24 hours (Rosling, 1994). In a population where cassava is the main staple food, a basic daily energy need of 1500 kcal can be obtained from consumption of 500g dry weight of cassava flour. Cassava flour with 25ppm cyanide may be used to prepare a safe cassava meal. Indonesia has set a safe level for cyanide in cassava at 40ppm (Tivana and Bvochoro, 2005). Since some cyanogens will be lost during preparation of a cassava flour meal, the residual cyanogenic potential values of 19 and 20ppm (dry weight) obtained after heap fermentation may be considered safe if the WHO safe level is revised upwards (Essers et al., 1995 and Tivana, 2005). Reported high cyanogenic potential values, up to 150ppm, of heap fermented cassava flour may have been Complimentary Contributor Copy Status of Cassava Processing and Challenges in the Coastal … 219 caused by shortcuts in the fermentation regime, or result from increased root cyanide levels due to drought or use of high cyanide cultivars (Tivana, 2005). Shortcuts in processing commonly occur when food supply is low or the product is for sale. It is important to develop further processing techniques to reduce cyanide, such as a combination of grating cassava roots, fermentation and sun drying or soaking of cassava roots in water and sun drying. Grating and crushing of cassava roots are very effective in removing cyanide because of the contact in the wet parenchyma between linamarin and the hydrolyzing enzyme, linamarase (Rosling, 1994; Githunguri, 2002). In countries like Mozambique, the cassava flour usually comes from plants that have been subjected to two years of drought. Under drought conditions the cyanogenic content of cassava roots is known to increase due to increased water stress on the cassava plant (Bokonga et al., 1994; Githunguri et al., 1998; Githunguri, 2002). This increased water stress may have caused an increase in the linamarin content of the roots. Serious drought may increase the cyanide intake of individuals, if non-efficient processing techniques are used, to such a degree as to precipitate konzo disease epidemic among the consumers as has been observed in Mozambique and parts of Congo (Rosling, 1987). How do we overcome the problem of high cyanide intake levels during drought? Obviously, it is not possible to eliminate the recurrent episodes of drought. Hence, the only possible solution is to reduce the cyanide intake of the populace. Ways to reduce the cyanide intake of the populace include: Improving early warning and food security; encouraging greater use of improved processing methods; improvement of the diet by introduction of other vegetables, pulses and fruits which would help in raising the sulphur containing protein intake which detoxifies cyanide in the blood system as thiocyanate; and a greater use of low cyanide cassava varieties (Cardoso et al., 1999). It seems as if poor soils and droughts increase toxicity (Cock, 1985; Githunguri, 2002). This is of considerable importance to food security programmes focusing on cassava. The very causes of food shortage i.e., drought and poor soils also increase the toxicity of the cassava grown (Rosling, 1987). Prevention of toxic effects from cassava consumption should be based on the fact that incidences of cassava toxicity have been reported only when contributing nutritional deficiencies are present and/or when extraordinary circumstances induce consumption of inadequately processed roots. The nutritional deficiencies are low intake of protein and iodine and the extraordinary circumstances are drought, hunger, war and severe poverty. It must nonetheless be remembered that cassava has saved affected populations in Mozambique and other cassava growing countries like Uganda from starvation under these very circumstances. To advise these populations to reduce cassava cultivation runs counter to common sense (Rosling, 1987). Information on possible acute intoxication must also be included in all forms of cassava promotion programmes, and this problem should thereby be possible to solve or at least control. To try to avoid cassava toxicity by persuading the farmers in these areas to change their staple crops is wishful thinking. New high yielding cassava varieties are an adequate short term solutions, as better food security will enable populations to process the roots adequately. Promotion of better processing is a medium-term solution, but in the long run, farming systems must be developed to increase productivity and to maintain soil fertility. In this way, a stable dietary situation may be created in which the problems with cassava toxicity can be solved. It should be noted that various methods for determination of cyanogens levels in cassava and its products and its metabolite thiocyanate have been developed. One of the Complimentary Contributor Copy 220 C. M. Githunguri, M. Gatheru and S. M. Ragwa simplest methods is the use of simple kits for the determination of the total cyanogens, acetone cyanohydrins and cyanide in cassava roots and cassava products (Egan and Bradbury, 1998; Bradbury et al., 1999). Whether cassava can be relied upon as a low cost staple food in urban centres and a source of steady real income for rural households will to a larger extent depend on how well it can be processed into safe forms and on how far it can be presented to urban consumers in an attractive form at prices which are competitive to those of cereals (Nweke et al., 2002). In some large cassava producing countries like Nigeria, the market for some processed products is highly limited to low income groups, while other forms of cassava, e.g., gari have a significant market value for middle and high income consumers. How far the market for cassava may be expended would therefore depend largely on the degree to which the quality of the various processed products can be improved to make them attractive to potential consumers without significant increase in processing costs. Cassava products processing and utilization is done mainly at the subsistence level (Kadere, 2002). At the coastal region, it is men who roast and sell cassava crisps. In both Eastern and Western Kenya, women dominate home-based processing while service processing like milling is male dominated. As processing becomes mechanized men tend to play a leading role. The few home-based processors sell their products directly to consumers or retailers. Tapioca Ltd. in Mazeras is the only factory that employs modern technology to produce cassava flour, starch and glue. Most cassava processing technologies are labour-based facing serious limitations in areas with labour shortages (Mbwika, 2002). Rudimental processing technologies like over reliance on sun-dried methods are rendered impossible during the rainy season. Peeling of cassava roots manually using a knife is time consuming, laborious, difficult to ensure quality control and wasteful. Figure 1 shows a trader processing cassava crisps along Mama Ngina Drive, Mombasa using a crude tool. The fine particles of cassava flour render current milling technologies wasteful. There is need to identify appropriate storage and processing technologies that are cheap, have low losses, improve shelf life and guarantees quality products. Efforts should be made to involve the food processing industry in making ready to eat cassava products available in supermarkets and retail outlets. Due to the enormous potential demand for cassava by the feeds, pharmaceutical, food, paper printing and brewing industries there is need to involve them in the research and development of this sub-sector.

STUDY METHODOLOGY

The study was conducted in the coastal (Coast Province), eastern (Central, Nairobi and Eastern Provinces) and western (Nyanza and Western Provinces) regions of Kenya where only the major processors were visited and interviewed randomly. Like in marketing studies it is not possible to predetermine the sample size. Randomly selected cassava processors were interviewed using a structured questionnaire. Data collected included information on characteristics of cassava processors, cassava products, and raw materials, processing equipment, utilities and constraints in cassava processing. The data collected were analyzed using the Statistical Package for Social Sciences (SPSS).

Complimentary Contributor Copy Status of Cassava Processing and Challenges in the Coastal … 221

Figure 1. A trader processing cassava crisps along Mama Ngina Drive, Mombasa. Sales are normally very high during public holidays and weekends.

RESULTS AND DISCUSSION

Characteristics of Cassava Processors

Characteristics of cassava processors in the three study regions are shown in Table 1. The average number of male employees was two in all the study regions while the average number of female employees was one in the coastal, three in eastern and one in western regions. Labour availability was not a problem in eastern and western regions but was a problem at the coast as reported by 25% of respondents. Concerning employees’ skills on processing, eastern region was leading with 69% of employees being skilled, followed by western (66%) and eastern (63%) regions. At the coast, 62.5% of the processors were sole proprietors while 37.5% were in partnership. In the eastern region, 66.7% of the processors were sole proprietors while 33.3% were in partnership. In the western region, the only processor interviewed was a company based in Busia. Figure 2 shows a motorized cassava chipper that is already in use in Mbeere and coast. At the coast, 75% of respondents had their own initial capital while in eastern 33% of respondents reported the same. Only 25% and 33% of respondents at the coast and eastern respectively had acquired their initial capital on credit. In western, the only respondent had acquired initial capital through own resources and credit. In the three study regions, all processors (100%) met their operating costs.

Complimentary Contributor Copy 222 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Figure 2. Motorized cassava chipper that farmers’ groups already use in Mbeere and coast region.

Table 1. Characteristics of cassava processors

Region Coast Eastern Western Characteristic Mean Number of employees Males 2 2 2 Females 1 3 1 Percent of respondents Business ownership Sole proprietorship 62.5 66.7 0 Partnership 37.5 33.3 0 Company 0 0 100 Labour availability Yes 75 100 100 No 25 0 0 Type of employees Skilled 63 69 66 Unskilled 37 31 34 Source of initial capital Own resources 75 33 0 Credit 25 33 0 Own resources & credit 0 0 100 Other sources 0 34 0 Source of operating capital Own resources 100 100 100 Credit 0 0 0 Own resources & credit 0 0 0

Complimentary Contributor Copy Status of Cassava Processing and Challenges in the Coastal … 223

Cassava Products

Table 2 shows the cassava products that were being processed in each of the three regions. In the coastal region (Mombasa), among the respondents interviewed, 50% made cassava crisps (Figure 2), 17% made cassava chapatti and 8% made cassava bhajia. In eastern region (Kibwezi), 50% of respondents made Nimix (composite flour) and 50% boiled cassava. In western region (Busia), 100% of respondents made composite flour (cassava mixed with other cereals). Figure 3 shows dry cassava chips ready for milling into flour in Busia. None of the processors had their products patented.

Figure 2. Processed cassava crisps ready for sale at Mama Ngina Drive, Mombasa. Apart retaild, markets, some are sold to big supermarkets like Nakumatt, Likoni.

Figure 3. Dry cassava chips ready for milling in Matayos Division, Busia District. This is the most common processing method practiced by farmers in this region.

In the coastal region, wholesale price of cassava crisps ranged between 30 and 80 shillings while retail price ranged between 50 and 100 shillings depending on the size of the package (Figure 4). The size of packages ranged between 100g and 250g. Other products Complimentary Contributor Copy 224 C. M. Githunguri, M. Gatheru and S. M. Ragwa were chapatti-mandazi, fried cassava, roasted cassava, and bhajia sold at Kshs. 10 per piece respectively. In eastern (Kibwezi), the composite flour commonly known as Nimix was being sold to wholesalers at 60 shillings and to retailers at 80 shillings. In Nairobi, a hotel was selling boiled cassava at 40 shillings per plate, while in western the composite flour was being sold at 50 shillings per kilogram. Except in the coastal region where 25% of respondents experienced closure due to lack of demand, the other regions the products were in high demand. Perception on market trend of cassava-processed products was recorded. Eighty two percent (82%) of respondents at the coast reported that the cassava market was rising while 9% reported a decreasing trend and 9% reported a constant trend.

Figure 4. Processed cassava crisps being sold at Mama Ngina Drive, Mombasa.

Table 2. Cassava products being processed in the coastal, eastern and western regions

Region Coast Eastern Western Product Percent respondents Nimix 0 50.0 0 Cassava crisps 50.0 0 0 Cassava chapati & mandazi 16.7 0 0 Fried cassava 25.0 0 0 Boiled cassava 0 50.0 0 Bhajia 8.3 0 0 Cassava flour 0 0 100.0

In eastern, 50% of respondents reported an increasing marketing trend while 50% reported constant trend. In western, the only respondent reported an increasing trend in cassava marketing. The increase in demand at the coast was attributed to high demand of cassava crisps in the supermarkets and tourists along the beach. In the coastal region, 75% of cassava products were sold locally along the beach and 25% in Mombasa city supermarkets. In eastern and western regions, all the cassava products were Complimentary Contributor Copy Status of Cassava Processing and Challenges in the Coastal … 225 sold locally. Direct clients of cassava products were recorded. In the coastal region 50% were consumers, 25% were retailers and 25% wholesalers. In eastern, 50% of clients were consumers and 50% were retailers. In western, direct clients of the only processor in the area were retailers.

Quality Control Standards for Cassava and Cassava Based Products

The minimum quality control standards of cassava and cassava based products varied with region and the product. In the coastal region, where the main product was cassava crisps, cleanliness of crisps (50%), use of fiber free cassava (10%), golden colour of crisps (20%), and sweet taste (20%) control standards were maintained. In the eastern region, the only processor maintained quality standards by indicating the ingredients of the composite flour (Nimix) on the package while in western the processor kept the standards by maintaining high hygiene. To ensure the standards were maintained, processors in the coastal region ensured thorough washing of cassava (30%), use of clean oil (20%) and use of white colour cassava roots (50%). In eastern, the standards were maintained by ensuring the ratios of ingredients were in the right proportions while in western, the standards were maintained by washing the cassava roots thoroughly. The effect of environmental regulations on the production of cassava and cassava-based products was reported by 25% of respondents in the coastal while in eastern and western regions, there were no environmental regulations.

RAW MATERIALS

The main raw materials for cassava and cassava-based products varied with region and the products processed. In the coastal region, the main raw materials were cassava roots (50%), cooking oil (25%), charcoal (19%) and salt (6%). The source of raw materials was mainly Kongowea market in Mombasa as shown in Figure 5. In eastern region, the main raw materials were cassava roots, cowpea leaves, pearl millet, pigeon peas, sorghum and sweetpotato leaves all in equal proportions of about 17%. In western region, the main raw material was cassava roots. In both eastern and western regions, the raw materials were sourced locally. In the coastal region, 73% of respondents reported that they obtained raw materials from the source while 27% had the raw materials delivered to them by agents. Similarly, in the eastern region, 50% of respondents reported that they obtained raw materials from the source while 50% had them delivered by agents. In western, the processor collected the raw materials from the source. Fifty percent of respondents at the coastal region reported that they could access raw materials when required while 100% of respondents in eastern reported that they could not access the raw materials when required. In the eastern region the unavailable raw materials are mainly cowpea and sweetpotato leaves during off-season periods. In the western region,

Complimentary Contributor Copy 226 C. M. Githunguri, M. Gatheru and S. M. Ragwa

100% of the respondents could access raw materials when required. Except in the coastal region where 12.5% of respondents had contractual arrangement with suppliers, none of the processors in the other regions had any contractual arrangement. Twenty five percent of the respondents at the coastal region had storage facilities while all respondents in the eastern and western regions had storage facilities.

Figure 5. Cassava tubers for both retail/wholesale market at Kongowea, Mombasa.

PROCESSING EQUIPMENT AND UTILITIES

Except in the eastern region (Figure 6) where the equipment was of foreign origin, all the other processing equipment in the three regions were locally fabricated. The average lifespan of the equipment ranged from 8 to 18 years. The average cost of various processing equipment ranged from 38,000 to 1,000,000 K. Shs. At the coastal region, 50% of the equipment was powered by electricity while 50% were both manually and wood powered. The power supply was 67% reliable and 33% unreliable. In eastern region, the equipment was diesel/petrol powered which was highly reliable while in western region the equipment was electrical and also very reliable. At the coastal and eastern regions, 50% of the processors had alternative sources of power. In eastern and western regions, all the processors interviewed had alternative sources of power. The average months of operation of processing equipment ranged from 10 months in the eastern region to 12 months in the coastal and western regions.

Complimentary Contributor Copy Status of Cassava Processing and Challenges in the Coastal … 227

Figure 6. A manual cassava chipper being promoted in Mbeere.

At the coastal region, 83% of the respondents had access to tap water whereas 27% had access to other sources of water (Table 3). In the eastern and western regions, all respondents had access to tap water. In all three regions, the water supply was reliable and processors paid for the water.

Table 3. Source of water and reliability for cassava processors in western, eastern and coastal regions

Tap water Other sources Reliability Region % respondents Coast 83 17 100 Eastern 100 0 100 Western 100 0 100

CONSTRAINTS IN CASSAVA PROCESSING

At the coastal region, the major constraints reported were market fluctuations (37.5%), availability of cassava (12.5%), lack of credit facilities (25%), competition from alternative products (12.5%) and city council regulations (12.5%). In the eastern region lack of market (50%) and capital (50%) were the major constraints reported while in the western region, lack of processing equipment (50%) and competition (50%) from other related products like maize and sweetpotatoes were the major constraints.

CONCLUSION

The major cassava products reported were cassava crisps, fried chips, composite flours (cassava mixed with cereals, legumes, leaves etc.). Clean and golden coloured crisps, fiber free cassava and sweet taste were preferred by consumers. Processors maintained high standards by thorough washing of cassava, use of clean oil and white cassava roots. In all the

Complimentary Contributor Copy 228 C. M. Githunguri, M. Gatheru and S. M. Ragwa regions none of the processors had their products patented. Processing of cassava products showed a rising trend in the three regions. The study shows that cassava products were marketed in local outlets like supermarkets, direct consumers, retailers and wholesalers. There is thus need to explore other outlets like manufacturers and export markets. Except for the eastern region, the coastal and western regions most processors could access raw materials throughout the year. A few processors in the coastal region had contractual arrangements with suppliers, whereas none of the processors in the other regions had contractual arrangements. In all three regions the processing equipment were locally fabricated except in the eastern region where the equipment was imported. The three regions had reliable sources of power for running processing equipment. Common sources of power supply included electricity, wood, diesel/petrol and manual. In all three regions the water supply was reliable and processors paid for the water. Generally, the major constraints reported were market fluctuations, inadequate supply of cassava, city council regulations, competition from other related products like maize and sweetpotatoes, lack of credit facilities, market and capital, and processing equipment.

REFERENCES

Ayinde, A. O. Dipeolu, K. Adebayo, O. B. Oyewole, L. O. Sanni, J. Adusei and A. Westby. 2004. A cost-benefit analysis of the processing of a shelf stable cassava fufu in Nigeria In: Book of Abstracts of the Sixth International Scientific Meeting of the Cassava Biotechnology Network. CIAT:Cali: Colombia. Azogu I, O Tewe, C Ezedinma and V Olomo. 2004. Cassava Utilisation in Domestic Feed Market, Root and Tuber Expansion Programme. Nigeria. pp 148. Bokonga, M., I. J. Ekanayake, A. G. O. Dixon, and M.C.M.Porto.1994.Genotype environment interactions for cyanogenic potential in cassava. Acta Horticulturae 375.131-139. Bradbury M.G., S. V. Egan and J. H. Bradbury. 1999. Picrate paper kits for determination of total cyanogens in cassava products. Journal of Science, Food and Agriculture. (79) 593- 601. Cardoso A. P., M. Ernest, J. Clifford and J. H. Bradbury. 1999. High levels of total cyanogens in cassava flour related to drought in Mozambique. Roots; volume 6 issue 2.4-6pp. Cock, J.H. 1985. Cassava. New Potential for a Neglected Crop. Westview Press / Boulder and London, 191pp. Egan, S. V. and J. H Bradbury. 1998. Simple kit for determination of the cynogenic potential of cassava flour. Journal of Science, Food and Agriculture. (76) 39-48. Essers, A.A., Ebong, C., van de Gritt, R., Nout, M. R., Otim-Nape, W. and Rosling, H. 1995. International Journal of Food Science and Nutrition. 46 (2), 126 – 136. EFDI-Technoserve. 2005. Assessment of different models of cassava processing enterprises for the south and South-East of Nigeria, including the Niger Delta. Draft Final Report submitted to IITA-CEDP, March 2005. Ezedinma, C., M. Patino, L. Sanni, R. Okechukwu, P. Ilona, M. Akoroda, A. Dixon. 2005. Investment options in the High Quality Cassava Flour (HQCF) Enterprise. Presented at

Complimentary Contributor Copy Status of Cassava Processing and Challenges in the Coastal … 229

the Stakeholders meeting on Strategies on sourcing high quality cassava flour – H. R. Albrecht Conference Center, IITA, Ibadan, Nigeria, Jan 2005. FAO/WHO. 1991. Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission, XII, Supplement 4, FAO/WHO, Rome, Italy. Githunguri, C.M., E. G. Karuri, J. M. Kinama, O. S. Omolo, J. N. Mburu, P. W. Ngunjiri, S. M. Ragwa, S. K. Kimani and D. M. Mkabili. 2006. Sustainable Productivity of the Cassava Value Chain: An Emphasis on Challenges and Opportunities in Processing and Marketing Cassava in Kenya and Beyond. A project proposal present to the Kenya Agricultural Productivity Project (KAPP) Competitive Agricultural Research Grant Fund, Research Call Ref No.KAPP05/PRC- CLFFPS –03. KAPP Secretariat 106p. Githunguri, C.M. 2002. The influence of agro-ecological zones on growth, yield and accumulation of cyanogenic compounds in cassava. A thesis submitted in full fulfillment for the requirements for the degree of Doctor of Philosophy in Crop Physiology, Faculty of Agriculture, University of Nairobi, 195pp. Githunguri, C. M, I. J. Ekanayake, J. A. Chweya, A. G. O. Dixon and J. Imungi. 1998. The effect of different agro-ecological zones on the cyanogenic potential of six selected cassava clones. Post-harvest technology and commodity marketing. IITA,71-76pp. Githunguri, C. M. 1995. Cassava food processing and utilization in Kenya. In: Cassava food processing. T. A. Egbe, A. Brauman, D. Griffon and S. Treche (Eds.) CTA, ORSTOM, pp119-132. Kadere, T.T. 2002. Marketing opportunities and quality requirements for cassava starch in Kenya. In Proceedings of regional Workshop on improving the cassava sub-sector, held in Nairobi, Kenya, April 2002, 8-18, pp. 81 - 86. Mbwika, J.M 2002. Cassava sub-sector analysis in the Eastern and Central African region. In Proceedings of regional Workshop on improving the cassava sub-sector, held in Nairobi, Kenya, April 2002, 8-18, pp. 8-18. Nweke, F. I., D. S. C. Spencer and J. K. Lynam. 2002. Cassava transformation. International Institute of Tropical Agriculture. 273p. Odongo G. O. (2008). Analysis of level of toxicity (cyanogenic potential) of various cassava varieties and cassava based products. A Trade Project Report in the Kenya Polytechnic University College Department of Applied Science submitted to the Kenya National Examination Council in partial fulfillment of the requirements for award of Diploma in Food Technology, 35pp. Onyango, C., P. W. Ngunjiri, T. J. Oguta and S. M. Wambugu. 2006. Small-Scale Processing Technologies for Selected Traditional and Horticultural Food Crops in Kenya. Kenya Industrial Research and Development Institute, 166pp. Rosling H.1987. Cassava and food security. A review of health effects of cyanide exposure from cassava and of ways to prevent these effects. A report for UNICEF African Household Food Security Programme 40pp. Rosling, H. 1994. Measuring effects in humans of dietary cyanide exposure from cassava. Cassava Safety. Acta Horticulturae 375, 271-283. Tivana, L. D. 2005. Study of heap fermentation and protein enrichment of cassava. MPhil. Thesis, University of Zimbabwe. 142pp. Tivana, L. D. and Bvochora, J. 2005. Reduction of cyanogenic potential by heap fermentation of cassava roots. Cassava Cyanide Diseases Network News, Issue No. 6 2005, 1p. 5.0 Budget. Complimentary Contributor Copy Complimentary Contributor Copy In: Handbook on Cassava ISBN: 978-1-53610-291-8 Editor: Clarissa Klein © 2017 Nova Science Publishers, Inc.

Chapter 12

CASSAVA WASTE: A POTENTIAL BIOTECHNOLOGY RESOURCE

Aniekpeno I. Elijah* Department of Food Science and Technology, University of Uyo, Uyo, Nigeria

ABSTRACT

Although cassava waste may pose serious environmental challenges if not properly disposed of, it could constitute important potential resource if properly harnessed especially by adopting modern biotechnology approach. In this study, plasmids extracted from bacterial isolates associated with cassava waste were explored, using molecular tools, in order to identify genes encoded on the plasmids as well as determine the industrial potentials of the genes borne on the plasmids. Bacterial species isolated from cassava peel (CP) and cassava wastewater (CW) from cassava processing centres in Abeokuta, Nigeria, were identified by aligning their 16S rRNA gene sequences with sequences in the GenBank. Plasmid DNA was extracted from the bacterial isolates, using the Pure Yield Plasmid Miniprep System (Promega, USA) and sequenced. The Open Reading Frame (ORF) Finder was used to identify ORFs on the plasmid DNAs. ORFs were translated and searched against publicly available archives [a non-redundant protein database of GenBank proteins, SWISS-PROT and cluster of orthologous groups (COG)] using the BLAST-P algorithm. Putative genes borne on the plasmids, as well as their products, were deduced from the plasmid nucleotide sequences. Plasmids were found on 14 bacterial isolates. Eight of the isolates (Lactobacillus plantarum, L. brevis, Bacillus coagulans, B. circulans, B. licheniformis, B. pumilus, Enterococcus faecalis and Pediococcus pentosaceus) were from CP while 6 isolates (Lactobacillus fallax, L. fermentum, L. delbruckii, Weisella confusa, Bacillus subtilis and Leuconostoc mesenteroides) were from CW. The gene, tanLpl - encoding tannase was detected on Lactobacillus plantarum plasmid while the gene (bgl1E) which encodes beta- glucosidase was found on Bacillus coagulans and Bacillus circulans plasmids. Other genes detected were hydroxynitrile lyase (HNL) gene on Bacillus licheniformis and Lactobacillus fermentum plasmids; poly-glutamic acid (PGA) synthesis regulator gene on Lactobacillus fermentum plasmid; glutamate synthase gene on Bacillus substilis plasmid; bacteriocin related genes on Lactobacillus fermentum, Lactobacillus fallax and Weisella confusa

* [email protected]. Complimentary Contributor Copy 232 Aniekpeno I. Elijah

plasmids as well as some hypothetical proteins. These enzymes and accessory proteins are all well known for their importance in the food industry. Furthermore, the hypothetical proteins may turn out to be hitherto unknown enzymes for important metabolites or structural proteins. The plasmids could constitute an easy source of genes for mass production of the enzymes and their products. This study, therefore, shows that cassava waste has potentials as an important biotechnology resource, especially for the food industry.

INTRODUCTION

Cassava process wastes, including peels, fibrous core and the carbohydrate rich pressing slurry, account for over 50% of the tuber on a wet weight basis (Adeneye and Sunmonu, 1994). Consequently, a large amount of cassava peel is generated annually. Hou et al. (2007) estimated that about 0.3～0.4 tonne of cassava peel is generated when 1 tonne of starch is produced. Indiscriminate disposal of these wastes contributes significantly to environmental pollution and aesthetic nuisance. With the projected total world cassava utilization of 275 million tonnes by 2020 (Arowolo and Adaja, 2012), resulting from ongoing effort at stimulating cassava production and utilization globally, a more challenging environmental concern is undoubtedly anticipated. Although domestic animals such as pigs, ruminants and poultry may feed on the peels, its use for this purpose is often limited, ultimately, by the high level of toxic cyanogenic glycosides which may constitute a health hazard to the animals. Cassava peel is made up of the rough, brown outer part which consists of lignified cellulosic material and the whiter inner portion which consists of parenchymatous material and contains most of the toxic cyanogenic glucosides. The peel is therefore rich in starch and can be used in some industrial processes. Cassava waste products contain almost 70% water and 30% dry weight. In the dry weight fraction, there is 3.5% protein, 10% crude fibre, 11% lignin, 14%, cellulose, and 27% hemicelluloses (Ratnadewi et al., 2016). There is also a small amount of poisonous cyanogenic glycoside present in cassava waste, which must be reduced to below 10 ppm to make it less poisonous. The major nutrients present in cassava waste are sugars and mineral salts. Cassava liquid waste contains nitrogen, sulphur, carbon and minerals (phosphorus, potassium, calcium, magnesium, zinc, manganese, copper, iron and sodium) (Barana, 2000). Hemicelluloses are the second highest component in cassava waste. Bioconversion of hemicelluloses gets high attention because of its benefit in many fields such as the generation of fuel and chemicals, delignification of paper pulp, clarification of juice, digestibility enhancement of animal feedstuffs in addition to the production of emerging prebiotics, i.e., xylooligosaccharides (Saha, 2003; Aachary et al., 2011). In starch processing, pulp waste is the main problem, especially in bigger factories, which produce massive quantities (Ubalua, 2007). Management of this waste is difficult, as it is not easily dried, due to its high moisture and starch contents (Srioth et al., 2000). This solid residue can also be ensiled. The ensiling process contributes to lower the cyanide level to a non-toxic level thus reducing the pH to about 4.0 and allowing lactic acid to build up, and the product can be used as animal feed (Sackey and Bani, 2007). Considerable research has been conducted and is currently being intensified to maximize the use of cassava waste for useful products. The production of animal feed from cassava peels has been well established (Iyayi and Lossel, 2000; Tweyongyere and Katongole, 2002;

Complimentary Contributor Copy Cassava Waste: A Potential Biotechnology Resource 233

Akinfala and Tewe, 2004). Cassava peels have been enriched in nutrient by fermentation using microorganisms such as yeast and lactic acid bacteria (Oboh, 2006), Aspergillus and Trichoderma species (Obadina et al., 2006). Cassava peels have also been used for the production of functional food (Raupp et al., 2004), ethanol (Adesanya et al., 2008), biofertilizer (Ogbo, 2010), as substrate for mushroom cultivation (Beux et al., 1995; Sonnenberg et al., 2014), and as raw material for xylooligosaccharide production (Ratnadewi et al., 2016). It has also been reported that cassava peels activated carbon is used in the treatment of oil refinery wastewater (Oghenejoboh et al., 2016). Similarly, cassava wastewater has been used for the production of butanol (Wang et al., 2012), organic acids (Pandey et al., 2007), volatile aromatic compounds (Damasceno et al., 2003), biosurfactant (Nitschke and Pastore, 2006) and has even been considered for the production of probiotic beverages (Avancini et al., 2007). However, the status of scientific knowledge in relation to cassava waste microbial genetic resources has been relatively superficial. Cassava waste is a known veritable source of important microorganisms, some of which may have industrial importance. Cassava solid waste degradation is generally initiated by mesophilic heterotrophs, which as the temperature rises, are replaced by thermophilic microorganisms (Ubalua, 2007). Oyeleke and Oduwole (2009) reported 16 strains of Bacillus species isolated from cassava dumpsites. These include Bacillus subtilis, B. macerans, B. megaterium, B. polymyxa and B. coagulans. Similarly, Akpomie et al. (2012) isolated Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, Streptococcus sp., Corynebacterium kutseri, Lactobacillus fermenti, Escherichia coli and Serratia marcescens. Earlier, Cuzin et al. (2001) had reported a new species of the genus Methanobacterium, namely Methanobacterium congolense spp. nov. The strain which is a non-motile, mesophilic, hydrogenotrophic, methanogenic bacterium, was isolated from an anaerobic digester used for the treatment of raw cassava-peel waste in Congo. Fungal species identified in decaying cassava peels include Aspergillus fumigatus and Aspergillus niger (Ogbo, 2010). In addition, Obadina et al. (2006) isolated A. flavus from fermenting cassava solid waste. Cassava starch fermentation wastewater is composed mainly of lactic acid bacteria with predominance of the genera Lactobacillus. Arotupin (2007) identified the microorganisms associated with cassava wastewater to consist of 5 bacteria, 5 moulds and 2 yeasts. The bacteria isolates were Aerococcus viridans, Bacillus substilis, Bacillus] spp. Corynebacterium manihot and Lactobacillus acidophilus, while the fungal (mould) isolates included Aspergillus fumigatus, A. niger, A. repens, Articulospora inflata and Geotrichum candidum. The yeast isolates were Candida utilis and Saccharomyces exiguus. Most of these isolates have been implicated during the processing of cassava into various products (Olowoyo et al., 2000; Akinyosoye et al., 2003). Ahaotu et al. (2011) isolated Alcaligenes faecalis, Lactobacillus plantarum, B. substilis, Leuconostoc cremoris, Aspergillus niger, A. tamari, Geotrichum candidum and Penicillium expansum from cassava wastewater. Out of these, Leuconostoc cremoris, Alcaligenes faecalis, Lactobacillus plantarum and Geotrichum candidum produced linamarase. Similarly, Adamafio et al., (2010) isolated from fermented cassava pulp juice, microorganism such as Aspergillus niger, Aspergillus flavus and Lactobacillus spp. which were capable of reducing the levels of cyanogenic glycosides in cassava peels to non-toxic levels as well as improving the nutritional value of the peels by increasing the protein content appreciably. Recently, Elijah et al. (2014) reported that Bacillus licheniformis and Bacillus substilis are the dominant bacterial species in cassava peel waste while while Lactobacillus fermentum and Complimentary Contributor Copy 234 Aniekpeno I. Elijah

Lactobacillus plantarum are the dominant bacterial species in cassava wastewater. It has also been reported that Aspergillus niger is the dominant fungal species in cassava peel waste while Saccharomyces cerevisiae and Candida krusei are the dominant species in cassava wastewater (Elijah and Asamudo, 2015). It is believed that cassava waste could constitute an important potential biotechnology resource if the diverse cassava waste microbial community is properly exploited especially by adopting modern biotechnology approach. In this study, plasmids extracted from bacterial isolates associated with cassava waste were explored, using molecular tools, in order to identify genes encoded on the plasmids as well as determine the industrial potentials of the genes borne on the plasmids.

MATERIALS AND METHODS

Sample Collection

Cassava peel (CP waste) from CP waste dumpsites and cassava wastewater (CWW) from CWW discharge outlets were collected from major cassava processing centres in Abeokuta, Ogun State, Nigeria and used for the study.

Isolation, Characterization and Identification of Bacterial Species of Bacteria

Bacterial isolates were obtained by seeding serially diluted cassava peel and cassava wastewater samples on appropriate growth medium. Discrete representative colonies were picked from the plates and streaked out on nutrient agar to obtain pure cultures which were transferred to slant and stored in a refrigerator at 4°C. The bacterial isolates were characterized using a culture dependent molecular method.

Bacterial Isolates Genomic DNA Extraction

Genomic DNA extraction from bacterial isolates was also carried out using the DNeasy Blood and Tissue Extraction Kit (Qiagen, USA) following the protocol provided by the manufacturer. Overnight cultures grown in tryptone-soy broth (TSB) were centrifuged for 10 min at 5000 x g, to harvest cells. The pellet was washed 3 times in TE buffer, resuspended in enzymatic lysis buffer (containing 2 mg/ml lysozyme, 25 Mm Tris HCl pH 8, 10 Mm EDTA, 25% sucrose) and incubated at 37°C for 30 min in an incubator (Uniscope SM9052, Surgifriend Medicals, England). Proteinase K and extraction buffer were added, mixed by vortexing and incubated at 56°C in a water-bath (Uniscope SM101 Shaking Water bath, Surgifriend Medicals, England) for 30 min. The DNA was precipitated with ethanol (96 – 100%, v/v) and transferred into the DNeasy Mini spin column for binding of DNA to the column, washed with two different 500 µl washing buffers and eluted with 200 µl elution buffer. The resulting DNA was stored at -20°C.

Complimentary Contributor Copy Cassava Waste: A Potential Biotechnology Resource 235

Plasmid DNA Extraction

Plasmid DNA was extracted from bacterial isolates using the Pure Yield Plasmid Miniprep System (Promega, USA) according to the manufacturer’s instruction. Pure isolates were inoculated in tryptone-soy broth (TSB) and incubated overnight at 37°C. Cells were pelleted (by centrifuging for 10 min at 5000 x g and the supernatant discarded) and resuspended in sterile distilled water prior to lysis. The bacteria culture (600 µl) was transferred into a 1.5 ml microcentrifuge tube. Cell lysis buffer (100 µl) was added and mixed by inverting the tube 6 times. Colour change of the solution from opaque to clear blue indicated complete lysis. About 350 µl of cold (+8°C) neutralization solution was added and mixed thoroughly by inverting the tube. The sample turned yellow when neutralization was complete, forming a yellow precipitate. The resulting suspension was centrifuged at 14,000 x g for 3 min and the supernatant (~900 µl) transferred to a Pure Yield Minicolumn placed into a Pure Yield collection tube and centrifuged at 14,000 x g in a microcentrifuge for 15 s. The flow-through was discarded and the minicolumn was placed in the same Pure Yield collection tube. Endotoxin Removal Wash (200 µl) was added to the column and centrifuged at 14,000 x g in a microcentrifuge for 15 s. About 400 µl of Column Wash Solution was added to the column centrifuged at 14,000 x g in a microcentrifuge for 30 s. The minicolumn was transferred to a clean 1.5 ml microcentrifuge tube; 30 µl of Elution Buffer was added directly to the minicolumn matrix and allowed to stand for 1 min at room temperature (29 - 32°C) after which it was centrifuged at 14,000 x g in a microcentrifuge for 15 s to elute the plasmid DNA which was stored at -20°C.

Amplification of the 16S rRNA Genes

The 16S rRNA gene from bacterial isolates’genomic DNA was amplified by Polymerase Chain Reaction (PCR) using bacterial universal primers (27F – AGAGTTTGAT CCTGGCTCAG and 1492R – GGTTACCTTGTTACGACTT). The amplification was carried out in a Techne TC-412 Thermal Cycler (Model FTC41H2D, Bibby Scientific Ltd, UK) in a 50 µl reactions containing 25 µl of 2 X PCR Master Mix (Norgen Biotek, Canada), 1.5 µl of template DNA (0.5 µg), 1 µl of both forward and reverse primers (2.5 µM of each) and 21.5 µl of nuclease free water in a PCR tube added in that order. PCR was carried out at an initial denaturation step at 94°C for 2 min, followed by 30 cycles at 94°C for 30 sec, 52°C for 30 sec and 72°C for 2 min, and a final extension step at 72°C for 5 min. PCR products (amplicons) as well as plasmid DNA were separated by electrophoresis on a 1% agarose TAE gel containing ethidium bromide and visualized by UV transillumination (Foto/UV 15, Model 3- 3017, Fotodyne, USA).

DNA Sequencing and Analysis

The bacteria 16S rRNA gene from the genomic DNA as well as the plasmid DNA were sequenced with 518F and 800R primers using ABI PRISM Big Dye Terminator cycle sequencer (Macrogen, USA). The gene sequences obtained were compared by aligning the

Complimentary Contributor Copy 236 Aniekpeno I. Elijah result with the sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) search program at the National Centre for Biotech Information (NCBI). The Open Reading Frame (ORF) finder (NCBI) was used to identify protein coding regions in the plasmid DNA sequence.

Plasmid Analysis

Nucleotide sequence analysis of plasmids extracted from some of the bacterial isolates showed that cassava waste has great potentials as important biotechnology resource for industrial applications as novel putative and useful genes were found on their plasmids. The putative genes carried on the plasmids of bacterial isolates from cassava waste, as well as their products, as deduced from plasmid nucleotide sequences are presented in Table 1. Out of the 52 bacterial isolates obtained from both cassava peel and cassava wastewater, plasmids were found only on 14 isolates. Eight of the isolates (Lactobacillus plantarum, L. brevis, Bacillus coagulans, B. circulans, B. licheniformis, B. pumilus, Enterococcus faecalis and Pediococcus pentosaceus) were obtained from cassava peel while 6 isolates (Lactobacillus fallax, L. fermentum, L. delbruckii, Weisella confusa, Bacillus subtilis and Leuconostoc mesenteroides) were obtained from cassava wastewater. Two open reading frames (ORF) encoding two genes were found on plasmid from Lactobacillus plantarum isolated from cassava peel: a hypothetical protein (ORFs 2: 1412- 1612), with an expect value (E-value) of 3e-25, whose region was identical (100% identity) to the conserved hypothetical protein region of Lactobacillus hilgardii ATCC 8290 (accession no. ZP_03954203.1) and the tanLpl gene (ORFs 1: 1 – 303) which encodes tannase (E-value: 3e-38). The open reading frame of the tanLpl gene, spanning 303 bp, encoded a 100 -amino- acid protein that showed 95% similarity to the tannase of Lactobacillus plantarum (BAG68453.1) with several commonly conserved sequences. The gene tanLpl, encoding a possibly novel tannase enzyme (tanLpl), has been identified in Lactobacillus plantarum ATCC 14917 isolated from pickled cabbage, cloned and expressed in E. coli (Iwamotoa et al., 2008). However, the amino acid sequence of the tanLpl (469 aa) reported by the authors was longer than 100aa reported in the present study, although it shared several highly conserved sequences, likely to include catalytic residues, with other known tannase. L. plantarum isolated from various fermented plant materials have also been shown to possess tannase activity (Nishitani et al., 2004). Tannase, or tannin acyl hydrolase (E.C. 3.1.1.20), catalyzes the hydrolysis of the ester bond and the depside bond present in hydrolyzable tannins such as tannic acid to release glucose and gallic acid (Lekha and Lonsane, 1997). The gallic acid, although most commonly used in the pharmaceutical industry for the production of the antibacterial drug trimethoprim (Bajpai and Patil, 1996), is also used as an important substrate for the synthesis of propyl gallate, an antioxidant, in the food industry (Lekha and Lonsane, 1997), and catechin gallates (Raab et al., 2007). A number of innovative applications of tannase have been reported, such as in the enhancement of antioxidant activity and in vitro inhibitory activity against the N-nitrosation of dimethylamine in green tea (Lu and Chen, 2007; 2008), the production of derivatives from prunioside-A with anti-inflammatory activities (Jun et al., 2007), the hydrolysis of epigallocatechin gallates (Battestin et al., 2008), and enzymatic treatment for the nutritive Complimentary Contributor Copy Cassava Waste: A Potential Biotechnology Resource 237 utilization of proteins and carbohydrates from peas (Urbano et al., 2007). Additionally, it is used to reduce the antinutritional effects of poultry and animal feed along with food detanification and industrial effluent treatment (Belmares et al., 2004). Tannic acid - an anti-nutrient which precipitates protein, thereby inhibiting its absorption and utilization, is present in cassava peel and in the tuber itself (Osuntogun et al., 1987). The presence of the organism carrying the gene encoding tannase in cassava waste could imply that this anti-nutrient can be eliminated naturally form cassava tuber and peel by fermentation. This gene could be cloned and successfully overexpressed in Escherichia coli, thereby solving the problem of limited availability of the enzymes for large scale industrial application. The plasmid from Bacillus coagulans carried two functional genes; a replication protein, RepA (ORFs 2: 473 – 775) with an E-value of 2e-58 and the bgl1E gene (ORFs 1: 1 - 249) which encodes beta-glucosidase (E-value of 1e-36). The replication protein was 95% identical to the replication protein region of Lactobacillus plantarum (accession no. YP_002117539.1) while the beta-glucosidase had 89% similarity to beta-glucosidase from an uncultured bacterium (accession no. ACM91556.1), with several commonly conserved sequences. Two novel genes (bgl1D and bgl1E) which encode 172- and 151-aa peptides respectively, have been identified by function-based screening of a metagenomic library from uncultured soil microorganisms and their corresponding recombinant putative beta-glucosidases biochemically characterized (Jiang et al., 2011). Lactobacillus brevis carried a plasmid with two ORFs (2 and 3). ORF 2 (26 - 850) translated into an HNL gene encoding hydroxynitrile lyase (E-value: 0.0). This region was closely identical (99% identity) to the (S)-hydroxynitrile lyase region of Manihot esculenta (accession no. P52705.3). ORF 3 (1005 – 1364) translated into abp118a gene encoding bacteriocin alpha peptide (E- value; 1e-35) which was 100% identical to abp118a acid bacteriocin alpha peptide from Lactobacillus salivarius UCC118 (YP _536804.1). Lactobacillus fermentum plasmid had 3 ORFs (1, 2 and 3) encoding HNL (77- 922) for hydroxynitrile lyase (E-value: 0.0), DNA-binding protein, Ptr (1157-1497) with an E- value of 4e-78 and a polyglutamic acid (PGA)-synthesis regulator, pgsR (1 - 207) with an E-value of 8e-20. The HNL gene was 100% identical to a chain A crystal structure of hydroxynitrile lyase from Manihot esculenta in complex with substrates acetone and chloroacetone, 1DWO. Ptr was 100% identical to DNA-binding protein Ptr, from Bacillus subtilis (NP_049444.1) while pgsR was 98% identical to PGA-synthesis regulator PgsR from Bacillus amyloliquefaciens (YP_003600424.1). The detoxification of cyanogenic glycosides is a two step process involving first a deglycosylation (regulated by β -glucosidases) resulting in a cyanohydrin. Finally, HNLs catalyse the last step of cyanogenesis, i.e., the breakdown of the cyanohydrin to release the corresponding aldehyde or ketone and cyanide. Beta-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) catalyzes the hydrolysis of β-glucosidic linkages of various oligosaccharides and glycosides to form glucose and a shorter/debranched oligosaccharide. It is a key rate-limiting enzyme of the cellulose hydrolyzing system in bacteria and fungi. As cassava contains various amounts of cyanogenic glucosides, bacteria with these enzymes can hydrolyse linamarin to glucose and acetone cyanohydrin and use the glucose for their

Complimentary Contributor Copy

Table 1. Putative genes and their products deduced from plasmid nucleotide sequence

Source Gene name ORF Length (aa) Best hit % identity E- value Proposed identity position (Organism, Genbank accession of gene product (range) number) Lactobacillus tanLpl 1 - 303 100 tannase (Lactobacillus 95 3e-38 Tannase plantarum plantarum, BAG68453.1) 1412- 1612 66 Conserved hypothetical protein 100 3e-25 Hypothetical (Lactobacillus hilgardii ATCC protein 8290, ZP_03954203.1) Bacillus coagulans bgl1E 1 - 249 82 beta-glucosidase (uncultured 89 1e-36 Beta-glucosidase bacterium, ACM91556.1|) RepA 473 – 775 100 Replication protein 95 2e-58 Replication protein (Lactobacillus plantarum, YP_002117539.1) Lactobacillus Hnl 26 - 850 274 (S)-hydroxynitrile lyase 99 0.0 Hydroxynitrile brevis (Manihot esculenta, P52705.3) lyase abp118a 1005 – 1364 108 Abp118 bacteriocin alpha 100 1e-35 Bacteriocin alpha peptide (Lactobacillus peptide salivarius UCC118, YP _536804.1) Lactobacillus Hnl 77- 922 281 A chain A, crystal structure of 100 0.0 Hydroxynitrile fermentum hydroxynitrile lyase from lyase Manihot esculenta in complex with substrates acetone and chloroacetone, 1DWO Ptr 1157-1497 114 DNA-binding protein Ptr 100 4e-78 DNA-binding (Bacillus subtilis, protein Ptr NP_049444.1) pgsR 1 - 207 68 PGA-synthesis regulator PgsR 98 8e-20 Polyglutamic acid (Bacillus amyloliquefaciens, (PGA)-synthesis YP_003600424.1) regulator Lactobacillus 1 -1793 598 glutamate synthase subunit 100 0.0 Glutamate synthase fallax beta (Bacillus subtilis subsp. subtilis str. 168, ZP_03591582.1) Complimentary Contributor Copy

Source Gene name ORF Length (aa) Best hit % identity E- value Proposed identity position (Organism, Genbank accession of gene product (range) number) Lactobacillus 207 – 395 62 hypothetical protein 53 3e-10 Hypothetical fallax CaO19.6256 (Candida protein albicans SC5314, XP_718844.1) Bacillus 1028-1246 72 glutamate synthase subunit 100 5e-16 Glutamate synthase licheniformis beta (Bacillus licheniformis DSM 13, YP_079323.1) 1306-1701 131 putative thioredoxin protein 100 2e-53 Putative (Bacillus licheniformis, thioredoxin protein NP_955636.1) Lactobacillus 1 – 834 277 bacteriocin secretion accessory 98 0.0 Bacteriocin delbrueckii protein (Lactobacillus secretion accessory salivarius ACS-116-V-Col5a, protein ZP_07205827.1) Lactobacillus LSL_1918 1024-1305 93 bacteriocin-like prepeptide 100 7e-40 Bacteriocin-like delbrueckii (Lactobacillus salivarius prepeptide UCC118, YP_536805.1) Bacillus circulans Dtur_1677 1 – 885 294 glycoside hydrolase family 99 0.0 Glycoside protein (Dictyoglomus hydrolase family turgidum DSM 6724, protein YP_002353563.1) DICTH_1569 1068 – 1205 45 6-phospho-beta-glucosidase 79 4e-13 6-phospho-beta- BglT (Dictyoglomus glucosidase thermophilum H-6-12, YP_002251384.1) Weisella confusa abp118b 47 - 253 68 Abp118 bacteriocin beta 90 9e-13 Bacteriocin beta peptide (Lactobacillus peptide salivarius UCC118, YP_536803.1) AbpIM 93 – 293 66 AbpIM bacteriocin immunity 100 3e-06 Bacteriocin protein (Lactobacillus immunity protein salivarius UCC118, YP_536802.1)

Complimentary Contributor Copy

Table 1. (Continued)

Source Gene name ORF Length (aa) Best hit % identity E- value Proposed identity position (Organism, Genbank accession of gene product (range) number) Bacillus subtilis LSL_1822 1 – 240 79 hypothetical protein 96 2e-34 Hypothetical LSL_1822 (Lactobacillus protein salivarius UCC118, YP_536710.1) 313 – 507 64 conserved hypothetical protein 94 7e-13 Hypothetical (Lactobacillus salivarius protein ATCC 11741, ZP_04009819.1) Bacillus pumilus BMQ_pBM60021 1 – 393 130 glycosyl transferase, group 2 100 1e-86 Glycosyl family protein (Bacillus transferase, megaterium QM B1551, YP_003569785.1) pPER272_0129 911 – 1038 43 RNA chaperone Hfq (Bacillus 100 4e-21 RNA chaperone cereus, YP _001966673.1) Pediococcus parA 1 – 656 219 plasmid partition protein 88 3e-123 Plasmid partition pentaceus homolog ParA protein (Corynebacterium glutamicum, NP_862298.1) Enterococcus KPHS_25200 1 - 986 329 unnamed protein product 91 0.0 Putative faecalis (Klebsiella pneumoniae subsp. dehydrogenase pneumoniae HS11286, YP_005226820.1 Leuconostoc LEUM_A23 1 - 659 220 site-specific recombinase, 85 2e-122 site-specific mesenteroides DNA invertase Pin related recombinase, DNA protein (Leuconostoc invertase Pin mesenteroides subsp. related protein mesenteroides ATCC 8293, YP_794196.1)

Complimentary Contributor Copy Cassava Waste: A Potential Biotechnology Resource 241 metabolism. Previous studies have demonstrated the potentials of Bacillus spp. (Ugwuanyi et al., 2007), and Lactobacillus spp. (Adamafio, 2010), for the detoxification of cyanogenic glycoside. The genes encoding these enzymes being borne on the plasmid can easily be manipulated for industrial applications. The polyglutamic acid (PGA) synthesis regulator, pgsR on the Lactobacillus fermentum plasmid is of great significance. PGA is a water-soluble, anionic, biodegradable, and edible biopolymer produced predominantly by bacteria belonging to Bacillus spp., such as B. licheniformis, B. subtilis, B. megaterium, B. pumilis, B. mojavensis and B. amyloliquefaciens (Bajaj and Singhal, 2011). It has multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. Sakai et al. (2000) showed that the addition of PGA had a de-bittering effect to substances having a bitter taste (amino acids, peptides, quinine, caffeine, minerals, etc.), is used for prevention of aging and improvement of textures of starch-based bakery products and noodles, and as an ice cream stabilizer. In addition, PGA is reported to increase bioavailability of calcium by increasing its solubility and intestinal absorption (Tanimoto et al., 2007). Till date, there is no report of production of PGA by Lactobacillus fermentum. The presence of polyglutamic acid (PGA) synthesis regulator gene in Lactobacillus fermentum plasmid seems to suggest the possible presence of PGA synthase, the gene that drives the synthesis of PGA. Ordinarily, a regulatory gene controls the expression of the gene. This is worth further investigation, considering the numerous applications of PGA. Plasmid from Lactobacillus fallax carried 2 genes. These included ORF 1(1- 1793) which encoded glutamate synthase (E-value: 0.0), 100% identical to glutamate synthase subunit beta from Bacillus subtilis subsp. subtilis str. 168 (ZP_03591582.1) and a hypothetical protein (ORF 2: 207 – 395), with an E- value of 3e-10, which was 53% identical to hypothetical protein CaO19.6256 from Candida albicans SC5314, (XP_718844.1). Two genes were also found on Bacillus licheniformis plasmid. The first one was a putative thioredoxin protein (ORF 1:1306 - 1701) with an E-value of 2e-53, which was 100% identical to putative thioredoxin protein from Bacillus licheniformis (NP_955636.1) while the second one was a glutamate synthase gene (ORF 2:1028 - 1246) with an E-value of 5e-16, 100% identical to glutamate synthase subunit beta from Bacillus licheniformis DSM 13 (YP_079323.1). Glutamate synthase is the enzyme responsible for biosynthesis of glutamate, an essential component of the major pathway for ammonia assimilation and a direct nitrogen donor for the biosynthesis of amino acids and other nitrogen-containing compounds. Among the metabolites of a bacterial cell, glutamate is of central importance, since it provides the link between carbon and nitrogen metabolism (Commichau et al., 2008). In Bacillus subtilis, glutamate is synthesized exclusively by the reductive amination of α-ketoglutarate by the enzyme glutamate synthase (Belitsky, 2002). This enzyme produces two molecules of glutamate from α- ketoglutarate and glutamine, the primary product of ammonium assimilation. Of these two molecules, one remains in the cycle, whereas the second can be used for protein biosynthesis or transamination reactions to provide the cell with nitrogen- containing compounds. In addition, glutamate is used in the manufacture of monosodium glutamate-an important food flavour enhancer, indicating that cassava waste has great potentials as important biotechnology resource for the food industry. The plasmid from Lactobacillus delbrueckii had 2 ORFs (1-834 and 1024-1305) encoding bacteriocin secretion accessory protein (E-value: 0.0) which was 98% identical to bacteriocin secretion accessory protein from Lactobacillus salivarius ACS-116-V-Col5a Complimentary Contributor Copy 242 Aniekpeno I. Elijah

(ZP_07205827.1) and a bacteriocin-like prepeptide (E-value:7e-40), 100% identical to bacteriocin-like prepeptide from Lactobacillus salivarius UCC118 (YP_536805.1) respectively. Similarly, Bacillus circulans plasmid had 2 ORFs (1 and 3) encoding a glycoside hydrolase family protein (ORF1:1 – 885; E-value: 0.0) which was 99% identical to glycoside hydrolase family protein from Dictyoglomus turgidum DSM 6724 (YP_002353563.1) and a 6-phospho-beta-glucosidase (ORF 3: 1068 – 1205; E-value: 4e-13) which was 79% similar to 6-phospho-beta-glucosidase BglT from Dictyoglomus thermophilum H-6-12 (YP_002251384.1). Two genes were also found on Weisella confusa plasmid; Abp118 bacteriocin beta peptide (ORF2: 47 – 253; E-value: 9e-13) which was 90% similar to Abp118 bacteriocin beta peptide from Lactobacillus salivarius UCC118 (YP_536803.1) and AbpIM bacteriocin immunity gene (ORF 3:93– 293; E-value: 3e-06) which was 100% identical to AbpIM bacteriocin immunity protein from Lactobacillus salivarius UCC118 (YP_536802.1). Bacteriocin related genes, including bacteriocin alpha peptide, bacteriocin secretion accessory protein, bacteriocin-like prepeptide, bacteriocin beta peptide and bacteriocin immunity protein are most often encoded on plasmids but are occasionally found on the chromosome (Riley, 2009). The activity of some bacteriocins depends on the complementary role of the alpha- and beta- peptides. The immunity gene encodes a protein conferring specific immunity to the producer cell that acts by binding to and inactivating the toxin protein, while the accessory protein appears to be required for secretion of the bacteriocin. The presence of these genes further revealed the hidden potentials in cassava waste. Previously, Lactobacillus lactis and Lactobacillus plantarum isolated from vegetable waste had been reported to be potent producers of bacteriocins (Lade et al., 2006). Similarly, Lactobacillus fermentum (Riaz et al., 2010), Lactobacillus fallax (Kostinek et al., 2005) and Weisella confusa (Ayeni et al., 2011) have been shown to produce bacteriocins. Bacillus substilis plasmid carried a gene for a hypothetical protein (ORF 1: 1 – 240; E- value: 2e-34), a region closely identical (96% similarity) to the hypothetical protein LSL_1822 region of Lactobacillus salivarius UCC118 (YP_536710.1) and a conserved hypothetical protein (ORF 1: 313- 507; E-value: 7e-13) which was 94% identical to the conserved hypothetical protein from Lactobacillus salivarius ATCC 11741 (ZP_04009819.1). Similarly, Bacillus pumilus plasmid carried two genes encoding a glycosyl transferase, group 2 family protein (ORF 1: 1 – 393; E-value: 1e-86) which was 100% identical to the glycosyl transferase group 2 family protein from Bacillus megaterium QM B1551 (YP_003569785.1), and an RNA chaperone (ORF 2: 911 – 1038; E-value: 4e-21), a region identical (100% identity) to the RNA chaperone Hfq of Bacillus cereus (YP_001966673.1). The Pediococcus pentaceus plasmid carried only one functional gene, a plasmid partition protein homolog, ParA (ORF1: 1 – 656; E-value: 3e-123). This region was somewhat identical (88% similarity) to the plasmid partition protein homolog ParA region of Corynebacterium glutamicum (NP_862298.1). Similarly, Enterococcus faecalis plasmid had an ORF (1:1 – 986; E-value: 0.0) which encoded an unnamed protein product. This region was 91% identical to an unnamed protein product region of Klebsiella pneumoniae subsp. pneumoniae HS11286 (YP_005226820.1). Also, the Leuconostoc mesenteroides plasmid had an ORF (1:1 – 659; E- value: 2e-122) encoding a site-specific recombinase, DNA invertase Pin related protein, 85% identical to the site-specific recombinase, DNA invertase Pin related protein from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 (YP_794196.1).

Complimentary Contributor Copy Cassava Waste: A Potential Biotechnology Resource 243

The gene encoding site-specific recombinase borne on the plasmid of Leuconostc mesenteroides isolated from cassava wastewater could be a useful tool for DNA recombination technology. Site-specific recombinases are able to recombine specific sequences of DNA with high fidelity without the need for cofactors (Dymecki, 2000). For this reason, they have been used effectively to create gene deletions, insertions, inversions, and exchanges in exogenous systems (Branda et al., 2004). This technology is quite attractive since it enables the precise integration of transgenes of interest into pre-defined integration sites, thereby allowing the prediction of the expression properties of a genetically manipulated cell. Lack of control over the copy number and position of the integrated DNA molecules in the chromosome(s) results in an unpredictable transgene expression pattern (Wirth et al. 2007). This affects not only the level of transgene expression and long-term stability but may also cause undesired disturbance of nearby host genes. Though most described site-specific recombinases are from prokaryotes, they are not limited to prokaryotes (Wirth et al. 2007), and so could function in eukaryotes, providing a useful biotechnological tool. Other genes isolated encoded on the bacterial plasmids such as replication protein (Rep A), DNA-binding protein, putative thioredoxin, glycoside hydrolase family protein, glycosyl transferase, RNA chaperone, plasmid partition protein (parA), putative dehydrogenase, site- specific recombinase, DNA invertase Pin related protein, and many hypothetical proteins whose functions are unknown play vital roles in maintaining the integrity of the cell. The unknown proteins are subjects for further research, to determine their identity and potential uses. Replication protein (Rep A) is one of the DNA replication accessory proteins which are universally found in nature. It is an essential protein for viability of the cell that participates in DNA replication, DNA repair (nucleotide excision repair), and homologous DNA recombination (Longhese et al,. 1994). DNA-binding proteins (DBP) are proteins that are composed of DNA-binding domains and thus have a specific or general affinity for either single or double stranded DNA. They include transcription factors which modulate the process of transcription, various polymerases, nucleases which cleave DNA molecules, and histones which are involved in chromosome packaging and transcription in the cell nucleus. They can incorporate such domains as the zinc finger nucleases, the helix-turn-helix, and the leucine zipper (among many others) that facilitate binding to nucleic acid. DBP are critically important in the regulation of a variety of essential cellular processes, such as genome replication, gene transcription, cell division, and DNA repair (Liu et al., 2012). Thioredoxin are a group of small (10- to 12-kDa) ubiquitous proteins which have a conserved CXXC catalytic site that undergoes reversible oxidation/reduction of both cysteine residues. The thioredoxin system plays several key roles in maintaining the redox balance inside the cell and responding to oxidative stress in all three domains of life (Hirt et al., 2002). In addition to functioning as an electron donor, thioredoxin is an essential component of the T7 DNA polymerase (Ye et al., 2007). RNA chaperones are proteins that interact with RNA molecules to solve the RNA folding problem (Schroeder et al., 2004), by preventing misfolding or by resolving misfolded species, thereby ensuring that RNA is accessible for its biological function. This is in contrast to proteins that help protein or RNA folding by catalyzing steps along the folding pathway or by stabilizing the final folded protein or RNA structure (Herschlag, 1995). RNA molecules have Complimentary Contributor Copy 244 Aniekpeno I. Elijah the tendency to fold into diverse secondary structures, and these alternative misfolded structures have to be resolved in order for the RNA molecules to function normally. Plasmid partition protein (parA) is critical to the survival of any bacterial species as it is required for the faithful inheritance of genetic information to the offspring. The stable maintenance of a plasmid in a bacterial cell depends on effective replication followed by partition of newly synthesized plasmid particles between newborn cells. Low copy number bacterial plasmids fulfill this requirement by encoding partitioning systems, similar to those found in their hosts. Plasmid partitioning systems have been divided into types I–III, based upon the homology of their filament-forming proteins to known protein families (Salje, 2010). Type I plasmid partitioning systems is distinguished by two proteins, often called ParA and ParB. The ParA-like protein is an ATPase, and the ParB-like protein is a site-specific DNA-binding protein that recognizes the partition site(s). ParA and ParB are required for two distinct functions in P1 partition: regulation of par gene expression and physical segregation of the plasmids (Bignell and Thomas, 2001). ParA’s regulatory role is as the transcriptional repressor of the par operon. ParB improves the repressor activity of ParA and is therefore a co-repressor.

CONCLUSION

This study has shown that cassava waste, on account of its rich microbial genetic resources, is an important biotechnology resource with great industrial potential that could be exploited for the benefit of mankind. The enzymes and accessory proteins are of great significance to the food industry. The hypothetical proteins may turn out to be hitherto unknown enzymes for important metabolites or structural proteins. Therefore, these plasmids constitute an easy source of genes for mass production of the enzymes and their products, using appropriate host cells such as E. coli.

REFERENCES

Aachary, A. A. and Prapulla, S. G. (2011). Xylooligosaccharides (XOS) as an emerging prebiotic: microbial synthesis, utilization, structural characterization, bioactive properties and applications. Comprehensive Reviews in Food Science and Food Safety, 10: 2–16. Adamafio, N. A., Sakyiamah, M. and Tettey, J. (2010). Fermentation in cassava (Manihot esculenta Crantz) pulp juice improves nutritive value of cassava peel. African Journal of Biochemistry Research, 4(3): 51-56. Adeneye, J. A. and Sunmonu, E. M. (1994). Growth of male WAD sheep fed cassava waste or dried sorghum brewer's grain as supplements to tropical grass/legume forage. Small Ruminant Research, 13: 243-249. Adesanya, O. A., Oluyemi, K. A., Josiah, S. J., Adesanya, R. A., Shittu, L. A. J., Ofusori, D. A., Bankole, M. A. and Babalola, G. B. (2008). Ethanol production by Saccharomyces cerevisiae from cassava peel hydrolysate. The Internet Journnal of Microbiology, 5(1): DOI: 10.5580/4f1.

Complimentary Contributor Copy Cassava Waste: A Potential Biotechnology Resource 245

Ahaotu, C. C., Ogueke, C. I., Owuamanam, N. N., Ahaotu and Nwosu, J. N. (2011). Protein improvement in gari by the use of pure cultures of microorganisms involved in the natural fermentation process. Pakistan Journal of Biological Science, 14: 933-938. Akinfala, E. O and Tewe, O. O. (2004). Supplementatal effects of feed additives on the utilization of whole cassava plant by growing pigs in the tropics. Livestock Research for Rural Development, 16: 10. Akinyosoye, F. A., Adeniran, A. H. and Oboh, G. (2003). Production of fungal amylase from agroindustrial wastes. Peer-reviewed Proceeding of the 16th Annual Conference of Biotechnology Society of Nigeria, 35-40. Akpomie, O. O., Akponah, E. and Okorawhe, P. (2012). Amylase production potentials of bacterial isolates obtained from cassava root peels. Agricultural Science Research Journal, 2(2): 95 – 99. Arotupin, D. J. (2007). Evaluation of microorganisms from cassava wastewater for production of amylase and cellulose. Research Journal of Microbiology, 2(5): 475-480. Arowolo, A. A., Adaja, J. I. (2012). Investigation of Manihot Esculenta and Manihot Duleis Production in Nigeria: An Econometric Approach. Journal of Applied Science and Environment, 3:18-24. Avancini, S. R. P., Faccin, G. L., Vieira, M. A., Rovaris, A. A., Podesta, R., Tramonte, R., de Souza, N. M. A. and Amante, E. R. (2007). Cassava starch fermentation wastewater: characterization and preliminary toxicological studies. Food and Chemical Toxicology, 45: 2273 – 2278. Ayeni, F. A., Sánchez, B., Adeniyi, B. A., de los Reyes-Gavilán, C. G., Abelardo Margolles, A. and Ruas-Madiedo, P. (2011). Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow's intestine. International Journal of Food Microbiology, 147: 97–104. Bajaj, I. and Singhal, R. (2011). Poly(glutamic acid) – An emerging biopolymer of commercial interest. Bioresource Technology, 102: 5551–5561. Barana, A. C. (2000). Avalicao de tratamento de manipuiera em biodigestores fase acidogenica e metanogenica. Botucatu: UNESP/FCA (TESE- Doutorado)., p. 95. Battestin, V., Macedo, G. A. and De Freitas, V. A. P. (2008). Hydrolysis of epigallocatechin gallate using a tannase from Paecilomyces variotii. Food Chemistry, 108: 228-233. Belitsky, B. R. (2002). Biosynthesis of amino acids of the glutamate and aspartate families, alanine, and polyamines. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. American Society for Microbiology, Washington, DC. p. 203–231. Belmares, R., Contreras-Esquivel, J. C., Rodríguez-Herrera, R., Coronel, A. R. and Aguilar, C. N. (2004). Microbial production of tannase: an enzyme with potential use in food industry. Lebensmittel-Wissenschaft und -Technologie, 37: 857–864. Beux, M. R., Soccol, C. R., Marin, B., Tonial, T. and Roussos, S. (1995). Cultivation of Lentinus edodes on the mixture of cassava bagasse and sugarcane bagasse. In: Roussos, S., Lonsane, B.K., Raimbault, M. and Viniegra-Gonzalez, G. (eds.) Advances in Solid State Fermentation. Kluwer Academic Publishers, Dordrecht, pp. 499 – 511. Bignell, C. R. and Thomas, C. M. (2001). The bacterial ParA-ParB partitioning proteins. Journal of Biotechnology, 91: 1-34.

Complimentary Contributor Copy 246 Aniekpeno I. Elijah

Branda, S. S., Gonza´lez-Pastor, J. E., Dervyn, E., Ehrlich, S. D., Losick, R. and Kolter, R. (2004). Genes involved in formation of structured multicellular communities by Bacillus subtilis. Journal of Bacteriology, 186(12): 3970–3979. Commichau, F. M., Gunka, K., Landmann, J. J. and Stulke, J. (2008). Glutamate metabolism in bacillus subtilis: gene expression and enzyme activities evolved to avoid futile cycles and to allow rapid responses to perturbations of the system. Journal of Bacteriology, 190(10): 3557–3564. Cuzin, N., Ouattara, A. S., Labat, M. and Garcia, J. (2001). Methanobacterium congolense sp. nov., from amethanogenic fermentation of cassava peel. International Journal of Systematic and Evolutionary Microbiology, 51: 489–493. Damasceno, S., Cereda, M.P., Pastore, G.M. and Oliveira, J.G. (2003). Production of volatile compounds by Geotrichum fragrans using cassava wastewater as substrate. Process Biochemistry., 39: 411 – 414. Dymecki S. M. (2000). Site-speciﬁc recombination in cells and mice. In: Joyner, A. L. (ed.) Gene targeting: A practical approach. Oxford: Oxford University Press. p 37–100. Elijah, A. I. and Asamudo, N. U. (2015). Molecular Characterization and Potential of Fungal Species Associated with Cassava Waste. British Biotechnology Journal, 10(4): 1-15. Elijah, A. I., Atanda, O. O., Popoola, A. R. and Uzochukwu, S. V. A. (2014). Molecular Characterization and Potential of Bacterial Species Associated with Cassava Waste. Nigerian Food Journal, 32 (2):56 – 65. Herschlag, D. (1995). RNA Chaperones and the RNA Folding Problem. Journal of Biological Chemistry, 270(36): 20871–20874. Hirt, R. P., Muller, S., Embley, T. M. and Coombs, G. H. (2002). The diversity and evolution of thioredoxin reductase: new perspectives. Trends in Parasitology, 18: 302–308. Hou, W., Chen, X., Song, G., Wang, Q. and Chang, C.C. (2007). Effect of copper and cadmium on heavy metal polluted water body restoration by duckweed (Lemna minor). Plant Physiology and Biochemistry, 45: 62–69. Iyayi, E. A and Lossel, D. M. (2000). Protein enrichment of cassava by-products through solid state fermentation by fungi. The Journal of Technology in Africa, 6(40): 116-118. Jiang, C., Li, S-X., Luo, F-F., Jin, K., Wang, Q., Hao, Z-Y., Wua, L-L., Zhao, G-C Maa, G- F., Shen, P-H., Tang, X-L. and Wua, B. (2011). Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning. Bioresource Technology, 102: 3272–3278. Jun, C. S., Yoo, M. J., Lee, W. Y., Kwak, K. C., Bae, M. S., Hwang, W. T., Son, D. H. and Chai, K. Y. (2007). Ester derivatives from tannase-treated prunioside A and their anti- inflammatory activities. Bulletin of the Korean Chemical Society, 28: 73-76. Kostinek, M., Specht, I., Edward, V. A., Schillinger, U., Hertel, C., Holzapfel, W. H. and Franz, C. M. A. P. (2005). Diversity and technological properties of predominant lactic acid bacteria from fermented cassava used for the preparation of Gari, a traditional African food. Systematic and Applied Microbiology, 28: 527–540. Lade, H. S., Chitanand, M. P., Gyananath, G. and Kadam, T. A. (2006). Studies on some properties of bacteriocins produced by Lactobacillus species isolated from agro-based waste. The Internet Journal of Microbiology, 2(1). Lekha, P. K. and Lonsane, B. K. (1997). Production and application of tannin acyl hydrolsase: state of the art. Advances in Applied Microbiology, 44: 215–260.

Complimentary Contributor Copy Cassava Waste: A Potential Biotechnology Resource 247

Liu, Y., Toh, H., Sasaki, H., Zhang, X. and Cheng, X. (2012). An atomic model of Zfp57 recognition of cpg methylation within a specific DNA sequence. Genes and Development, 26: 2374-2379. Longhese, M. P., Plevani, P. and Lucchini, G. (1994). Replication factor A is required in vivo for DNA replication, repair, and recombination. Molecular and Cellular Biology, 14: 7884-7890. Lu, M. J. and Chen, C. (2007). Enzymatic tannase treatment of green tea increases in vitro inhibitory activity against N-nitrosation of dimethylamine. Process Biochemistry, 42: 1285-1290. Lu, M. J. and Chen, C. (2008). Enzymatic modification by tannase increases the antioxidant activity of green tea. Food Research International, 41: 130-137. Nishitani, Y., Sasaki, E., Fujisawa, T. and Osawa, R. (2004). Genotypic analyses of Lactobacilli with a range of tannase activities isolated from human faeces and fermented foods. Systematic and Applied Microbiology, 27: 109–117. Nitschke, M., Pastore, G.M. (2006). Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Biores. Technol., 97: 336 – 341. Obadina, A. O., Oyewole, O. B., Sanni, L. O., and Abiola, S. S. (2006). Fungal enrichment of cassava peels protein. African Journal of Biotechnology, 5 (3): 302-304. Oboh, G. (20060. Nutrient enrichment of cassava peels using a mixed culture of Saccharomyces cerevisiae and Lactobacillus spp. Solid media fermentation. Electronic Journal of Biotechnology, 9(1): 46-49. Ogbo F. C. (2010). Conversion of cassava wastes for biofertilizer production using phosphate solubilizing fungi. Bioresource Technology, 101: 4120–4124. Oghenejoboh, K. M., Otuagoma, S. O. and Ohimor, E. O. (2016). Application of cassava peels activated carbon in the treatment of oil refinery wastewater – a comparative analysis. Journal of Ecological Engineering, 17(2): 52–58. Olowoyo, O. O., Akinyosoye, F. A. and Adetuyi, F. C. (2000). Micro-organisms associated with some cassava (Manihot esculenta, Crantz) products. J. Res. Rev. Sci. 2: 10-14. Osuntogun, B. A., Adewusi, S. R. A., Telek, L. and Oke, O. L. (1987). The effect of tannin content on the nutritive value of some leaf protein concentrates. Human Nutrition: Food Science and Nutrition, 41(F):41-46. Oyeleke, S. B. and Oduwole, A. A. (2009). Production of amylase by bacteria isolated from a cassava waste dumpsite in Minna, Niger State, Nigeria. African Journal of Microbiology Research, 3 (4): 143 – 146. Pandey, J., Ganesan, K. and Jain, R.K. (2007). Variations in T-RFLP profiles with differing chemistries of fluorescent dyes used for labelling the PCR primers. Journal of Microbiological Methods., 68: 633 – 638. Raab, T., Bel-Rhlid, R., Williamson, G., Hansen, C. E. and Chaillot, D. (2007). Enzymatic galloylation of catechins in room temperature ionic liquids. Journal of Molecular Catalysis, 44: 60-65. Ratnadewi, A. A. I., Santosoa, A. B., Erma Sulistyaningsih, E. and Wuryanti Handayania, W. (2016). Application of Cassava Peel and Waste as Raw Materials for Xylooligosaccharide Production using Endoxylanase from Bacillus subtilis of Soil Termite Abdomen. Procedia Chemistry, 18: 31 – 38.

Complimentary Contributor Copy 248 Aniekpeno I. Elijah

Raupp, D. d-S., Rosa, D. M., Marques, S. H. d-P. and Banzatto, D. A. (2004). Digestive and functional properties of a partially hydrolyzed cassava solid waste with high insoluble fiber concentration. Scientia Agricola (Piracicaba, Braz.) 61(3): 286 – 291. Riaz, S., Nawaz, S. K. and Hasnain, S. (2010). Bacteriocins produced by Lactobacillus fermentum and Lactobacillus acidophilus can inhibit cephalosporin resistant E. coli. Braziliann Journal of Microbiology, 41: 643-648. Riley, M. A. (2009). Bateriocins, biology, ecology and evolution. Encyclopedia of Microbiology. (Moselio Schaechter, Ed.), Oxford: Elsevier, pp. 32-44. Sackey, I. S. and Bani, R. J. (2007). Survey of waste management practices in cassava processing to gari in selected districts of Ghana. Journal of Food Agriculture and Environment, 5 (2): 325-328. Saha, B. C. (2003). Hemicelluloses bioconversion. Journal of Industrial Microbiology and Biotechnology, 30: 279–291. Sakai, K., Sonoda, C., Murase, K. (2000). Bitterness relieving agent. J. P. patent WO0021390. Salje, J. 2010. Plasmid segregation: how to survive as an extra piece of DNA. Critical Reviews in Biochemistry and Molecular Biology, 45(4): 296–317. Schroeder, R., Barta, A. and Semrad, K. (2004). Strategies for RNA folding and assembly. Nature Reviews Molecular Cell Biology, 5: 908–919. Sonnenberg, A. S. M., Baars, J. J. P., Obodai, M. and Asagbra, A. (2014). Cultivation of oyster mushrooms on cassava waste. Proceedings of the 8th International Conference on Mushroom Biology and Mushroom Products (ICMBMP8), 286 – 291. Srioth, K., Chollakup, R., Chotineeranat, S., Piyachomkwan, K. and Oates, C. G. (2000). Processing of cassava waste for improved biomass utilization. Bioresource Technology, 71(1): 63-69. Tanimoto, H., Fox, T., Eagles, J., Satoh, H., Nozawa, H., Okiyama, A., Morinaga, Y., Susan, J. and Fairweather-Tait, S. J. (2007). Acute effect of poly-glutamic acid on calcium absorption in post-menopausal women. Journal of the American College of Nutrition, 26 (6): 645–649. Tweyongyere, R and Katongole, I. (20020. Cyanogenic potential of cassava peels and their detoxification for utilization as livestock feed. Veterinary and Human Toxicology, 44(6): 366-369. Ubalua, A. O. (2007). Cassava wastes: Treatment options and value addition alternatives. African Journal of Biotechnology, 6 (18): 2065-2073. Ugwuanyi, J.O., Harvey, L.M., McNeil, B. (2007). Linamarase activities in Bacillus spp. Responsible for thermophilic aerobic digestion of agricultural wastes for animal nutrition. Waste Management, 27: 1501-1508. Urbano, G., Lopez-Jurado, M., Porres, J. M., Frejnagel, S., Gomez-Villalva, E., Frias, J., Vidal-Valverde, C. and Aranda, P. (2007). Effect of treatment with α-galactosidase, tannase or a cell-wall-degrading enzyme complex on the nutritive utilization of protein and carbohydrates from pea (Pisum sativum L.) flour. Journal of the Science of Food and Agriculture, 87: 1356-1363. Wang, W., Xie, L., Luo, G. and Lu, Q. (2012). Optimization of biohydrogen and methane recovery within a cassava ethanol wastewater/waste integrated management system. Bioresource Technology, 120: 165 – 172.

Complimentary Contributor Copy Cassava Waste: A Potential Biotechnology Resource 249

Wirth, J., Chopin, F., Santoni, V., Viennois, G., Tillard, P., Krapp, A., Lejay, L., Daniel- Vedele, F. and Gojon, A. (2007). Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana. Journal of Biological Chemistry, 282: 23541–23552. Ye, J., Cho, S- H., Fuselier, J., Li, W., Beckwith, J. and Rapoport, T. (2007). A Crystal structure of an unusual thioredoxin protein with a zinc finger domain. Journal of Biological Chemistry, 282(48):34945–34951.

BIOGRAPHICAL SKETCH

Aniekpeno Isaac Elijah, PhD

Affiliation: Department of Food Science and Technology, University of Uyo, Uyo, Akwa Ibom State, Nigeria

Education: Ph.D. Food Microbiology and Biotechnology (2013) - Federal University of Agriculture, Abeokuta, Nigeria M.Sc. Food Processing and Preservation (2004) - Michael Okpara University of Agriculture, Umudike, Nigeria B. Sc. Brewing Science and Technology (2000) - University of Uyo, Nigeria

Research and Professional Experience: Senior Lecturer 2013 till date Lecturer I 2010 – 2013 Lecturer II 2007 – 2010 Assistant Lecturer 2005 - 2007 Teaching and supervision of postgraduate and undergraduate students

Professional Appointments: (i) Secretary, Nigerian Institute of Food Science and Technology South East Chapter, Nigeria - June 2013 till Feb., 2016 (ii) Co-ordinator, Nigerian Institute of Food Science and Technology, Uyo Zone, Nigeria- June 2013 till date (iii) Secretary LOC, Nigerian Institute of Food Science and Technology, South East Chapter Food Summit - June 2014

Honors: Best Graduating Student, Faculty of Natural and Applied Sciences, University of Uyo – 2000

Publications: 1. Elijah, A.I. and Asamudo, N. U. 2015. Molecular characterization and potential of bacterial species associated with cassava waste. British Biotechnology Journal, 10(4):1-15.

Complimentary Contributor Copy 250 Aniekpeno I. Elijah

2. Elijah, A.I., Atanda, O.O., Popoola A.R and Uzochukwu, S.V.A. 2015. Diversity and techno-functional properties of bacterial species associated with cassava waste. Proceedings of the 2nd International Conference on Food and Biosystems Engineering, Mykonos Island, Greece, 28th to 31st May 2015. 3. Elijah, A.I., Atanda, O.O., Popoola A.R and Uzochukwu, S.V.A. 2014. Molecular characterization and potential of bacterial species associated with cassava waste. Nigerian Food Journal, 32(2): 57-66. 4. Elijah, A.I., Atanda, O.O., Popoola A.R and Uzochukwu, S.V.A. 2014. Genes of industrial importance on cassava waste bacteria plasmids. Proceedings of the 17th IUFoST World Congress of Food Science and Technology, Montrael, Canada, 17th - 21st August, 2014. 5. Umo-udofia, S. J., Edem, V. E. and Elijah, A. I. 2014. Effect of soaking time and storage period on the moisture content of smoked bonga fish (Ethmalosa fimbriata). Book of extended abstract for the 1st NIFST South-East Chapter Food Summit, Uyo, 38 – 40. 6. Umo-udofia, S. J., Edem, V. E. and Elijah, A. I. 2014. Microbilogical quality and sensory attributes of smoked bonga fish (Ethmalosa fimbriata) obtained from Oron, Akwa Ibom State, Book of extended abstract for the 1st NIFST South-East Chapter Food Summit, Uyo, 40 – 43. 7. Ojimelukwe, P., Elijah, A., Ekong, U. and Nwokocha, K. 2013. Effect of different preservatives on the shelf-life of Kunun zaki A traditional fermented cereal based non-alcoholic beverage. Nigerian Journal of Agriculture, Food and Environment, 9(1): 76 – 79. 8. Adamu, L., Edeghagba, B., Abiola, O., Elijah, A. I. and Ezeokoli, O. 2013. Antimicrobial activity of extracts of Jatropha curcas and Calotropis procera leaves against pathogenic isolates from motorcycle helmets in Lagos metropolis. International Journal of Current Microbioliology and Applied Science, 2(12): 292- 302. 9. Elijah, A. and Ojimelukwe, P. 2013. Use of botanicals in palm wine preservation. Lambert Academic Publishing, Deutsshland, Germany.

Chapter 13

POTENTIAL USES OF CASSAVA PRODUCTS AND ITS FUTURE CHALLENGING OPPORTUNITIES

Reddy T. Ranjeth Kumar, Kim Hyun-Joong* and Park Ji-Won 1Lab. Of Adhesion and Bio-Composites, Program in Environmental Materials Science, College of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea

ABSTRACT

Cassava is the third largest source of food carbohydrates in the tropics after rice and maize. Cassava is a major staple food in the developing world, providing a basic diet for over half a billion people. Cassavas are multipurpose commercial products that have many potential uses, such as in bio-fuels, animal feed, medicines, bio-composite, food packaging and so on. Apart of from these uses, processed cassava serves as an industrial raw material for the production of adhesives, bakery products, dextrin, dextrose, glucose, lactose and sucrose. This chapter elucidates the uses of cassava products and its future challenging opportunities.

Keywords: cassava, products, applications

INTRODUCTION

Cassava is the most important cultivated crop in the tropics after rice and corn. Sometimes, cassava is branded as a “third world crop”. It belongs to the family Euphorbiaceae and is referred to as Manihotesculenta Crantz. (Euphorbiaceae) botanically, as shown in Figure 1. The genus Manihot comprises 98 species, of which M. esculenta is the

* Corresponding authors address: Prof. Hyun-Joong Kim. Lab. Of Adhesion & Bio-Composites, Program in Environmental Materials Science, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea. Email: [email protected]. Complimentary Contributor Copy 252 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won most widely cultivated member [1]. Cassava originated in South America and subsequently has been distributed to tropical and subtropical regions of Africa and Asia. The enlarged tuberous root of the cassava plant contains carbohydrates of a highly nutritional value, with only rice and sugar cane having more carbohydrates [2]. The derived products from cassava are the staple food for more than 1 billion people due to the high starch content. By the 1920- 30s, cassava had attained its present status as one of the major African staple food crops, and currently, the total annual production of approximately 85 million tons is greater than that of any other crop in Africa. At the end of the 20th century, cassava had a vital role in the economic life of sub-Saharan Africa, both as a reliable food source for rural and urban populations and as an important source of income through the sale of fresh and processed produce. It was estimated that, on average, each African eats nearly 80 kg of cassava annually. Due to many superior properties and potential nutritional value, cassava has been the focus of research and development and has been identified as a good commodity source for the wider growth of the economy [3-4]. The cassava plant can be continuously harvested, growing and yielding well under conditions of marginal soil and low acidic rainfall. Moreover, this is an attractive energy crop due to its high carbohydrate content, superior starch conversion for ethanol, high water-use efficiency, and high rate of CO2 fixation. All of these characteristics make it a commonly grown, low-cost crop that is well suited for small-scale biofuel feedstock production. Cassava has been used for bioethanol production in Brazil and Asia for several years, and several studies have investigated this potential in sub-Saharan Africa [5]. Cassava is categorized into two types: sweet and bitter. Due to deterioration by pests, animals, and thieves, most cultivators prefer the bitter type of cassava. The roots and tubers of both the sweet and bitter varieties contain toxins and anti-nutritional factors. Before consumption of cassava, it must be prepared properly. Often, improper preparation of cassava can leave enough residual cyanide to cause acute cyanide intoxication and goiters and may even cause ataxia or partial paralysis. The more toxic varieties of cassava are a fallback resource (a “food security crop”) in times of famine in some places [6]. According to United States Department of Agriculture, Agricultural Research Service, National Nutrient Database for Standard Reference Release 28 basic report 11134, the nutritional content of raw cassava is listed in Table 1 [7].

Figure 1. Cassava plant [6].

Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 253

Table 1. Nutrient information of raw cassava

Nutrient Unit Value per 100 g Proximate Water g 59.68 Energy kcal 160 Protein g 1.36 Total lipid (fat) g 0.28 Carbohydrate, by difference g 38.06 Fiber, total dietary g 1.8 Sugars, total g 1.7 Minerals Calcium, Ca mg 16 Iron, Fe mg 0.27 Magnesium, Mg mg 21 Phosphorus, P mg 27 Potassium, K mg 271 Sodium, Na mg 14 Zinc, Zn mg 0.34 Copper, Cu mg 0.1 Manganese, Mn mg 0.384 Selenium, Se µg 0.7 Vitamins Vitamin C, total ascorbic acid mg 20.6 Thiamin mg 0.087 Riboflavin mg 0.048 Niacin mg 0.854 Vitamin B-6 mg 0.088 Folate, total µg 27 Vitamin A, IU IU 13 Vitamin E (alpha-tocopherol) mg 0.19 Vitamin K (phylloquinone) µg 1.9

BIO-COMPOSITES WITH CASSAVA

There is lot of interest in replacing nondegrable materials with degradable materials in many areas. Most studies have focused on biopolymers, such as natural biopolymers, synthetic biodegradable polymers and biopolymers that are formed by microbial fermentation and are used for making biodegradable composites, and among those biopolymers, natural biopolymers, such as starch, show good promise for making biodegradable composites. Starch is a promising candidate due to its many superior properties, such as its low cost, renewable, recyclable, biodegradable, thermoplastic behavior and abundant availability. Conversely, there are also some drawbacks to the use of starches, such as high water solubility, poor melting processability, and difficulty in processing and brittleness, which necessitates the use of a plasticizer to make them suitable for engineering applications. Starch Complimentary Contributor Copy 254 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won can be processed into thermoplastic starch by breaking its structure under high temperature and shear stress; this causes intermolecular rearrangement due to de-structuring of the starch chains. In this de-structuring of the starch chains, plasticizers, such as glycerin, play a vital role. Furthermore, the properties of this thermoplastic starch can change depending on the concentration of plasticizer used. For example, the cassava starch glass transition temperature (Tg) is reported to be 131.9°C, and this temperature decreases with the addition of glycerol. At 30% glycerol content, the value of Tg is 62.2°C. The tensile properties are also reported to change accordingly [8]. Moreover, the mechanical properties of starch could be improved by using reinforcement with natural fibers, such as from kenaf, jute, sugarcane fiber, flax, sisal, bagasse and other cellulose fibers. Composites with this reinforcement of fibers are referred to as “bio- composites” or “green composites”. These composites should be considered eco-friendly composites because of their superior biodegradability and compostability without any damage to the environment. Additionally, natural fibers are not only used to enhance the properties of biopolymers but have other advantages as well, such as their abundant availability, renewability and low cost. The enhancement of properties was observed in the preparation of bio-composites with cassava starch and green coconut fiber. In this study, composites were prepared using a compression molding process, and their characterization regarding their tensile properties and water absorption properties were analyzed before and after thermal treatment of the matrix and composites. The tensile strength and Young’s modulus of the composites containing 30% of coir fibers in a thermoplastic starch matrix was shown to be enhanced by 212.2% and 366.7%, respectively, for treated thermoplastic starch composites compared with untreated thermoplastic starch composites. Conversely, the water uptake, moisture absorption and swelling properties of thermoplastic starch decreased with an increase in the coir fiber content [9]. Souza et al. observed the influence of glycerol and clay nanoparticles on the tensile, barrier and glass transition temperatures of cassava starch biodegradable films. The incorporation of a lower content of glycerol and sugar-derived plasticizers were compatible with the starch to enhance the tensile and barrier properties compared with a higher content these additives. In addition, the tensile and barrier properties were significantly influenced by adding both glycerol and clay nanoparticles, whereas the permeability was greatly decreased when clay nanoparticles were present, and the glass transition temperature did not change [10]. Matsui et al., analyzed cassava bagasse-kraft paper composites with the addition of starch acetate. Cassava bagasse, which is extracted from the industrial production of cassava starch and used to obtain a cardboard composite, is used in small-scale artisan production of recycled paper. A mixture of 90% cassava bagasse and 10% kraft paper was used for the production of these composites. However, the addition of kraft fiber to cassava bagasse improves the properties of the material. These materials have similar characteristics to the molded fiber packaging made using recycled paper, such as the material used in egg boxes. The prepared composites were immersed in water to study the water absorption properties, and the composites showed slight resistance to direct water contact. The effect of starch acetate on the absorption of water mass was approximately half that of the materials without impregnation. However, the impregnation had little influence on the tensile strength of the tested samples. Starch acetate is, therefore, an attractive additive for use in the manufacture of waterproof materials, such as disposable trays [11].

Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 255

Additionally, the effect of chitosan was observed on montmorillonite (MMT) that was incorporated into cassava starch composite films. Chitosan acts as a compatibilizing agent to maintain the homogeneous dispersion of clay particles in a starch matrix. Due to hydrophilicity, chitosan plays a significant role between the starch matrix and montmorillonite. As a result, a low MMT content in cassava starch improves the tensile properties of composite films. Moreover, an increase in the chitosan content of the composite films increases the surface hydrophobicity and water vapor transmission and decreases moisture absorption [12]. Farias et al. prepared and characterized blended composites with cassava bagasse and low-density polyethylene (LDPE). The incorporation of cassava bagasse in the LDPE demonstrated a positive result with an enhancement of the elastic modulus value from 131.90 to 186.2 MPa, with up to a 30% reinforcement of cassava bagasse with LDPE [13]. A similar effect was observed in the preparation and characterization of cassava fiber reinforced with polypropylene (PP) and polybutylene succinate (PBS) composites. Maleic anhydride- polypropylene (MAPP) was used as a compatibilizer to improve the interfacial strength between the fibers and matrix. An increase in the fiber content of the composites increased the Young’s modulus and flexural strength in both the PP and PBS matrix-reinforced composites to levels that were nearly 50% greater than the pure matrix composites. From this result, fiber reinforced composites show a better stiffness compared with pure composites. Conversely, the tensile strength and flexural strength decreased with an increase in the fiber content. Moreover, the thermal stability of the composites increased with the presence of MAPP in both PP and PBS composites. This result suggests the enhancement of the interfacial interaction and compatibility due to the treatment with the compatibilizer [14]. Modified Eucalyptus pulp cellulose fibers with a deposition of silica (SiO2) nanoparticles were used in cassava starch bio-composites. The modified and unmodified pulp fibers composites were prepared with 5% and 10% nanoparticles by weight, and the tensile strength and moisture adsorption were analyzed. The addition of modified fibers improved the tensile strength by 183% compared with that of the thermoplastic starch (TPS) composites, whereas the moisture adsorption decreased by 8.3%. These properties of unmodified fibers were greater than of the modified fiber composites because of the poor interaction between the modified fiber and matrix [15]. Moraes et al. introduced a tape-casting technique for the formation of cassava starch-based films. Generally, the extrusion process for the preparation of cassava starch films will not result in good properties due to the greater shear rates applied. Instead of this extrusion process, the tape-casting technique improves the shear rate by having an adjustable blade at the bottom of the spreading device. The films were prepared with varying concentrations of starch, glycerol and cellulose fibers. Moreover, the results have shown a satisfactory viscosity with a low shearing rate. These flow properties and interactions at the liquid–solid interface have shown that suspensions with fibers are suitable to be processed using the tape-casting technique [16]. Some other researchers used cassava root peel and bagasse as natural fillers in the preparation of bio-composites. The mechanical and optical properties of these composites were studied. The addition of filler resulted in a significant change in the properties of the composites. The filler addition increased the UV- barrier capacity and the opacity of the composite materials. Peel and bagasse fillers were added to the TPS in the range of 0.5to1.5%. The peel-reinforced composites had a greater tensile strength with a 0.5% addition of TPS, whereas bagasse addition (1.5%) increased the elastic modulus by 260% and the maximum tensile stress of the TPS composites by 128%, Complimentary Contributor Copy 256 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won which makes it the most efficient reinforcing agent due to its high residual starch content and lower proportion of smaller particles [17]. Moreover, compression molding has been used for the preparation of pregelatinized cassava starch/kaolin composites. The composites obtained have been studied with respect to their structure and properties. The mechanical and thermal properties were observed to be better than those of TPS. The tensile strength of the composites was greater with a 10 wt% kaolin content but decreased with additional kaolin content; however, the tensile strength was still greater than that of TPS. Similarly, the thermal degradation temperature was greater with 60 wt.% kaolin added to the TPS. This improvement in thermal stability by the addition of kaolin was because kaolin acts as a heat barrier [18].

WHAT IS BIO-FUEL ?

Bio-fuels from renewable resources have recently gained significance as an alternative to finite fossil fuels and a potential solution to ending our dependence on declining natural resources. Bio-fuels are most often used as a motor fuel and are mainly used an additive for gasoline. Bio-fuels are a domestically produced alternative fuel and are made from corn; cellulosic feedstocks, such as crop residues and wood; or can be made from any crop or plant that contains natural sugar (beet and cane). Crops, such as corn, wheat and barley, contain starch that can be easily converted in to sugar, and most trees and grasses are made of cellulose, which can also be converted into sugar. The renewable fuel standard (RFS2) as part of the Energy Independence and Security Act of 2007 (EISA) requires the annual U.S. consumption of biofuels to be 36 billion gallons by the year 2022. Of this amount, 15 billion gallons are to be conventional biofuels most likely achieved using ethanol from corn. The remaining 21 billion gallons are to be advanced biofuels from feedstocks other than corn, 16 billion gallons of which are to be cellulosic biofuels derived from cellulose, hemicellulose, or lignin. Feedstocks for these advanced biofuels include plant residues (e.g., corn stover, cereal grain straw, and forestry residues), dedicated energy crops (e.g., switchgrass, energy cane, and hybrid poplar), and other sources of biomass (e.g., municipal solid waste and algae). Another 1 billion gallons of advanced biofuels are to be biomass-based diesel, which is primarily biodiesel from soybean oil, other vegetable oils, and animal fats. Meeting RFS2 targets would increase the share of renewable fuel by volume to approximately one-quarter of the U.S. gasoline by 2022 [19]. Ethanol, which is also called ethyl alcohol, is produced by a fermenting and distilling biomass. Most significantly, ethanol can be used as a bio-fuel or fuel additive from renewable energy resources. It is the base of most of alcoholic beverages and is used in many pharmaceutical and beauty products. The significance of ethanol is reduced deforestation, free of air pollutant, contains a greater octane rating than petrol as a fuel, is an excellent raw material for synthetic chemicals, improves economic development in rural areas, provides more jobs, increases revenue from producing countries, and reduces adverse foreign trade balances. The general processing of ethanol is a four-step process:

Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 257

1. Feedstocks (crop or plant) are ground up for easier processing; 2. Sugar is dissolved from the ground material or the starch or cellulose is converted to sugar; 3. Microbes feed on the sugar producing ethanol and carbon dioxide as by products; and 4. Ethanol is purified to achieve the correct concentration.

ETHANOL PRODUCTION BY CASSAVA

Cassava has great potential as a raw material for ethanol production because it contains a large amount of starch and cellulosic substances that can be hydrolyzed and fermented to make ethanol. The production of ethanol using cassava follows a five-step process:

Grinding; 1. Liquefaction; 2. Saccharification; 3. Fermentation; and 4. Distillation.

Figure 2. Ethanol production process using cassava.

Complimentary Contributor Copy 258 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won

1. Grinding and Liquefaction First, the collected cassava roots and other materials are milled into flour. Generally, in the industry, this is referred to as “meal” and is processed without separating out various component parts of the grain. To form a “mash” of the product, the meal is slurried with water at 90∼95°C. 2. Saccharification Enzymes are added to the mash to convert starch to dextrose and sugar. Ammonia is added for pH control and as a nutrient for yeast. The mash is processed in a high-temperature cooker to reduce bacterial levels prior to fermentation. 3. Fermentation After the saccharification process, the mash is cooled and transferred to fermenters where yeast is added. The yeast Saccharomyces cerevisiae is activated. Due to this microbial activity, the conversion of sugar to ethanol and carbon dioxide (CO2) begins. 4. Distillation

Finally, water is evaporated in the distillation process, which increases the purity of ethanol. Kosugi et al. produced ethanol from cassava pulp via fermentation with a surface engineered yeast strain displaying glucoamylase. The significance of this process is that without the addition of amylolytic enzymes, Saccharomyces cerevisiae Kyokai no. 7 (strain K7) fermented both the starch and glucose in pretreated samples and produced ethanol at 91% and 80% of the theoretical yield from 5% and 10% cassava pulp, respectively [20]. Klanarong et al. reported that good cultivation practices are important to improve the yield of cassava roots and significantly increased the yield from 22 to 60 tons/hectare (ha). The process of cultivation is composed of many aspects, including soil plowing, high stake quality, weed control, good planting and harvesting period, land conservation with organic fertilizers and water irrigation. Globally, the production of cassava is approximately 200 million tons per annum, with an average yield of 12 tons/ha and a total acreage of 18.5 million ha. This report suggests that if cassava root productivity increases, for example, by 5 tons/ha, approximately 90 million tons of roots will be produced, which can be converted to 15,000 ML of ethanol using the simultaneous saccharification and fermentation (SSF) process, which is a current production process in which cooked and enzymatically liquefied cassava materials are subjected to saccharification enzymes and yeasts in concert. Instead, an uncooked process of a granular starch-hydrolyzing enzyme has been introduced to improve the ethanol production efficiency [21]. The energy efficiency of cassava fuel ethanol was estimated by China, and the net energy value [NEV] and net renewable energy value [NREV] were indicated to be 7.475 MJ/L and 7.881 MJ/L, respectively. The solar energy trapped is greater than the energy used in the processes. Moreover, the ethanol fuel produced from cassava can be used for transportation. Through fuel ethanol production, cassava can produce 9.8 J of fuel ethanol by consuming an input of 1 J of petroleum fuel and other forms of energy inputs, such as coal. Nevertheless, this cassava fuel ethanol can be an alternative for gasoline and other reduced oil imports because, based on the estimation of the cassava output in 2003, this fuel ethanol can substitute for 166.107 million liters of gasoline. This potential cassava output can replace 618.162 million liters of gasoline. These results indicate that the energy efficiency of cassava fuel is greater than those of gasoline, diesel and corn fuel ethanol products, but less than that

Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 259 of biodiesel [22]. Elias et al. produced bio-ethanol by utilizing cellulosic cassava waste. Generally, the chemical composition in cellulosic cassava waste was determined to consist of protein, fiber, insoluble carbohydrates and residual starch content. After the SSF process, dilute HCl was more helpful in converting the cellulosic materials into reducing sugars. Finally, the results showed the obtained fuel ethanol from cassava cellulosic waste to be 2.7 g ethanol/15 g of cellulosic waste, which is equal to 32.4% alcohol [23]. Wei et al. studied the influence of the genotype, growth type and harvest time on cassava stem starch contents and the yield of ethanol production. The design of the experiment contained 3 varieties, 3 locations and 5 harvest times in Guangxi, China. While comparing the cassava stem starch with the root starch, the stem starch content was significantly affected by all parameters, and the location greatly affected the stem starch content compared with variety and harvest timing, whereas root starch was only affected by the location. Additionally, the soil properties were significantly correlated with the starch and sugar contents in both the stem starch and root starch. This promising result showed that stem starch can be used without reducing root starch and can result in approximately 26% ethanol production [24].

Figure 3. Accumulations of (■) lactic acid and (○) acetic acid and (▲) the final alcohol degree during 87 repeated batches [30].

Marco et al. applied genetic algorithms (GAs) to study of the economic viability of alcohol production from cassava root from 2002 to 2013. This GA suggested the hydrolysis occurred for starch concentrations from 8.0 to 22 g/L at temperatures ranging from 30 to 60°C. After the SSF, a significant effect was observed in the production of ethanol and was 88% under the best conditions for starch hydrolysis at 23.4 g/L and 61.9°C and for 111.0 min. The cost of alcohol as determined by the GA was between 0.04 and 0.62 US $/L, and the alcohol sale price were between 0.10 and 0.88 US $/L during this complete study. Furthermore, this method demonstrated that the method is environmental friendly because the implemented (GA) company will acquire 7.8 billion carbon credits, which are equal to 72.477

Complimentary Contributor Copy 260 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won

US $/year. These results show improvements in bioethanol production in industries through the use of GA [25]. Conversely, Papong et al. analyzed the life-cycle energy and environmental cost of bioethanol production from cassava in Thailand. In this analysis, for 1 L of 99.5% cassava- based bioethanol production, the energy and material inputs and water, air and solid-waste emissions were estimated. A major portion of energy consumption takes place at the stage of ethanol production, which requires 78% of the total energy of usage. Therefore, steam and coal were used for power in this process. This results in a major environmental impact compared with the remaining stages. The results of the net energy gain (NEG) and net energy ratio (NER) values for cassava-based ethanol were found to be -3.72 MJ/L and 0.85, respectively. To increase the NEG and decrease the environmental impact, the alternative solution to coal could be the use of biogas recovered from wastewater treatment [26]. To create a negative energy balance, Ramasamy et al. studied ethanol production from cassava starch using co-immobilized cells. To achieve the fermentation of liquefied starch, these researcher used Saccharomyces diastaticus and Zymomonas mobilis co-immobilized cells. They observed that the co-immobilized cells produced high concentrations of ethanol compared with immobilized cells of S. diastaticus during the batch fermentation of liquefied cassava starch. The immobilized cells of S. diastaticus + yeast could produce 37.5 g/l ethanol from 150 g/l of liquefied cassava starch, but the significant improvement that was demonstrated in the co-immobilized cells produced 46.7 g/1 ethanol. This indicates the mixed culture fermentation results in a greater concentration of ethanol compared with free cells. The ethanol concentration was increased to 53.5 g/l in the repeated batch fermentation using co-immobilized cells [27]. A similar study conducted by Shanavas et al. reported novel eco- friendly enzymes that were used to optimize bioethanol production from cassava starch. In this process, Spezyme and Stargen enzymes were introduced in the SSF process. Spezyme was active even at 90°C, and 10% (w/v) of starch was hydrolyzed at levels of 20.0 mg (280 amylase activity units) at a pH 5.5 for 30 min. Stargen was active at room temperature (30 ± 1 0C) and hydrolyzed the 10% (w/v) of the starch of 100 mg (45.6 granular starch hydrolyzing units). The greatest yield of ethanol was obtained by Spezyme using rapid Saccharification fermentation using Stargen plus a yeast system. The fermentation efficiency for 1 kg of starch was shown to be approximately 98.4%. The specific advantage of the new process was that the reaction could be completed within 48.5 h at 30 ± 1°C) [28]. In the process of achieving bioethanol fuel production, the cost of the feedstock, energy and enzymes used in pretreatment prior to fermentation will dominate. To increase the economic viability and decrease the complexity, non-conventional feedstocks have been proposed as alternatives. These feedstock bioconversion deployments downsize the cost. Cassava starch has been proposed to be a non-conventional feedstock for bioethanol production. The co-culture and monoculture of both Aspergillus sp. and Saccharomyces cerevisiae have been proposed for bioethanol production. The production of bioethanol in co-culture is greater than monoculture production. The efficiency of ethanol is approximately 91-95%. This process efficiently reduces the energy expense [29]. Moreover, the chemical modification of the fibrous matrix was used along with SSF with immobilized Saccharomyces cerevisiae 1308. Without filtration, cassava hydrolysate was used for ethanol fermentation. After seven repeated SSF processes, the utilization of starch obtained by the immobilized cells was approximately 83.5%. The utilization of starch by immobilized cells was 2.1% greater than that of free cells, with an inoculation quantity of 15% (v/v) under the same fermentation conditions. The Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 261 enzyme feeding strategy suggests that the utilization of starch is 85.9% at 35°C. After 6 months and approximately 87 batches of SSF, ethanol production from cassava using immobilized yeast in a fibrous-bed bioreactor is suggested to be feasible and may meet the demands of industrial production, as shown in Figure 3 [30].

CASSAVA IN MEDICINE

Starch is a biodegradable polymer and is a promising carrier for drug delivery. Starch has been used in various fields, biomedical, agriculture among others. However, many drugs are released quickly due to the considerable swelling and quick enzymatic degradation of unmodified native starch from biological systems. The chemical modification of starch includes the esterification of native starch, which is referred to as acetylated starch, and this process improves the properties of the starch. The chemical modification of starch into starch acetates will improve the hydrophobicity and decrease the hydrophilicity. In drug delivery applications, this modified starch acetate has been extensively used. Raj et al. investigated cassava starch acetate (CSA)–polyethylene glycol (PEG)–gelatin (G) nanocomposites as a novel controlled drug delivery system for anticancer drugs. These nanocomposites were used to entrap cisplatin (CDDP). In this investigation, the authors prepared CSA-CDDP, CSA- CDDP-PEG and CSA-CDDP-PEG-G nanocomposites. Then, 0.1 mg of a 10%, 20%, 30%, 40%, or 50% drug loaded sample was suspended in a definite volume (10 ml) of phosphate buffer saline (PBS) at various pH values at 37°C. The resulting suspension was placed in an incubated shaker at 120 rpm for a definite time period (1 h), and five-milliliter aliquots were taken out of the dissolution medium at appropriate time intervals (30 min) and were replaced by the same volume of fresh PBS buffer to maintain the volume of the release medium constant. The amount of drug released was observed using a UV spectrophotometer at 290 nm. The results suggested that the CDDP releasing environment in an acidic medium is faster than in a basic medium from CSA, CSA–PEG and CSA–PEG–G nanocomposites. This is because the binding action plays a vital role by attacking the H+ or Cl- between the drug and carboxyl group in cassava starch acetate nanocomposites. In the body’s environment, the Cl- concentration is very high (95∼105 mM) and relatively stable in circulation, and a more acidic environment means more H+, which can speed up the release of CDDP from coated polymeric nanocomposites [31]. Simi et al. prepared hydrophobic grafted and cross-linked cassava starch nanoparticles for drug delivery applications. Indomethacin was used as a model drug and was loaded in cassava starch nanoparticles. Suitable amounts of cassava starch nanoparticle and indomethacin were dissolved in DMSO. In in vitro drug release studies, a dialysis membrane with a molecular weight of 12,000 g/mol was used and a small quantity of the drug-loaded starch nanoparticles were stirred with phosphate buffer at a pH 7.4 at room temperature. At regular intervals, a certain amount of medium was replaced with fresh phosphate buffer. The amount of indomethacin released was determined using a UV- visible spectrophotometer at 320 nm. The greatest amount of drug loading in nanoparticles was identified to be 16%. The controlled drug release was studied at pH 7.4. The surface cross-linking of the starch nanoparticles slowed drug release from the nanoparticle. This significant slowing of drug release in a buffer at pH 7.4 demonstrated that the starch nanoparticle can be used as a good carrier for drugs [32].

Complimentary Contributor Copy 262 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won

Rajan et al. observed the enzymatic modification of cassava starch using bacterial lipase. For this enzymatic modification of cassava starch, Burkholderia cepacia (lipase PS) of an industrial lipase was used with two acyl donors, lauric acid and palmitic acid. The degree of substitution (DS) of the reactions performed using palmitic acid in a liquid state and using microwave esterification resulted in 62.08% (DS 1.45) and 42.06% (DS 0.98), respectively. The estimation of the α-amylase starch digestibility of the unmodified starch was 76.5%, which was reduced to 18% through modification (DS 1.45). These significant changes in the α-amylase digestion due to the hydrophobicity of the starch esters, in turn, reduce the swelling power. This is suitable for biomedical applications, carriers for controlled release of drugs and bioactive agents. Therefore, enzymatic esterification is ecofriendly [33]. Moreover, for the efficient drug delivery of curcumin (CUR), cassava starch crosslinked with N, N- methylenebisacrylamide (MBA) microspheres were used. This drug release effect was studied in treating tumor cell lines. For colon cancer treatment, the microspheres were shown to be a capable device for the controlled release of CUR, which, in general, decreases the pH of the colon. The CUR-loaded microspheres presented a much greater activity against Caco-2 and HCT-116 tumor cell lines than free CUR [34].

CASSAVA IN FOOD PACKAGING

Souza et al. prepared cassava starch composite films by incorporating cinnamon essential oil and evaluated the antibacterial activity, microstructure, and mechanical and barrier properties. It is necessary to maintain the quality and shelf life of food products through the use of active packaging films. The effect of an increase in the cinnamon essential oil, glycerol and emulsifier contents in the composites films decreased the tensile strength (TS) and elongation at break (E), and these values varied from 2.32 ± 0.40 to 1.05± 0.16 MPa and from 264.03 ± 35.06 to 191.27 ± 22.62%, respectively, indicating a loss of macromolecular mobility. The barrier properties of the composite films increased with an increase in the amount of essential oil incorporated. The water vapor permeability (WVP) and oxygen permeability coefficient (OPC) of the films with cinnamon essential oil varied from 9.78±1.40 to 14.79±2.76 g mm m-2d-1kPa-1and from 27.50 ± 0.60 to 143.47 ± 8.30 ×109 cm3m-1d-1Pa-1, respectively. Furthermore, all films, which contained different amounts of essential oils, showed effective antimicrobial activity against P. commune and E. amstelodami, which are fungi that are commonly found in bread products [35]. It was observed that the effect of kaolinite on cassava starch composites films influences its glass transition temperature (Tg), transparency, UV-vis blocking, water uptake, and decomposition temperatures. As the clay content increased up to 10%, the Tg of the composites decreased. This clay content acted as a plasticizer by reducing the interactions between the polymer chains, which promotes their mobility. Furthermore, the clay content had a barrier effect on water uptake at a low relative humidity, but at a greater relative humidity, the composite films increased their water uptake. Moreover, perfect UV-vis blocking was evident at kaolinite loads between 2% and 6%. These properties of the composite films work effectively for food packaging [36]. Zainuddin et al. prepared and characterized cassava starch bio-composites reinforced with cellulose nanocrystals (CNC) from kenaf fibers. An increase in the CNC content in the thermoplastic starch (TPS) of up to a reinforcement of 6% caused the tensile

Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 263 strength and modulus to increase. The thermal stability of the CNC composites reinforced with TPS varied from 280 to 385°C.Whereas, for pure cassava starch, the thermal stability ranged from 273 to 335°C. The thermal stability was attributed to the pyrolysis of the starch yielding products, such as carbon monoxide, volatile organic compounds, and carbonaceous residues. Moreover, water uptake was significantly decreased by the CNC composites reinforced with TPS compared to TPS. These composites were useful for food packaging applications [37]. An edible film was made of agar (AG), cassava starch (CAS), normal rice starch (NRS), and waxy (glutinous) rice starch (WRS) and was tested for the application of edible packaging. The water vapor permeability (WVP) of the films depends on the water vapor pressure gradient and achieves a constant value at RH values greater than 84%. Among these films, AG-based films have a better moisture sorption property compared with CAS films at the same RH conditions. These films had high WVP and hygroscopicity, which restricted their potential use as moisture barrier packaging. Moreover, some foods will retard the water loss during short-term storage. The composed films could be used in the same way. Conversely, all of the films had good mechanical properties except for the WRS-based film. The tensile strength of the NRS, CAS and AG films varied from 28 MPa to 42 MPa and was 8.5 MPa for the WRS film. Specifically, the AG- and CAS-based films plasticized with glycerin were clear, homogeneous, transparent, flexible and easy to handle. Such films are useful for the integrity of the products and the functional properties of the films themselves [38]. The cyanogenesis (SRRC) downstream processing approach was used to reduce cassava-borne environmental waste and to develop peeled (BP) and intact (BI) bitter cassava as biopolymer derivatives. Using the SRRC, the BI approach resulted in a greater yield reduction of approximately 16% waste with no environmental impact caused by the discarded residues. The reduction of the cyanogen content of the cassava gave promising results for industrial applications. The casting method was applied to produce transparent films from both BP and BI derivatives. BI films were more transparent and homogeneous, less soluble, less permeable to moisture, less hydrophilic, more permeable to oxygen and carbon dioxide, sealable, and had a lower cost than the BP films [39]. Bangyekan et al. analyzed the changes in the properties of chitosan-coated cassava starch films. The tensile and barrier properties of the composite films changed significantly with an increase in the concentration of chitosan in the coating. A shift in the starch diffraction peak was likely due to a change in its chain orientation caused by a hydrogen-bonding interaction between the chitosan and starch molecules resulting in good adhesion. The tensile and barrier properties of the composite films increased when increasing the concentration of chitosan [40]. Souza et al. added antioxidant additives from mango and acerola pulps [41] and yerba mate extract [42] into cassava starch bio-based films. These films were investigated for their feasibility of incorporation in a biodegradable matrix. In addition, these films were used to pack palm oil (maintained for 45 and 90 days) under accelerated oxidation conditions (63% relative humidity and 30°C) to simulate a storage experiment. These additives significantly improved the shelf life with a decreasing oxidation effect. The antioxidant activity was obtained due to the presence of phenolic and flavonoid compounds. These results suggest that the incorporation of antioxidant additives into starch-based films will improve the quality of food by degrading the antioxidant action. Furthermore, due to the many superior properties of cassava starch, researchers identified the use of acetylated starch nanoparticles (NPAac) as reinforcements in TPS films. These TPS films were prepared by selecting different portions Complimentary Contributor Copy 264 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won of NPAac, and the mechanical, thermal and barrier properties of the obtained films were studied for food packaging applications. The results of these films reveal a significant improvement in the addition of NPAac as a reinforcement. The Young’s modulus and thermal stability increased by 162% and 15%, respectively, with the addition of 0.5% (w/w) NPAac. Similarly, the WVP was lowered by 41% for the film with 1.5% (w/w) NPAac. These results indicate the reinforcement with NPAac shows significant improvement compared with TPS [43]. To utilize every part of the cassava, Versino et al. used the remaining fibrous residue from cassava starch extraction as a reinforcement for fully biodegradable starch-based composite films. However, the reinforcement of the TPS filler significantly increases the apparent viscosity and storage modulus without segregation of the filler particles. The films were formed using various filler ratios from 0 to 3% (w/w) along with glycerol. The homogeneous films were obtained for all TPS composites films. Reinforced films exhibited a UV-barrier capacity and adequate water vapor barrier properties (14.6 ± 0.7 10−11 g/m s Pa) and tensile strengths (18.01 ± 0.19 MPa) when 25% (w/w) glycerol was added as a plasticizer. The addition of the filler to the TPS increases the mechanical resistance, and furthermore, the obtained eco-compatible materials could be heat-sealed, which indicates their suitability for packaging development [44]. In addition, the edible cassava starch and soy protein concentrate coatings were proven to extend the shelf life of toasted groundnut. The combination of cassava starch and soy protein concentrate with 20% glycerol was applied to toasted groundnut by dip coating. The chemical indices, oxidative rancidity, and sensory parameters were evaluated using standard procedures. The thiobarbituric acid, peroxide and moisture uptake values were greater than 100% cassava starch-coated groundnuts, whereas the blended coated toasted groundnut had lower values. Moreover, the blended film resulted in greater color, texture, taste and overall acceptability scores compared with the 100% cassava starch and control. These combinations of edible coatings extended the shelf life of toasted groundnuts that were stored in ambient (27 ± 1oC) conditions for 14 days compared with uncoated toasted groundnuts [45]. The extension of the shelf life also depends on protection from microbial contaminations, and control is achieved using active packaging films. Franciele et al. prepared active packaging films by incorporating oregano essential oil (OEO) with cassava starch-chitosan blended films. The physicochemical and antimicrobial properties of the obtained films were evaluated. The disc inhibition zone method was applied against Bacillus cereus, Escherichia coli, Salmonella enteritidis, and Staphylococcus aureus. The tensile strength (21.95 ± 1.98 to 1.43 ± 0.26 MPa), elongation at break (%) (21.95 ± 1.98 to 48.4 ± 5.32), Young’s modulus (140.36 ± 5.3 to 18.9 ± 2.61 MPa) and WVP (1.39 ± 1.56 to 0.62 ± 0.15 10−11 g/m s Pa) varied with an increase in the concentration of the OEO from 0∼1% in the cassava starch- chitosan blended films. The mechanical properties and WVP decreased compared with the control film. The WVP values decreased with the incorporation of the OEO when increasing its concentration. Correspondingly, the incorporated OEO films demonstrated an effective inhibition against all tested microorganisms, as shown in Figure 4 [46].

Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 265

Figure 4. Inhibition zones for (a) cassava starch film, (b) cassava starch-chitosan film, and (c-f) cassava starch-chitosan films with 1% OEO incorporated against the microorganisms (c) S. enteritidis, (d) E. coli, (e) B. cereus, and (f) S. aureus. (Because in a and b no inhibition zones were observed, only one representative plate for each case is shown.) [46]

FUTURE CHALLENGING OPPORTUNITIES FOR CASSAVA

There are many potential industrial uses for cassava, such as the development of cassava beers and as a substitute for wheat flour in many countries; for example, Nigeria is likely to save nearly N163 billion annually and create approximately 3 million jobs by using 20% cassava flour blended in wheat flour. Still, many countries have not explored large-scale commercial applications due to the reduced shelf life of cassava roots. It is important to reduce the time gap between production and marketing because most cassava is wasted in this period. The identification of new seed varieties is important to reduce disease to achieve an adequate improvement in yield. Extensive research into breeding is needed to improve the post-harvest storability in the fields as soon as possible. There is a need to find molecular markers for the progeny from crosses. Progeny identification gives the root characteristics while improving the agronomic characteristics and incorporating virus resistance as well as increasing the yield in cold climates. Expanded research on mechanically harvesting cassava is needed. Extensive information is necessary to educate farmers about soil conditions, fertilizers and so on to produce a greater cassava crop yield. The exchange of knowledge is important with respect to processing cassava from different countries and their marketing activities. The global production of ethanol by cassava is still low, and the benefits of cassava need to be explored by using various media and other communications.

Complimentary Contributor Copy 266 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won

The reduction of the moisture content of cassava products is important to enhance the shelf life of products after a short period of processing. The elimination of toxic glycosides in cassava chips is required before use as a commercial feed for animals. Transgenic programs for cassava are needed to meet the exciting challenge with respect to product development and a quick delivery process [47].

CONCLUSION

We conclude that, the cassava is a being multipurpose commercial products having a many potential uses like bio-fuel, animal feed, medicinal, bio-composite, and food packaging use etc. Moreover, this cassava product has significant usage in food industry and a major staple food in the developing world, providing a basic diet for over half a billion people. In the production of ethanol, it is possible to produce 95% of pure ethanol by conversion. The preparation of bio-composites using cassava starch has shown good mechanical and barrier properties for the application of food packaging. The remarkable note identified in drug releasing effect with using cassava starch as a crosslinking agent. Still there is need to identification for increase shelf life of cassava, due to this it is unexplored in commercial applications at many other countries. The extensive research needed on transgenic cassava for improving yield and reducing disease to become more commercial crop in worldwide.

REFERENCES

[1] Blagbrough, I. S., Bayoumi, S. A., Rowan, M. G., and Beeching, J. R. (2010). Cassava: an appraisal of its phytochemistry and its biotechnological prospects. Phytochemistry, 71(17): 1940-1951. [2] Ngudi, D. D., Kuo, Y. H., and Lambein, F. (2003). Cassava cyanogens and free amino acids in raw and cooked leaves. Food and Chemical Toxicology, 41(8): 1193-1197. [3] Sornyotha, S., Kyu, K. L., and Ratanakhanokchai, K. (2010). An efficient treatment for detoxification process of cassava starch by plant cell wall-degrading enzymes. Journal of bioscience and bioengineering, 109(1): 9-14. [4] Legg, J. P., and Thresh, J. M. (2000). Cassava mosaic virus disease in East Africa: a dynamic disease in a changing environment. Virus research, 71(1): 135-149. [5] Kristensen, S. B. P., Birch-Thomsen, T., Rasmussen, K., Rasmussen, L. V., and Traoré, O. (2014). Cassava as an energy crop: A case study of the potential for an expansion of cassava cultivation for bioethanol production in Southern Mali. Renewable Energy, 66:381-390. [6] http://sattvamji.blogspot.kr/2014/10/78-cassava-or-maniok-root-one-more.html [7] https://ndb.nal.usda.gov/ndb/foods/show/2907?fgcd=&manu=&lfacet=&format=&cou nt=&max=35&offset=&sort=&qlookup=Cassava%2C±raw [8] Ramírez, M. G. L., Satyanarayana, K. G., Iwakiri, S., de Muniz, G. B., Tanobe, V., and Flores-Sahagun, T. S. (2011). Study of the properties of biocomposites. Part I. Cassava starch-green coir fibers from Brazil. Carbohydrate Polymers, 86(4): 1712-1722.

Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 267

[9] Lomelí-Ramírez, M. G., Kestur, S. G., Manríquez-González, R., Iwakiri, S., de Muniz, G. B., and Flores-Sahagun, T. S. (2014). Bio-composites of cassava starch-green coconut fiber: Part II—Structure and properties. Carbohydrate polymers, 102: 576-583. [10] Souza, A. C., Benze, R. F. E. S., Ferrão, E. S., Ditchfield, C., Coelho, A. C. V., and Tadini, C. C. (2012). Cassava starch biodegradable films: Influence of glycerol and clay nanoparticles content on tensile and barrier properties and glass transition temperature. LWT-Food Science and Technology, 46(1): 110-117. [11] Matsui, K. N., Larotonda, F. D. S., Paes, S. S., Luiz, D. D. B., Pires, A. T. N., and Laurindo, J. B. (2004). Cassava bagasse-Kraft paper composites: analysis of influence of impregnation with starch acetate on tensile strength and water absorption properties. Carbohydrate Polymers, 55(3): 237-243. [12] Kampeerapappun, P., Aht-ong, D., Pentrakoon, D., and Srikulkit, K. (2007). Preparation of cassava starch/montmorillonite composite film. Carbohydrate Polymers, 67(2): 155-163. [13] Farias, F. O., Jasko, A. C., Colman, T. A. D., Pinheiro, L. A., Schnitzler, E., Barana, A. C., and Demiate, I. M. (2014). Characterisation of Cassava Bagasse and Composites Prepared by Blending with Low-Density Polyethylene. Brazilian Archives of Biology and Technology, 57(6): 821-830. [14] Tantatherdtam, R., Tran, T., Chotineeranat, S., Lee, B. H., Lee, S. N., Sriroth, K., and Kim, H. J. (2009). Preparation and Characterization of Cassava Fiber-Based Polypropylene and Polybutylene Succinate Composites. Kasetsart J (Nat Sci), 43: 245- 251. [15] Raabe, J., Fonseca, A. D. S., Bufalino, L., Ribeiro, C., Martins, M. A., Marconcini, J. M., and Tonoli, G. H. D. (2015). Biocomposite of cassava starch reinforced with cellulose pulp fibers modified with deposition of silica (SiO 2) nanoparticles. Journal of Nanomaterials, 2015: 6. [16] de Moraes, J. O., Scheibe, A. S., Sereno, A., and Laurindo, J. B. (2013). Scale-up of the production of cassava starch based films using tape-casting. Journal of Food Engineering, 119(4): 800-808. [17] Versino, F., López, O. V. and García, M. A. (2015). Sustainable use of cassava (Manihot esculenta) roots as raw material for biocomposites development. Industrial Crops and Products, 65: 79-89. [18] Kaewtatip, K., and Tanrattanakul, V. (2012). Structure and properties of pregelatinized cassava starch/kaolin composites. Materials & Design, 37: 423-428. [19] Keeler, B. L., Krohn, B. J., Nickerson, T. A., and Hill, J. D. (2013). US federal agency models offer different visions for achieving renewable fuel standard (RFS2) biofuel volumes. Environmental science & technology, 47(18): 10095-10101. [20] Kosugi, A., Kondo, A., Ueda, M., Murata, Y., Vaithanomsat, P., Thanapase, W., and Mori, Y. (2009). Production of ethanol from cassava pulp via fermentation with a surface-engineered yeast strain displaying glucoamylase. Renewable Energy, 34(5): 1354-1358. [21] Sriroth, K., Piyachomkwan, K., Wanlapatit, S., and Nivitchanyong, S. (2010). The promise of a technology revolution in cassava bioethanol: From Thai practice to the world practice. Fuel, 89(7): 1333-1338.

Complimentary Contributor Copy 268 Reddy T. Ranjeth Kumar, Kim Hyun-Joong and Park Ji-Won

[22] Dai, D., Hu, Z., Pu, G., Li, H., and Wang, C. (2006). Energy efficiency and potentials of cassava fuel ethanol in Guangxi region of China. Energy Conversion and Management, 47(13): 1686-1699. [23] Elemike, E. E., Oseghale, O. C. and Okoye, A. C. (2015). Utilization of cellulosic cassava waste for bio-ethanol production. Journal of Environmental Chemical Engineering, 3(4): 2797-2800. [24] Wei, M., Zhu, W., Xie, G., Lestander, T. A., and Xiong, S. (2015). Cassava stem wastes as potential feedstock for fuel ethanol production: A basic parameter study. Renewable Energy, 83: 970-978. [25] Benvenga, M. A. C., Librantz, A. F. H., Santana, J. C. C., and Tambourgi, E. B. (2016). Genetic algorithm applied to study of the economic viability of alcohol production from Cassava root from 2002 to 2013. Journal of Cleaner Production, 113: 483-494. [26] Papong, S., and Malakul, P. (2010). Life-cycle energy and environmental analysis of bioethanol production from cassava in Thailand. Bioresource technology, 101(1): S112-S118. [27] Amutha, R., and Gunasekaran, P. (2001). Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus. Journal of bioscience and bioengineering, 92(6): 560-564. [28] Shanavas, S., Padmaja, G., Moorthy, S. N., Sajeev, M. S., and Sheriff, J. T. (2011). Process optimization for bioethanol production from cassava starch using novel eco- friendly enzymes. Biomass and Bioenergy, 35(2): 901-909. [29] Moshi, A. P., Hosea, K. M., Elisante, E., Mamo, G., Önnby, L., and Nges, I. A. (2016). Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of inedible wild cassava flour to bioethanol. Journal of bioscience and bioengineering, 121(4): 457-463. [30] Liu, Q., Cheng, H., Wu, J., Chen, X., Ying, H., Zhou, P., and Chen, Y. (2014). Long- Term Production of Fuel Ethanol by Immobilized Yeast in Repeated-Batch Simultaneous Saccharification and Fermentation of Cassava. Energy & Fuels, 29(1): 185-190. [31] Raj, V. and Prabha, G. (2015). Synthesis, characterization and in vitro drug release of cisplatin loaded Cassava starch acetate–PEG/gelatin nanocomposites. Journal of the Association of Arab Universities for Basic and Applied Sciences. [32] Simi, C. K., and Abraham, T. E. (2007). Hydrophobic grafted and cross-linked starch nanoparticles for drug delivery. Bioprocess and biosystems engineering, 30(3): 173- 180. [33] Rajan, A., and Abraham, T. E. (2006). Enzymatic modification of cassava starch by bacterial lipase. Bioprocess and Biosystems Engineering, 29(1): 65-71. [34] Pereira, A. G., Fajardo, A. R., Nocchi, S., Nakamura, C. V., Rubira, A. F., and Muniz, E. C. (2013). Starch-based microspheres for sustained-release of curcumin: preparation and cytotoxic effect on tumor cells. Carbohydrate polymers, 98(1): 711-720. [35] Souza, A. C., Goto, G. E. O., Mainardi, J. A., Coelho, A. C. V., and Tadini, C. C. (2013). Cassava starch composite films incorporated with cinnamon essential oil: Antimicrobial activity, microstructure, mechanical and barrier properties. LWT-Food Science and Technology, 54(2): 346-352.

Complimentary Contributor Copy Potential Uses of Cassava Products and Its Future Challenging Opportunities 269

[36] Mbey, J. A., Hoppe, S., and Thomas, F. (2012). Cassava starch–kaolinite composite film. Effect of clay content and clay modification on film properties. Carbohydrate Polymers, 88(1): 213-222. [37] Zainuddin, S. Y. Z., Ahmad, I. and Kargarzadeh, H. (2013). Cassava starch biocomposites reinforced with cellulose nanocrystals from kenaf fibers. Composite Interfaces, 20(3): 189-199. [38] Phan, T. D., Debeaufort, F., Luu, D., and Voilley, A. (2005). Functional properties of edible agar-based and starch-based films for food quality preservation. Journal of Agricultural and Food Chemistry, 53(4): 973-981. [39] Tumwesigye, K. S., Oliveira, J. C., and Sousa-Gallagher, M. J. (2016). New sustainable approach to reduce cassava borne environmental waste and develop biodegradable materials for food packaging applications. Food Packaging and Shelf Life, 7: 8-19. [40] Bangyekan, C., Aht-Ong, D., and Srikulkit, K. (2006). Preparation and properties evaluation of chitosan-coated cassava starch films. Carbohydrate Polymers, 63(1): 61- 71. [41] Souza, C. O., Silva, L. T., Silva, J. R., López, J. A., Veiga-Santos, P., and Druzian, J. I. (2011). Mango and acerola pulps as antioxidant additives in cassava starch bio-based film. Journal of agricultural and food chemistry, 59(6): 2248-2254. [42] Reis, L. C. B., de Souza, C. O., da Silva, J. B. A., Martins, A. C., Nunes, I. L., and Druzian, J. I. (2015). Active biocomposites of cassava starch: the effect of yerba mate extract and mango pulp as antioxidant additives on the properties and the stability of a packaged product. Food and Bioproducts Processing, 94: 382-391. [43] Teodoro, A. P., Mali, S., Romero, N., and de Carvalho, G. M. (2015). Cassava starch films containing acetylated starch nanoparticles as reinforcement: Physical and mechanical characterization. Carbohydrate polymers, 126: 9-16. [44] Versino, F., and García, M. A. (2014). Cassava (Manihot esculenta) starch films reinforced with natural fibrous filler. Industrial Crops and Products, 58: 305-314. [45] Chinma, C. E., Ariahu, C. C., and Abu, J. O. (2014). Shelf Life Extension of Toasted Groundnuts through the Application of Cassava Starch and Soy Protein-Based Edible Coating. Nigerian Food Journal, 32(1): 133-138. [46] Pelissari, F. M., Grossmann, M. V., Yamashita, F., and Pineda, E. A. G. (2009). Antimicrobial, mechanical, and barrier properties of cassava starch− chitosan films incorporated with oregano essential oil. Journal of Agricultural and Food Chemistry, 57(16): 7499-7504. [47] Taylor, N., Kent, L., and Fauquet, C. (2004). Progress and challenges for the deployment of transgenic technologies in cassava.

Chapter 14

UTILIZATION OF MODIFIED CASSAVA FLOUR AND ITS BY-PRODUCTS

Setiyo Gunawan *, Zikrina Istighfarah, Hakun Wirawasista Aparamarta, Firdaus Syarifah and Ira Dwitasari Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia

ABSTRACT

Cassava is an important component in the diets of more than 800 million people around the world. It is kind of tropic and sub-tropic plant. It is able to grow in less- nutrition soil. In a dry land, cassava sheds its leaves to keep it damp and produces new leaves in the rainy season. Otherwise, cassava can not survive in cold weather but it can grow very well in the area with pH 4-8. Cassava needs at least 5 months in the summer for producing ripe cassava. The aim of this chapter is to discuss the proximate composition, production, application, and modification process of cassava roots as well as their future perspective. The typical important parameters for proximate composition of cassava are protein, lipids, fibre, starch, cyanide acid and ash contents. The carbon to nitrogen ratio (C/N ratio) of dried fresh cassava roots is also important parameter for microbial activities within fermentation process. The development of new utilization techniques of cassava roots has gained increasing importance in chemical, food, and pharmaceutical industries, due to their content of economically-valuable compounds, the necessity of environmental friendly process, global food and energy security. There are several different methodologies for enhancing detoxification and improving the quality of cassava flour, such as fermentation process (liquid, solid state, submerged, culture and spontaneous fermentations), different microorganisms (yeast, fungi and bacteria) and different additional nutrients (with and without nutrients). Moreover, lactid acid is produced as by-product during the fermentation. This is also interesting topic due to the potential application of lactic acid for the production of biodegradable polymers. Another, the analysis methods of the compounds in cassava roots are also a challenging

* Corresponding Author address; Email: [email protected]. Complimentary Contributor Copy 272 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al.

work. Few analytical methods are available to provide a detailed and simpler analysis. It is of great interest if new utilization of cassava roots and analysis methods of the compounds in cassava roots are available to establish all products during the fermentation.

Keywords: cassava, cyanide acid, fermentation, lactic acid, protein, starch

INTRODUCTION

Cassava came from the tropical part of America continent, precisely from Brazil. Its deployment reaches all around the world, among others are Africa, Madagascar, India, Indonesia, and China (Achi and Akoma, 2006). This plant is growing well under critical conditions of climate and soil, high resistance to diseases, and flexibel harvesting date, allowing farmers to keep the roots in the ground until needed. Moreover, both leaves and roots can be processed into many kinds of foods. Cassava roots have a high content of fiber and carbohydrate but low protein content. Cassava leaves even have higher protein content than that of cassava roots, because they contain amino metionin acid (Richana, 2012).

CLASSIFICATION OF CASSAVA

According to Lingga (1986), depend on its age, cassava divided into two groups: short- lived and long-lived cassavas. Short-lived cassava only has 5-8 months of age from planting to harvesting. At this range, cassava production is optimum. If it is harvested longer than its effective age, the roots are woody. Another, long-lived cassava has 12-18 months of age. Before this range, its tubers become too small. As a defense mechanism against predators, cassava produces two cyanogenic glucosides: linamarin and a small amount of lotaustralin (methyl linamarin) as can be seen in Figure 1. These cyanogens are distributed widely throughout the plant, with large amounts in the leaves and the root cortex (skin layer), and generally smaller amounts in the root parenchyma (interior) (Montagnac and Davis, 2009). Hydrogen cyanide (HCN) is released from the cyanogenic glycosides when fresh plant material is macerated as in chewing, which allows enzymes and cyanogenic glycosides to come together, releasing hydrogen cyanide. Consumption of cassava and its products that contain large amounts of cyanogens may cause cyanide poisoning with symptoms of vomiting, nausea, dizziness, stomach pains, weakness, headache and diarrhea and occasionally death (Cardoso et al., 2005; Nhassico et al., 2008). Cumbana et al. (2007) reported that high cyanide acid is thought to be the major contributing cause of konzo, an irreversible paralysis of the legs in women of child-bearing age and children. Another, there are many varieties of cassava with cyanide content which varies based on the suitability for growing and nutrients consumed. Kobawila et al. (2005) reported that according to the cyanide content in cassava roots, cassava is classified into 3 categories: high- , moderate-, and non-toxics varieties with more than 100, 50-100, and less than 50 mg/kg, respectively. Akintonwa et al. (1994) mentioned that, the deathly dose consuming of cyanide

Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 273 acid is 0.5 mg per kg of body weight. They also mentioned that bitter cassava variety is more resistant to drought, more readily available and cheaper. The cyanide acid content of bitter cassava is about 50-400 mg/kg, meanwhile the sweet one is 15-50 mg/kg. In so-called sweet cassava, the roots contain only a small amount of cyanogens, therefore after peeling, these roots can be safely boiled and eaten, as occurs in the South Pacific, such as Manihot esculenta Cranz. The bitter taste of bitter cassava is due to high content of cyanide, such as Manihot utilissima Pohl and Manihot glaziovii Muell. These roots must be processed before consumption to reduce the amount of toxic cyanogens to a safe level. The World Health Organization (WHO) has set the safe level of cyanide content in cassava flour at 10 ppm (FAO, 1991; SNI, 2011).

CH2OH

O H CH3 H O C OH H

OH H CN

H OH (a)

CH2OH

O H CH3 H O C CH OH H 3

OH H CN

H OH (b)

Figure 1. Linamarin (a) and lotaustralin (b) structures.

PROXIMATE COMPOSITION OF CASSAVA

Indonesia is a tropical region that is rich in natural resources, one of which is tuber root, such as cassava (M. esculenta). Cassava roots are tuber or root of a tree shaped like a cylinder that ends narrowed with an average diameter of 2-5 cm and a length of about 20-50 cm as can be seen in Figure 2 (Jayasuriya, 2015). Cassava is one of foodstuff which has been consumed for long time by Indonesian people. In Indonesia, it was introduced by Portuguese in 16th century. Furthermore, it was planted commercially around 1810s and spread in 1914-1918 (Lingga, 1990). It become an alternative substitute of staple food because Indonesia was experiencing rice shortage. Cassava reaches the top 4th on staple food list in developing countries after rice, wheat, and corn (Bokanga, 1995). It gives nutrition and energy for more than 800 million people

Complimentary Contributor Copy 274 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al.

(Bokanga, 1995). Recently, cassava becomes the 3rd staple food after rice and corn in Indonesia. In 2005, Indonesia was the 3rd largest cassava roots producer countries (13.300.000 tons) after Brazil (25.554.000 tons), and Thailand (13.500.000 tons), then followed by Nigeria (11.000.000 tons), India (6.500.000 tons) from total world production (122.134.000 tons). Indonesia produced 21.756.991 tons cassava roots in 2008, and then increased up to 24.044.025 tons in 2011, however decreased to 23.627.955 tons in 2013 (Statistic Indonesia, 2014).

Figure 2. Manihot esculenta Cranz.

Cassava’s nutrition consist of carbohydrate, lipid, protein, fiber, and ash. The proximate, minerals, and anti-nutrients compositions of dried cassava roots are shown in Table 1, 2 and 3, respectively. Cassava roots are considered as low price and quality raw materials, such as low in protein, minerals and vitamins contents (Onwueme, 1978; Aletor, 1993). Hahn (1992) reported that the low price of cassava roots is also affected by the properties of fresh cassava roots are easily damaged due to the presence of tannic acid, a substance that can cause colors in processed cassava products. Cassava roots contain a little protein, but that protein does contain essential amino acids, such as methionine, cystine and cysteine. Protein is one of bio-macromolecule that has important role for living things. According to Adams and Ray (1988), protein is molecular unit of amino acids containing carbon, hydrogen, oxygen, nitrogen, and sulphur. Amino acids Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 275 itself is divided into two: acid (oxygen, carbon, sulfur) and amino (nitrogen and hydrogen) which attach in carbon atom. Main function of protein is forming cell structure and biocatalyst for chemical reactions in metabolism.

Table 1. Proximate composition (wt.%) of cassava roots (Manihot esculenta)

No Protein Lipids Fiber Ash Carbohydrates References 1 3.20 0.80 2.67 0.80 92.53 Depkes RI (1981) Westby and Choo, 2 2.63 0.79 2.63 1.84 92.11 1991 Akindahunsi et al. 3 3.60±0.10 3.60±0.10 3.70±0.2 1.90±0.2 87.2±0.2 (1999) Boonnop et al. 4 2.80-3.20 2.30-2.70 6.1-7.8 NAa NA (2009) Apea-Bah et al. 5 0.31-1.41 0.77-4.62 0.70-2.20 0.87-1.67 53.60-75.50 (2011) 6 2.37 0.40 7.48 NA 90.00 Ahaotu et al. (2011) 7 0.49 0.13 0.15 0.24 98.40 De Silva, 2013 8 1.46 NA 5.61 0.90 92.03 Moshi, 2014 9 3.38 0.68 4.34 NA 91.61 USDA, 2014 10 - 89,4% - 98,4% 82% Ogunnaike, 2015 Gunawan et al., 11 1.93±0.04 0.66±0.01 4.24±0.05 0.69±0.03 92.48±1.14 2015 aNot available.

Lipids are molecules found in living things. They are substances that dissolve in organic solvents, such as chloroform, hydrocarbon and alcohol instead of water. They may contain acyglycerols, free fatty acids, gums, plant pigments, wax esters, and aldehydes. Fibre also consists of polysaccharides, such as cellulose, hemicellulose, lignin, pectin, and other components associated with the fibrous carbohydrates. Moreover, carbohydrates consist of sugars, and starches. It was observed that starches content in cassava roots is about 83-89.4% (Montagnac and Davis, 2009; Moshi, 2014; Gunawan et al., 2015). Starch consists of two separable fraction, soluble fraction called amylose and unsoluble fraction called amylopectin (Hee-Young, 2005). Starch composed of at least three main components: 15-30% amylose, 70-85% amylopectin and 5-10% other materials (Greenwood and Munro, 1979). The quantity of inorganic materials or minerals knowns as ash. Moreover, carbon to nitrogen ratio is very important for microbial activities within fermentation process. Microorganisms use the carbon from organic matters (carbohydrates) as a source for energy and require nitrogen from protein and other nutrients for reproduction. Gunawan et al. (2015) reported that carbon to nitrogen ratio of dried fresh cassava is 28.41. The variability of proximate, minerals, and anti-nutrients compositions of cassava roots was attributed to cassava cultivar, harvesting age, and diversity of agronomic factors. Anti-nutritions are subtances contained in food that lead poisoning in human when consumed, such as cyanide, phytate, and tannin. Cyanide is the most toxic factor and should be limited for consumption. Cyanide is harmfull for cell metabolism by hampering Cytrochrome oxidase in human body. Peeling and cooking treatments ensure that cassava is able to be consumed safely (Nuwamanya et al., 2008).

Complimentary Contributor Copy 276 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al.

Phytate is another molecule with high concentratiom found in cassava (Table 3). Phytate structure is shown in Figure 3. It is able to bind cation, such as magnesium, calcium, iron, zinc. It also bothers mineral absorption (Montagnac, 2009). Moreover, polyphenol (tanin) increases as the age of cassava (Figure 4). Polyphenol in the human body forms unsaturated complex molecule with divalen ion, such as iron, zinc, and copper. Tanin also deactivate thyamine, and bothers the starch and protein digestion. The other anti-nutrient molecule is oxalate, affects bioavailability and inhibit the digestion system in stomach.

Table 2. Minerals and vitamin contents (ppm) of Cassava roots (Manihot esculenta)

Depkes RI, Akindahunsi et Boonnop et al., Moshi, USDA, Gunawan, et al., Component 1981 al., 1999 2009 2014 2014 2015 Ca 33 167.1 1.31-1.35 NAa 16 38.55 P 40 NA 0.7-0.73 42 27 NA Fe 0.7 22.5 NA 1,4 0.27 2.95 Cu NA NA NA 2,6 0.10 NA K NA 555.5 NA 1 271 NA Mg NA 277.5 NA 6,3 21 NA Mn NA NA NA 24 0.38 NA Na NA 513.7 NA 1,9 14 NA Zn NA 57.5 NA 9,6 0.34 NA Vitamin B1 0.06 NA NA NA 0.09 NA Vitamin C 30 NA NA NA 20.6 NA aNot available.

Table 3. Anti-nutrient content of Cassava roots (Manihot esculenta) (mg/Kg)

No HCN Tannin Phytate Oxalate References 1 14.9 0.1 662.8 NA Akindahunsi et al., 1999 2 21 NA a NA NA Moshi, 2014 3 13.83 NA NA NA Ogunnaike, 2015 4 17.5 ± 1.26 NA NA NA Gunawan, et al., 2015 aNot available.

H2O3PO H2O3PO

H2O3PO

Figure 3. Phytate structure. Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 277

Figure 4. Tanin structure.

FERMENTATION

Word “fermentation” came from Latin language “fervere” which means to boil. Fermentation is a metabolic process that converts sugar to acids, gases, or alcohols. It is also used more broadly to refer to the bulk growth of microorganisms on a growth medium, often with the goal of producing a specific chemical product. In definition, fermentation is a chemical changes process in organic substrate through enzyme activity produced by microorganisms. Microorganisms can ferment various substrates; the end-products depend on the particular microorganisms, the substrates, and the enzymes that are present and active. Chemical analyses of these end-products are useful in identifying microorganisms. The two main types of fermentation are alcoholic and lactic acid fermentation (Tortora et al., 2010). In alcoholic fermentation, substrates are converted into ethanol with the production of carbon dioxide, whereas in lactic fermentation, substrates are converted into lactic acid, and there is no production of carbon dioxide. Alcohol fermentation is carried out by a number of bacteria and yeasts. The ethanol and carbon dioxide produced by the yeast Saccharomyces (sak-ii-ro- mi'ses). They are waste products for yeast cells but are useful to humans. Moreover, two important of lactic acid bacterias are Streptococcus and Lactobacillus. Food fermentation is a product of microorganism’s activities, such as bacteria, leavened, and mold. Microorganisms can produce favorable changes either adverse changes. The most substantial fermentation microorganisms of food are lactic acid bacteria, acetic acid bacteria, and alcohol leavened (Suprihatin, 2010). Liquid, submerged and solid state fermentations are age-old techniques based on water level that used for the preservation and manufacturing of foods. During the second half of the twentieth century, liquid state fermentation developed on an industrial scale to manufacture vital metabolites, such as antibiotics. It is an ideal for the growing of unicellular organisms,

Complimentary Contributor Copy 278 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al. such as bacteria or yeasts. Submerged fermentation is a method of manufacturing biomolecules in which enzymes and other reactive compounds are submerged in a liquid, such as alcohol, oil or a nutrient broth. Another, solid state fermentation uses culture substrates with low water levels (reduced water activity), which is particularly appropriate for mould. The methods used to grow filamentous fungi using solid state fermentation allow the best reproduction of their natural environment. Depend on microorganisms origin, fermentation can be divided into two groups: spontaneous and culture fermentations. Spontaneous fermentation, food fermentation in which no microorganism added in the form of neither starter nor yeast, such as lafun production, Nigerian traditional food. Natural microorganisms which are active in the fermentation process can grow well spontaneously because the ambient condition is good for the growth. Lactobacillus spp. and Leuconostoc spp. are some of the organism that are involved in spontaneous fermentation. Besides, organisms which contribute in culture fermentation are Saccharomyces cerevisiae, Rhizopus oryzae, Aspergillus niger, Endomycopsis burtonii, Mucor sp., Candida utilis, Saccharomycopsis fibuligera, and Pediococcus, sp. The hydrolysis of cyanogenic glucosides takes place during the fermentation. The cyanide content decreases during the fermentation by more than 70% through the activities of the bacterial produced linamarase, allowing the hydrolysis of cyanogenic glucosides. Certain lactic bacterias present in the environment of fermentation that are resistant to the strong cyanide concentrations of between 200 and 800 ppm.

UTILIZATION OF CASSAVA

As a commodity, cassava can be processed to produce many kind of products, such as cassava leaves, cassava chip, cassava flour, sweeteners, starch, ethanol, and modified cassava flour (mocaf).

1. Cassava Roots and Leaves as Vegetables

Both the cassava roots and leaves are edible and most commonly eaten as vegetables. Young tender cassava leaves are a good source of dietary proteins and vitamin K. Cassava leaves contain 17-34% of proteins in dry basis and pH around 8.5 (Kobawila et al., 2005). Vitamin-K has a potential role in bone mass building by promoting osteotrophic activity in the bones (Buitrago, 1990). It also has established role in the treatment of Alzheimer's disease patients by limiting neuronal damage in the brain (Allison, 2001). Cassava roots are popular ingredients in fries, stew-fries, soups, and savory dishes all over the tropic regions. In general, cassava roots are fried in oil until brown and crisp and served with salt, and pepper seasoning as a snack (Bempong et al., 2014). Moreover, there are also used as the ingredients of animal feeds (Oppong-Apane, 2013). Cassava roots are sun-dried for one to two days until they have final dry matter content of less than 85%. They are valued as a good roughage source for ruminants, such as cattle.

Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 279

2. Cassava Starch (Tapioca Starch)

Cassava starch, also known as tapioca starch, is a starchy white flour that has a slight sweet flavor to it. It is extracted from the cassava roots through a process of washing and pulping. The wet pulp is then squeezed to extract a starchy liquid. Once all the water evaporates from the starchy liquid, the tapioca starch remains (FAO, 2016). It is used in both its original form and its other modified forms in various industries: food and beverage, textile, adhesives, paper, plywood, medicine, and biodegradable material industries. The food industries constitute one of the largest consumers of starch and starch products. Cassava starch is used for producing instant noodle, sago, seasoning sauce and monosodium glutamate. It is also used for producing glucose and fructose which are used as the sweeteners in the beverage industry. One of the advantages of cassava starch when compared to corn starch is the absence of the undesired “cereal flavor.” This makes cassava starch preferred for application in many processed foods, with particular interest in bland flavored products. Other factors that make cassava starch as a key ingredient for food industry and also for other kinds of applications are its particular physicochemical behavior when cooked in aqueous dispersion, producing high clarity and high viscosity pastes (Che et al., 2007), as well as presenting a low gelatinization temperature and low tendency to retrogradation when compared to cereal starches. The gelatinization temperature is lower and its apparent viscosity higher than that of corn starch at the same concentration, what represents an advantage for some applications. After cooking, cassava starch paste has lower tendency to retrogradation, what is often a desired property for industrial uses. Although cassava starch has several advantages when compared to corn starch, it also has limitations like being unstable to cooking and to acidity, as other native starches (Takizawa et al., 2004). In the textile industry, cassava starch is used in three main areas: sizing, finishing and printing. Properties of the starch used are abrasion resistance, flexibility, ability to form a bond to the fiber, ability to penetrate the fiber bundle to some extent and ability to have enough water holding capacity so that the fiber itself does not rob the size of its hydration. Moreover, cassava starch is a popular base for adhesives, particularly those designed to bond paper in some form to itself or to other materials, such as glass, mineral wool, and clay (Agboola et al., 1990). It can be also used as a binder or adhesive for non-paper substances, such as charcoal in charcoal briquettes, mineral wool in ceiling tiles and ceramics before firing. Cassava starch adhesives are more viscous and smoother working. They are fluid, stable glues of neutral pH that can be easily prepared and can be combined with many synthetic resin emulsions. In the pharmaceutical industry, cassava strach is used as a disintegrant and binder. Disintegrants enable tablets and capsules to break down into smaller fragments (dissolve) so that the drug can be released for absorption. Starch is used to influence or control the characteristics, such as texture, moisture, consistency and shelf stability. It can be used to bind or to disintegrate, to expand or to densify, to clarify or to opacify, to attract moisture or to inhibit moisture, to produce smooth texture or pulpy texture, and soft coatings or crisp coatings. It can be used to stabilize emulsions or to form oil resistant films (Miyazaki et al., 2006). Another, cassava starch can be transformed as product by mean of adding the biodegradable substance to be in place of plastic.

Complimentary Contributor Copy 280 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al.

3. Cassava Flour

Cassava flour is an alternative to traditional wheat flours and has a variety of uses in baking. It helps bind gluten free recipes and improves the texture of baked goods. It helps add crispness to crusts and chew to baked goods. It is an extremely smooth flour, which makes for a great thickener in sauces, pies and soups since it never discolors and contains no discernible taste or smell.

Table 4. The proximate composition of wheat, cassava and modified cassava flours (SNI standard, 2011)

Composition Wheat flour Cassava flour MOCAF Moisture, % Max 14.5 Max 12 Max 13 Protein, % Min 7.0 NA Min 7.0 Fibre, % Max 2.0 Max 4.0 Max 2.0 Lipid, % NAa NA NA Carbohydrate, % 60-70% 75% NA Ash, % Max 0.7 Max 1.5 Max 1.5 Max 5.0 NA Max 4.0 Total Titrable Acidity (mg KOH / 100 g) (mL NaOH 1N/100 g) HCN (mg/kg) Max 10 Max 10 Max 10 aNot available.

Cassava flour is from freshly harvested roots. The roots are peeled, washed and cut into chips. The cassava chips are sun‐dried, milled into a fine powder and packaged in moisture proof materials. The chipping method is faster and requires the use of only the chipping machine before drying; however, its use is limited to cassava of low cyanogenic potential. The use of casava flour for baking, pastry production and other catering purposes has also been developed and demonstrated to home caterers, bakers and industrial food processors (Falade and Akingbala, 2008). However, it contains a lower protein content than that of wheat flour as can be seen in Table 4 (SNI standard, 2011).

4. Fermented Cassava a. Modified Cassava Flour (Also Knowns as Lafun and Gari Flours in West Africa) Modified cassava flour (mocaf) is a product derived from cassava that uses a principle of modifying cassava flour in fermentation, which produces distinctive characteristics, so it can be used as a food ingredient with a very wide scale. Preliminary experimental results showed that mocaf could be used as raw materials from a variety of foods, ranging from noodles, bakery, cookies and semi-moist food, since the application has a spectrum similar to wheat flour, rice and other starchy materials. Advantages of mocaf: mocaf has aroma and flavor better than that of regular cassava flour, has more color than that of usual cassava flour, and has relatively low prices compared to wheat or rice flours (Sulistyo and Nakahara, 2014). Moreover, it contains a higher protein content than that of cassava flour. Mocaf is expected to be one of the wheat flour alternative substitutes. In Indonesia, mocaf has a good prospect because of the cassava availability and low cost production. There Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 281 are several different methodologies for fermentation process of cassava (liquid, solid state, submerged, culture and spontaneous fermentations), different microorganisms (yeast, fungi and bacteria) and different additional nutrients (with and without nutrients) for enhancing detoxification and improving the quality of cassava flour. The typical important parameters for proximate composition of mocaf are protein, lipids, fibre, starch, cyanide acid and ash contents as shown in Table 4 (SNI standard, 2011). Mocaf can be produced by different methods depending on the locality. The nutrients comparison of mocaf are shown in Table 5. Lafun flour is one of mocaf from south-western part of Nigeria as can be seen in Figure 5 (Rauf, 2015). The flour is made by allowing peeled tuberous roots of cassava steeped in water to ferment naturally (spontaneous fermentation). It is produced by submerged fermentation of peeled sliced cassava roots in water for 3-5 days or by immersing peeled or unpeeled cassava in a stream or stationary water or in an earthenware vessel. It was fermented until the roots become soft after which the fermented cassava was subjected to sun-drying and milling to powder/flour. It was also found that C. manihot, Lactobacillus spp. and Leuconostoc spp. are some of the organisms that are involved in fermentation of cassava to Lafun (Ogunnaike, 2015). The flour is usually prepared as stiff porridge using boiling water, prior to being consumed with soup (Podonou et al., 2009). Gari flour (also known as garri, garry, or gali) is one of mocaf from west africa as can be seen in Figure 6 (International Starch Institute, 2014). Gari flour color is browner than that of lafun. Cassava roots are peeled, washed, and grated or crushed to produce a mash. The mash is placed in a porous bag and weights are placed on the bag for a day to a few days to press excess water out. It is involving spontaneous and solid state fermentations. When the cassava has become dry enough, it is ready for the next step. It is then sieved and fried in a large clay frying pot with or without palm oil. The resulting dry granular garri can be stored for long periods. It may be pounded or ground to make fine flour (Oduah, 2015). b. Bikedi Bikedi is one of Congo traditional food from fermented cassava roots, involving spontaneous and submerged fermentations. In the first step, freshly harvested cassava roots are peeled and cleaned in the water. They are then immersed in the water for fermentations for 3-6 days at ambient temperature (28 to 32°C). In the second step, the whole roots of cassava (unpeeled) are immersed in the water for fermentations for 3-6 days at ambient temperature (28 to 32°C). After fermentation, the softened cassava product, bikedi, is removed from the water. It contains acid condition with pH around 4 (Kobawilla et al., 2005). c. Ntoba Mbodi Ntoba mbodi is one of Congo traditional food from fermented cassava leaves, involving spontaneous and submerged fermentations. Two weeks to 3 months old cassava leaves were harvested. After harvesting, the cassava leaves are exposed to the sun at ambient temperature for 2-3 h. Stalks and petioles were removed and the leaves cut in fragments. Cut leaves are then cleaned in water, then drained and finally packed in the clean leaves of papaw (Papaya carica) at the rate of 150 g per package for fermentation for 2-4 days. After fermentation, the product, ntoba mbodi, is obtained (Kobawila et al., 2005). Mokemiabeka (2011) reported that proximate composition of Ntoba mbodi are 25% protein, 20% fiber, 13.29% ash and others.

Complimentary Contributor Copy

Table 5. Nutrients comparison of modified cassava flour

Ogunnaike, Composition Gunawan, et al., 2015 Oboh and Akindahunsi, 2003 Oboh et al., 2002 et al., 2015 Moisture, % 8.83 9.61 10.42 12.6 NAa NA NA NA Protein, % 8.58 2.29 4.72 1.94 10.9 6.3 12.2 7.3 Fibre, % 2.92 2.53 2.20 2.0 NA NA NA NA Lipids, % 2.55 3.29 3.36 NA 4.5 3.0 5.7 4.0 Starch, % 55.40 71.00 48.2 82.46 77.9 84.5 NA NA Ash, % 0.49 0.47 0.42 1.0 NA NA NA NA HCN, mg/kg 1.8 3.28 3.17 13.2 9.5 9.1 9.1 4.1 Odor Normal Normal Normal Normal Normal Normal Normal Normal Color White White White White White Browner White Browner Mikroorganism L. plantarum S. cereviseae R. oryzae natural S. cereviseae S. cereviseae A. niger A. niger Submerged Submerged Submerged Submerged Submerged Solid state Solid state Submerged Submerged a Not Available.

Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 283

Figure 5. Lafun Flour.

Figure 6. Gari flour. d. Foofoo (Also Known as Fufu, Foufou, Fufuo, and Fofo) Foofoo is one of Nigeria traditional food from fermented cassava roots, involving spontaneous and submerged fermentations. The proximate composition of foofoo can be seen in Table 6. Foofoo production includes peeling, washing and steeping or submerging of cassava roots in water for 3- 4 days. During this period, the retted cassava tubers are softened. The softened pulpy mass is then disintegrated in water and passed through a coarse sieve. This separates the fiber from starch which is allowed to sediment, then the water is decanted. It is then packed into cloth bags and excess water is squeezed out. The resulted meal is white and crumbly which is usually cooked before being eaten. Foofoo is reconstituted by stirring in boiling water to form dough and eaten with favoured sauces (Abriba et al., 2012).

Complimentary Contributor Copy 284 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al. e. Chikwangue (Cassava Bread) Chikwangue is one of Congo traditional food from fermented cassava roots, which are mainly consumed in the form of cassava bread as can be seen in Figure 7 (Naliaka, 2015). It contains 10.3% protein for composition cassava bread and soya at 80:20 and 12.4% protein for composition cassava bread and peanut at 80:20 (Tajudeen, 2013). Naliaka (2015) reported that proximate of chikwangue is 51.4% moisture, 0% lipid, 1% fibre, and 45% carbohydrates. Chikwangue production includes peeling, washing and submerging of cassava tubers in water for 3-4 days. It is involving spontaneous and submerged fermentations. Chikwangue is a popular starchy food from a recipe for cooking a roll of cassava dough (often times called 'cassava bread') inside a multi-layered wrapping of large fresh leaves (about 7 leaves), tied snugly with palm fibers or string to hold steam inside during cooking.

Table 6. The proximate composition of foofoo flours

Ojo et al., 2014 Blessing et al., Abriba et al., Composition Oven dried Sun Dried Solar Dried 2013 2012 Moisture, % 8.700.16 8.500.23 8.000.19 NAa 71.13% Protein, % 1.170.32 1.020.41 1.210.27 3.09% 0.14% Fibre, % 0.230.20 0.170.13 0.150.10 0.36% NA Lipid, % 0.720.11 0.620.09 0.700.10 0.18% 0.009% Carbohydrate, % 88.830.27 88.390.33 88.620.22 95.41% 28.64% Ash, % 0.350.21 0.300.17 0.320.11 0.96% 0.09% a Not Available

Figure 7. Chikwangue.

Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 285 f. Tapai Tapai is one of Indonesian traditional food from fermented starchy substrates, such as cassava and white rice, involving solid state and culture fermentations by yeast as shown in Figure 8 (Astawan and Mita, 1991). It contains 0.5% protein, 0.1% lipid, 56.1% moisture and 42.5% carbohydrates (Ganjar, 2003). Cassava is wrapped with leaf and softened (Saleh, 2015). The culture fermentation involves many kind of microorganisms, such as S. Cerevisiae, R. oryzae, E. burtonii, Mucor sp., C. utilis, S. fibuligera, and Pediococcus, sp. (Ganjar, 2003). Tapai fermentation consists three stages of decomposition: (1) starch molecules are split into 10 dextrin and monosaccharaides, using enzymatic hydrolysis process, (2) monosaccharaides are modified into organic acids and alcohol, (3) organic acids are reacted with alcohol forming unique taste of tapai (ester) (Hidayat, 2006).

Figure 8. Tapai. g. Bioethanol Cassava is used for producing alcohol for the liquor manufacture and the disinfectant. Production of bioethanol is also a global requirement to address the question of energy crisis. Ethanol has the advantages of being renewable, providing cleaner burning and producing no green-house gases compared to conventional fossil fuel. Attempts have been made to use agricultural starch based renewable resources (cassava roots) to produce ethanol. Bioethanol production consists of three major processes as can be seen in Figure 9. At the first step, cassava is obtained from agricultural crops. During the second step, cassava is converted into fermentable sugars by enzymatic degradation (α-amylase and glucoamylase exogenously) or by acid hydrolysis. These simple sugars are then fermented to produce ethanol by yeast, S. cerevisiae. At the final step, ethanol is recovered from the fermentation broth by distillation.

Complimentary Contributor Copy 286 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al.

CH2OH CH2OH

H H O H H

OH OH

OH Enzyme OH OH

H OH +H 0 O 2 H OH

HOCH 2 OH O HOCH2 O OH OH

H CH2OH H CH2OH OH H OH H Sucrose Fructose (a) CH2OH CH2OH O H+ or OH O O OH OH OH lactase OH OH

CH2OH CH2OH HO O O OH OH + OH OH HO OH OH (b)

H2COH

o H O OH H OH C +Yeast OH H + 2CO2 CH2 C O OH OH CH3 CH3 H OH 2 Pyruvate 2 Ethanol Glucose (c)

Figure 9. Production of bioethanol from cassava: Enzyme degradation(a), acid hydrolysis (b), and ethanol production (c).

ANALYSIS

The typical important parameters for proximate composition of mocaf are protein, lipids, fibre, starch, cyanide acid and ash contents. A summary of most analytical techniques used in the quantification of compounds in mocaf is given in Table 7. Moisture content was analyzed using a Halogen Moisture Analyzer. A sample was weighed accurately in a clean and dried crucibile (W1). Then the crucible was introduced into o the Halogen Moisture Analyzer and held at 105 C until constant weight was achieved (W2). The moisture content (M) was calculated as Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 287

M = (W1-W2) / (W1) × 100% (1)

A Soxhlet extractor, equipped with a condenser system, was employed to measure lipids content. The sample was wrapped in filter paper and placed inside the Soxhlet extractor. Neutral lipids, such as fatty acids and acylglycerols, were extracted from the sample with hexane as the solvent. After a predetermined time, the extraction process was stopped, which was designated as the crude oil. The lipids content (L) was calculated as

L = {[weight of hexane extract, g] / [weight ofsample, g]} × 100% (2)

The proteins content was determined by analyzing its nitrogen content (AOAC, 2003). Total protein was determined by multiplying the amount of nitrogen by the 6.25 correction factor (FAO, 2003). A dried sample (W) was transferred into a Kjeidahl digestion flask. Then, a certain volume of concentrated HCl were added to the digestion flask. The flask was swirled in order to mix the contents thoroughly, then placed on heater to digest untill the mixture became clear. Then, distilled water was added and swirled in order to mix the contents thoroughly. Then, a certain volume of NaOH 30% solution and some drops of phenolptalein indicator were added into the Kjeidahl digestion flask. Distillation was finished when there was no flow from digestion flask. The NH3 produced was collected as NH4OH in a conical flask containing 4% boric acid solution with few drops of Conway indicator. The distillate was titrated againts standard HCl 1 N. The crude proteins content (P) was calculated as

P = 6.25 ×[(V1-V2) × N ×0.014 × f / W] × 100% (3) where V1, V2, N, f, W were the sample titration reading, blank titration reading, HCl normality, sample dilution, and sample weight, respectively. The 0.014 was the mili equivalent of nitrogen. Ash content was determined by AOAC (2003). A clean and empty evaporating dish was o heated in a muffle furnace at 600 C for 1 h, cooled in a desiccator and weighed (W1). A sample was weighed into an evaporating dish. The sample was heated in a muffle furnace at 550 oC for 6 h until it was charred. The result appeared as a gray white ash. It indicates that complete oxidation of all organic matters in the sample had occured. The evaporating dish was cooled and weighed (W2). The percent ash (A) was calculated as

A = (W2 - W1) / sample weight × 100% (4)

Fibers content was determined by AOAC (2003). Moisture free sample was digested using dilute H2SO4 followed by dilute KOH solution. The undigested residue was ignited in a muffle furnace. Weight loss after ignition was considered as crude fiber. A 0,5 g sample (W1) was placed in an evaporating dish along with 150 ml of H2SO4 and some drops of acetone as foam suppresser. The mixture was heated at 100oC until it started to boil. Then, the temperature was reduced to 45oC for 0.5 h. The sediment was filtered and rinsed with distilled water to remove any remaining acid. Afterwards, the same procedure was repeated using KOH instead of sulfuric acid. Filter paper with sediment was dried in an oven at 150oC for 1 h, then transferred into a desiccator, and then weighed (W2). The sediment and filter paper

Complimentary Contributor Copy 288 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al. were placed in an evaporating dish and heated in a furnace for 3-4 h, then transferred into a desiccator and then weighed (W3). The crude fibers content (F) was calculated as

F = (W2 – W3) / W1× 100% (5)

Acid method (AOAC,2003) was used to determine starch content of washed and fermented cassava. Briefly, a crushed sample was put into a flask containing distilled water. The mixture was stirred for 1 h and filtered using a Whatman 42 filter paper. The solid phase on the filter paper was washed with distilled water. Furthermore, the solid residue on the filter paper was washed with diethyl ether, and allowed the ether to evaporate. Afterwards, it washed again with 10% ethanol for further release of soluble carbohydrates. The filtrate containing soluble carbohydrates was discarded. The solid residue was transferred into a flask containing distilled water. Then, 25% HCl was added to the flask and heated at 100°C for 2.5 h. After cooled, the mixture was neutralized with 45% NaOH solution. The mixture was filtered by vacuum filtration. The resulting solid phase on the filter paper was wasshed with distillate water. Furthermore, sugar content of the filtrate was analyzed for according to the 3,5-dinitrosalicylic acid (DNS) method. The percentage of starch was determined by multiplying glucose content by factor number of 0.9. The cyanide acid content of fermented cassava was determined by titration (SNI, 2011). Briefly, a fermented cassava was transferred into a Kjeldahl digestion flask containing distilled water. The flask was swirled to mix the contents thoroughly for 2 h and heated to recover cyanide acid as distillate. The distillate was collected in a conical flask containing 2.5% NaOH solution, NH4OH solution and 5% KI solution. The resulting mixture was titrated against 0.02 N AgNO3 until there was a turbidity. A blank was also run through all steps above. The cyanide acid content (HCN) was calculated from the amount of AgNO3 used for titration.

HCN (mg/kg) = (V1-V2) × N × 27 / (V3× W) (6) where V1, V2, V3, N, and W were the blank titration reading, sample titration reading, distillate volume, AgNO3 normality, and sample weight, respectively. The 27 was the molecular weight of cyanide acid. The lactic acid content of filtrate obtained from fermentation was determined by total titrable acidity (GEA Niro, 2006). Briefly, a sample was transferred into a flask. A few drops of phenolphthalein as indicator was added to the flask. Then, the sample was titrated against 0.2 N NaOH. The lactic acid content (TTA) was calculated as:

TTA (mg/ml) = N × V1 × 0.090 / V2 (7) where N, V1, and V2 were the NaOH Normality, NaOH volume, and sample volume. The 0.090 was the milli equivalent of lactic acid.

Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 289

Table 7. Summary of most analytical techniques used in quantification of compounds in mocaf

Compounds Techniques References Moisture Gravimetric method Gunawan et al., 2015 Lowry Method (AOAC) Sulistyo and Nakahara 2013; Emmanuel et al., 2015 Protein Kjeldahl method (AOAC) Nuwamanya et al.., 2011; Moshi et al., 2014; Gunawan et al., 2015; Ogunnaike et al., 2015; Oduah et al., 2015; Soxhlet exraction method Nuwamanya et al..,2011; Gunawan et al., Lipids (AOAC) 2015; Ogunnaike et al., 2015; Oduah et al., 2015; Fiber AOAC Gunawan et al., 2015 DNS colorimetric method Sulistyo and Nakahara 2013 (AOAC) Acid method, DNS colorimetric Gunawan et al., 2015 Starch method Acid method, HPLC method Moshi et al., 2014 Spectrophotomeric method Nuwamanya et al..,2011 Titration (AOAC) Hidayat et al., 2002; Emmanuel et al., HCN 2015 Titration (SNI) Gunawan et al., 2015 Titration Method (AOAC) Abriba et al., 2012; Sulistyo and Nakahara, 2013; Emmanuel et al., 2015; Lactic acid Gunawan et al., 2015; Ogunnaike et al., 2015; Oduah et al., 2015;

LACTIC ACID

Lactic acid is a carboxylic acid widely used as preservative, acidulant, and/or flavouring in food industry. It is also used as a raw material for the production of lactate ester, propylene glycol, 2,3-pentanedione, propanoic acid, acrylic acid and acetaldehyde. The demand for lactic acid production has dramatically increased due to its application as a monomer for poly-lactic acid synthesis, a biodegradable polymer used as a plastic in many industrial applications (Milkos et al., 1994; Barrera et al., 1995; Quintero et al. 2012). In recent years, development of biological control should help improve the safety of products by controlling mycotoxin contamination. Data has actually shown that many lactic acid bacterias can inhibit mould growth and that some of them have the potential to interact with mycotoxins. Dalié et al. (2010) summarized that lactic acid bacterias are promising biological agents for food safety. Moreover, lactic acid can be produced either by fermentation or chemical synthesis. The former route has received considerable interest, due to environmental concerns and the limited nature of petrochemical feedstocks. Thus, 90% of lactic acid produced worldwide is obtained by fermentation. This process comprises the bioconversion of carbohydrates into

Complimentary Contributor Copy 290 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al. lactic acid in the presence of a microorganism. Cassava rots are promising low-cost substrate as a carbon source for lab and eventually large scale lactic acid biosynthesis. Fermentation couses substantial modification to the physicochemical characteristic of cassava roots. pH is an important parameter in determining the quality of modified cassava flour. pH of 4 or less indicates appreciable level of fermentation and hence starch breakdown. Fermentation also imparts characteristic aroma, flavour and sour taste to the flour. The effect of fermentation time and culture type (L. plantarum, S. cereviseae, and R. Oryzae) on the pH profile of cultivation is shown in Table 8. It can be seen that a relatively high pH is indicate of an unfermented cassava roots and pH value of culture decreases by increasing fermentation time. However, pH profile of culture varies according to the fermentation time within the culture type studied. The final pH for each culture is within the range of 3.87-4.37. Previous work reported that pH profile was decreased with time as a result of more lactate production and accumulation when cultivating R. oryzae without pH control. The final pH for each culture was within the range of 2.0–4.5 (Phrueksawan, 2012).

Table 8. pH profile of cultivation of L. plantarum, S. cereviseae, and R. oryzae on cassava roots

Fermentation time, pH (h) L. plantarum S. cereviseae R. oryzae 0 6.02±0.54 6.02±0.54 6.02±0.54 12 4.14±0.05 4.18±0.01 4.33±0.50 24 3.98±0.01 4.10±0.02 4.17±0.01 36 3.89±0.02 4.15±0.01 4.11±0.01 48 3.87±0.03 4.17±0.01 4.12±0.01 60 3.89±0.01 4.17±0.02 3.99±0.01 72 3.90±0.01 4.26±0.01 4.02±0.01 84 3.97±0.02 4.30±0.01 3.94±0.03 96 3.93±0.01 4.37±0.15 3.87±0.06

Table 9. The lactic acid production from L. plantarum, S. Cereviseae, and R. Oryzae cultivations on cassava fermentation

Fermentation time, Lactic acid concentration (mg/mL) (h) L. plantarum S. cereviseae R. oryzae 0 0.02 ±0.00 0.02±0.00 0.01±0.01 12 0.30±0.03 0.32±0.02 0.42±0.01 24 0.49±0.05 0.45±0.01 0.51±0.01 36 0.56±0.02 0.49±0.01 0.57±0.03 48 0.68±0.01 0.52±0.01 0.58±0.01 60 0.73±0.01 0.52±0.02 0.61±0.04 72 0.75±0.01 0.53±0.02 0.61±0.04 84 0.84±0.01 0.54±0.01 0.76±0.01 96 0.90±0.01 0.55±0.01 0.89±0.01

Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 291

To verify the above results, the lactic acid content is an important parameter to measure. The effect of fermentation time and culture type (L. plantarum, S. cereviseae, and R. Oryzae) on the lactic acid production is shown in Table 9. It can be seen that conversion from glucose to lactic acid is the critical step in the fermentation on cassava roots. L. plantarum, S. Cereviseae, and R. Oryzae cultivations produced lactic acid within the range of 0.55-0.90 mg/mL. This cause a rapid drop in pH, the environment then became selective against less acid-tolerant microorganisms. Wang et al. (2010) reported that the high L-lactic acid concentration (175.4 g/L) was obtained using 275 g/L of cassava powder concentration (total sugar of 222.5 g/L). This is the highest L-lactic acid concentration from cassava fermentation. It provides an efficient L-lactic acid production process with cheap raw bioresources, such as cassava powder.

REFERENCES

Abdallah, M.A., Lei, Z.M., Li, X., Greenwold N., Nakajima, S.T., Jauniaux, E., Rao, C.V., 2004. Human Fetal nongonadal tissues contain human chorionic gonadotropin/ luteinizing hormone receptors. J. Clin. Endocrinol. Metab. 89, 952-956. Abriba, C., Henshaw, E.E., Lenox, J, Eja, M., Ikpoh, I.S., Agbor, B.E., (2012). Microbial succession and odour reduction during the controlled fermentation of cassava tubers for the production of ‘foofoo,’ a staple food consumed popularly in Nigeria. J. Microbiol. Biotech. Res., 2, 500-506. Achi, O.K. and Akoma, N.S., (2006). Comparative Assessment of Fermentation Techniques in the Processing of Fufu, a Traditional Fermented Cassava Product. Pak. J. Nutr. 5, 224- 229. Adams, A. and Ray, C., (1988). Catering Technology, 1st ed. London: B.T. Bastford Ltd. Agboola, S. O., Akingbala, J. O., Oguntimein, G. B. (1990) Processing of Cassava Starch for Adhesive Production. Nr. 1 S. 12-15. Ahaotu, I., Ogueke, C. C., Owuamanam, C. I., Ahaotu, N. N. and Nwosu, J. N. (2011). Protein improvement in gari by the use of pure cultures of microorganisms involved in the natural fermentation process. Pakistan J. Biol. Sci. 14, 933 – 938. Akihandusi, A.A., Oboh, G., Oshodi, A.A. (1999). Effect of Fermenting Cassava with Rhizopus oryzae on the Chemical Composition of Its Flour and Gari Products. Riv. Ital. Sostanze Grasse, 76, 437-440. Akintonwa, A., Tunwashe, O., Onifade, A., (1994). Fatal and Non-Fatal Acute Poisoning Attributed to Cassava–based Meal. Acta Hortic., 375, 285–288. Aletor, V.A., (1993). Allelochemichal in Plant Food and Feeding Stuffs: Nutrional, Biochemichal and Physiophatological Aspects in Animal Production. Veterinary and Human Toxicology Journal. 35: 57-67. Aletor, V.A (1993) Cyanide in Garri: Distribution of Total, free and bound hydro cyanide acid (HCN) in commercial garri and the effect of fermentation time on its residual cyanide content. Intern. J. Food Sc. Nutr.44, 181-287. Allison, A. C., (2001). The Possible Role of Vitamin K Deficiency in The Pathogenesis of Alzheimer’s Disease and in Augmenting Brain Damage Associated with Cardiovascular Disease. Medical Hypotheses. 57(2), 151–155.

Complimentary Contributor Copy 292 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al.

AOAC, (2003). Official Methods of Analysis. 17th edition, 2 revision. AOAC International. Gaithersburg, Maryland, USA. Apea-Bah, F., Oduro I., Ellis, W. and Safo-Kantanka, (2011). W.J.D.S. 6, 43-54. Aplin, J.D., (1991). Implantation, trophoblast differentiation and hemochorial placentation, Mechanistic evidence in vivo and in vitro. J. Cell. Sci. 99, 681-692. Aplin, J.D., Haigh, T., Jones, C.J., Church, H.J., Vićovac, L., (1999). Development of cytotrophoblast columns from explanted first-trimester human placental villi, role of fibronectin and integrin alpha5beta1. Biol. Reprod. 60, 828-38. Badan Standarisasi Nasional. 2009. SNI-7125-2009 Standar Mutu Tepung Terigu. Jakarta. Badan Standarisasi Nasional. 2011. SNI-7622-2011 Standar Mutu Tepung MOCAF. Jakarta. Bahado-Singh, R.O., Oz, A.U., Kingston, J.M., Shahabi, S., Hsu, C.D., Cole, L.A., (2002). The role of hyperglycosylated hCG in trophoblast invasion and the prediction of subsequent preeclampsia. Prenat. Diagn. 22, 478-83. Bempong, E.K., Constance, A.S., Sarkodie, N.A., (2014). Enhancing Food Tourism in Ghana Through Indigenous Dishes: A Case Study of the Brong-Ahafo Region, Ghana. I.J.I.A.R, 2, 83 – 96. Berndt, S., Blacher, S., d'Hauterive, PS., Thiry, M., Tsampalas, M., Cruz, A., Pequeux, C., Lorquet, S., Munaut, C., Noel, A., Foidart, J.M., (2009). Chorionic gonadotropin stimulation of angiogenesis and pericyte recruitment. J. Clin. Endocrinol. Metab. 94, 4567-4574. Blessing, N., Ishola, Chikwendu, O. (2013). Effect of blending on the Proximate, Pasting and Sensory Attributes of Cassava-African yam Bean Fufu Flour. I.J.S.E.R. 3, 2250-3153. Bokanga, M., (1995). Biotechnology and Cassava Processing in Africa. Food Technol. 49, 88-90. Boonnop, K., M. Wanapat, N. Nontaso, and S. Wanapat. (2009). Enriching nutritive value of cassava root by yeast fermentation. Sci. Agric. 66, 629-633. Buitrago, A.J.A., (1990). La Yuca en la Alimentaciόn Animal. Centro Internacional de Agricultural Tropical (CIAT), Cali, Colombia. 446-449. Burton G.J., (2004). Placental oxidative stress, From miscarriage to preeclampsia. J. Soc. Gynecol. Invest. 11, 342-52. Cardoso, A.P., Mirione, E., Ernesto, M., Massaza, F., Cliff, J., Haque, M.R. and Bradbury, J.H., (2005). Processing Cassava Roots to Remove Cyanogens. J. Food Comp. Anal., 18(5): 451-460. Che, L. M. Li, D., Wang, L., Chen, X. D., and Mao, Z. (2007). Micronization and hydrophobic modification of cassava starch. Int. J. Food Prop. 10, 527-536. Cumbana, A., Mirione, E., Cliff, J. and Bradbury, J.H., (2007). Reduction of Cyanide Content of Cassava Flour in Mozambique by The Wetting Method. Food Chem. 101: 894-897. Dalié, D.K.D., Deschamps, A.M., Richard-Forget, F. (2010). Lactic acid bacteria – Potential for control of mould growth and mycotoxins: A review. Food Control 21: 370–380. Emmanuel, N.O., Olufunmi, A.O., Elohor, E.P. (2015). Effect of Fermentation Time on the Physico –Chemical, Nutritional and Sensory Quality of Cassava Chips (Kpo-Kpo Garri) a Traditional Nigerian Food. American Journal of BioScience. 3, 59-63 Health Department (Depkes) Of Indonesia (1981). Composition list of food resource. Bhatara Karya Aksara, Jakarta. FAO/WHO. (1991). Joint FAO/WHO food standards programme. Codex Alimentarius Commission XII (Suppl. 4). Rome; FAO. Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 293

FAO (2016). Cassava Processing. http://www.fao.org/docrep/ x5032e/ x5032E02.htm. Indonesian Statistics., (2014). Perkembangan Produksi Singkong Indonesia Tahun 2009- 2013. http://www.bps.com. Falade, K. O., and Akingbala, J. O. (2008). Improved Nutrition and National Development Through the Utilization of Cassava in Baked Foods. Using Food Science and Technology, IUFoST: Chapter 10. Ganjar I., (2003). Tapai from Cassava and Sereals, First International Symposium and Workshop on Insight into the World of Indigenous Fermented Foods for Technology Development and Food Safety; Bangkok, pp.1 – 10. Greenwood, C.T. and Munro, D.N. (1979). Carbohydrates. R.J. Priestley, ed. Effects of Heat on Food stuffs. London: Applied Science Publ. Ltd. Gunawan, S., Widjaja, T., Zullaikah, S., Ernawati, L., Istianah, N., Aparamarta, H.W. dan Prasetyoko, D. (2015). Effect of Fermenting Cassava with Lactobacillus plantarum, Saccharomyces cerevisiae, and Rhizopus oryzae on the Chemical Composition of Their Flour. Int. Food Res. J. 22, 1280-1287. Hahn, S.K., (1992). An Overview of Traditional Processing and Utilizatin of Cassava in Africa. In Hahn, S.K., Reynolds, L. and Egbunike, G.N. (Eds). Cassava as Live Stock Feed in Africa, p. 16-27. Ibadan: International Institute for Tropical Agriculture (IITA). Hee-Young. (2005). Effects of Ozonation and Addition of Amino Acids On Properties of Rice Starches. A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Hidayat, N., Padaga, M.C., and Suhartini, S. (2006). Mikrobiologi Industri. Andi, Yogyakarta. International Starch Institute. (2014). Gari. http://www.starch.dk/isi/ starch/gari.asp. Jayasuriya, C. (2015). Singkong Obat Ajaib untuk Kanker. http://akinarishop.com/berita/ detail/singkong-obat-ajaib-untuk-kanker-4774.html. Kobawila, S.C., Louembe, D., Keleke, S., Hounhouigan, J., and Gamba, C., (2005). Reduction of The Cyanide Content During Fermentation of Cassava Roots and Leaves to Produce Bikedi and Ntoba Mbodi, Two Food Products from Congo. Afr. J. Biotechnol. 4, 689-696. Lingga. P., (1990). Bertanam Ubi – Ubian. Jakarta: Penebar Swadaya. Miyazaki, M. R., Hung, P. V., Maeda, T. and Morita, N. (2006), Recent advances in application of modified starches for bread making. Trends Food Sci. Tech., 17, 591-599. Myatt, L., (2002). Role of placenta in preeclampsia. Endocrine 19, 103-111. Mokemiabeka, S., Dhellot, J., Kobawila, S.C., Diakabana, P., Ntietie-Loukombo, R.N., Nyanga-Koumou, A.G., and Louembe, D. (2011). Softening and Mineral Content of Cassava (Manihot esculenta Crantz) Leaves During the Fermentation to Produce Ntoba mbodi. Adv. J. Food Sci. Technol. 3, 418-423. Montagnac, J.A. and Davis, C.R. (2009). Nutritional Value of Cassava for Use as a Staple Food and Recent Advances for Improvement. Compr. Rev. Food, vol. 8. Moshi, A. P. (2015). Production of Bioethanol from Wild Cassava Manihot glaziovii through Various Combinations of Hydrolysis and Fermentation in Stirred Tank Bioreactors. Sweden. British Journal. 5. Naliaka, T.K. (2015). Chikwangue. https://commons.wikimedia.org/wiki/ File:Chikwangue.

Complimentary Contributor Copy 294 S. Gunawan, Z. Istighfarah, H. Wirawasista Aparamarta et al.

Nhassico D., Muquingue H., Cliff J., Cumbana A., Bradbury J.H. (2008) Rising African cassava production, diseases due to high cyanide intake and control measures. J. Sci. Food Agr, 88, 2043–2049. Nuwamanya, E., Baguma, Y., Kawuki, R.S. and Rubaihayo, P.R. (2011). Quantification of Starch Physicochemical Characteristics in A Cassava Segregating Population. Afr. Crop Sci. J. 16, 191-202. Oboh, G. and Akindahunsi, A.A. (2003). Biochemical changes in cassava products (flour and gari) subjected to Saccharomyces cereviseae solid media fermentation. Food Chem. 82, 599-602. Oboh, G., Akindahunsi, A.A. and Osodhi, A.A. (2002). Nutrient and Antinutrient content of Aspergillus niger fermented cassava product (flour and gari). Journal of Food Composition and Analysis 15: 617-622. Oduah, N.O., Adepoju, P.A., Longe, O., Elemo, G.N. and Oke, O.V. (2015). Effects Of Fermentation On The Quality And Composition Of Cassava Mash(Gari). International Journal of Food Nutrition and Safety. 6, 30-41 Onwueme, I.C. (1978). The Tropical Tuber Crops: Yams, Cassava, Sweet Potato and Cocoyams. Wiley, NewYork, ISBN: 9780471996071, pp: 210. Oppong-Apane, K., (2013). Cassava as Animal Feed in Ghana: Past, Present and Future. Accra: Food and Agriculture Organization of the United Nations. Ogunnaike, A. M., Adepoju, P. A., Longe, A. O., Elemo, G. N., Oke, O.V. (2015). Effets of Submerged and Anaerobic Fermentation on Cassava Flour (Lafun). Afr J Biotechnol. 14, 961-970. Ojo, A., Abiodun, O.A., Odedeji, J.O., and Akintoyese, O.A. (2014). Effects of Drying Methods on Proximate and Physico-chemical Properties of Fufu Flour Fortified with Soybean. Br. J. Appl. Sci. Technol. 4, 2079-2089. Padonou S.W, Hounhouigan J.D, Nago M.C (2009). Physical, chemical and microbiological characteristics of lafun produced in Benin. Afr J Biotechnol. 8, 3320-3325. Phrueksawan, P., Kulpreecha, S., Sooksai, S. and Thongchul, N. (2012). Direct fermentation of L(+)-lactic acid from cassava pulp by solid state culture of Rhizopus oryzae. Bioprocess Biosyst. Eng. 35, 1429-1436. Quintero, J. E., Alejandro A. C., Carlos M. G, Rigoberto R. E., Ana M.T. L. (2012). Lactic acid production via cassava-flourhydrolysate fermentation. Vitae, Revista De La Facultad De Quimica Farmaceutica, 19: 287-293. Rauf, W., (2015). List of Products by Manufacturer Mac. Phillips Foods. http://www.indopak.se/28_ Richana, N., (2012). Budidaya Singkong. Bandung: ITB. Saleh, Z. (2015). Hukum Tape. http://tarbijahislamijah.com/hukum-tape-tapai. Shiu, P., Gunawan, S., Hsieh, W.H., Kasim, N.S., Ju, Y.H. (2010). Biodiesel production from rice bran by a two-step in-situ proces. Bioresour. Technol. 101, 984-989. Suprihatin. 2010. Teknologi Fermentasi. Surabaya: UNESA Press. Sulistyo, J and Nakahara, K. (2014). Physicochemical properties of modified cassava starch prepared by application of mixed microbial starter. Int. j. res. agric. food, 2, 1-8. ISSN 2311 -2476. Silva, A.D.S., Mota, T.A., Fernandes, M.G., and Kassab, S.O. Spatial distribution of Bemisia tuberculata (Hemiptera: Aleyrodidae) on cassava crop in Brazil. Rev. Colomb. Entomol. 39, 93-196. Complimentary Contributor Copy Utilization of Modified Cassava Flour and Its By-Products 295

Takizawa, F. F., Silva, G. O., Konkel, F. E., Demiate, I. M. (2004) Characterization of tropical starches modified with potassium permanganate and lactic acid. Braz. arch. biol. technol., 47, 921-931. Tortora, G., Funke, B.R., and Case, C.L. (2010). Microbiology: An Introduction, 10th edition. San Francisco: Pearson Education Inc., 141-147. USDA National Nutrient Database for Standard Reference. (2014). Proximate Nutrient Composition of Cassava. http://www.nal.usda.gov./fnic/food comp/search/. Wang, L., Feng, Z., Wang, X., Wang, X., Zhang, X. (2010). DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26, 136– 138. Westby, A., Choo, B.K. (1994). Cyanogen Reduction during Lactic Fermentation of Cassava. Acta Hortic, 375, 15 - 209.

Chapter 15

RECENT ADVANCES IN THE DEVELOPMENT OF BIODEGRADABLE FILMS AND FOAMS FROM CASSAVA STARCH

Giordana Suárez1 and Tomy J. Gutiérrez2,* 1School of Chemistry, Faculty of Sciences, Central University of Venezuela, Caracas, Venezuela 2Composite Materials Group, Institute of Materials Science and Technology (INTEMA) (CONICET-UNMdP), Faculty of Engineering, National University of Mar del Plata and National Research Council (CONICET), Mar del Plata, Argentina

ABSTRACT

Currently eco-friendly polymeric materials are made from different biopolymers. In this sense, special attention has brought the use of starch at industrial level, since can be processed as conventional polymers. In the same way, one of the starches most used for developing biodegradable films and foams for use as packing material has been cassava (Manihot esculenta) starch, due to its high production and performance, which makes it be a promising material for replacement of polymers obtained from the petrochemical industry. At regard, in this chapter will be reviewed and discussed recent advances related to the development of biodegradable films and foams made from cassava starch.

Keywords: cassava, eco-materials, films, foams, packaging, starch, thermoplastic materials

* Corresponding Author address: Institute of Materials Science and Technology, Faculty of Engineering, National University of Mar del Plata and National Research Council (CONICET), PO Box B7608FLC, Solís 7575, Mar del Plata, Argentina. Tel.: +54 223 481 6600 int 321; fax: +54 223 481 0046. E-mail address: [email protected]; [email protected]. Complimentary Contributor Copy 298 Giordana Suárez and Tomy J. Gutiérrez

INTRODUCTION

The synthetic polymers have replaced metals, glasses, ceramics and wood in many applications, chiefly in the area of packaging. The main commodity plastics, the so-called “big five”, which are polyethylene (PE), poly(propylene) (PP), polystyrene (PS), poly(vinyl chloride) (PVC) and poly(ethylene terephthalate) (PET) have revolutionized the packaging industry in a variety of forms such as films, flexible bags, rigid containers and foams (Soykeabkaew et al. 2015). In this context, our dependence on plastic in our daily life is very high, since we use almost 100 million tons annually, which has preoccupied the world population, since these materials are highly polluting and contribute to global overheating of planet earth (Iriani et al. 2015). As an alternative have emerged mainly biodegradable materials from starch, and specifically cassava starch has had much attention for their extraordinary properties in this regard. Therefore, in the present chapter will be reviewed and discussed recent advances related to the development of biodegradable films and foams made from cassava starch.

1. CASSAVA

Cassava (Manihot esculenta) belongs to Euphorbiaceae family and responds to various names such as cassava, tapioca, mandioca or yuca (Montaldo 1991). Its cultivation is done with the purpose of being a food plant or for industrial purposes, being noted its use increasingly in the industriy. Due to its high starch content, cassava is used, along with corn, as the main sources of raw material for starch extraction (Thomas and Atwell 1999). There are two types of cassava: bitter cassava and sweet cassava. The first, more developed, rich in starch and with a higher content of linamarin (cyanogenic glucoside), whereas the second is generally for direct consumption. An alternative to minimize losses in this area is starch production, which exhibits characteristics of particular interest in industrial applications, due to its high purity, neutral taste, easy swelling and solubilization, and considerable development of viscosity. Cassava is considered as the third most significant food source for those who live in tropical areas. It is also the fifth most produced starch crop around the worlds (Edhirej et al. 2015). It is a type of plant which has different purposes of use. It is used to produce various foods, biofibers, bio-composites and bio-polymers. Besides, now is used as renewable energy source. The intention is to focus on the importance of cassava as biodegradable material to various industrial applications such as the food production, cassava films and foams, food packaging and the incorporation of organic or inorganic natural nanofillers in polymeric matrices based on cassava starch. On the other hand, studies carried out have allowed to evaluate the composition of cassava roots, yielding values of 70.25% moisture, 13.13% sugar, 1.12% protein, 0.14% fat, 1.11% fiber and 0.54% ash. It is also considered that bitter varieties have on average 30% of starch (Grace 1987). Due to high starch content in their roots, the bitter varieties are grown for industrial purposes.

Complimentary Contributor Copy Recent Advances in the Development of Biodegradable Films … 299

2. CASSAVA STARCH

Among the most studied biopolymers is found cassava starch, which has the advantages of coming from renewable sources and being biodegradable, inexpensive (US$ 0.25-0.60/ kg), and widely available (da Silva et al. 2013). In this sense, cassava starch is extracted from the plant roots by various processes that are described in Figure 1. In the case of cassava starch, have been reported moisture content values ranging from 8.2 to 14% (Whistler and Paschal 1967; Wurzburg 1972; Matos 1996a). Whistler and Paschal (1967), have indicated that moisture can vary up to values of 15% depending on storage conditions. However, values greater than 18% bring problems mainly due to growth of molds and other microorganisms, as well as avoiding the flow of material due to caking of the particles (Radley 1976). Matos (1996b) and Matos and Pérez (2003) indicated that chemical analysis of the cassava starch revealed a high purity (~ 98%), for both research works. Pérez (1994), reported a total starch content of 99.5%. The fat presented a value of 0.17%, ash 0.11%, crude protein 0.75% and crude fiber 0.49%. Similar results were also reported by Gutiérrez et al. (2014).

Figure 1. Flow chart for cassava starch production (Pérez et al. 1993).

Complimentary Contributor Copy 300 Giordana Suárez and Tomy J. Gutiérrez

3. BIODEGRADABLE FILMS FROM CASSAVA STARCH

Starch is one of polysaccharides used to obtain biodegradable films because of its ability to form a continuous matrix, its low permeability to oxygen (Liu 2005; Dole et al. 2004), and compared to other non-starch films, its lower cost. However, like other hydrocolloids, when compared to plastic polymers, starch films exhibit several drawbacks, such as their hydrophilic character and poor mechanical properties. Nonetheless, films based on starch are transparent, odorless, tasteless, and colorless (Mali et al. 2004, Jiménez et al. 2012). Biodegradable starch films can be obtained from the native starch or its components, amylose and amylopectin, by two main techniques: solution casting and subsequent drying (wet method) and thermoplastic processing (dry method) (Paes et al. 2008). Native, modified or pre-gelatinized starches have also been used to obtain starch films (Pagella et al. 2002; López et al. 2008). As mentioned previously, starch films can be formed from a film-forming dispersion, or an emulsion, which contains a high percentage of water. Otherwise, starch films may be obtained by using a dry process (thermoplastic or thermal processing) in which the water content is lower when compared to the wet process. A dry process can be used with those raw materials which present thermoplastic properties; this means that they become soft (melted or rubbery) at a temperature lower than decomposition temperature and so, they can be molded into a determined shape when submitted to a thermal/mechanical process. Although starch does not present this characteristic in its native state, it is capable of becoming a thermoplastic material if it is treated correctly. According to Carvalho (2008), thermoplastic starch (TPS) is generally produced by processing a starch-plasticizer(s) mixture in an extruder at temperatures between 140 and 160 °C at high pressure and high shear. Additionally, batch mixers can also be used, operating in conditions similar to those of the extrusion process. The result of the process, in which starch granules are disrupted and mixed with one or a mixture of plasticizers is the TPS. The presence of plasticizers (not only water) is necessary in order to obtain a rubbery material, without brittleness, when equilibrated at ambient relative humidity (Forssell et al. 1997). In order to obtain starch-based films, an essential requirement which must be considered is that, if native starch is used, the granules have to be disrupted previously through a gelatinization process in an excess of water media (> 90% w/w, Carvalho 2008), where they undergo an irreversible order–disorder transition, or destructuration. Starch gelatinization is a process in which granules swell, depending on the available water, provoking the breakage of the amylopectin matrix and releasing the amylose. In other words, it can be considered as a first step, in which the solvent diffuses through the starch granules and a second, in which the melting of the starch crystallites takes place (Carvalho 2008). Although the gelatinization process seems to be simple, it is a very complex process. According to Ratnayake and Jackson (2007), the gelatinization process initiates at low temperatures and continues until that the granules are completely disrupted. These authors studied seven types of starch by scanning electron microscopy and observed differences in the granule structure when were treated at different temperatures. Furthermore, they summarized the gelatinization as a three- stage process during which different structural events take place:

Complimentary Contributor Copy Recent Advances in the Development of Biodegradable Films … 301

1. The water absorption by starch granules promotes an increase in starch polymer mobility in amorphous regions. 2. Starch polymers in the amorphous regions rearrange often forming new intermolecular interactions. 3. With increasing hydrothermal effects, the polymers become more mobile and lose their intermolecular interactions and overall granular structure.

In order to ensure sustainability in the plastics sector it is essential to develop new biopolymers with new routes and/or synthesis processes that enable highly efficient procurement implementation within the sector. It is also necessary to reduce the cost of biopolymers as well as improve their mechanical and thermal properties. In this regard has been implemented as synthesis route the reactive extrusion. Reactive extrusion (REX) is a process that combines the mass and heat transportation operations with simultaneous chemical reactions taking place inside the extruder for the purpose of modifying the properties of existing polymers or for producing new ones. REX is increasingly becoming a powerful technique to develop and fabricate a variety of novel polymeric materials in a highly efficient and flexible way. This combination of chemical reactions and transport phenomena in an extruder provides a large opportunity window for polymeric prototyping (Tzoganakis et al. 1989).

3.1. Properties of Cassava Film

Alves el at. (2007) have observed and studied the properties of cassava films and some important results were obtained. It was found that the elongation, Young’s modulus and tensile strength decreased when the glycerol concentration was increased. On the other hand, water solubility, moisture content and water vapor permeability of the cassava film increased with the escalating glycerol concentration. Therefore, the glycerol content affected both the mechanical and water barrier properties.

3.1.1. Physicochemical Properties of Cassava Films

3.1.1.1. Moisture Content Table 1 shows the moisture content (MC) of the cassava starch biodegradable films. The moisture content was found to be high when the glycerol content was high (45 wt%) and low when the amount of glycerol was low (30 wt%). The values of moisture content for the films ranged in between 11.8% and 41.1% (Alves el at. 2007).

3.1.1.2. Water Solubility Table 1 shows the water solubility (WS) of cassava films. Increasing the amount of glycerol in the film led to increased solubility of the cassava in water. The values of solubility ranged in between 23.0% and 32.1% (Alves el at. 2007).

3.1.1.3. Water Vapour Permeability Table 1 lays out water vapour permeability of the cassava films. The water vapour permeability of the cassava films increased with the enhanced amount of glycerol in the film.

Complimentary Contributor Copy 302 Giordana Suárez and Tomy J. Gutiérrez

The values of water vapor permeability ranged in between 3.28x10-10 g/m.Pa and 4.47x10-10 g/m.Pa (Alves el at. 2007).

3.1.2. Mechanical Properties of Cassava Films As has been mentioned earlier, presence of glycerol has a direct influence on the percent elongation, Young’s modulus and tensile strength of the cassava films. Increasing the amount of glycerol resulted in the decrement of Young’s modulus (E) and tensile strength (σ) (Bangyekan et al. 2006). This is because of certain alterations on the structure of starch network upon addition of glycerol. The network became under stress and also less dense when glycerol was amalgamated. Therefore, the flexibility of film improved because of the ease in movement of polymer chains. The percent elongation (ε) of the films also decreases with the increase in glycerol content. Due to this property of the films, the starch films became more ductile i.e., easily breakable instead of becoming more brittle. These three properties have been shown in the table as a function of the glycerol contained (Silva et al. 2008).

3.1.3. Other Properties of Cassava Films Gutiérrez et al. (2015a,b) evaluated two starches (cassava and cush-cush yam) with different amylose contents for edible films forming plasticized with glycerol, determining that a more open and compact structure it is associated with the greater hydrogen bonding interaction between the amylose and the glycerol, thus allowing obtain films with greater elongation. Therefore, open and compact structures are related to improved hydrogen bonding interactions between glycerol and starch. Moreover, due to hydrophilic nature of the starch different alternatives have been used to overcome this, including: starch modification, addition of natural fillers and catalysts, blends with other polymers, plasticizer rate variation, photochemical modifications, among others (Gutiérrez and González 2016). It is also worth noting that the starch-based products has another problem and is the problem of aging, i.e., their properties are modified during storage. Since alternatives to solve the problems inherent in the films based on cassava starch are equal to options to solve the problems of cassava starch foams, therefore, in the next section will be explained.

Table 1. Moisture contents, water solubility, water vapor, and mechanical properties of cassava films, as a function of glycerol content

Glycerol (%) WSP (x 10-10 WS (%) MC (%) σ(MPa) ε (%) E (MPa) g/m.s.Pa) 30 3.28 23.0 11.8 2.4 49.4 46.3 35 4.22 26.0 22.2 2.1 41.9 32.2 40 4.39 29.2 24.8 1.4 28.8 14.7 45 3.39 32.1 41.1 1.2 26.8 14.0

Complimentary Contributor Copy Recent Advances in the Development of Biodegradable Films … 303

4. BIODEGRADABLE FOAMS FROM CASSAVA STARCH

Polystyrene foam is most common in single-use plastic packaging due to its high strength, low density, good thermal insulation property and low cost (Kaisangsri et al. 2012). However, polystyrene foams may require several hundred years to degrade and can cause serious environmental pollution (Kaisangsri et al. 2014). In this sense, increasing environmental concerns on the global waste problem have motivated the global interests in producing environmentally friendly products from renewable resources (Soykeabkaew et al. 2015). Biodegradable packaging produced from renewable sources is an alternative to conventional plastic packaging. Agromaterials such as cassava starch is a very promising raw material to reduce our dependence on polystyrene (Iriani et al. 2015). The starch-based foams have emerged as polystyrene replacements, and currently the the aim in this field is to improve the drawbacks of the starch itself, since are well known these problems, i.e., poor mechanical properties, high hydrophilicity and changes under temperature conditions (Shogren et al. 2002; Mello and Mali 2014; Palma-Rodríguez et al. 2016). To overcome these limitations, many research groups have tried to improve the properties of starch foams by using modified starch derivatives, e.g., starch acetate, cationic starch, pregelatinized starch and cross-linked starch (Pornsuksomboon et al. 2014). The results have suggested that differences in botanic source, granule dimension, moisture, protein, fiber content, as well as the amylose/amylopectin ratio influence on the resulting foams (Soykeabkaew et al. 2015). In addition, Shogren et al. (2002) has shown that foams made from chemically modified starch have shorter baking times, are lighter and show a higher elongation at break than unmodified starch. Guan and Hanna (2006), have indicated that the degree of cross-linking and degree of substitution may affect the baking time, the density, the mechanical properties, and water absorptivity of starch foams. Kaewtatip et al. (2014) have claimed that the thermal stability and morphology of starch foams depends on the type of the substituent in the starch derivative. Pornsuksomboon et al. (2014) have made starch foams from native cassava starch/cross- linked starch blends, which were prepared by baking in a hot mold. The native cassava starch/cross-linked starch ratio was varied in the series of 100/0, 80/20, 60/40, 50/50, 40/60, 20/80, and 0/100. The authors investigated the effect of the native cassava starch/cross-linked starch ratio on the properties of foams including density, morphology, water adsorption, impact strength and thermal stability. The mixture of cross-linked starch resulted in a higher density of the foams. The impact strength of the blend foams were between 232 and 268 J/m2. The water adsorption for the modified starch foams (7.3%) and the blend foams (11-12%) was lower than for the native cassava starch foam (13.9%). Also, higher density values (~0.15 g/cm3) have been reported for potato starch foams than those made from cassava starch (~0.12 g/cm3) (Soykeabkaew et al. 2015). Similarly, in order to enhance water resistance and strength of the foams, various biodegradable polymers, for example, poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), poly(vinyl alcohol) (PVA), chitosan, poly(hydroxyester ether) (PHEE), poly(hydroxybutyrate-co-valerate) (PHBV), poly(butylenes-succinate) (PBSA), butanediol- terephthalate-adipate terpolymer (PBAT), cellulose acetate (CA), poly(ester amide) (PEA) have been blended with starches (Soykeabkaew et al. 2015). For example, the water resistance of baked starch-based foams was improved by the addition of hydrophobic

Complimentary Contributor Copy 304 Giordana Suárez and Tomy J. Gutiérrez materials such as monostearyl citrate, latex and PCL (Shey et al. 2006). Shey et al. (2006) found that latex could be added to the batter of baked starch foams to increase their flexibility and moisture resistance. Iriani et al. (2015), have also displayed that the addition of PVA up to 30% can increase starch-baked foam compressibility and tensile strength The best formulation to produce starch-baked foam is cassava starch: corn hominy: PVA = (75:25):30%. Likewise, to improve the mechanical properties of starch foams have been added various fillers such as fibers, clay, proteins, wax, ethylene-vinyl acetate copolymer and calcium carbonate (Pornsuksomboon et al. 2014; Soykeabkaew et al. 2015). Additionally, the nano- scale fillers have opened the new windows to the development of starch-based foams. Due to the nano-sized effect, various properties of the foams have shown to be effectively improved at low filler content (Mansourighasri et al. 2012; Matsuda et al. 2013). It is very well observed that a wide range of the starch-based foam properties can be achieved and tuned by the selection of appropriate processing technique as well as the set of input ingredients, the types of starches, blend sand additives/fillers. The success and continuity in developing of starch-based foams will make possible to many more of their new applications (Soykeabkaew et al. 2015). However, the hydrophilic characteristics of the starch foam can cause incompatibility with some hydrophobic fillers and they are also prone to aggregate during foam production process (Pornsuksomboon et al. 2014). Kaisangsri et al. (2014) reported that the increased kraft fiber content (5-15 wt%) in cassava starch foam trays caused their flexural and compressive strengths to increase. Soykeabkaew et al. (2004) found that addition of 10% jute or flax fibers to the cassava starch- based foams significantly improved the flexural strength due to the cross-link reaction between starch and fibers. On the contrary, the addition of some kraft fibers into starch foam results in high density, and some types of kraft fiber produce a dark color. The density of starch-based foam blended with cassava, wheat, jute, flex and softwood fiber was 0.1-0.3 g/cm3 (Glenn et al. 2001; Soykeabkaew et al. 2004; Carr et al. 2006). Kaisangsri et al. (2012) conducted a study to improve quality of cassava starch-based foam for application in fresh cut fruits. The kraft fiber at 0, 10, 20, 30 and 40% (w/w of starch) and chitosan at 0, 2, 4 and 6% was mixed with cassava starch. Hot mold baking was used to develop the cassava starch-based foam by using a baking machine controlled temperature at 250 oC for 5 min. Results showed that foam produced from cassava starch, 30% kraft fiber (w/w of starch) and 4% chitosan had properties similar to polystyrene foam. Tensile strength and elongation of starch-based foam were 944.40 kPa and 2.43%, respectively, but the water absorption index and water solubility index were greater than the polystyrene foam. Vercelheze et al. (2012) found that the addition of sugarcane bagasse and clay (Na- MMT) decreased the density and stress at break and increased the strain at break values of the starch foams. Fiber and Na-MMT acted as reinforcing fillers that improved the foaming ability of the starch pastes, resulting in more expandable materials. Another foam made from starch, cellulose and protein isolates from sunflower produced a foam with a density ranging 3 from 0.45 to 0.58 g/cm , while starch, cellulose fiber and CaCO3 produced foam with a density 0.63 to 1.3g/cm3 (Schmidt 2006). Vercelheze et al. (2013), in another study found that the addition of fibers and Na-MMT resulted in less dense and less rigid trays compared to control samples (only starch). Complimentary Contributor Copy Recent Advances in the Development of Biodegradable Films … 305

Later on, Mello and Mali (2014) also reported that addition of 5-15 wt% malt bagasse (particles < 0.3 mm) in cassava foams resulted in high tensile strength of approximately 13 MPa, thicknesses ranged from 2.16 to 2.24 mm and densities ranged from 0.415 to 0.450 g/cm3. Each tray produced in this study had a good appearance, adequate expansion, and a homogeneous distribution of malt bagasse in the polymeric matrix. The addition of the lignocellulosic material at concentrations up to 15% (w/w) decreased the initial moisture adsorption rate of the trays. According to authors the main application of these trays is for dry food packaging that have short shelf-life. Kaisangsri et al. (2014) demonstrated that the addition of kraft, zein, and gluten could improve flexural and compressive strength of the cassava starch foam trays. Moreover, the water absorption and water solubility index of blended cassava starch foams with zein and gluten proteins were low. Although adding palm oil into cassava starch foams increased the water resistance, their flexural and compressive strength decreased. These findings demonstrate that blended cassava starch foam trays with kraft, gluten and/or zein could be used as an alternative to polystyrene foam trays for oily and less moist foods. The starch foam trays should further improve and develop their expansion, water resistance and their possible application to moist foods. Moreover, water is also an important component because it acts as the blowing agent in the expansion process. Starch pastes also must have certain rheological characteristics such that the foam does not collapse during water evaporation. Pastes with low water content were very viscous and result in less expandable and higher density foams, and the presence of fibers and other solids in the formulations increase the viscosity of the mixture, which decreases its foaming ability (Cinelli et al. 2006). Other research indicated that water addition during extrusion process may less en the magnitude of radial expansion (Yu et al. 2006, Ma et al. 2008, Sarazin et al. 2008) and volumetric expansion (Jawaid and Klali 2011). One characteristic of foam property is density, and lower density is better for packaging. Foam density is affected by starch, fiber, synthetic polymer and interactions between the materials (Mali et al. 2010). The addition of fiber tends to limit expansion ability and result in stiff materials, which do not support air cell growth in their foams. It was also reported that a reduction in foam density was achieved with the addition of corn fiber (Glenn et al. 2001, Cinelli et al. 2006). On the other hand, the starch-based foams can be produced by many techniques such as hot-press technology, extrusion processes and baking starch/water batters in heated closed molds (Shogren et al. 2002; Iriani et al. 2015), but essentially the creation of starch-based foam can be divided into two main steps: starch gelatinization and water evaporation from batter. In this sense, the mixture is expanded and forms a foam dewatered to a moisture content of 2-4% (Soykeabkaew et al. 2004; Kaisangsri et al. 2012; Vercelheze et al. 2012). Each technique has its own processing parameters which affect the product properties including shapes, cellular structures, density and mechanical properties. For example, increasing barrel temperature and screw speed usually increases the number of cells, and as a result, generally increases expansion ratio of the extruded foams leading to a decrease in foam density (Soykeabkaew et al. 2015). However, the starch-foam products can be designed to suit a particular application via appropriate processing systems. Usually, extrusion processing system produces the foam with large cell size and that leads to the production of low density products. This can be useful for the application where the weight is the high priority such as packaging for transportation (Soykeabkaew et al. 2015). Researchers have produced starch Complimentary Contributor Copy 306 Giordana Suárez and Tomy J. Gutiérrez puff, this kind of product is known also as plate expanded by extrusion, gelatinized starch puff or baking with water.The products are formed in the extruder by the swelling and expansion of starch through the action of high temperature and water vapor to form starch foam (Poovarodom and Praditduang 1999). Clean Green Company in Minneapolis, MN, USA, has produced “starch foam” by extrusion of wheat starch and polyvinyl alcohol. “Eco-Foam”, a product of National Starch, using waxy corn as raw material. In European countries, the baking technology is also at a commercial scale. Packaging products, such as fast food utensils, are available in the market using both cereal and potato starches. The marketing of biodegradable packaging products are supported in the EU. Cassava starch has been successfully expanded under extrusion conditions. Due to its low bulk density, a little modification is needed so that its moisture content is increased. Twin screw extrusion Figure 2 is recommended for direct expansion of cassava starches. Cassava starch can also be used as the raw material for plate expanded or baking products. Cassava starch can be expanded in moulds, for 1-3 min. at 200-240 oC, to form into package utensils such as bowls. About 10% additives, including calcium carbonate, agar, or emulsifier is needed to improve the properties (Parra et al. 2004). In applications that required the well-molded shape such as disposal containers, baking or compression process may be well suited as the foam is expanded within a well-closed mold. Microwaved foams allow thick cell walls which can be suitable for the application where strength and stiffness are essential. Whereas, the applications which required micro-scale and uniform cells with interconnectivity such as scaffold or drug delivery system, the freeze drying and solvent exchange processes should be more suitable. Lastly, a novel supercritical fluid extrusion technology seems to be more versatile and controllable, thus, a wide range of foam structures with desired properties can be better designed for several uses.

Figure 2. Extrusion of cassava foam (Parra et al. 2004).

CONCLUSION AND OUTLOOKS

Films based on cassava starch have been widely studied for food packaging applications. Nonetheless, the same has not happened with foams based on cassava starch. These materials are promising as a replacement for plastic materials obtained from the petrochemical industry, which would allow the production of “green” materials. However, several well-known disadvantages are produced during the storage of these thermoplastic materials. For this

Complimentary Contributor Copy Recent Advances in the Development of Biodegradable Films … 307 reason, its study this on top of researches in the field of polymers. To improve the mechanical and physicochemical properties of these materials have been added various natural fillers within these native or modified matrices with different types of plasticizers. Even cassava starch mixtures with other biopolymers have been made to overcome the hydrophilic nature of starch. However, these investigations are still booming because of its importance. In the future early, a large part of plastic materials known to date will be made from cassava or corn starch, since its high production and yields that make attractive in polymer and food industries. Additionally, due to characteristics of the cassava starch, this can be applied under the conventional-polymers processing technology, i.e., extrusion. This together with the reactivity that present the starch, gives rise to the possibility to reactive reactions using the extruder as a chemical reactor. Therefore, this will be the future trends in the development of polymeric materials based on cassava starch.

ACKNOWLEDGMENTS

The authors would like to thank Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (Postdoctoral fellowship internal PDTS-Resolution 2417), Universidad Nacional de Mar del Plata (UNMdP) for the financial support and to Dr. Mirian Carmona- Rodríguez.

REFERENCES

Alves, V.D., S. Mali, A. Beléia, M.V.E. Grossmann. 2007. “Effect of glycerol and amylose enrichment on cassava starch film properties”. Journal of Food Engineering, 78(3): 941- 946. Bangyekan, C., D. Aht-Ong, K. Srikulkit. 2006. “Preparation and properties evaluation of chitosan-coated cassava starch films”. Carbohydrate Polymers, 63(1): 61-71. Carr, L.G., D.F. Parra, P. Ponce, A.B. Lugao, P.M. Buchler. 2006. “Influence of fibers on the mechanical properties of cassava starch foams”. Journal of Polymers and the Environment, 14(2): 179-183. Carvalho, A.J. 2008. “Starch: major sources, properties and applications as thermoplastic materials”. In: Monomers, polymers and composites from renewable resources. M.N. Belgacem & A. Gandini (Eds.). Elsevier, Amsterdam. Pp. 321-342. Cinelli, P., E. Chiellini, J.W. Lawton, S.H. Imam. 2006. “Foamed articles based on potato starch, corn fibers and poly (vinyl alcohol)”. Polymer Degradation and Stability, 91(5): 1147-1155. da Silva, A., L.M. Nievola, C.A. Tischer, S. Mali, P. Faria‐Tischer. 2013. “Cassava starch‐based foams reinforced with bacterial cellulose”. Journal of Applied Polymer Science 130(5): 3043-3049. Dole, P., C. Joly, E. Espuche, I. Alric, N. Gontard. 2004. “Gas transport properties of starch based films”. Carbohydrate Polymers, 58(3): 335-343. Edhirej, A., S.M. Sapuan, M. Jawaid, N.I. Zahari. 2015. “Cassava: Its polymer, fiber, composite, and application”. Polymer Composites. 1-16.

Complimentary Contributor Copy 308 Giordana Suárez and Tomy J. Gutiérrez

Forssell, P.M., J.M. Mikkilä, G.K. Moates, R. Parker. 1997. “Phase and glass transition behaviour of concentrated barley starch-glycerol-water mixtures, a model for thermoplastic starch”. Carbohydrate Polymers, 34(4): 275-282. Glenn, G.M., W.J. Orts, G.A.R. Nobes. 2001. “Starch, fiber and CaCO3 effects on the physical properties of foams made by a baking process”. Industrial Crops and Products, 14(3): 201-212. Glenn, G.M., W.J. Orts. 2001. “Properties of starch-based foam formed by compression/explosion processing”. Industrial Crops and Products, 13(2): 135-143. Grace, M.R. 1987. Cassava Processing. Food and Agriculture Organization (FAO). pp. 55-61. Roma. Guan, J., M.A. Hanna. 2006. “Selected morphological and functional properties of extruded acetylated starch-cellulose foams”. Bioresource technology, 97(14): 1716-1726. Gutiérrez, T.J., E. Pérez, R. Guzmán, M.S. Tapia, L. Famá. 2014. “Physicochemical and functional properties of native and modified by crosslinking, dark-cush-cush yam (dioscorea trifida) and cassava (manihot esculenta) starch”. Journal of Polymer and Biopolymer Physics Chemistry, 2(1): 1-5. Gutiérrez, T.J., G. González. (2016). Effects of exposure to pulsed light on surface and structural properties of edible films made from cassava and taro starch. Food and Bioprocess Technology. 9(8): Gutiérrez, T.J., M.S. Tapia, E. Pérez, L. Famá. 2015. “Structural and mechanical properties of edible films made from native and modified cush-cush yam and cassava starch”. Food Hydrocolloids 45: 211-217. Gutiérrez, T.J., N.J. Morales, E. Pérez, M.S. Tapia, L. Famá. 2015. “Physico-chemical properties of edible films derived from native and phosphated cush-cush yam and cassava starches”. Food Packaging and Shelf Life 3: 1-8. Iriani, E.S., T.T. Irawadi, T.C. Sunarti, N. Richana, I. Yuliasih. 2015. “Effect of corn hominy and polyvinyl alcohol on mechanical properties of cassava starch-baked foam”. Polymer- Plastics Technology and Engineering 54(3): 282-289. Jawaid, M.H.P.S., H.A. Khalil. 2011. “Cellulosic/synthetic fibre reinforced polymer hybrid composites: A review”. Carbohydrate Polymers, 86(1): 1-18. Jiménez, A., M.J. Fabra, P. Talens, A. Chiralt. 2012. “Effect of re-crystallization on tensile, optical and water vapour barrier properties of corn starch films containing fatty acids”. Food Hydrocolloids, 26(1): 302-310. Kaewtatip, K., M. Poungroi, B. Holló, K.M. Szécsényi. 2014. “Effects of starch types on the properties of baked starch foams”. Journal of Thermal Analysis and Calorimetry, 115(1): 833-840. Kaisangsri, N., O. Kerdchoechuen, N. Laohakunjit. 2012. “Biodegradable foam tray from cassava starch blended with natural fiber and chitosan”. Industrial Crops and Products 37(1): 542-546. Kaisangsri, N., O. Kerdchoechuen, N. Laohakunjit. 2014. “Characterization of cassava starch based foam blended with plant proteins, kraft fiber, and palm oil”. Carbohydrate Polymers 110: 70-77. Liu, Z. 2005. “Edible films and coatings from starch”. In: Innovations in food packaging. Han, J.H. (Ed.). London: Elsevier Academic Press. Pp. 318-332. López, O.V., M.A. García, N.E. Zaritzky. 2008. “Film forming capacity of chemically modified corn starches”. Carbohydrate Polymers, 73(4): 573-581. Complimentary Contributor Copy Recent Advances in the Development of Biodegradable Films … 309

Ma, X., P.R. Chang, J. Yu. 2008. “Properties of biodegradable thermoplastic pea starch/carboxymethyl cellulose and pea starch/microcrystalline cellulose composites”. Carbohydrate Polymers, 72(3): 369-375. Mali, S., F. Debiagi, M.V. Grossmann, F. Yamashita. 2010. “Starch, sugarcane bagasse fibre, and polyvinyl alcohol effects on extruded foam properties: A mixture design approach”. Industrial Crops and Products, 32(3): 353-359. Mali, S., M.V.E. Grossmann, M.A. Garcı́a, M.N. Martino, N.E. Zaritzky. 2004. “Barrier, mechanical and optical properties of plasticized yam starch films”. Carbohydrate Polymers, 56(2): 129-135. Mansourighasri, A., N. Muhamad, A.B. Sulong. 2012. “Processing titanium foams using tapioca starch as a space holder”. Journal of Materials Processing Technology 212(1): 83-89. Matos, M.E. 1996a. Aprovechamiento integral y uso industrial de la yuca (Manihot esculenta Crantz). Seminario Especial de Grado. Facultad de Ciencias. Universidad Central de Venezuela. Caracas, Venezuela. Matos, M.E. 1996b. Modificación por métodos químicos de fosfatación, acetilación y doble derivación de almidón de yuca. Tesis de Maestría en Ciencias y Tecnología de Alimentos. Facultad de Ciencias. Universidad Central de Venezuela. Caracas, Venezuela. Matos, M.E., E. Pérez. 2003. “Characterization of native and modified cassava starches by scanning electron microscopy and X-ray diffraction techniques”. Cereal Foods World. 48(2): 78-81. Matsuda, D.K., A.E. Verceheze, G.M. Carvalho, F. Yamashita, S. Mali. 2013. “Baked foams of cassava starch and organically modified nanoclays”. Industrial Crops and Products 44: 705-711. Mello, L.R., S. Mali. 2014. “Use of malt bagasse to produce biodegradable baked foams made from cassava starch”. Industrial Crops and Products 55: 187-193. Montaldo, A. 1991. Cultivo de raíces y tubérculos tropicales. Instituto Interamericano de Cooperación para la Agricultura. San José, Costa Rica. Segunda Edición. 408 pp. Paes, S.S., I. Yakimets, J.R. Mitchell. 2008. “Influence of gelatinization process on functional properties of cassava starch films”. Food Hydrocolloids, 22(5): 788-797. Pagella, C., G. Spigno, D.M. De Faveri. 2002. “Characterization of starch based edible coatings”. Food and Bioproducts Processing, 80(3): 193-198. Palma-Rodríguez, H.M., J.D.J. Berrios, G. Glenn, R. Salgado-Delgado, A. Aparicio-Saguilán, A.I. Rodríguez-Hernández, A. Vargas-Torres. 2016. “Effect of the storage conditions on mechanical properties and microstructure of biodegradable baked starch foams”. CyTA- Journal of Food 14(3): 415-422. Parra, D.F., C.C. Tadini, P. Ponce, A.B. Lugão. 2004. Mechanical properties and water vapor transmission in some blends of cassava starch edible films. Carbohydrate Polymers, 58(4): 475-481. Pérez, E. 1994. Caracterización de las propiedades funcionales de almidones nativos y modificados por métodos físicos de extrusión, deshidratación con doble tambor e irradicación gamma y microondas. Trabajo de ascenso. Facultad de Ciencias. ICTA. Universidad Central de Venezuela. Pérez, E., Y.A. Bahnassey, W.M. Breene. (1993). A simple laboratory scale method for isolation of amaranth starch. Starch‐Stärke, 45(6): 211-214.

Complimentary Contributor Copy 310 Giordana Suárez and Tomy J. Gutiérrez

Poovarodom, N., S. Praditduang. 1999. “The development of package from cassava starch”. Packaging Directory. Thailand, The Thai Packaging Association, 41-42. Pornsuksomboon, K., K.M. Szécsényi, B. Holló, K. Kaewtatip. 2014. “Preparation of native cassava starch and cross‐linked starch blended foams”. Starch‐Stärke 66(9-10): 818-823. Radley, J. 1976. Examination and analysis of starch and atarch products. Applied Science Publichers LTD, London. 220 pp. Ratnayake, W.S., D.S. Jackson. 2007. “A new insight into the gelatinization process of native starches”. Carbohydrate Polymers, 67(4): 511-529. Sarazin, P., G. Li, W.J. Orts, B.D. Favis. 2008. “Binary and ternary blends of polylactide, polycaprolactone and thermoplastic starch”. Polymer, 49(2): 599-609. Schmidt, V.C. 2006.Desenvolvimento de embalagens biodegradáveis a partir da fécula de cassava, calcário e fibra de celulose. Dissetac, ao de Mestrado em Engenharia de Alimentos. Universidade Federal de Santa Catarina (UFSC), Flori-anópolis, Brazil. Shey, J., S.H. Imam, G.M. Glenn, W.J. Orts. 2006. “Properties of baked starch foam with natural rubber latex”. Industrial Crops and Products, 24(1): 34-40. Shogren, R.L., J.W. Lawton, K.F. Tiefenbacher. 2002. “Baked starch foams: starch modifications and additives improve process parameters, structure and properties”. Industrial Crops and Products, 16(1): 69-79. Silva, R., E.M. Aquino, L.P. Rodrigues, A.R. Barros. Materia 2008 (Rio de Janeiro), 13(1): 154. Soykeabkaew, N., C. Thanomsilp, O. Suwantong. 2015. “A review: Starch-based composite foams”. Composites Part A: Applied Science and Manufacturing 78: 246-263. Soykeabkaew, N., P. Supaphol, R. Rujiravanit. 2004. “Preparation and characterization of jute-and flax-reinforced starch-based composite foams”. Carbohydrate Polymers, 58(1): 53-63. Thomas, D., W. Atwell. 1999. Starch structure. En Starches: Practical Guide for the Food Industry. Eagan Press. St. Paul. MN, EEUU. 1-12 pp. Tzoganakis, C. 1989. “Reactive extrusion of polymers: a review”. Advances in Polymer Technology, 9(4): 321-330. Vercelheze, A.E., F.M. Fakhouri, L.H. Dall’Antônia, A. Urbano, E.Y. Youssef, F. Yamashita, S. Mali. 2012. “Properties of baked foams based on cassava starch, sugarcane bagasse fibers and montmorillonite”. Carbohydrate Polymers 87(2): 1302-1310. Vercelheze, A.E.S., A.L. Oliveira, M.I. Rezende, C.M. Muller, F. Yamashita, S. Mali. 2013. “Physical properties, photo-and bio-degradation of baked foams based on cassava starch, sugarcane bagasse fibers and montmorillonite”. Journal of Polymers and the Environment 21(1): 266-274. Whistler, R., E. Paschall. 1967. Starch chemistry and technology. Academic Press. New York and London. Vol. II. Wurzburg, O.B. 1972. Starch in the food industry. Vol. I. Chap. 8. In: Hanbook of food additives. Segunda Edición. E.E. Furia. Editorial CRC Press. Boca Raton. Yu, L., K. Dean, L. Li. 2006. “Polymer blends and composites from renewable resources”. Progress in Polymer Science, 31(6): 576-602.

Complimentary Contributor Copy Recent Advances in the Development of Biodegradable Films … 311

BIOGRAPHICAL SKETCH

Tomy J. Gutiérrez

Affiliation: Composite Materials Group, Institute of Materials Science and Technology (INTEMA) (CONICET-UNMdP), Faculty of Engineering, National University of Mar del Plata and National Research Council (CONICET), Mar del Plata 7600, Argentina.

Education: Degree in Chemistry. Central University of Venezuela. (12/07/2007). Degree in Education (Chemistry). Central University of Venezuela. (07/18/2008). Specialist in International Hydrocarbons Negotiation. National Experimental Polytechnic University of the Armed Forces. (07/06/2011). M.Sc. in Food Science and Technology. Central University of Venezuela. (10/31/2013). Ph.D. in Food Science and Technology. Central University of Venezuela. (24/04/2015). Doctoral Candidate in Metallurgy and Materials Science. Central University of Venezuela. (2015). Ph.D. in Materials Science. National University of Mar del Plata. (12/06/2016).

Business Address: [email protected]; [email protected]

Research and Professional Experience: Food Science and Technology Polymers Science and Technology Petroleum and Natural Gas

Publications Last 3 Years:

2014 (1): 1.- Tomy J. Gutiérrez*, Elevina Pérez, Romel Guzmán, María Soledad Tapia, Lucía Famá. (2014). Physicochemical and functional properties of native and modified by crosslinking, dark-cush-cush yam (Dioscorea Trifida) and cassava (Manihot Esculenta) starch. Journal of Polymer and Biopolymer Physics Chemistry, 2(1):1-5. doi: 10.12691/jpbpc-2-1-1.

2015 (6): 1.- Tomy J. Gutiérrez*, Noé J. Morales, Elevina Pérez, María Soledad Tapia, Lucía Famá. (2015). Physico-chemical study of edible films based on native and phosphating cush-cush yam and cassava starches. Food Packaging and Shelf Life, 3, 1-8. doi: 10.1016/j.fpsl.2014.09.002. 2.- Tomy J. Gutiérrez*, María Soledad Tapia, Elevina Pérez, Lucía Famá. (2015). Structural and mechanical properties of native and modified cush-cush yam and cassava starch edible films. Food Hydrocolloids, 45, 211-217. doi: 10.1016/j.foodhyd.2014.11.017.

Complimentary Contributor Copy 312 Giordana Suárez and Tomy J. Gutiérrez

3.- Tomy J. Gutiérrez*, María Soledad Tapia, Elevina Pérez, Lucía Famá. (2015). Edible films based on native and phosphated 80:20 waxy:normal corn starch. Starch-Stärke, 67, 90-97. doi: 10.1002/star.201400164. 4.- Tomy J. Gutiérrez*, Noé J. Morales, María Soledad Tapia, Elevina Pérez, Lucía Famá. (2015). Corn starch 80:20 “waxy”:regular, “native” and phosphated, as bio-matrixes for edible films. Procedia Materials Science, 8, 304-310. doi: 10.1016/j.mspro.2015.04.077. 5.- Tomy J. Gutiérrez, Elevina Pérez*. (2015a). Chapter 1. Significant quality factors in the chocolate processing: cocoa post harvest, and in its manufacture. In: Chocolate: Cocoa Byproducts Technology, Rheology, Styling, and Nutrition. Editor Elevina Pérez Sira. Editorial Nova Science Publishers, Inc. NY. EE.UU. ISBN: 978-1-63482- 355-5. pp. 1-47. 6.- Yuniesky González Muñoz*, Tomy J. Gutiérrez. (2015b). Chapter 7. Evaluation of the sensory quality of chocolate. In: Chocolate: Cocoa Byproducts Technology, Rheology, Styling, and Nutrition. Editor Elevina Pérez Sira. Editorial Nova Science Publishers, Inc. NY. EE.UU. ISBN: 978-1-63482-355-5. pp. 167-189.

2016 (6): 1.- Tomy J. Gutiérrez*, Romel Guzmán, Carolina Medina Jaramillo, Lucía Famá. (2016). Effect of beet flour on films made from biological macromolecules: native and modified plantain flour. International Journal of Biological Macromolecules, 82, 395-403. doi: 10.1016/j.ijbiomac.2015.10.020. 2.- Tomy J. Gutiérrez*, Jusneydy Suniaga, Antonio Monsalve, Nancy L. García. (2016). Influence of beet flour on the relationship surface-properties of edible and intelligent films made from native and modified plantain flour. Food Hydrocolloids, 54, 234- 244. doi: 10.1016/j.foodhyd.2015.10.012. 3- Carolina Medina Jaramillo, Tomy J. Gutiérrez, Silvia Goyanes, Celina Bernal, Lucía Famá*. (2016). Biodegradability and plasticizing effect of yerba mate extract on cassava starch edible films. Carbohydrate Polymers, 151, 150-159. doi: 10.1016/j.carbpol.2016.05.025. 4.- Tomy J. Gutiérrez*, Gema González. (2016). Effects of exposure to pulsed light on surface and structural properties of edible films made from cassava and taro starch. Food and Bioprocess Technology,. doi: 10.1007/s11947-016-1765-3. 5.- Tomy J. Gutiérrez, Paula González Seligra, Carolina Medina Jaramillo, Lucia Famá*, Silvia Goyanes*. (2016). Effect of filler properties on the antioxidant response of thermoplastic starch composites. In: Handbook of Composites from Renewable Materials. Editors Vijay Kumar Thakur, Manju Kumari Thakur, Michael R. Kessler. WILEY-Scrivener Publisher. EE.UU. ISBN: 978-1-119-22362-7. 6.- Melina Bracone, Danila Merino, Jimena González, Vera A. Alvarez, Tomy J. Gutiérrez*. (2016). Chapter 6. Nanopackaging from natural fillers and biopolymers for the development of active and intelligent films. In: Natural Polymers: Their Derivatives, Blends and Composites. Editors Saiqa Ikram and Shakeel Ahmed. Editorial Nova Science Publishers, Inc. NY. EE.UU. ISBN: 978-1-63485-831-1.

Chapter 16

CASSAVA CULTIVATION, PROCESSING AND POTENTIAL USES IN GHANA

Richard Bayitse1,*, Ferdinand Tornyie1,† 2, and Anne-Belinda Bjerre ‡, PhD 1 Council for Scientific and Industrial Research, Institute of Industrial Research, Accra, Ghana 2 Danish Technological Institute, Taastrup, Denmark

ABSTRACT

This review highlights the traditional and improved methods of cassava production and processing in Ghana. It also explains the geographical distribution of cassava production and utilisation. Facts and figures from agricultural production in Ghana is used to analyse production trends as well as the contribution of cassava to Agricultural Gross Domestic Production. Most importantly, cassava is a staple food crop and accounts for about 152.9 kg per capita consumption. Making it one of the most processed crop into gari, fufu powder and kokonte to increase its shelf life. Additionally, it can be used as an industrial crop because of its high starch content. These process technologies have contributed to the reduction of post-harvest losses in cassava production in Ghana. The residue generated from cassava processing has a huge potential in biorefinery. The review also brings into focus current research works in cassava residue utilisation, reviewing technologies for converting this valuable feedstock which is a mixture of cassava peels, trimmings and cuttings into sugar platform in a biorefinery for the production of major products such as ethanol, lactic acid and protein.

INTRODUCTION

Cassava (Manihot esculenta Cralztz) is a starchy root crop which is an essential food eaten mainly by developing countries. The root tuber and leaves are edible and serve as

* [email protected]. † [email protected]. ‡ [email protected]. Complimentary Contributor Copy 314 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre source of nutritional food for about 500 million people and more worldwide. It is an important crop in developing countries because, it is a major food for households, drought tolerant, fairly resistance to plant disease, and extremely flexible in its cultivation, management requirements and harvesting cycles (FAO, 2002; Meridian Institute, 2009). Cassava is said to be the highest producer of carbohydrates when it comes to staple crops. According to the United Nations Food and Agriculture Organisation (FAO), cassava is graded fourth food crop in the developing countries, next to rice, maize and wheat (FAO, 2002). Cassava which is consumed in all the 10 regions of Ghana was introduced from Brazil, to the tropical areas of Africa by the Portuguese during the 16th and 17th centuries (Jones, 1959). During its introduction in Ghana, it was grown around trading ports, forts and castles and it was a major food that was eaten by slaves and the Portuguese as well. Around the second half of the 18th century, cassava had become the most commonly grown and eaten by majority of people along the coastlines of Ghana (Adams, 1957). Cassava cropping then spread from the coastlines of the country to all over the country progressively until it became a major staple food in most parts of the country following a serious drought in the year 1982/1983 when most crops failed dramatically (Korang-Amoakoh et al., 1987). Cassava then became a central food in Ghana that was eaten by various ethnic groups, processed in various forms. Currently, cassava occupies an important position in Ghana's agricultural economy and contribute about 46% of agricultural Gross Domestic Product (GDP). Cassava accounts for a daily calorie intake of 30% in Ghana and is grown by almost every farming family (FAO, 2000). Cassava as a food security crop can be used in various forms. It can be eaten raw by cooking, pounded into fufu or semi processed. Some processed forms include, gari, tapioca and flour for konkonte. It is also used as animal feed. Gari is exported to neighboring West African countries. Cassava is harvested at the farm and the tuber transported to a processing facility. These process technologies have contributed to the reduction of postharvest losses in cassava production in Ghana. The residue generated from cassava processing has a huge potential in biorefinery because it can easily be hydrolysed by appropriate enzymes into fermentable sugars.

PATTERN OF CASSAVA PRODUCTION

Ghana is the sixth producer of cassava (15,113,000MT) in the world in terms of value and in terms of volume as in the year 2015, the third in Africa and the second among producers of fresh cassava roots in West Africa (FAO, 2015). Cassava is cultivated in all the 10 regions of Ghana. The five leading producers by regions on average over the past three years (2012-2014) included, Eastern: 4,307,372.22MT, Brong Ahafo: 3,460,907.08MT, Ashanti: 2,435,915.22MT, Central: 1,813,888.18MT, Northern: 1,403,454.35MT. Average area cropped per year between 1999 and 2004 was about 750,000 hectares, yielding about 10 million metric tonnes, increasing to 889,000 hectares in 2011 and producing 14 million metric tonnes (SRID-MoFA, 2012). However, in 2012 the total land area for cultivation dropped to 869,000 hecares but with slight increase in yield to 14.5 million metric tonnes of cassava increasing to 889,000 hectares of cultivated land in 2014 producing 16.5 million metric tonnes of cassava (SRID-MoFA, 2012, 2014). The bulk of the nation's cassava is produced in

Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 315 the south and middle part of Ghana, which accounts for roughly 78% of the total cassava production. Currently, Eastern region is the largest producer of cassava in Ghana accounting for 3 years average of 4.3 million metric tonnes spanning 2012-2014 (SRID-MoFA, 2014). Mean annual growth rate of area planted with cassava increased by 1.24% between 2003- 2005 and 2006-2008 and a marginal decrease of 0.22% between 2009-2011 and 2012-2014 (SRID-MoFA, 2014). The decline is predicted to continue next year due to slight drought in the Sub-Sahara African (SSA) region. This is a treat to Ghana’s food security being that human population keeps increasing and cassava is a major staple food in the country (FAO, 2015).

CASSAVA CULTIVATION

Over the years, cassava has been recognised as a major crop in Ghanaian agricultural and Africa in general. Although cassava was considered as a food security crop in most places where it had not previously been grown, notably in dry areas and marginal lands, the focus has gradually changed and it become a commercial crop for most farmers. This is due to the ability of the crop to withstand drought and thrive under harsh conditions (FAO, 2002). The major cassava planting season is mainly during the rainy season from April to November. With the intervention of new varieties in Ghana, Cassava is harvested approximately 12 months after planting. The largest percentage of the cassava root harvest comes onto the market in the early part of the wet season (May to July) before planting begins. Harvesting during the dry season (November to March) is in small quantities (Sam & Deppah, 2009). Mix cropping is common in Ghana and cassava is often mix cropped with maize, cocoyam, yam and cowpea. The crop is also rotated with some of these mix crops when farmers observe decline in soil fertility or productivity, the land is cropped to cassava for a period ranging between 12 to 18 months after which the maize/cowpea rotation is resumed. The total land area used for cassava cultivation increased by 18.5% since 2005. This increase in land use for cassava cultivation is as a result of its importance for industrial applications (FAO, 2002). Generally, the crop needs a warm and humid climate to grow with temperatures averaging 25-27ºC. The tropical lowlands with altitude below 150 m with annual rainfall from 500 mm to 5,000 mm are most suitable for higher root yield. Because the plant is resistance to prolong drought it is able to thrive in regions where annual rainfall is low or where seasonal distribution is irregular (USDA NRCS, n.d.). The crop is also able to grow on poor and degraded soil because it can withstand low pH, high level of exchangeable aluminum and low concentration of phosphorus in the soil matrix (Howeler, 2001). The agro- ecological regions of Ghana have mean annual rainfall varying between 800 mm and 2,200 mm (SRID-MoFA, 2014) making them very suitable for cassava cultivation. The soil pH vary from one ecological zone to the other but generally are in the range of 3.5 to 7.8. Ghana has a tropical climate with wet and dry seasons. The rainfall distribution is bimodal in the Forest, Transitional and Coastal Zones, giving rise to major and minor growing season; whiles Guinea Savannah and Sudan Savannah have unimodal distribution resulting in a single growing season (SRID-MoFA, 2014).

Complimentary Contributor Copy 316 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre

The land is prepared for planting before the major rain starts in June. The slash and burn is carried out on virgin lands where planting is done for the first time. But lands which have been cultivated before are cleared without burning before planting is done. Hoes are used to make either small mounds or ridges for planting the cassava stick cuttings. Cutlasses and hoes are also used for weeding and cutting of planting materials. Currently tractors are not popularly used but with the inception of commercial farming, it is becoming a very useful tool for cassava cultivation. Stem cuttings of 20 cm lengths with an average weight of 0.085 kg/stem cutting are used as planting materials and are not pre-treated before planting. For traditional farms, about 3,240 stem cuttings are used per hectare, whiles commercial farms use 10,000 stem cuttings/hectare. Application of herbicides are not common for cassava cultivation for now but may be used when commercial farming takes off in full. Because cassava cultivation is rain fed, irrigation is not used for now. Mulching in cassava cultivation is done basically by allowing the slashed grasses mostly Andropogon on the field for some few days and then ploughed into the soil using hoes. This is done only once before planting. In most of the traditional farms fertilizers are not applied, but with the introduction of new concept, Integrated Crop Management (ICM), farmers are encouraged and trained on fertilizer application to increase yield. Demonstration farms are set up to teach farmers. Fertilizer application is done by using hand and containers. In cassava cultivation pruning is not common or not done, but weeding is done at intervals to prevent weed growth. When the plant is young this is done more frequently depending on the type of grass found at the farm and when the plant is grown, the frequency is reduced and at full maturity, the farms are not weeded. An average of 13 people are mostly involved in the cultivation of 1 hectare of cassava farm using about 78 man hours.

CASSAVA VARIETIES DEVELOPED AND CULTIVATED IN GHANA

Over the years, there has been increase research of improved varieties of cassava in Ghana. The National Agricultural Research Systems (NARS) have released about 24 improved cassava varieties since 1993, which are high yielding, disease and pest resistant and mature early. Currently, Crop Research Institute of Council for Scientific and Industrial Research, Ghana, has released 11 improved varieties (CSIR-CRI, 2014). In Ghana, farmer’s preference for the variety they choose for cultivation is based on; yield, in-soil storage (longevity), disease resistance (Acheampong et al., 2013), utilisation purpose (multiple usages or the food type the cassava will be processed into) and readily available as planting materials. Nevertheless, new variety adoption by small scale farmers is very low leading to low outputs and incomes. There is low adoptability of high yielding improved cassava varieties in Ghana over the past 15 years due to low understanding of the varieties (Acheampong et al., 2013) and their management practices, and availability of planting materials. Farmer’s selection for varieties they cultivate is also based on the market value for the various varieties. For examples there is high value for cassava varieties that are used for preparing a local food called ‘fufu’ (pounded boiled cassava) because it is one of the delicacies for Ghanaians especially those in the south.

Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 317

FACTORS AFFECTING CASSAVA PRODUCTION

Cassava production is faced with serious biotic constraints, such as diseases and pests, poor handling of planting materials, poor agronomic practices, limited technical know-how in cropping new varieties and poor postharvest handling and processing. The major factor that affects cassava production in SSA is pests of cassava; green mite and the variegated grasshopper. The main diseases affecting cassava are cassava mosaic disease (CMD), cassava bacterial blight, cassava anthracnose disease and root rot. In Africa, 50% of yield losses can be attributed to pests, disease and poor cultivation practices (FAO, 2002). Over the years, cassava production worldwide had been increasing remarkably at 4% p.a overtaking world population growth, however, due to unfavourable weather conditions, cassava production growth rate is predicted to reduce in 2015. This is probably going to be the first time phenomenon in virtually ten years, which could lead to the SSA production estimate of 163 million tonnes; a 3 million tonne drop from 2014. This drop over the SSA has reflected in the 0.2% drop in cassava production in Ghana in the year 2014. The drop has been partly attributed to El Niño and uncertain demand for cassava non-food products (FAO, 2013). Mechanisation, development of new technologies and new improve varieties of cassava and making planting material available at the right time will strengthen cassava production in the near future in Ghana. One of the main causes of low productivity of cassava in Ghana is the continuous use of indigenous, low yielding crop varieties (FAO, 2002; SRID- MoFA, 2014).

POLICY ON CASSAVA PRODUCTION IN GHANA

The Ministry of Food and Agriculture (MoFA) is the lead ministry of Ghana government that is tasked with the responsibility of developing and executing policies and strategies for the development of the agriculture sector. Over the years, MoFA has been involved and leading various agriculture policies to improve agriculture production in Ghana. A lot of interventions have been made over the years by various organisations to develop cassava in the country however, government policies relegated the crop in favour of export crops such as cocoa, coffee and maize (Kleih et al., 2013). In the early 1930’s research works on cassava was directed toward high yields, low HCN content and excellent cooking qualities. Consequently, research and development (R&D) in cassava have focused on new high yielding varieties and improved pest, disease and drought resistance varieties. Since 1984, various projects have been rolled to improve cassava production in the country. Some of the interventions included: Ecologically Sustainable Cassava Plant Protection (ESCaPP) project; National Root and Tuber Crops Improvement Project (NTRCIP-1988) as a component of the International Fund for Agricultural Development (IFAD) sponsored Ghana Smallholder Rehabilitation and Development Programme (SRDP); National Agricultural Research Project (NARP); Medium-Term Agricultural Development Project (MTADP) in 1991; Food and Agriculture Sector Development Policy (FASDEP II) and the Medium Term Agriculture Sector Investment Plan (METASIP 2010-15); Root and Tuber Improvement and Marketing Programme (RTIMP) funded by IFAD and West African Agricultural Productivity Programme (WAAPP) to mention few (Kleih et al., 2013).

Complimentary Contributor Copy 318 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre

CASSAVA HARVESTING AND PROCESSING

Cassava as a staple food crop in Ghana can be eaten fresh by boiling in water and or pounded into traditional food called “fufu.” To achieve food security and to develop the agro- based economy in the rural areas to improve their living conditions, processing of cassava becomes very important. Cassava-based industrial food products have a potential of boosting the local economy. For industrialisation of cassava, it is necessary to improve upon the traditional methods of processing to improve the quality of product as well as to prolong the shelf life. Cassava harvesting is full of drudgery especially during the dry season when the soil is much firm and this has been a major constraint to commercial farmers. Cassava harvesting is usually manual by; cutting stem about 0.75meters above ground/leaving it uncut, the stem is held with both hands and pulled up to bring out the root from the soil. In instances that some of the roots remains in the soil, a hoe or cutlass is use to dig them out. In 2013, a mechanical cassava harvester called TEK mechanical harvester which is tractor drawn device was introduced. The device was tested and functions much better in the dry season when soils are much firmer, than during the wet season when soils are loose (WAAPP/PPAAO, 2013). The technology is yet to be adapted for commercial purpose.

TRADITIONAL CASSAVA PROCESSING

Agro-processing activities in the rural levels are responsible for the preservation and distribution of most of Ghana’s agricultural produce. These activities play a major role in the post-harvest food system and are mainly carried out by rural women (IFAD, 2007) who employ very old and reliable traditional techniques in the processing of root and tuber crops. Traditional methods employed are simple and easy to use for their level of production. The equipment used for the traditional processes are cheaper compared to what is used for modern high technology processes. However, these traditional technologies produce products of relatively low quality coupled with high labour (Dziedzoave, et al. 1999; Westby, 2002). There are six (6) operational units involved in traditional cassava processing; peeling, chipping, grating, fermentation, sieving and frying/drying/roasting.

PEELING

Cassava is peeled to remove none edible outer covering which is commonly known to contain most of the toxic cyanogenic glucosides. Peeling is usually done manually with hand using knife. Peeling is done either by slitting along the length of one side of the root with a knife followed by using the fingers to roll back the peels from the fleshy portion of the root, or by using the knife to slice the outer covering entirely from the flesh. Hand peeling is slow and laborious but it is the only method available now and used for cassava peeling in Ghana.

Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 319

Figure 1. Peeling of cassava by women at cassava processing facility at Bawjiase, Ghana.

CHIPPING

Chipping is done to reduce the thickness of the cassava tuber thereby exposing the maximum surface area of the starchy flesh to facilitate quick drying. The drying process is affected by the size of the slice. It is known that thick slices take much longer time to dry because the rate of moisture diffusion from the inside is slower and the time for complete drying is longer. Usually sun-drying systems are effective when the chips are dried by passing air over them than by the direct effects of the sun’s rays. For efficient drying, the chip’s shape should allow air to readily circulate through a large mass of them.

FERMENTATION AND WATER REMOVAL

Traditional operations normally combine both fermentation and water removal in one unit operation. The grated mash is put into jute bags, baskets, or any perforated material that allows water to drain and left to ferment for 1-5 days. During the fermentation, the sacks containing the cassava mash are twisted tightly and put on wooden boards with heavy stones pilled on them to press and remove the water (James et al., 2012; Quaye, Gayin, Yawson and Plahar, 2009). The fermentation process can be reduced by adding a starter culture in the form of seeding with previously fermented liquor. The fermentation process affects the quality of the product in terms of taste, colour and texture and must be properly controlled.

GRATING

In traditional set up, grating is done manually with hand. But power operated graters of different models are also manufactured locally and used. Hand grating is a cumbersome

Complimentary Contributor Copy 320 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre operation and is normally done after washing and allowing excess water to drain from the cassava flesh to prevent the cassava from being slippery during grating. The manual grater is made up of galvanised metal sheet or a piece of flattened can or tin, punched with about 3mm diameter nails leaving a raised jagged flange on the underside. This grating surface is fixed on a wooden frame forming a dome shape or flat and the cassava pieces pressed against the jagged side of the metal and rubbed vigorously with strong downward movements. It is not possible to completely grate a whole cassava piece, 3% to 5% of the cassava had to be left un- grated. A skilful person is able to produce only about 20 kg/hour (Quaye et al., 2009).

SIEVING

After pressing to remove the water, the relatively dry cassava mash is broken up and sieved to remove the large lumps and fibre to obtain a homogenous product. This is done by using sieves made from bamboo, palm leaves or raffia cane by rubbing and pressing the broken lump on the sieve with the palm. Mechanical sieves are also available and used in small commercial operations.

FRYING/ROASTING AND DRYING

Frying of gari is a combination of two processes namely; roasting and drying. At the rural set up frying of gari is done in shallow aluminum pans, or in earthenware pans, over an open wood fire. The sieved cassava mash is spread thinly in the pan in 2-4kg batches depending on the size of the frying pan. A piece of calabash is often used to press the mash against the hot surface of the pan but scraped quickly and stirred constantly to keep the material moving to prevent it from burning until frying is completed at about 80° to 85°C. The quick heating partially gelatinises the gari which is dried during frying. The process takes 30-35 minutes, with the moisture content of the final product reduced to about 18% (Quaye et al., 2009).

GARI PROCESSING

Gari is one of the most popular processed cassava products in all the cassava producing districts in Ghana. Traditional processing of gari from fresh cassava is made up of various unit operations of peeling, washing, grating, pressing and fermentation, sieving and roasting. The peeled tubers are washed thoroughly with water and grated by rubbing on the rough surface of a perforated galvanised metal sheet fixed to a wooden board support. The grated cassava mash is packed into jute bags and the open ends tied securely with rope. The loaded bags are then packed on wooden racks and heavy stones placed on them to squeeze out the starchy juice. After which fermentation is done for a period of about two days. The pressed fermented dough is dried in the Sun and sieved with traditional sieves. The sieved grains are roasted over fire in open cast iron frying pan with quick stirring until cooked and crisp. The roasted mass is again sieved to remove lumps, and packaged for storage and marketing (James et al., 2012; Quaye et al., 2009).

Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 321

Figure 2. Frying of gari using improved cook-stove in Kete Krachi, Ghana.

“KOKONTE” PROCESSING

Traditional processing of cassava into “kokonte” requires less effort as compared to gari processing. The peeled roots are cut into small pieces and dried in the sun for 3 -6 days, depending on the sun’s intensity. Smaller pieces dry faster than the bigger one. Fermentation is achieved during drying, and this provided the desired aroma to the dried product. The potential of mould growth is reduced when drying is done rapidly. The dried product has a long shelf life and could be stored for several weeks as whole chips. This intermediate product is milled into flour and used in the preparation of a cooked traditional meal (Quaye et al., 2009).

AGBELIMA PROCESSING

Traditional processing of cassava to fermented cassava dough is normally called “agbelima.” The unit processes involved is similar to gari processing as described earlier but the pressed and fermented product is not fried. This pressing and fermentation enhance the storage properties of the dough but only for a few days. The fermented dough is used for the preparation of Ghanaian dishes like (Akple or banku) and “Yakeyake.”

Complimentary Contributor Copy 322 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre

PRODUCT DEVELOPED WITH HIGHLY QUALITY CASSAVA FLOUR (HQCF)

In Ghana, several products have been developed using HQCF. A composite proportion of 1:4 of HQCF to wheat flour is use to bake bread adding other ingredients like milk, sugar, margarine, salt, nutmeg, baking powder etc. Pastries are made using cassava flour by replacing 75% of wheat flour in sponge cakes and chiffon cakes, 50% in butter cakes and cookies, and 25% in doughnuts and spaghetti. Noodles are also made from cassava flour by replacing 25-50% of the rice starch used and it gives a softer and elastic nature to the noodles (Dziedzoave et al., 2003). Cassava flour is used in the brewery industry to produce beer as in the Root beer; a popular beer in Ghana. The HQCF is also used as binding agents in food and plywood industry, and as adhesives to replace maize starch in starch-based adhesives.

INTEGRATED CASSAVA PROCESSING PLANT

Over the years, various cassava processing machines have been designed to aid in one or two stages in the processing line. CSIR-Institute of Industrial Research, Ghana and other institutions have been instrumental in design and manufacturing of cassava processing machines with effort to integrate the various machines. An Integrated Cassava Processing Plant is designed to process 10-25metric tones of fresh cassava tubers into traditional fermented derivatives including gari, kokonte, agbelima vis-à-vis relatively new product of unfermented high quality cassava flour. Another unique feature of the plant is the incorporation of an animal feed processing unit that converts the cassava peels into animal feeds supplements to promote rearing of goats, sheep, cows etc. Three main machine incorporated into traditional process techniques to ensure production of unfermented high quality cassava flour are dryer for drying the sieved fine dough into flakes, hammer mill for milling and sieving the dried flour flakes and sifter to capture and finally sieve and grade the finely milled unfermented high quality cassava flour (Hahn, 2006; Selormey, et al. 2006).

HOW THE INTEGRATED CASSAVA PROCESSING PLANT TECHNOLOGY WORKS

The technology involves peeling of cassava manually and carefully to ensure total removal of the peels without peeling off a greater portion of the flesh in which the starch tissues are contained. The peeled cassava is washed thoroughly with clean water in three (3)- segment washing trough. Grating is done with a diesel-engine driven horizontal shaft Grater and/or a motorised vertical shaft Grater. The cassava mash can be fermented and used to process gari. The cassava mash captured in jute sacks are pressed with the help of a single manual screw and two double screw manual presses to dewater the cassava mash. The pressed dough is fed into the horizontal shaft drum Grater to disintegrate dough into fine granules and then sieved with a sieve which consists of a rotating drum with mesh. The roughages retained by the screen are considered by-product and used mostly as animal feed. The fine dough is dried with an electric dryer and after drying, the cooled flakes is fed into Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 323 the mechanise hammer mill consisting of stainless steel hammer. The milled flakes are drown out of the hammer mill into a stainless steel hammer mill-blower-sifter-cyclone, falling by gravity into a hopper of a sifter guided with a slide to avoid overloading. Medium and coarse flour particles, which are not sieved are fed back into the hammer mill for further milling to obtain the fine high unfermented cassava flour. To make kokonte, after peeling and washing the cassava, the cassava is fed into motorises chipping machine to chip the cassava into smaller sizes and dried using a hybrid solar dryer to form kokonte. The peels are roughly milled and fed into a motorised feed mixing machine to make animal feed supplement (Dziedzoav, et al., 2006; Selormey, et al., 2006).

QUALITY IMPROVEMENT

Cassava roots are an excellent source of carbohydrates. However, this food source has three major deficiencies: poor shelf-life, low content of protein and free amino acids, and high content of the poisonous cyanogenic glucosides (CNG): linamarin (96%) and lotaustralin (4%) (Cooke & Coursey, 1981). These cyanogens are distributed widely throughout the plant, with large amounts in the leaves and the root cortex (skin layer) and, generally, smaller amounts in the root parenchyma (interior). The designation of bitter and sweet varieties of cassava depends on the associated levels of toxicity (Sundaresan et al., 1987). Consumption of cassava products with high cyanogens levels may cause acute intoxications (Mlingi et al., 1992), aggravate goiter (Bourdoux et al., 1982) and, in severe circumstances, induce paralytic diseases (Tylleskar et al., 1992). To avoid dietary cyanide exposure, the glycosides and their metabolites, collectively known as cyanogens, must be removed by processing before consumption. Available research data confirms that peeling, first substantial process step lowers cassava toxicity, as the CNG distributed in large amounts in the root cortex (skin layer) is removed (Cooke & Coursey, 1981). Additionally, grating of the pulp, as the second step in processing, enables linamarin to have contact with its hydrolytic enzyme (linamarase), resulting in hydrolysis and subsequent removal of the breakdown products (Sornyotha et al., 2010). Fermentation is another process operation which has been observed to detoxify cassava. Fermentation experiment conducted by Lambri et al., (2013) confirmed that cultured microorganism played significant role in cynogen detoxification in cassava. They further concluded that yeast Saccharomyces cerevisiae, followed by Oenococcus oeni and Lactobacillus plantarum V22 were more effective in degrading linamarin after 24 hours than mixed cultures. These findings confirm the results of other researches (Tweyongyere & Katongole, 2002), regarding the fermentation of cassava roots soaked in water in which microbial growth was shown to be essential for the efficient elimination of cyanogens (Westby & Choo, 1994).

Complimentary Contributor Copy 324 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre

POTENTIAL UTILISATION OF CASSAVA RESIDUES

Cassava is harvested at the farm and the tuber transported to a processing facility. Cassava stalk and leaves are the major residues generated at the farm level. In 2014 about 3.7 million MT of cassava stalk was estimated to have been generated at farms using crop residue ration of 0.192 (Koopmans & Koppejan, 1997; OECD/IEA, 2010; SRID-MoFA, 2014). Cassava stalk is used as planting material and excess is sometimes burnt or left at the farm to decay. Stalks of new improved varieties are sold to farmers and in subsequent years enough is generated and excess becomes waste. Cassava leaves are also used for food by both humans and animals because of its nutritional value. Consumption of cassava leaves by humans is limited, but that of animals is prominent in areas of livestock rearing especially goats and sheep. Cassava leaves are considered as a good source of supplementary protein which can be used for preparing dishes in order to add variety to the diet as well as nutrition. The digestibility and nutritional value of cassava leaves have been investigated by Eggum, (1970) and Ravidran et al., (1987) who found it to be 80% for the protein in young leaves and 67% for the protein of older ones. Cassava leaves are good source of minerals. They are particularly rich in Ca, Mg, Fe, Mn and Zn. They are also rich in ascorbic acid and vitamin A and contain significant amount of riboflavin. But considerable losses of vitamins particularly of ascorbic acid occur during processing (Ravindran, n.d.). Cassava leaf yields amounting to as much as 4.60 MT dry matter per hectare may be produced as a by- product at root harvest (Ravindran & Rajaguru, 1988). Apart from cassava stalks and leaves generated at the farm levels, tons of cassava residue are generated at processing facilities. The cassava residue is composed of peels and trimmings. Peels normally consist of the thin pericarp and the thicker ring. Most processes remove both the pericarp and the thicker ring along with some pulp adhered to the peels. Analysis of the chemical composition of cassava peels indicates the following: dry matter 86.5–94.5%; organic matter 81.9–93.9%; crude protein 4.1–6.5%; hemicellulose and cellulose 34.4%; and lignin 8.4% (Kongkiattikajorn & Sornvoraweat, 2011). The composition of cassava residue make it a good resource for biorefinery. Composition analysis of the cassava residue by Bayitse et al., (2015) indicated that 47.16% was made up of starch, 2.40% protein and 83.41% glucose. Glucose is one of the major raw material in biorefinery and can be used to produce ethanol, lactic acid, and lysine. Cassava peel has some amount of crude protein as specified in composition analysis by Bayitse et al., (2015). The protein content of the cassava peel can be enhanced using solid state fermentation to make it more valuable in animal feed formulation. Solid state fermentation of cassava residue with Trichoderma pseudokoningii was conducted for 12 days. The fermentation was carried out at temperature of 24 °C and a pH of 5.0. Urea and ammonium sulphate were used as nutrient sources and moisture content varied at 60 and 70%. Protein content of the unfermented cassava residue was increased from 8.4 to 12.5% when urea was used with initial moisture content of 70% w/v. This study showed that a maximum of 48.1% protein enrichment was achieved using urea as a source of nutrient for the growth of the fungi, whiles ammonium sulphate achieved 36.9% protein enrichment under the same condition (Bayitse et al., 2015).

Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 325

Figure 3. Schematic diagram of biorefinary process of cassava peel (Bayitse, et al., 2015).

ETHANOL PRODUCTION

Ethanol produced from lignocellulosic biomass is a potential alternative transportation fuel to none-renewable fossil fuels. Currently, the prevalent technique for cellulosic ethanol production is an enzyme based process because it is more environmentally friendly and produces a better hydrolysis yield than acid hydrolysis. Therefore, present cellulosic ethanol research is driven by the need to reduce the production cost (Mielenz, 2001). The enzyme based process primarily includes three steps such as biomass pretreatment, enzymatic hydrolysis and fermentation. Following the pretreatment, the enzymatic hydrolysis process can be designed in various ways. It can be run separately; separate hydrolysis and fermentation (SHF) or simultaneously; simultaneous saccharification fermentation (SSF). For either process, the key cost element to consider is that of the enzyme (Saddler & Gregg, 1998). For this reason, it is important to use the enzymes as efficiently as possible by creating a favourable environment in the hydrolysis step. This outcome could be realised by optimising operation methods (batch, fed-batch or continuous) and process parameters such as solid loading. In addition to enzyme concentration, solid loading is another important physical parameter that can affect the efficiency of cellulose hydrolysis. Although low solid loading could achieve high cellulose conversion, it would result in low yield of sugar concentrations for fermentation and ethanol for distillation thereby increasing ethanol recovery cost (Kongkiattikajorn, 2012). Also, low solid loading would increase both the capital cost of equipment and the operation costs in order to reach certain ethanol production capacity. Therefore, high solid loading is preferable and economically practical than low solid loading. However, the problems of sugar inhibitions and mixing with high solid loading need to be solved properly (Kongkiattikajorn, 2012).

Complimentary Contributor Copy 326 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre

In fed-batch fermentation, solids and/or enzymes are added into reactors stepwise and solids are gradually degraded; thereby making the mixture more fluid creating adequate room for more solids to be added (Koppram and Olsson, 2014). As a result, fed-batch is expected to be a better procedure than batch on dealing with the situation of high solid loading and low enzyme concentration. Additionally, fed- batch can generate high glucose concentration for fermentation and finally yield high ethanol concentration for distillation resulting in significantly decrease of ethanol production cost (Ballesteros et al., 2002). Bacteria, yeasts and fungi are able to ferment xylose to ethanol. However, research showed yeasts are favourable for producing higher ethanol yields from xylose than the others. To date, the most extensively studied xylose- fermenting yeasts include Candida shehatae, Pachysolen tannophilus and Pichia stipitis. C. shehatae and P. stipitis are the best native ethanol producers from xylose, with yields approaching the theoretical maximum of 0.51 g ethanol /g xylose (Chu & Lee, 2007). The Baker's yeast Saccharomyces cerevisiae is normally accepted as safe microorganism for use in industrial wine making, brewing and baking processes to produce ethanol and CO2 from fermentable sugars respectively (van Zyl et al.,1989). Glucose fermentation is an anaerobic process that is used industrially for the production of ethanol with minimal formation of biomass and glycerol. Despite the efficiency of S. cerevisiae in glucose fermentation, it cannot utilise xylose effectively as a sole carbon source to ferment xylose to ethanol despite having a full xylose metabolic pathway (Batt et al., 1986). Ethanol yields and productivity from xylose fermentation by naturally occurring pentose-fermenting yeasts are significantly lower than glucose fermentation by S. cerevisiae, suggesting that there is considerable scope for improvement in xylose fermentation biotechnology (Chu & Lee, 2007). Recently, Olanbiwoninu and Odunfa, (2015) hydrolysed cassava peel into fermentable sugars using organic acid pre-treatment before enzyme hydrolysis. This process could add additional cost to the fermentation process but this has shown the potential of bioconversion of cassava peel into fermentable sugar. Bayitse et al., (2015) in their work to bioconvert cassava peel into fermentable sugars, evaluated enzymatic hydrolysis of cassava peel using cellulase and beta-glucanase enzymes and their mixtures at three different enzyme loadings with time. The pH of the medium used for hydrolysis was 5 and the temperature was 50 °C. They reported that efficiency of the hydrolysis using beta-glucanase was better than cellulase and glucose recovery of 69% was realised when beta-glucanase dosage was increased to 10% (v/w) at 48 h which rose to 73% at 120 h, releasing 11.19 g/l and 12.17 g/l of glucose respectively. Less than 20% of glucose was hydrolysed at 10% (v/w) cellulase at 120 h releasing 2.6 g/l glucose. The optimum experimental condition for hydrolysis of cassava peel was established at 120 h when glucose recovery increased to 88% for enzyme mixture of 5% (v/w) cellulase + 10% (v/w) beta-glucanase producing 14.67 g/l glucose in the hydrolysate. To obtain high concentration of ethanol from cassava peel, Kongkiattikajorn, (2012) pretreated cassava peel with acid to remove noncellulose components, and then subjected it to simultaneous saccharification and fermentation (SSF). An ethanol concentration as high as 7.62 g/L was realized with 2.5% dry matter (DM) using batch SSF, producing 84.34% overall ethanol yield. He further investigated a fed-batch process using a high solid concentration. Dry substrate was pretreated with steam and dilute sulfuric acid at 135°C under pressure of 15 lb/in 2, and then added at different amounts during the first 24 h, to yield a final dry matter content of 20% (w/v). Fed batch SSF conditions with cellulase loading of 100 FPU/g, Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 327 xylanase 25 IU/g, pectinase 25 IU/g and amylase with amyloglucosidase loading of 50 and 75 U/g, respectively, yeast (Saccharomyces cerevisiae) loading of 2 g/L and substrate supplementation every 4 h yielded the highest ethanol concentration of 58.73 g/L after 72 h. This corresponded to a 76.47% overall ethanol yield. The upscale process for ethanol production using cassava peel was conducted by Bayitse et al., (2015). They pretreated the cassava peel by wet milling followed by simultaneous saccharification and fermentation. Their findings suggested that intermittent wet milling with α-amylase has increased glucose concentration over five-fold from the initial concentration of 1.36 mg/L. Simultaneous saccharification using the optimal condition of enzymes (amyloglucosidase, β-glucanase and cellulase) has increased the glucose concentration in the hydrolysate from 5.5 g/L to 75.5 g/L after wet-milling. Fermentation was carried out for 72 hours, but the optimum was reached after 24 hours without additional nutrient supplements. High-Performance Liquid Chromatography analysis of the fermented broth recorded 46.52 g/L of ethanol which represented 98% of theoretical ethanol yield.

LACTIC ACID PRODUCTION

Industrial scale production of lactic acid demands availability of sustainable cheap raw materials with low level of contamination. Biomass as raw material in the form of starch (corn, wheat, potato, cassava, rice and sweet sorghum) and lignocelluloses (corn cobs, waste paper and woody materials) can be used as a substrate for fermentation of lactic acid (Oh et al., 2005; Richter & Berthold, 1998). Biomass from agricultural crop residues can be put into two major categories. The primary category is obtained as a by-product of agricultural post-harvesting activities, normally from the harvesting and processing of staple crops for domestic use. The secondary category is generated from industrial processing of agricultural crops. Cereal crop mills and food processing industries are directly involved in biowaste generation from agricultural residues (Mohammed et al., 2013). Lactic acid production can be done either by fermenting sugars or hydrolysates containing sugars. It can also be produced by converting starchy or cellulosic materials using lactic acid producing microorganisms. Simultaneous hydrolysis and fermentation with saccharifying enzymes is widely deployed. The use of hydrolysate is preferred to refined sugars for solid state or submerged fermentation of lactic acid (John et al., 2006). The hydrolysis of starch or cellulose to sugar is a high energy utilisation process which can increase the cost of production. Woiciechowski et al., (1999) in their study of hydrolysis of cassava bagasse starch by acid and enzyme reported that, both methods were quite efficient when considering one or the other parameter like the percentage of hydrolysis, time and cost of the chemicals and energy consumption. Although acid hydrolysis is time saving and cost effective, there is always a neutralising step after acid hydrolysis. This will increase the level of salts in the medium and affect the microbial growth and production of lactic acid. Conventional fermentative production of lactic acid from starch materials requires a pretreatment process that involves gelatinisation and liquefaction, which is carried out at a temperature between 90 and 130 °C for 15 min followed by long time enzymatic

Complimentary Contributor Copy 328 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre saccharification to glucose at a higher temperature, and subsequent conversion of glucose to lactic acid by fermentation. Anuradha et al., (1999) conducted batch experiments to establish optimum operating conditions for the simultaneous saccharification and fermentation (SSF) of starch to lactic acid using Lactobacillus delbrueckii. They developed a predictive model for SSF by combining the kinetics of saccharification and fermentation. Their results showed that saccharification rate was always higher for SSF than in simple saccharification (SS) at all substrate concentrations. Nwokoro, (2014) produced L-lactic acid using cultures of Rhizopus oligosporus and Lacto- bacillus plantarum from cassava peel. He hydrolysed cassava peels for 1 hour in both NaOH and HCl by boiling after which the hydrolysates were neutralised to a pH of 6.2. He reported that there were proportional increase in reducing sugar with increasing concentrations of alkali or acid. Higher concentration of reducing sugar (402 mg/g) was realised in the acid hydrolysate as compared with 213 mg/g reducing sugar concentration in alkali hydrolysate. He further added 0.5% ammonium sulphate solution to the hydrolysates and inoculated with either single or mixed cultures of R. oligosporus and L. plantarum and incubated for 48 hours for lactic acid production. He concluded that the best lactic acid production of 50.2 g/100 g substrate was observed in a mixed culture fermentation of acid hydrolyzed peels as compared with 36.4 g/100g substrate of alkali hydrolysed peels. However, unhydrolysed cassava peels inoculated with a mixed culture of the microorganisms produced only 4.6 g/100g substrate. His conclusion also buttress the point that the lactic acid bacteria need reducing sugar to produce lactic acid. Fibrous residue is a major waste produce during cassava starch extraction. Because of the high starch content (60-65% on dry weight basis) and organic matter of cassava fibrous residue (CFR), research has been conducted to utilise it for the production of lactic acid (LA) in semi solid state fermentation using Mann Rogassa Sharpe medium containing [5% (wv (- 1))] CFR in lieu of glucose [2% (wv (-1))] as the carbon source. Response Surface Methodology (RSM) was used to evaluate the effect of main variables, i.e., incubation period, temperature and pH on LA production. The experimental results showed that the optimum incubation period, temperature and pH were 120 hours 35 degrees C and 6.5, respectively. Maximum starch conversion by Lactobacillus plantarum MTCC 1407 to LA was 63.3%. The organism produced 29.86 g of (L+) LA from 60 g of starch present in 100 g of CFR. The LA production yield was 49.76%. (Ray et al., 2009).

CONCLUSION

Cassava has been recognised as a major crop in Ghanaian agricultural systems and has been grown in almost all the 10 regions in the country. The major cassava planting season is mainly during the rainy season from April to November. With the intervention of new varieties in Ghana, cassava is harvested approximately 12 months after planting. The bulk of the nation's cassava is produced in the south and middle part of Ghana, which accounts for roughly 78% of the total cassava production. Currently, Eastern region is the largest producer of cassava in Ghana accounting for 3 years average of 4.3 million metric tonnes spanning 2012-2014. The total land area used for cassava cultivation increased by 18.5% since 2005.

Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 329

This increase in land use for cassava cultivation is as a result of its importance for industrial applications. Most importantly, cassava is a staple food crop and accounts for about 152.9 kg per capita consumption, making it one of the most processed crop into gari, fufu powder, Highly Quality Cassava Flour (used for bakery products) and kokonte to increase its shelf life. Additionally, it can be used as an industrial crop because of its high starch content. These process technologies have contributed to the reduction of post-harvest losses in cassava production in Ghana. The residue generated from cassava processing has a huge potential in biorefinery for the production of major products such as ethanol, lactic acid and protein.

REFERENCES

Acheampong, P., Owusu, V. and Gyiele K. N. (2013). 203-Farmers Preferences for Cassava Variety Traits: Empirical Evidence from Ghana. In Fourth International Conference of the African Association of Agricultural Economists (pp. 1–21). Hammamet, Tunisia. Adams, C. (1957). Activities of Danish Botanists in Guinea 1738–1850. Transactions of the Historical Society of Ghana III. Part 1. Anuradha, R., Suresh, A. K. and Venkatesh, K. V. (1999). Simultaneous saccharification and fermentation of starch to lactic acid. Process Biochemistry, 35(3-4), 367–375. http://doi.org/10.1016/S0032-9592(99) 00080-1. Ballesteros, M., Oliva, J. M., Manzanares, P., Negro, M. J. and Ballesteros, I. (2002). Ethanol production from paper material using a simultaneous saccharification and fermentation system in a fed-batch basis. World Journal of Microbiology and Biotechnology, 18(6), 559–561. http://doi.org/10.1023/A:1016378326762. Batt, C. A., Carvallo, S., Easson, D. D., Akedo, M. and Sinskey, A. (1986). Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae. Biotechnol Bioeng, 28, 549–53. Bayitse, R., Hou, X., Laryea, G., Aggey, M., Oduro, W., Selormey, G. and Bjerre, A.-B. (2015). Ethanol process from biowaste c5 and c6. Brussels. Retrieved from http://www.biowaste4sp.eu. Bayitse, R., Hou, X., Bjerre, A.-B. and Saalia, F. K. (2015). Optimisation of enzymatic hydrolysis of cassava peel to produce fermentable sugars. AMB Express, 5(1), 60. http://doi.org/10.1186/s13568-015-0146-z. Bayitse, R., Hou, X., Laryea, G. and Bjerre, A.-B. (2015). Protein enrichment of cassava residue using Trichoderma pseudokoningii (ATCC 26801). AMB Express, 5(1), 80. http://doi.org/10.1186/s13568-015-0166-8. Bourdoux, P., Seghers, P., Mafuta, M., Vandrpas-Rivera, M., Delange, F. and Ermans, A. M. (1982). Cassava products: HCN content and detoxification process. In E. A. Delange F, Iteke FB (Ed.), Nutritional factors involved in the goitrogenic action of cassava (pp. 51– 58). Ottawa, Canada: IDRC-184e. IDRC. Chu, B. C. H. and Lee, H. (2007). Genetic improvement of Saccharomyces cerevisiae for xylose fermentation. Biotechnology Advances, 25(5), 425–441. http://doi.org/10.1016/ j.biotechadv.2007.04.001.

Complimentary Contributor Copy 330 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre

Cooke, R. D. and Coursey, D. G. (1981). Cassava: a major cyanide containing food crop. In F. Vennesland, B; Conn, E; Knowles, C; Westley, J; Wissing (Ed.), Cyanide in biology (pp. 93–114). London: Academic Press. CSIR-CRI. (2014). The National Centre of Specialisation for Root and Tuber Crops Research. (R. Baning, I.S, Adu-Donyinah, Ed.). Kumasi, Ghana.: CSIR-Crops Research Institute. Dziedzoav, N. T., Abass, A. B., Amoa-Awua, W. K. A. and Sablah, M. (2006). Quality Management Manual for Production of High Quality Cassava Flour. Dziedzoave, N. T., Ellis, W. O., Oldham, J. H. and Osei-Yaw, A. (1999). Subjective and Objective assessment of agbelima (Cassava dough) quality. Food Control, 10(2), 63–67. Dziedzoave, N. T., Graffham, A. J. and Boateng, E. (2003). Training manual for the production of cassava-based bakery products. CSIR-FRI, Accra, Ghana. Eggum, R. O. (1970). “The protein quality of cassava leaves.” Br. J. Nutr., 24, 761–768. Retrieved from sciencedirect.com. FAO. (2000). A review of cassava in Africawith country case studies on Nigeria, Ghana,the United Republic of Tanzania, Uganda and Benin. Retrieved December 22, 2014, from http://www.fao.org/docrep/009/ a0154e/A0154E01.htm. FAO. (2002). Protein sources for the animal industry. FAO Expert Consultaion and Workshop. http://doi.org/Expert Consultation and Workshop Bangkok, 29 April - 3 May 2002. FAO. (2002). The State of Food and Agriculture. Rome. FAO. (2013). Cassava Farmer Field Schools: Resource material for facilitators in sub- Saharan Africa. FAO. (2015). Food Outlook, Biannual report on global food markets. Hahn, S. K. (2006). An overview of traditional processing and utilization of cassava in Africa. Howeler, R. H. (2001). Cassava mineral nutrition and fertilization. Cassava: Biology, Production, and Utilization, 115–147. http://doi.org/10.1079/97 80851995243.0115. IFAD. (2007). Improving marketing strategies in Central and West Africa. Rural Poverty Portal,. James, B., Okechukwu, R., Abass, A., Fannah, S., Sanni, L., Fomba, S. and Lukombo, S. (2012). Producing Gari from Cassava: An illustrated guide for smallholder cassava processors. International Institute of Tropical Agriculture (IITA): Ibadan, Nigeria. John, R. P., Nampoothiri, K. M. and Pandey, A. (2006). Solid-state fermentation for l-lactic acid production from agro wastes using Lactobacillus delbrueckii. Process Biochemistry, 41(4), 759–763. http://doi.org/10.1016/ j.procbio.2005.09.013. Jones, W. O. (1959). Manioc in Africa. (F. R. Institute, Ed.). Stanford, USA: Stanford University. Kleih, U., Phillips, D. and Wordey, M. T. (2013). Cassava Market and Value Chain Analysis Ghana Case Study Final Report (Anonymised version) February 2013, (January). Retrieved from http://www.value-chains.org/dyn/bds/docs/859/GhanaCassavaMarket Study-FinalFebruary 2013_anonym.pdf. Kongkiattikajorn, J. (2012). Ethanol Production from Dilute-Acid Pretreated Cassava Peel by Fed-Batch Simultaneous Saccharification and Fermentation. International Journal of the Computer, the Internet and Management, 20(2), 22–27.

Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 331

Kongkiattikajorn, J. and Sornvoraweat, B. (2011). Comparative Study of Bioethanol Production from Cassava Peels by Monoculture and Co-Culture of Yeast. Kasetsart J. (Nat. Sci.), 274, 268–274. Koopmans, A. and Koppejan, J. (1997). Agricultural and forest residues generation, utilisation and availability. Retrieved February 12, 2013, from wgbis.ces.iisc.ernet.in/energy/ HC270799/RWEDP/acrobat/p_residues.pdf. Koppram, R. and Olsson, L. (2014). Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings. Biotechnology for Biofuels, 7(1), 54. http://doi.org/10.1186/1754-6834-7-54. Korang-Amoakoh, S., Cudjoe, R. A. and Adams, E. (1987). Biological control of cassava pests in Ghana. Prospects for the integration of other strategies. Lambri, M., Fumi, M. D., Roda, A. and De Faveri, D. M. (2013). Improved processing methods to reduce the total cyanide content of cassava roots from Burundi. African Journal of Biotechnology, 12(19), 2685–2691. http://doi.org/10.5897/AJB2012.2989. Meridian Institute. (2009). Science and Innovation for African Agricultural Value Chains: Lessons learned in transfer of technologies to smallholder farmers in Sub-Saharan Africa. New Growth International. Mielenz, J. R. (2001). Ethanol production from biomass: technology and commercialization status. Current Opinion in Microbiology, 4(3), 324–329. http://doi.org/10.1016/S1369- 5274(00)00211-3. Mlingi, N. L. V., Poulter, N. H. and Rosling, H. (1992). An outbreak of acute intoxi-cation from insufficiently processed cassava in Tanzania. Nutr. Res., 12, 677–687. Mohammed, Y. S., Mokhtar, A. S., Bashir, N. and Saidur, R. (2013). An overview of agricultural biomass for decentralized rural energy in Ghana. Renewable and Sustainable Energy Reviews, 20, 15–25. http://doi.org/10.1016/j.rser.2012.11.047. Nwokoro, O. (2014). Production of L-lactic acid from Cassava peel wastes using single and mixed cultures of Rhizopus oligosporus and Lactobacillus plantarum. Chemical Industry and Chemical Engineering Quarterly, 20(4), 457–461. http://doi.org/10.2298/CICEQ 130325027N. OECD/IEA. (2010). Sustainable production of second-generation biofuels, potential and perspectives in major economies and developing countries, Information paper. Retrieved February 12, 2010, from www.iea.org/ papers/2010/second_generation_biofuels.pdf. Oh, H., Wee, Y.-J., Yun, J.-S., Ho Han, S., Jung, S. and Ryu, H.-W. (2005). Lactic acid production from agricultural resources as cheap raw materials. Bioresource Technology, 96(13), 1492–8. http://doi.org/10.1016/ j.biortech.2004.11.020. Olanbiwoninu, A. A. and Odunfa, S. A. (2015). Production of Fermentable Sugars from Organosolv Pretreated Cassava Peels. Advances in Microbiology, (February), 117–122. Quaye, W., Gayin, J., Yawson, I. and Plahar, W. (2009). Characteristics of various cassava processing methods and the adoption requirements in Ghana. Journal of Root Crops, 35(1), 59–68. Retrieved from http://www.researchgate.net/publication/228308346_ Characteristics_of_Various_Cassava_Processing_Methods_and_the_Adoption_Requirem ents_in_Ghana/file/9fcfd50fd8dea34263.pdf. Ravidran, V., Kornegay, E. T., Rajaguru, A. S. B. and Notter, D. (1987). “Cassava leaf meal as a replacement for coconut oil meal in pig diets.” Journal of the Science of Food and Agriculture, 41, 45–53. Retrieved from sciencedirect.com. Complimentary Contributor Copy 332 Richard Bayitse, Ferdinand Tornyie and Anne-Belinda Bjerre

Ravindran, V. and Rajaguru, A. S. B. (1988). “Effect of stem pruning on cassava root yield and leaf growth.” Sri Lankan Journal of Agricultural Science, 24(2), 32–37. Retrieved from sciencedirect.com. Ravindran, V. (n.d.). Preparation of cassava leaf products and their uses as animal feeds. Retrieved February 11, 2013, from Fao.org/ag/aga/agap/ frg/ahpp95/95111.pdf>. Ray, R. C., Sharma, P. and Panda, S. H. (2009). Lactic acid production from cassava fibrous residue using Lactobacillus plantarum MTCC 1407. Journal of Environmental Biology, 30(5 SUPPL.), 847–852. Richter, K. and Berthold, C. (1998). Biotechnological Conversion of Sugar and Starchy Crops into Lactic Acid. Journal of Agricultural Engineering Research, 71(2), 181–191. http://doi.org/10.1006/jaer.1998.0314. Saddler, J. N & Gregg, D. (1998). Ethanol production from forest products wastes. In J.. Bruce, A and Palfreyman (Ed.), Forest products biotechnology (pp. 183– 207). London: Taylor & Francis Ltd. Sam, J. and Deppah, H. (2009). West African Agricultural Productivity Programme – Ghana Baseline Survey Report. Selormey, G., Amoah, J. Y. and Aggey, M. (2006). Diversification of cassava(Manihotesculenta Crantz)utilization in Northern Ghana – A case study: Integrated cassava processing pilot plant in Salaga-Kpembe.” In Proceedings of the 3rd National Conference on Agriculture Engineering. Kumasi, Ghana.: Kwame Nkrumah University of Science and Technology. Sornyotha, S., Kyu, K.L. and Ratanakhanokchai, K. (2010). An efficient treatment for detoxification process of cassava starch by plant cell wall-degrading enzymes. J. Biosc. Bioeng., 109, 9–14. SRID-MoFA. (2012). Agriculture in Ghana (Facts and figures 2012). Ministry of Food and Agriculture. Retrieved from https://www.itu.int/ITU-D/ict/facts/2011/material/ICTFacts Figures2011.pdf. SRID-MoFA. (2014). Agriculture in Ghana (Facts and Figures). Ministry of Food and Agriculture. Retrieved from http://facts/mofa.gov.gh/site/wp-content/uploads/.../mofa_ facts_and_figures.pdf. Sundaresan, S., Nambisan, B. and Easwari Amma, C. S. (1987). Bitterness in cassava in relation to cyano-glucoside content. Indian J. Agric. Sci., 57, 34–40. Tweyongyere, R. and Katongole, I. (2002). Cyanogenic potential of cassava peels and their detoxification for utilization as livestock feed. Vet. Hum. Toxicol., 44, 366–369. Tylleskar, T., Banea, M., Bikangi, N., Cooke, R. D., Poulter, N. H. and Rosling, H. (1992). Cassava cyanogens and konzo, an upper motoneuron disease found in Africa. Lancet, 339, 208–211. USDA NRCS. (n.d.). Plant Guide. Louisiana and Pacific Islands. Retrieved from . van Zyl, C., Prior, B. A., Kilian, S. G. and Kock, J. L. (1989). D-xylose utilization by Saccharomyces cerevisiae. Journal of General Microbiology, 135(11), 2791–8. Retrieved from http://www.ncbi.nlm.nih. gov/pubmed/2515242. WAAPP/PPAAO. (2013). Mechanical cassava harvester. Retrieved July 28, 2016, from www.waapp.org.gh. Westby, A. and Choo, B. (1994). Cyanogen reduction during the lactic fermentation of cassava. Acta Horticulturae, 375, 209–215. Complimentary Contributor Copy Cassava Cultivation, Processing and Potential Uses in Ghana 333

Westby, A. (2002). Cassava utilization, storage and small-scale processing. In A. C. Hillocks, R. J., Thresh, J. M. and Bellotti (Ed.), Cassava biology, production and utilization (pp. 281–300). Wallingford, UK: CABI Publishing. Woiciechowski, A. L., Soccol, C. R., Ramos, L. P. and Pandey, A. (1999). Experimental design to enhance the production of l-(+)-lactic acid from steam-exploded wood hydrolysate using Rhizopus oryzae in a mixed-acid fermentation. Process Biochemistry, 34(9), 949–955. http://doi.org/ 10.1016/S0032-9592(99)00012-6.

Chapter 17

POTENTIAL USES OF CASSAVA BAGASSE FOR BIOENERGY GENERATION BY PYROLYSIS AND COPYROLYSIS WITH A LIGNOCELLULOSIC WASTE

Luciano I. Gurevich Messina1,3, Pablo R. Bonelli1,3 and Ana L. Cukierman1,2,3,* 1Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Depto. de Industrias, Programa de Investigación y Desarrollo de Fuentes Alternativas de Materias Primas y Energía (PINMATE), Ciudad Universitaria. Buenos Aires, Argentina 2 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Depto. de Tecnología Farmacéutica, Cátedra de Tecnología Farmacéutica II. Buenos Aires, Argentina 3Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina

ABSTRACT

Cassava (Manihot esculenta) bagasse is a fibrous by-product generated in the tuber processing. After washing and peeling, the cassava is grated and then water is added in order to extract the starch. The mixture is filtered such that a rich starch solution and a wet solid residue can be separated. This slurry, known as bagasse, comprises up to 20% of the weight of the processed cassava. In addition, as the extraction of starch from cassava is less efficient than those based on processing of potato or maize, the bagasse contains around 50-70% of starch on a dry basis. As it has no important use, with the exception of animal feed, the bagasse is usually rejected to water courses increasing the environmental pollution. Therefore, several strategies are being studied to find useful applications for this by-product. Pyrolysis of the bagasse and copyrolysis, namely the thermal degradation of mixtures of the bagasse and lignocellulosic biomass in inert atmosphere, could be an appealing possibility to employ this waste in order to generate

* E-mail: [email protected]; [email protected]. Complimentary Contributor Copy 336 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman

green energy and/or other value-added products. In particular, growing attention is paid to the liquid products arising from pyrolysis/copyrolysis, commonly known as bio-oils, since they show many of the advantages of liquid fuels, such as inexpensive storage and transportation, and high energy density. In this scenario, the processes of pyrolysis of cassava starch, the major constituent of dry cassava bagasse, and of copyrolyisis of the starch with peanut hulls, an abundant lignocellulosic residue, were studied by performing experiments in a fixed-bed reactor at different process temperatures (400ºC – 600ºC). The pyrolysis of the starch led to a higher maximum yield of bio-oils that took place at a lower temperature than the copyrolysis (57 wt% at 400ºC vs. 49 wt% at 500ºC). Physichochemical characterization of the three kinds of pyrolysis/copyrolysis products with emphasis on the bio-oils was carried out mainly by proximate and ultimate analyses, Karl-Fischer titration, Fourier-transformed infrared spectroscopy, N2 adsorption, scanning electronic microscopy, and gas chromatography (GC-TCD and GC-MS). While the pyrolysis of the starch resulted in bio-oils with less nitrogen content, the copyrolysis produced bio-oils with lower content of oxygen and higher carbon percent. Water content of the bio-oils increased with rising process temperatures and it was lower for the liquids resulting from the pyrolysis of the starch. Also, the bio-oils arising from the pyrolysis of the starch presented more sugar compounds and fewer phenols. Besides, the pyrolysis of the starch led to a lower yield of solid products (bio-chars) than the copyrolysis. They showed greater high heating values (up to 35 MJ/kg) than those arising from the latter process in agreement with their larger carbon content and lower presence of ash. In addition, the bio-chars produced at the highest process temperature presented an incipient pore development, suggesting their possible use as rough adsorbents or as intermediary for further upgrading to activated carbons. Furthermore, the pyrolysis of cassava starch and copyrolysis with peanut hulls generated gases, principally CO2, CO, CH4 and H2, that could help to sustain the processes.

Keywords: cassava bagasse, cassava starch, pyrolysis, copyrolysis, bioenergy, bio-oils

1. INTRODUCTION

Growing demand of energy worldwide along with depleting reserves of fossil fuels has led to the search of renewable alternative sources. Biomass as an energy source has several advantages because its use is essentially carbon neutral and it provides a convenient way of storing energy compared to other renewable energies. Energy generation from biomass or bioenergy has great potentialities for sustainable supply of electricity, domestic heat, fuels for transport, and process heat for industrial facilities, with beneficial impact on the environment, particularly due to reduction in greenhouse gas emissions related to fossil fuels employment (Cherubini and Strømann, 2011). Biomass is considered as the most important source of energy (Frau et al., 2015). Contribution of bioenergy to the global energy matrix is currently around 10% (~50 EJ) and estimations forecast a technical potential higher than 1500 EJ in 2050 (Ullah et al., 2015). Among possible biomass sources, agricultural and agro-industrial residues have received increasing attention in the search of alternatives to the relatively more conventional forestry and/or wood wastes. The former ones encompass agricultural wastes such as straw, stem, stalk, leaves, husk, shell, peel, lint, seed/stones, pulp, stubble, arising from cereals, like rice, wheat, maize or corn, sorghum, barley, millet, cotton, groundnut, jute, legumes, coffee, cacao, olive, tea, fruits and palm oil. Likewise, agro-industrial residues are derived from the

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 337 processing of a particular crop or animal product. This category includes materials like molasses, bagasse, oilseed cakes, maize milling by-products and brewer’s wastes, among others (Menon and Rao, 2012; Long et al., 2013). In particular, cassava (Manihot esculenta) is a root crop whose industrial processing generates large amounts of residue. It is classified as the fifth most abundant starch crop produced in the world and the third most important food source for inhabitants of tropical regions (Debiagi et al., 2015). Copious solid and liquid wastes emerge from processing of cassava tubers for the large-scale production of starch. The bagasse, namely the fibrous by- product of the root containing part of the starch that was not previously extracted and fiber, is an important residue. After washing and peeling, the cassava is grated and then water is added in order to extract the starch. The mixture is filtered such that a rich starch solution and a wet solid residue can be separated. This slurry, known as bagasse, comprises up to 20% of the weight of the processed cassava tuber. In addition, as the extraction of starch from cassava is less efficient than those based on processing of potato or maize, the bagasse contains around 50-70% of starch on a dry basis. As it has no important use, with the exception of animal feed, cassava bagasse is usually rejected to water courses without any treatment leading to serious environmental pollution in areas where starch industries are located (Jyothi et al., 2005). Several strategies are being studied to find useful applications for cassava bagasse, such as the production of lactic acid by bacteria, production of ethanol, and development of biodegradable packaging (Pandey et al., 2011; Debiagi et al., 2015; Zhang et al., 2016). Despite cassava bagasse can be considered as a rich solar energy reservoir due to cassava’s easy regeneration capacity, in comparison to other agricultural residues, its conversion has been mostly investigated through the biochemical route, while thermochemical processes have been scarcely explored. Bio-energy generation via thermochemical conversion has advantages in comparison to the biochemical route, involving higher reaction rates and superior capacity to destroy organic matter (Zhang et al., 2010). Another advantage in the same direction is the relatively low ash content characterizing cassava bagasse (Pandey et al., 2000). Main thermochemical conversion processes for generation of bioenergy or energy carriers from biomass include combustion, pyrolysis, gasification, and liquefaction. In particular, pyrolysis is currently considered as the most promising thermochemical process to produce energy from biomass (Tripathi et al., 2016). Pyrolysis consists in the thermal degradation of biomass in an oxygen-depleted atmosphere, leading to products often lumped into three groups: permanent gases, pyrolytic liquids, and a carbon enriched solid product (char or bio-char) (Cukierman et al., 2012). The liquid products, known as bio-oils, are of great interest because of their potential as bio-fuels, presenting, in general, the advantages of other liquid fuels, such as a low transport cost, high energetic density, and the feasibility of being employed in combined cycle gas turbine to generate electricity (Fan et al., 2011). However, the high water content of the bio-oils and the large amount of oxygenated compounds restrict their utilization and cause difficulties for their direct combustion (Jacobson et al., 2013). On the other hand, co-pyrolysis, namely thermal degradation of mixtures of two or more wastes in inert atmosphere, has been increasingly investigated in the last years in order to ensure availability of biomass sources and to take advantage of possible synergic effects. A few studies additionally report that co-pyrolysis of biomass with some biopolymers might represent an option to reduce water content of bio-oils and to increase their yield (Cornelissen Complimentary Contributor Copy 338 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman et al., 2009; Abnisa and Wan Daud, 2014). Accordingly, thermal degradation of mixtures of cassava bagasse and lignocellulosic biomass could be an appealing possibility to employ these wastes for the sustainable generation of green energy and/or other value-added products. In this scenario, the present chapter examines the process of pyrolysis of cassava starch, as the major compound of dry cassava bagasse, and of mixtures of equal proportions of the starch with peanut hulls, as an abundant, representative lignocellulosic residue generated in the processing of this crop, which, as cassava, is also widely grown in tropic and subtropic regions. It focuses on yields and physicochemical properties of the three kinds of products obtained from assays for the pyrolysis of the starch, the peanut hulls, and of their mixtures (50 wt%) performed in a fixed-bed reactor for different process temperatures. Special emphasis is placed on possible synergetic effects which could lead to improve yield and/or properties of the bio-oils.

2. EXPERIMENTAL SECTION

2.1. Materials

Cassava (Manihot esculenta) starch, labeled as CS, was provided by Bernesa S.A.C.I., Argentina. The cassava starch powder was processed by wet granulation, and then milled and screen-sieved. Fractions of particle diameter between 250 µm and 500 µm were reserved for the pyrolysis assays. In order to explore the copyrolysis of the starch with a lignocellulosic biomass, hulls from commercial peanut (Arachis hypogaea), abbreviated as PH, were employed. They were cleaned, milled, and screen-sieved. The same particle size as for the starch was employed for the experiments. A mixture in equal proportions of the cassava starch and the lignocellulosic biomass, designated as CS/PH, was prepared by physical mixing and utilized in the copyrolysis assays. Main properties of cassava starch and peanut hulls are displayed in Table 1. Proximate analysis of the samples was performed by thermogravimetric analysis (TA Instruments SDT Q600), according to American Society of Testing and Materials (ASTM) standards 5142. An automatic elemental analyzer (Carlo Erba model EA 1108) was used to determine elemental composition of the samples. Besides, amylose content of the starch was analyzed by standardized iodine colorimetry, according to ISO 6647-2:2007. The absorbance of the starch-iodine mixture was measured at 620 nm. Amylopectin content was determined by difference. To assess the content of main biopolymers constituting the hulls, the Van Soest analysis was executed.

2.2. Bench-Scale Pyrolysis Experiments

A fixed bed reactor was utilized to carry out the pyrolysis/copyrolysis assays. The equipment mainly consisted of an AISI 316 stainless steel fixed bed reactor (2.5 cm I.D., 110 cm total length) with a special device which enabled to support a basket built in stainless steel mesh. The latter was used as a container of the samples. The reactor was externally heated by

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 339 an electrical furnace driven by a Yokogawa UT350 temperature controller. The basket with the sample, constituting the solid fixed-bed, was centrally placed in the heated bottom zone of the reactor. A chromel-alumel thermocouple was located at the geometrical center of the basket to record the process temperature. At the reactor outlet, a series of flasks immersed in a cooling bath, using isopropyl alcohol at -10°C as solvent, enabled condensation and collection of the condensable volatiles generated with the thermal degradation course. Non- condensable vapors, after passing through the condensation system, were sampled periodically using Teflon gas bags for further analysis by gas chromatography, as detailed in the next subsection.

Table 1. Chemical characteristics of the cassava starch (CS) and the peanut hulls (PH)

Characteristic / Sample CS PH Proximate Analysis (wt%)a Volatile matter 92.5 73.6 Ash 0.2 20.5 Fixed carbond 7.3 5.9 Ultimate Analysis (wt%)b Carbon 44.4 49.6 Hydrogen 6.3 6.5 Nitrogen 0.1 1.8 Oxygend 49.2 42.1 Biopolymer composition (wt%) Amyloseb 22.9 Amylopectinb,d 77.1 Ligninc 30.9 Cellulosec 54.6 Hemicellulosec 14.5 a Dry basis. b Dry and ash-free basis. c Neutral detergent fiber and ash-free basis. d Estimated by difference.

To avoid partial combustion of the samples, all the installation was purged by flowing N2 (300 cm3 min-1) for 1 h. Afterwards, the heating system was connected and the desired temperature was set. Once the pre-established temperature was attained, the basket containing the sample was displaced to the heated zone of the reactor. After the holding time, heating was cut-off and the basket was immediately shifted towards the upper (non-heated) part of the reactor, keeping the N2 stream. Once at ambient temperature, the basket was removed from the reactor. The residual solid and the accumulated liquid products contained in the flasks were weighed to determine product yields. These products were then carefully stored in closed containers for further characterization. Percent yields were calculated as weight of product per total weight of raw sample. Gas yields were obtained by difference from overall mass balances. From preliminary experiments, it was found that the process temperature was the variable that had the greatest influence on the products yields. Also, it was determined that after 30 min the volatiles generation was negligible, indicating almost complete conversion. Other conditions, such as the particle size or the N2 flow rate, had a weak influence on the process

Complimentary Contributor Copy 340 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman yields. Therefore, the following pre-established operating conditions were selected to conduct 3 -1 the pyrolysis/copyrolysis assays: temperature = 400-600°C, N2 flow rate = 300 cm min , particle diameter = 250-500 µm; samples’ masses = 10-15 g, holding time = 30 min.

2.3. Characterization of the Pyrolysis Products

Dichloromethane was used to extract the organic phase from the bio-oils (volume ratio solvent/bio-oil: 2:1). Elemental composition of the organic phase of the bio-oils was determined by ultimate analyses, as depicted above. Furthermore, their higher heating value (HHV) was also measured using a Parr 1341 oxygen bomb calorimeter. The samples’ pH was determined with an Orion 290A portable pH meter. Water content of the liquid samples was measured by volumetric Karl-Fischer titration (Methrom Herisau Karl Fisher Automat E 547) following ASTM E 203. Additionally, total phenol and sugar contents of the bio-oil were evaluated using the Folin-Ciocalteu and the phenol-sulphuric assays, respectively. Fourier transform infrared spectroscopy (FT-IR) analysis of the organic fractions of the bio-oils was carried out using a Perkin-Elmer IR Spectrum BXII spectrometer within the range 600-4000 cm-1 and an attenuated total reflection (ATR) device made of SeZn. Also, chemical compounds of the bio-oils were identified using a Trace GC Ultra chromatograph coupled with a Thermo Scientific EM/DSQ II mass spectrometer (GC-MS). The employed capillary column was a Rxi-5ms (length: 30 m; ID: 0.25 mm) and helium was used as carrier gas. Electron ionization (potential: 70 eV) was applied and the measured mass range varied from 30 to 500 m/z. Regarding the bio-chars, elemental analysis was performed, employing the aforementioned instrument and Fe3O4 as a combustion catalyst. FT-IR assays using the KBr pellet method were carried out using the aforementioned instrument. N2 adsorption- desorption isotherms at -196°C were determined for the bio-chars with an automatic Micromeritics ASAP-2020 HV volumetric sorption analyzer. Before carrying out the measurements, the samples were outgassed at 120 ºC for two hours. Textural properties were assessed from the isotherms, according to conventional procedures depicted in detail in previous studies (Basso et al., 2005; Bonelli et al., 2007). Moreover, the bio-chars were examined by scanning electronic microscopy (SEM) in a Zeiss Supra 40 microscope equipped with a field emission gun. The samples were placed on an aluminium holder, supported on conductive carbon tape and sputter coated with Au-Pd. Non-condensable gases, after flowing through the condensation system, were sampled periodically using Teflon gas bags, and further analyzed with a Shimadzu GC-8 gas chromatograph supplied with a thermal conductivity detector and a concentric packed Altech CTR I column (6 ft x ¼ in). Argon as carrier gas and a column temperature of 25°C were employed. All the experiments were performed at least by triplicate and average values are informed. Differences between replicates were less than 5% in all cases.

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 341

3. RESULTS AND DISCUSSION

3.1. Yields of the Pyrolysis/Copyrolysis Products

Yields of the three kinds of pyrolysis products for the pyrolysis of CS at different temperatures are displayed in Figure 1. Bio-char yields show a decreasing trend with increasing temperatures attributable to a preponderance of volatilization reactions. The similar yields obtained at 500ºC and 600ºC suggest that volatilization is almost complete at 500ºC. This is in agreement with TGA studies reported for this biopolymer (Marques et al., 2006; Sin et al., 2011). Unlike pyrolysis of lignocellulosic biomass (Akhtar and Amin, 2012; Bridgwater, 2012; Kim et al., 2014), which shows the maximum bio-oil yield at approximately 500ºC, pyrolysis of CS leads to maximum bio-oil yields at lower temperatures (400ºC). This would be due to the fact that volatilization of this biopolymer is less important than the cracking of the pyrolysis vapours at higher temperatures, resulting in a higher gases production and in a decrease in bio-oil yields.

60 55 50 45 Bio-char 40 Bio-oil 35 30 Gases

Yields [wt%] Yields 25 20 15 10 5 350400450500550600650

Temperature [ºC]

Figure 1. Effect of the process temperature on products yields for the pyrolysis of the cassava starch.

Figure 2 shows the yields for the three pyrolysis products obtained from the copyrolysis of CS/PH. It can be seen that the maximum bio-oil yield is achieved at a relatively higher temperature (500ºC) compared to that for the pyrolysis of CS. Volatilization of the solid is less complete for the mixture than for the pure starch, so the increase of temperature would promote vapor generation. At 600ºC, cracking reactions should be favored and, thus, gas yield is noticeably increased (Neves et al., 2011). Nevertheless, bio-oil yields are always lower than those obtained from the pyrolysis of CS. On the other hand, more bio-char is generated from the copyrolysis. This would be due to the more refractory nature of the hulls, owing to the presence of lignin which is thermally resistant to decompose (Bonelli et al., 2007; Yang et al., 2007; Collard and Blin, 2014).

Complimentary Contributor Copy 342 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman

40 Bio-char

35 Bio-oil 30 Gases

Yields Yields [wt%] 25

15 350400450500550600650

Temperature [ºC]

Figure 2. Effect of the process temperature on products yields for the copyrolysis of the mixture of cassava starch and peanut hulls.

3.2. Properties of the Pyrolysis/Copyrolysis Products

3.2.1. Bio-Oils Elemental composition, HHV and pH of the bio-oils resulting from the pyrolysis of CS and the copyrolysis of CS/PH are displayed in Table 2. Bio-oils arising from the copyrolysis of the mixture have lower oxygen content than those generated by the pyrolysis of the starch. This could be probably related to the minor oxygen content of the hulls (Table 1). The presence of oxygenated compounds in bio-oils is undesirable since they augment their instability. They also contribute to diminish their miscibility with conventional fuels, such as fuel-oils, because of their high polarity (Lehto et al., 2014). In addition, the higher oxygen content of the bio-oils resulting from the pyrolysis and their lower carbon content result in a lower HHV for these liquids, with the exception of the bio-oils produced at 600ºC. This bio- oil presents more hydrogen and less nitrogen than the one generated by the copyrolysis of the mixture at 600ºC, which could lead to enhance its HHV. Regarding the nitrogen content, the bio-oils generated by the pyrolysis of the cassava starch showed a lower value, probably due to the low nitrogen content of the raw CS. Bio-oils with little content of nitrogen are more suitable as their further combustion would generate less NOx which is related to negative environmental impacts, such as acid rain (Cao et al., 2010). pH values for the bio-oils are in the typical range reported in the literature (Chiaramonti et al., 2007; Bridgewater, 2012). In particular, bio-oils have a great amount of formic and acetic acid (Oudenhoven et al., 2013). Besides, it has been reported that bio-oil acidity is mainly caused by decomposition of the different polysaccharides (cellulose, hemicellulose, amylose and amylopectin) (Collard and Blin, 2014). As the CS/PH mixture has lignin, which does not contribute significantly to generate carboxylic acids, this may be reflected in the higher pH value of the bio-oil arising from the mixture. Also, the pyrolysis of polysaccharides with a minor degree of polymerization would result in more carboxylic acids since the rupture of the glucose ring seems to be favored (Patwardhan et al., 2009). Amylose and amylopectin

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 343 have a lower degree of polymerization than the one of cellulose and, consequently, thermal decomposition of the former biopolymers would produce more acids.

Table 2. Elemental composition, higher heating value, and pH of the different bio-oils generated from the pyrolysis of cassava starch (CS) and the copyrolysis of the starch and the peanut hulls (CS/PH), at different temperatures

CS CS (500ºC) CS CS/PH CS/PH CS/PH Bio-oil (400ºC) (600ºC) (400ºC) (500ºC) (600ºC) Ultimate Analysis [wt%]a C 50.4 49.5 51.7 54.1 53.5 53.2 H 7.0 7.0 7.5 7.4 7.8 6.2 N 0.4 0.0 0.2 0.7 1.7 2.9 Ob 42.4 43.5 40.7 37.8 37.0 37.7 HHV [MJ/kg]a 22.4 21.0 23.5 23.8 24.1 21.9 pH 2.7 2.7 2.8 3.0 2.9 2.8 a Organic phase. b Estimated by difference.

Figure 3 displays the influence of the process temperature on the contents of water, sugars and phenols of the bio-oils. Bio-oils produced by the pyrolysis of CS result in less water content than those originated from the CS/PH mixture. This mixture is richer in ash which could catalyze ring scission reactions that also generate water (Fahmi et al., 2008; Mourant et al., 2011). It is also seen that the increase in process temperature leads to raise water content of the bio-oils derived from both the pyrolysis of the individual starch and the mixture. In accordance with other authors (Akhtar and Amin, 2012; Lin et al., 2013), this suggests that high temperatures promote dehydration reactions. The presence of water in the bio-oils contributes to reduce their energy density and also to decrease the values of adiabatic flame temperature and combustion rate. On the other hand, water lowers bio-oil viscosity (Chiaramonti et al., 2007; Lehto et al., 2014). The pyrolysis of CS generates more sugars than the copyrolysis of the CS/PH mixture. Since CS is almost completely constituted by amylopectin and amylose which are formed by glucose rings, the pyrolysis of cassava starch would produce diverse anhydrosugars, such as levoglucosan. Levoglucosan in bio-oil could be used for the synthesis of chiral polymers and to obtain fermentable carbohydrates in order to produce bio-ethanol (Bennett et al., 2009; Yang et al., 2013). Instead, the thermal degradation of hemicellulose and lignin that compose the hulls might generate other compounds such as furans and phenols (Lin et al., 2013; Collard and Blin, 2014). Furthermore, the minerals present in the hulls might act as catalyst of reactions that break down the glucose ring. Considering the process temperature effect, the sugar content of the bio-oils generated by the pyrolysis of individual cassava starch drops noticeably at the highest temperature investigated. This is probably due to ring scission reactions that are favored at high temperatures. The behavior, however, is not so clear in the case of the copyrolysis. Bio-oils generated from the copyrolysis present a greater amount of phenolic compounds because of the hulls’ lignin. Temperature has no noticeable effect on phenol content of the bio-oils. The phenolic compounds could be applied to produce phenol formaldehyde resins, glues, and intermediaries in the synthesis of different pharmaceutical products (Fele Žilnik and Jazbinšek, 2012).

Complimentary Contributor Copy 344 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman

65 60 55 CS 50 CS/PH

45 Water [wt%]. 40 35 350400450500550600650 Temperature [ºC] (a)

] 20 3 CS 15 CS/PH 10

Sugars [g/dm 5

0 350400450500550600650 Temperature [ºC] (b)

25 ]

3 20 CS 15 CS/PH 10

Phenols [g/dm Phenols 5

0 350 400 450 500 550 600 650 Temperature [ºC] (c)

Figure 3. Contents of water (a), sugars (b) and phenols (c) in the bio-oils generated from the pyrolysis of the cassava starch (CS) and the copyrolysis of the starch and the peanut hulls (CS/PH), at different temperatures. Sugars and phenols were determined in the aqueous phase.

Figure 4 presents the FT-IR spectra of the bio-oils produced from the pyrolysis of the cassava starch and from the copyrolysis of the starch and the peanut hulls at different process temperatures. All the samples exhibit similar spectra characterized by a strong absorption band at 3400 cm-1, corresponding to O-H stretching, absorption peaks at 3000 to 2800 cm-1, attributable to C-H stretching of methyl and methylene groups, and an overlap of weak and strong bands at the region from 1750 to 1000 cm-1 (Ben Hassen-Trabelsi et al., 2014). The absorption peak at 1750 cm-1, corresponding to carboxyl and carbonyl groups, is mainly due to the presence of carboxylic acids, ketones and aldehydes. Also, it can be seen that the Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 345 absorption bands at 1595 cm-1 and at 1500 cm-1, that are assigned to the presence of aromatic rings, are more pronounced for the bio-oil arising from the mixture. This is expectable since lignin decomposition generates phenolic compounds. These peaks are noticeable in the bio-oil arising from the cassava starch pyrolysis as they contain furans which are also products of the thermal decomposition of polysaccharides. Furthermore, bands at 1080 to 1000 cm-1 corresponding to aromatic and aliphatic ethers are due to the presence of lignin products, such as syringol or guaiacol, and of sugars arising from the decomposition of starch and holocellulose.

CS (600ºC)

CS (500ºC) Transmitance[%]

CS (400ºC)

3600 3100 2600 2100 1600 1100 600 -1 Wavenumber [cm ] (a)

CS/PH (600ºC)

CS/PH (500ºC) Transmitance[%]

CS/PH (400ºC)

3600 3100 2600 2100 1600 1100 600 -1 Wavenumber [cm ] (b)

Figure 4. FT-IR spectra of the bio-oils produced from the pyrolysis of the cassava starch (a) and from the copyrolysis of the starch and the peanut hulls (b), at different temperatures.

In Figure 5 there are represented the total ion chromatograms of the bio-oils produced from the pyrolysis of CS and the copyrolysis of CS/PH mixture at a process temperature of 500ºC. The main compounds, identified by means of retention times (RT) and data of mass spectra found in the literature (Ohra-aho and Linnekoski, 2015; NIST Mass Spec Data Center, 2016) are listed in Table 3.

Complimentary Contributor Copy 346 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman

CS/PH

5 10 15 20 25 30 35 40 45 Retention time [min]

Figure 5. Total ion chromatograms (TIC) for the bio-oils generated from the pyrolysis of cassava starch (CS) and the copyrolysis of the starch and peanut hulls (CS/PH). Process temperature = 500ºC.

Table 3. Main compounds detected in bio-oils by GC/MS analysis

RT [min] Compound 6.7 phenol 8.1 2-hydroxy-3-methyl-2-cyclopenten-1-one 8.8 p-cresol 9.7 2-methoxyphenol (guaiacol) 10.9 5-hydroxymethylfurfural 13.5 2-methoxy-4-methylphenol (creosol) 15.0 catechol 15.3 1,4-anhydro--D-pyranose 15.7 levoglucosenone 17.2 1,4:3,6-dianhydropyranose 18.6 2,6-dimethoxyphenol (syringol) 20.5 vanillin 21.6 2-methoxy-4-propenylphenol (eugenol) 24.4 2-methoxy 4-propylphenol 35.0 2,6-dimethoxy 4-propylphenol 35.6 levoglucosan 40.1 1,6--D-glucofuranose

As can be inferred from the TIC corresponding to the bio-oil arising from the pyrolysis of the mixture, the main products of the pyrolysis of holocellulose would be 2-hydroxy-3- methyl-2-cyclopenten-1-one, 5-hydroxymethylfurfural, levoglucosan, and 1,6--D- glucofuranose. Regarding the lignin pyrolysis products, the most important peaks would correspond to compounds derived from coniferyl alcohol, such as guaiacol and eugenol, and from synapyl alcohol, such as syringol (Menon and Rao, 2012). The TIC for the bio-oil generated by the pyrolysis of CS is quite different since the peaks assignated to phenolic compounds, arising from lignin degradation, are not observable. In addition, besides the aforementioned compounds likely derived from holocellulose, other compounds are also detected: 1,4-anhydro--D-pyranose, levoglucosenone and 1,4:3,6- dianhydropyranose. The two latter evolve from levoglucosan, thus suggesting that secondary

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 347 reactions which partially transform this anhydrosugar have taken place. Although 1,4- anhydro--D-pyranose is usually derived from hemicellulose, it has been detected among the pyrolysis products of corn starch (Patwardhan et al., 2009).

3.2.2. Bio-Chars Ash content, elemental composition, and high heating value of the bio-chars arising from the pyrolysis of the cassava starch and its copyrolysis with peanut hulls are displayed in Table 4. It can be seen that the ash content of the bio-chars derived from the copyrolysis is higher than those derived from the pyrolysis of the starch. This is in agreement with the lower ash content of the raw cassava starch (Table 1). On the other hand, the mixture which contains hulls possessing a higher ash content results in bio-chars with greater ash content. The process temperature has no noticeable effects on the ash content of the bio-chars derived from CS. By contrast, a slight increase in the ash content of the bio-chars arising from CS/PH mixtures with rising temperatures is found (Neves et al., 2011).

Table 4. Ash content, elemental composition, and higher heating value of the different bio-chars generated from the pyrolysis of cassava starch (CS) and the copyrolysis of the starch with the peanut hulls (CS/PH), at different temperatures

CS CS CS CS/PH CS/PH CS/PH Bio-char (400ºC) (500ºC) (600ºC) (400ºC) (500ºC) (600ºC) Ash [wt %, dry 0.8 1.0 0.9 6.0 8.8 9.6 basis] Ultimate analysis [wt %, dry and ash-free basis] C 80.0 94.4 93.5 78.6 85.7 87.8 H 3.3 2.1 2.0 3.2 3.0 2.2 N 0.5 0.8 0.9 0.5 2.4 1.9 Oa 16.2 2.7 3.6 17.7 8.9 8.1 HHV [MJ/kg] 30.1 35.1 34.5 29.3 32.3 32.3 a Estimated by difference.

Regarding the elemental composition, pyrolysis of the cassava starch resulted in bio- chars with higher carbon content and lower oxygen content than those derived from the mixture. This could be due to the more refractory nature of the lignin present in the hulls, reducing the release of oxygenated volatile compounds. These differences in elemental composition lead to a higher value of the HHV of the bio-chars generated by the cassava starch pyrolysis. Nitrogen content of these bio-chars is also lower as the raw starch presents less elemental nitrogen than the hulls. Furthermore, at lower process temperatures, more oxygen is present in the bio-chars since the thermal degradation process is far from completion (Aysu y Küçuk, 2014). Nitrogen adsorption isotherms of the bio-chars generated from the pyrolysis of the cassava starch and the copyrolysis of the mixtures at different process temperatures are shown in Figure 6. Furthermore, the textural properties of these bio-chars are detailed in Table 5. As may be observed in Figure 6, the shape of the isotherms corresponding to all the samples present typical characteristics of isotherms type I according to IUPAC classification, indicating that all the solids mostly contain micropores (Rouquerol et al., 1999). The bio- chars generated at 400ºC and the one arising from cassava starch pyrolysis at 500ºC, do not Complimentary Contributor Copy 348 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman show pore development. However, the other three samples present a slight pore development, probably due to a partial gasification of the solids with steam or CO2 released in the starch pyrolysis (Raavendran and Ganesh, 1998). The derived bio-chars could be used as rough adsorbents, soil enhancer and CO2 capture agent in soil, or they could be activated to generate activated carbons by further steam or CO2 gasification (Cukierman y Bonelli, 2015).

CS 400ºC 500ºC 600ºC

0.45 30 0.4

/g] 25 3 0.35 0.3 20 0.25 15 0.2 0.15 10 0.1 5 Adsorbed volume [cm 0.05 0 0 00.10.20.30.40.50.60.70.80.91

Relative pressure (P/P0) (a) CS/PH 400ºC 500ºC 600ºC

7 /g]

3 6 5 4 3 2 1 Adsorbed volume [cm 0 00.10.20.30.40.50.60.70.80.91

Relative pressure (P/P0) (b)

Figure 6. Nitrogen adsorption isotherms (-196ºC) for the bio-chars generated from: (a) the pyrolysis of the cassava starch and, (b) the copyrolysis of the starch and the peanut hulls, at different temperatures.

Table 5. Textural properties of the differtent bio-chars produced from the pyrolysis of cassava starch (CS) and from the copyrolysis of the starch and the peanut hulls (PH) at different temperatures

CS CS (500ºC) CS CS/PH CS/PH CS/PH Bio-char (400ºC) (600ºC) (400ºC) (500ºC) (600ºC) 2 SBET [m /g] 0.3 1.5 81 2.8 17 16 3 Vt [cm /g] 0.001 0.002 0.04 0.003 0.008 0.01 Rm [nm] 2.5 2.0 1.0 2.1 0.9 1.2

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 349

Figure 7 exhibits the FT-IR spectra of the bio-chars derived from the pyrolysis of cassava starch and the copyrolysis of the mixture at the different process temperatures. Unlike the bio- oils (Figure 4), it may be seen that the spectra of the bio-chars present few peaks indicating scarce presence of functional groups (Yin et al., 2013). All the spectra show absorption peaks -1 -1 at 1600 cm (C=C stretching) and at 1370 cm (CH3 deformation). Most of the bands assigned to oxygenated groups vary in intensity with the temperature. The O-H bond stretching absorption band, at 3350 cm-1, is only noticeable for the bio-oils generated at a process temperature of 400ºC. At higher process temperatures, the peak becomes weak. The band at 1730 cm-1, corresponding to C=O bond stretching of carbonyl and carboxyl groups, also vanishes with increasing temperatures. Interestingly, this peak is more pronounced for the bio-chars derived from pyrolysis of CS than for those arising from the copyrolysis of the CS/PH mixture. However, the intensity of the peak assigned to ether bonds, at approximately 1110 cm-1, does not change remarkably with the temperature, suggesting that these bonds are quite resistant to thermal decomposition.

CS (600ºC)

CS (500ºC) Transmitance [%] Transmitance

CS (400ºC)

3600 3100 2600 2100 1600 1100 600 Wavenumber [cm-1] (a)

CS/PH (600ºC)

CS/PH (500ºC) Transmitance[%]

CS/PH (400ºC) 3600 3100 2600 2100 1600 1100 600 Wavenumber [cm-1] (b)

Figure 7. FT-IR spectra of the bio-chars produced from: (a) the pyrolysis of the cassava starch and, (b) the copyrolysis of the starch and the peanut hulls, at different temperatures. Complimentary Contributor Copy 350 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman

SEM micrographs of both the bio-char generated by the pyrolysis of CS and the one derived from the copyrolysis of CS/PH at a process temperature of 500ºC are displayed in Figure 8. As seen in Figure 8.a., the bio-char shows large sheets with circular marks. These ones could be probably caused by the sudden decomposition of the spherical particles that formed the cassava starch. On the other hand, pyrolysis of the mixture resulted in a bio-char (Figure 8.b) which shares characteristics of the aforementioned bio-char and those of the solid product resulting from the pyrolysis of that kind of lignocellulosic biomasses (Bonelli et al., 2001). It looks like the sheets generated by the starch pyrolysis coated the partial fibrous structure of the bio-chars derived from the hulls.

Figure 8. SEM micrographs (2000x) of the bio-chars produced from: (a) the pyrolysis of the cassava starch and, (b) the copyrolysis of the starch and the peanut hulls at 500ºC.

3.2.3. Gases Main gases generated by the pyrolysis of cassava starch and its copyrolysis with peanut hulls were CO, CO2, CH4, and H2. The presence of C2H4 and C2H6 has not been detected in this work with the instrument employed, even though they have been reported by some other authors in the literature (Horne and Williams, 1996; Couhert et al., 2009). Moles of each gas produced at a certain time are calculated according to: t   ii dtQ C G (1) 0 being Gi, the total generated moles of the generic gaseous species i, Ci, the molar concentration of the gaseous species i, and Q, the total volumetric flow of gas. In Figure 9 there are represented the generated moles for each gas over the reaction time for the pyrolysis of cassava starch and its copyrolysis with the peanut hulls, at 500ºC, respectively. Generation of CO and CO2 is mainly attributed to the thermal decomposition of the polysaccharides. The main source of the former would be carbonyl groups, while the latter may be associated with decarboxylation reactions. Thus, CO should be mainly generated by the cellulose pyrolysis, while CO2 may be principally derived from degradation of hemicellulose (Yang et al., 2007). The occurrence of decarboxylation reactions is linked to the polymerization degree and the cristallinity of the solids employed (Collard and Blin,

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 351

2014). As starch has a polymerization degree similar to hemicellulose and its cristallinity is lower than that of cellulose, these characteristics could explain the major CO generation. On the other hand, CH4 and H2 are mainly generated by lignin pyrolysis. Therefore, the copyrolysis of CS/PH produces slightly more of these two gases.

CS 7

5 CH 4 H 4 2 CO 2 3 CO

molgasesof /kg sample 1

0 0 5 10 15 20 25 30 t [min]

CS/PH 5

4 CH 4 3 H 2 CO 2 2 CO

molgasesof /kg sample 1

0 0 5 10 15 20 25 30 t [min]

Figure 9. Production of main gases arising from the pyrolysis of the cassava starch (CS) and from the copyrolysis of the starch and the peanut hulls at 500ºC.

In addition, the HHV of the gas stream is calculated taking into account the total moles produced of each species per unit of sample mass and the heat of combustion of each species, according to the following equation:

 0.802G CH4[MJ/kg] 0.286G PCS H2  0.283GCO (2)

The calculated HHV values are 0.9 MJ/kg for the gases generated by the pyrolysis of CS, and 1.5 MJ/kg for the gases generated by the copyrolysis of CS/PH. The combustion of these

Complimentary Contributor Copy 352 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman gases could supply part of the heat necessary for the development of the processes (Bridgwater, 2012).

CONCLUSION

The feasibility of the thermochemical conversion of cassava bagasse was investigated, exploring the pyrolysis process of its main constituent (cassava starch) and copyrolysis of the starch with peanut hulls, in equal proportions, at temperatures from 400ºC to 600ºC. Yields and characteristics of the three kinds of pyrolysis products (bio-oils, bio-chars and gases) were determined. Temperature had a considerable effect on the products yields of both the pyrolysis of cassava starch and the copyrolysis with peanut hulls. In both cases the rise of temperature led to an increase of gases generation and to a reduction of the bio-char produced. The maximum bio-oil generation took place at different temperatures for the starch and the mixture: while the yield for the pyrolysis of cassava starch occurred at 400ºC, the maximum yield of bio-oil for the copyrolysis happened at 500ºC. Furthermore, different maximum liquid yields were achieved, being 57 wt% for the former, and 49 wt% for the latter. Over the whole range of temperatures investigated, the pyrolysis of cassava starch resulted in a greater bio-oil generation and yielded less bio-char than the copyrolysis. Copyrolysis of the starch and the hulls led to bio-oils with a higher carbon content and a lower oxygen content, which would improve their stability and miscibility with conventional fuels. Also, in most cases, the employment of the mixture contributed to raise the high heating value of the bio-oils attaining values up to ~24 MJ/kg. In turn, pyrolysis of the starch yielded bio-oils with less nitrogen content, a favourable characteristic in terms of reducing NOx emissions in case of further combustion. Regarding water generation, the pyrolyisis of starch redounded in bio-oil quality as they presented less water. The increase of process temperature augmented water concentration in the bio-oils. Despite exhibiting almost the same functional groups, the bio-oils arising from the pyrolysis of cassava starch were different to the ones originated from the copyrolysis. Other derivatives from the constituent polysaccharides and no phenolic compounds were detected. The bio-chars generated from the pyrolysis of the starch presented less ash content than those arising from the copyrolysis, becoming more suitable for combustion in steam boilers as less fouling would occur. Carbon content of the bio-chars produced by the pyrolysis of the starch was higher, leading to greater high heating value (up to 35 MJ/kg). These bio-chars also showed less nitrogen content. In agreement with the observed FT-IR spectra, oxygen content of the bio-chars decreased with increasing temperature. The bio-chars arising from the highest process temperature presented an incipient pore development. Accordingly, they could be employed as rough adsorbents or be further upgraded to activated carbons. The pyrolysis of cassava starch and copyrolysis with peanut hulls generated gaseous compounds, principally CO2, CO, CH4 and H2. The presence of the hulls in the mixture allowed a major generation of the three latter, leading to increase the high heating value of the gaseous stream. The gases could aid to the energy sustainability of the pyrolysis/copyrolysis process. Overall, present results contribute to the understanding of the thermochemical

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 353 conversion of residues and mixtures, such as cassava bagasse and peanut hulls, into green energy vectors.

ACKNOWLEDGMENT

The authors gratefully acknowledge Agencia Nacional de Promoción Científica y Tecnológica – Fondo para la Investigación Científica y Tecnológica (ANPCYT-FONCYT), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and Universidad de Buenos Aires (UBA) from Argentina, for financial support.

REFERENCES

Abnisa, F., Wan Daud, W.M.A. 2014. A review on co-pyrolysis of biomass: An optional technique to obtain a high-grade pyrolysis oil. Energy Conversion and Management 87, 71–85. Akhtar, J., Saidina Amin, N. 2012. A review on operating parameters for optimum liquid oil yield in biomass pyrolysis. Renewable and Sustainable Energy Reviews 16, 5101–5109. Aysu, T., Küçük, M.M. 2014. Biomass pyrolysis in a fixed-bed reactor: Effects of pyrolysis parameters on product yields and characterization of products. Energy 64, 1002–1025. Basso, M.C., Cerrella, E.G., Buonomo, E.L., Bonelli, P.R., Cukierman, A.L. 2005. Thermochemical conversion of Arundo donax into useful solid products. Energy Sources, 27, 1429–1438. Ben Hassen-Trabelsi, A., Kraiem, T., Naoui, S., Belayouni, H. 2014. Pyrolysis of waste animal fats in a fixed-bed reactor: Production and characterization of bio-oil and bio- char. Waste Management 34, 210–218. Bennett, N.M., Helle, S.S., Duff, S.J.B. 2009. Extraction and hydrolysis of levoglucosan from pyrolysis oil. Bioresource Technology 100, 6059–6063. Bonelli, P.R., Della Rocca, P.A., Cerrella, E.G., Cukierman, A.L. 2001. Effect of pyrolysis temperature on composition, surface properties and thermal degradation rates of Brazil Nut shells. Bioresource Technology 76, 15–22. Bonelli, P.R., Buonomo, E.L., Cukierman, A.L. 2007. Pyrolysis of sugarcane bagasse and copyrolysis with an Argentinean subbituminous coal. Energy Sources. Part A: Recovery, Utilization, and Environmental Effects, 29, 731–740. Bridgwater, A.V. 2012. Review of fast pyrolysis of biomass and product upgrading. Biomass and Bioenergy 38, 68–94. Cao, J.-P., Zhao, X.-Y., Morishita, K., Wei, X.-Y., Takarada, T. 2010. Fractionation and identification of organic nitrogen species from bio-oil produced by fast pyrolysis of sewage sludge. Bioresource Technology 101, 7648–7652. Chiaramonti, D., Oasmaa, A., Solantausta, Y. 2007. Power generation using fast pyrolysis liquids from biomass. Renewable and Sustainable Energy Reviews 11, 1056–1086. Cherubini, F., Strømann, A.H. 2011. Principles of Biorefining. In “Biofuels: Alternative Feedstocks & Conversion Processes” Editors: A. Pandey, C. Larroche, S.C. Ricke, C.-G.

Complimentary Contributor Copy 354 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman

Dussap, E. Gnansounou., Academic Press, Amsterdam, The Netherlands, Chapter 1, 3– 24. Collard, F.-X., Blin, J. 2014. A review on pyrolysis of biomass constituents: Mechanisms and composition of the products obtained from the conversion of cellulose, hemicelluloses and lignin. Renewable and Sustainable Energy Reviews 38, 594–608. Cornelissen, T., Jans, M., Stals, M., Kuppens, T., Thewys, T., Janssens, G.K., Pastijn, H., Yperman, J., Reggers, G., Schreurs, S., Carleer, R. 2009. Flash co-pyrolysis of biomass: The influence of biopolymers. Journal of Analytical and Applied Pyrolysis 85, 87–97. Couhert, C., Commandre, J.-M., Salvador, S. 2009. Is it possible to predict gas yields of any biomass after rapid pyrolysis at high temperature from its composition in cellulose, hemicellulose and lignin? Fuel 88, 408–417. Cukierman, A.L., Nunell, G.V., Fernández, M.E., De Celis, J., Kim, M.R., Gurevich Messina, L., Bonelli P.R. 2012. Thermochemical processing of wood from invasive arboreal species for sustainable bioenergy generation and activated carbons production. In “Invasive Species: Threats, Ecological Impact and Control Methods.” Editors J. J. Blanco and A. Fernandes. Nova Science Publishers Inc., N.Y., USA. Chapter 1, 1–46. Cukierman, A.L., Bonelli, P.R. 2015. Potentialities of biochars from different biomasses for climate change abatement by carbon capture and soil amelioration. In “Advances in Environmental Research” Editor J.A. Daniels. Nova Science Publishers Inc., N.Y., USA. Volume 44, 57–79. Debiagi, F., Marim, B.M., Mali, S. 2015. Properties of cassava bagasse and polyvinyl alcohol biodegradable foams. Journal of Polymers and the Environment 23, 269–276. Fahmi, R., Bridgwater, A.V., Donnison, I., Yates, N., Jones, J.M. 2008. The effect of lignin and inorganic species in biomass on pyrolysis oil yields, quality and stability. Fuel 87, 1230–1240. Fan, J., Kalnes, T.N., Alward, M., Klinger, J., Sadehvandi, A., Shonnard, D.R. 2011. Life cycle assessment of electricity generation using fast pyrolysis bio-oil. Renewable Energy 36, 632–641. Fele Žilnik, L., Jazbinšek, A. 2012. Recovery of renewable phenolic fraction from pyrolysis oil. Separation and Purification Technology 86, 157–170. Frau, C., Ferrara, F., Orsini, A., Pettinau, A. 2015. Characterization of several kinds of coal and biomass for pyrolysis and gasification. Fuel 152, 138–145. Horne, P. A., Williams, P.T. 1996. Influence of temperature on the products from the flash pyrolysis of biomass. Fuel 75, 1051–1059. Jacobson, K., Maheria, K.C., Kumar Dalai, A. 2013. Bio-oil valorization: A review. Renewable and Sustainable Energy Reviews 23, 91–106. Jyothi, A. N., Sasikiran, K., Nambisan, B., Balagopalan, C. 2005. Optimisation of glutamic acid production from cassava starch factory residues using Brevibacterium divaricatum. Process Biochemistry 40, 3576–3579. Kim, T.-S., Oh, S., Kim, J.-Y., Choi, I.-G., Choi, J.W. 2014. Study on the hydrodeoxygenative upgrading of crude bio-oil produced from woody biomass by fast pyrolysis. Energy 68, 437–443. Lehto, J., Oasmaa, A., Solantausta, Y., Kytö, M., Chiaramonti, D. 2014. Review of fuel oil quality and combustion of fast pyrolysis bio-oils from lignocellulosic biomass. Applied Energy 116, 178–190.

Complimentary Contributor Copy Potential Uses of Cassava Bagasse for Bioenergy Generation … 355

Lin, T., Goos, E., Riedel, U. 2013. A sectional approach for biomass: Modelling the pyrolysis of cellulose. Fuel Processing Technology 115, 246–253. Long, H., Li, X., Wang, H., Jia, J., 2013. Biomass resources and their bioenergy potential estimation: A review. Renewable and Sustainable Energy Reviews 26, 344–352. Marques, P.T., Lima, A.M.F., Bianco, G., Laurindo, J.B., Borsali, R., Le Meins, J.-F., Soldi, V. 2006. Thermal properties and stability of cassava starch films cross-linked with tetraethylene glycol diacrylate. Polymer Degradation and Stability 91, 726–732. Menon, V., Rao, M. 2012. Trends in bioconversion of lignocellulose: Biofuels, platform chemicals & biorefinery concept. Progress in Energy and Combustion Science 38, 522– 550. Mourant, D., Wang, Z., He, M., Wang, X.S., Garcia-Perez, M., Ling, K., Li, C.-Z. 2011. Mallee wood fast pyrolysis: Effects of alkali and alkaline earth metallic species on the yield and composition of bio-oil. Fuel 90, 2915–2922. Neves, D., Thunman, H., Matos, A., Tarelho, L., Gómez-Barea, A. 2011. Characterization and prediction of biomass pyrolysis products. Progress in Energy and Combustion Science 37, 611–630. NIST Chemistry WebBook. 2016. http://webbook.nist.gov/chemistry. Ohra-aho, T., Linnekoski, J. 2015. Catalytic pyrolysis of lignin by using analytical pyrolysis- GC–MS. Journal of Analytical and Applied Pyrolysis 113, 186–192. Oudenhoven, S.R.G., Westerhof, R.J.M., Aldenkamp, N., Brilman, D.W.F., Kersten, S.R. A. 2013. Demineralization of wood using wood-derived acid: Towards a selective pyrolysis process for fuel and chemicals production. Journal of Analytical and Applied Pyrolysis 103, 112–118. Pandey, A., Soccol, C.R., Nigam, P., Soccol, V.T., Vandenberghe, L.P., Mohan, R. 2000. Biotechnological potential of agro-industrial residues. II: cassava bagasse. Bioresource Technology 74, 81–87. Pandey, A., Larroche, C., Ricke, S.C., Dussap, C.-G., Gnansounou., E. 2011. Biofuels: Alternative Feedstocks & Conversion Processes. Academic Press, Amsterdam, The Netherlands. Patwardhan, P.R. Satrio, J.A., Brown, R.C., Shanks, B.H. 2009. Product distribution from fast pyrolysis of glucose-based carbohydrates. Journal of Analytical and Applied Pyrolysis 86, 323–330. Raveendran, K., Ganesh, A. 1998. Adsorption characteristics and pore-developement of biomass- pyrolysis char. Fuel 77, 769–781. Rouquerol, F., Rouquerol, J., Sing, K. 1999. Adsorption by powders and porous solids. Principles, methodology and applications. Academic Press Ed., Londres. Sin, L.T., Rahman, W. A.W.A., Rahmat, A. R., Mokhtar, M. 2011. Determination of thermal stability and activation energy of polyvinyl alcohol–cassava starch blends. Carbohydrate Polymers 83, 303–305. Tripathi, M., Sahu, J.N., Ganesan, P. 2016. Effect of process parameters on production of biochar from biomass waste through pyrolysis: A review. Renewable and Sustainable Energy Reviews 55, 467–481. Ullah, K., Kumar Sharma, V., Dhingra, S., Braccio, G., Ahmad, M., Sofia, S. 2015. Assessing the lignocellulosic biomass resources potential in developing countries: A critical review. Renewable and Sustainable Energy Reviews 51, 682–698.

Complimentary Contributor Copy 356 Luciano I. Gurevich Messina, Pablo R. Bonelli and Ana L. Cukierman

Yang, H., Yan, R., Chen, H., Lee, D.H., Zheng, C. 2007. Characteristics of hemicellulose, cellulose and lignin pyrolysis. Fuel 86, 1781–1788. Yang, Z., Liu, X., Yang, Z., Zhuang, G., Bai, Z., Zhang, H., Guo, Y. 2013. Preparation and formation mechanism of levoglucosan from starch using a tubular furnace pyrolysis reactor. Journal of Analytical and Applied Pyrolysis 102, 83–88. Yin, R., Liu, R., Mei, Y., Fei, W., Sun, X. 2013. Characterization of bio-oil and bio-char obtained from sweet sorghum bagasse fast pyrolysis with fractional condensers. Fuel 112, 96–104. Zhang, L., Xu, C., Champagne, P. 2010. Overview of recent advances in thermo-chemical conversion of biomass. Energy Conversion and Management 51, 969–982. Zhang, M., Xie, L., Yin, Z., Khanal, S.K., Zhou, Q. 2016. Biorefinery approach for cassava- based industrial wastes: Current status and opportunities. Bioresource Technology 215, 50–62.

Chapter 18

TREND IN THE TRADE OF CASSAVA PRODUCTS IN THE COASTAL, EASTERN AND WESTERN REGIONS OF KENYA

ABSTRACT

The potential to increase cassava products utilization is enormous if the available recipe range can be increased. A marketing survey was conducted in Mombasa, Nairobi and Busia urban centres. In Mombasa and Nairobi, marketing of cassava products was done daily. In Busia, daily marketing accounted for 22% while 78% was through a local market that opens twice a week. In Mombasa, 100% of cassava products were mainly sold at the main market (Kongowea). In Nairobi, 94% of respondents sold their products in local markets (Gikomba and Kibera) and 6% to hotels. In Busia, 50% sold their products at the main market and 50% in secondary markets. Sale of cassava products in Mombasa, Busia and Nairobi dates as early as 1956, 1962 and 1987, respectively. In Mombasa, cassava crisps and fried fresh cassava constituted 8% and fresh roots 92%. In Nairobi, boiled cassava constituted 6%, flour 25% and dried chips 69% of products being traded in. In Busia cassava flour constituted 33% and dried chips (for milling) 67% of the products sold. In Mombasa, the average price of a fresh root was 13 shillings during scarcity and 8 shillings during abundance. In Nairobi, a 2-kg tin (gorogoro) was sold at 69 and 55 shillings during scarcity and abundance, respectively. In Busia, the average price of a gorogoro was 35 and 31 shillings during scarcity and abundance, respectively. In Mombasa, the majority of those marketing cassava products were males while in Nairobi and Busia females dominated. The main products sold in Mombasa were crisps, fried chips, and fresh roots. In Nairobi, the main products were boiled cassava, flour and dry chips. In Busia, flour and dried chips were the main products. In Mombasa the major customers were final consumers, retailers and processors. In Nairobi major customers

* Email: [email protected], Cellphone: +254 726959592. Complimentary Contributor Copy 358 C. M. Githunguri, M. Gatheru and S. M. Ragwa

were final consumers, wholesalers, retailers and millers. In Busia customers were final consumers, wholesalers, retailers and processors. In Mombasa and Busia the principal suppliers of cassava products were both male and female while in Nairobi it was women. One of the main supply constraint reported was lack of cassava during scarcity. Competition from maize was cited in Mombasa and Nairobi. Costly transport was reported in Mombasa and Busia. In Mombasa, lack of credit was also cited. In Busia, other important constraints recorded were lack of sorghum and finger millet for blending cassava, and unfavourable weather for drying of cassava chips.

INTRODUCTION

Cassava is a major food security crop across sub-Saharan Africa. In Kenya cassava is grown in over 61,000 ha with an annual production of about 738,000 tons (MoA, 2009; GoK, 2010). Kenya produced approximately 800,000 metric tons of cassava valued at 3.8 billion K. Shs in 2006 and has the potential to produce more than 2 million metric tonnes per year (MoA, 2011). Cassava is produced mainly in western, Coast and eastern Kenya while production in the other regions is relatively low. Cassava is a highly drought tolerant crop and as such there is high potential for its increased production in the arid and semi-arid lands, which comprise about 80% of Kenyan land mass. Though cassava is considered a food security crop in the sub-Saharan Africa, its production in Kenya is low compared to other crops like maize, beans, and sorghum. Its consumption is low especially in the central region of Kenya where it is considered a poor man’s crop and is usually consumed during periods of food scarcity. Despite its high production in the coastal and western regions of Kenya, utilization is limited to human consumption. Utilization in Kenya is limited to roasting and boiling of fresh roots for consumption in most growing areas. However, in Nyanza and Western provinces of Kenya, roots are also peeled, chopped into small pieces (cassava chips), dried and milled into flour for ugali. In order to promote its production, which has been decreasing in recent years, there, is need to explore and identify other uses of cassava (MoA, 2011). It is estimated that Africa produces about 42% of the total tropical world production of the crop (FAO, 1990). Cassava can grow in marginal lands, requires low inputs, and is tolerant to pests and drought (Githunguri et al., 1998; Nweke et al., 2002; Githunguri et al. 2006a). Despite its great potential as a food security and income generation crop among rural poor in marginal lands, its utilization remains low in Kenya. In addition, it can be safely left in the ground for a period of 7 to 24 months after planting and then harvested as needed. Cassava is the second most important food root crop after Irish potato in Kenya. However due to its narrow production base it is ranked number 36 out of 50 in KARI’s 1991 priority setting exercise (KARI, 1995). Available statistics on cassava production in the country show a slow but steady increase in production. Cassava production in the country is concentrated in three main regions; Coastal, Central and Western region. Western and Coastal regions are the main cassava producing areas, producing over 80% of the recorded cassava output in the country (MoA, 1999). The importance of cassava as a food and cash crop in central Kenya is however increasing. The potential to increase cassava products utilization is enormous if the available recipe range can be increased (Githunguri, 1995). It has also been demonstrated that it is very important to present cassava to urban consumers in attractive forms at affordable prices,

Complimentary Contributor Copy Trend in the Trade of Cassava Products … 359 which are competitive to those of cereals (Nweke et al., 2002; International Fertilizer Development Center (IFDC), 2012). The International Institute of Tropical Agriculture (IITA) has officially recognized cassava as a new cash commodity, which will help raise foreign exchange and be a vital food source throughout Africa. The Amsterdam-based Common Fund for Commodities has also recognized cassava as an internationally tradable commodity (Nweke, 2002). The Intergovernmental Group on Grains has adopted cassava as a commodity (Githunguri et al., 2006b; Nweke, 2002). Globally, the traditional use of cassava is changing from primarily human consumption to processing industrialized products (MoA, 2011). In Asia, cassava is a diversified fully commercial crop. Here, its roots are converted into an array of products - human food from the roots, and starch, flour, ethanol and animal feed for industry. In Latin America and the Caribbean, traditional processing and markets have now been dominated by industrial processing. In Brazil for example, starch and ethanol production from cassava is on the increase. The Far East, especially China is emerging as a major world market for starch and pellets. Europe, Latin America and Asia have seen the most cassava consumption increase using animal feed for their industries. Africa however continues to lag behind with 90% of its cassava still being consumed as human food (MoA, 2011). Marketing is still a major challenge for the cassava sub-sector especially for the dried chips. Cassava marketing in the country is undeveloped and like in most food crops not efficiently organized. Cassava producers sell fresh roots at farm gate or at the nearby markets. Buyers are mainly middlemen, local traders or neighbours who do not have cassava on their farms. The middlemen and local traders in turn sell the fresh roots in local markets directly to consumers or other retailers. Formal price/market information on cassava does not exist unlike other major food crops whose prices are provided through the print and electronic media. Cassava growers and traders get information on cassava prices through inquiry and previous market conditions (Makokha and Tunje, 2000). The demand for processed cassava products in Kenya has not been well documented. Some import and export statistics of cassava starch however point to a possibility of a potential cassava starch market in the country (Wambugu and Mungai, 2000). Import export trade for cassava products in Kenya is only documented for cassava starch. The level of trade is however, very small and there is scope for expansion. Exports of cassava starch from Kenya have been mainly to Tanzania, Uganda, Portugal, and South Africa. Unconfirmed reports also indicated informal trade of cassava between Kenya and Uganda along the common border. There are no organized marketing associations either by farmers or traders. Marketing is mainly done by producers as individuals in nearby markets or sold to middlemen who then transport cassava for sale in local or district market centres. According to Githunguri et al. (2008a, 2008b, 2009a and 2009b), the quality characteristics mostly preferred for cassava products are white colour, fibre-free, good taste, high dry matter content, medium size, good texture and low moisture content. Processing of cassava fresh roots would help to increase shelf-life, reduce transportation problems and costs, and remove cyanogens. It also improves palatability, adds value and extends market especially to medium income urban consumers (Nweke et al., 2002). Whether cassava can be relied upon as a low cost staple food In urban centres and a source of steady real income for rural households will to a larger extent depend on how well it can be processed into safe forms and on how far it can be presented to urban consumers in an attractive form at prices which are competitive to those of cereals. In some large cassava producing countries like Nigeria, the market for some processed Complimentary Contributor Copy 360 C. M. Githunguri, M. Gatheru and S. M. Ragwa products is highly limited to low income groups, while other forms of cassava, e.g., gari have a significant market value for middle and high income consumers. How far the market for cassava may be expended would therefore depend largely on the degree to which the quality of the various processed products can be improved to make them attractive to potential consumers without significant increase in processing costs. Cassava processing is therefore an important factor in marketing because an introduction of improved post-harvest handling facilities could lead to a substantial increase in proportion of cassava marketed (Nweke et al., 2002). Improved processing hygiene and packaging could improve their shelf life and make them attractive and acceptable in a wider market. Cassava products processing and utilization is done mainly at the subsistence level (Kadere, 2002). At the coastal region, it is men who roast and sell cassava crisps. In both Eastern and Western Kenya, women dominate home- based processing while service processing like milling is male dominated. As processing becomes mechanized men tend to play a leading role. The few home-based processors sell their products directly to consumers or retailers. Tapioca Ltd. in Mazeras is the only factory that employs modern technology to produce cassava flour, starch and glue. Most cassava processing technologies are labour-based facing serious limitations in areas with labour shortages (Mbwika, 2002). Rudimental processing technologies like over reliance on sun-dried methods are rendered impossible during the rainy season. Peeling of cassava roots manually using a knife is time consuming, laborious, difficult to ensure quality control and wasteful. The fine particles of cassava flour render current milling technologies wasteful. There is need to identify appropriate storage and processing technologies that are cheap, have low losses, improve shelf life and guarantees quality products. Efforts should be made to involve the food processing industry in making ready to eat cassava products available in supermarkets and retail outlets. Due to the enormous potential demand for cassava by the feeds, pharmaceutical, food, paper printing and brewing industries there is need to involve them in the research and development of this sub-sector. The Kenya Bureau of Standards needs to develop cassava products standards so that several industrial concerns can accept them as important inputs. Cassava utilization can be improved by availing cassava marketing information in audio and print media. Marketing is still a major challenge for the cassava sub-sector especially for the dried chips. Cassava marketing in the country is undeveloped and like in most food crops not efficiently organized. Cassava producers sell fresh roots at farm gate or at the nearby markets. Buyers are mainly middlemen, local traders or neighbours who do not have cassava on their farms. The middlemen and local traders in turn sell the fresh roots in local markets directly to consumers or other retailers. Import export trade for cassava products in Kenya is only documented for cassava starch. The level of trade is however, very small and there is scope for expansion. Exports of cassava starch from Kenya have been mainly to Tanzania, Uganda, Portugal, and South Africa. Unconfirmed reports also indicated informal trade of cassava between Kenya and Uganda along the common border. There are no organized marketing associations either by farmers or traders. Marketing is mainly done by producers as individuals in nearby markets or sold to middlemen who then transport for sell to local or district market centers. There is need to carry out a comprehensive marketing study on cassava and support its marketing in Kenya if cassava is to play its rightful role in the food security and industrialization (KARI, 1995).

Complimentary Contributor Copy Trend in the Trade of Cassava Products … 361

Markets refer to types of places where people shop e.g., (store, supermarket) and not a location where people shop and contrary to popular belief marketing begins from production all the way to the final consumer. Policy objectives of many governments are increasingly geared towards commercialization and thus developed markets are important for commercialization justifying the case for processing. This is due to the simple reason that unprocessed output or limited utilization option results in underutilization after harvest and thus processing will increase usable physical volume and economic value of existing production (Scott 1995). Identifying markets for improved or new processed products important critical in efforts to increase incomes, generate employment and in reducing post- harvest losses. Although a wide variety of product market options maybe available, identification of the few with the greatest commercial promise requires a systemic appraisal which this study was designed to do for the cassava sub-sector in Kenya. Thus the broad goal and objective of this study was to evaluate the status and the market potential for cassava and its processed products while the specific objectives were:

i. to determine supply and demand situation of cassava and cassava products in cassava grown regions of Kenya; ii. to characterize cassava producers (farmers) and determine marketing constraints and opportunities in the cassava sub-sector; iii. to determine the status of cassava processing and evaluate market potential of cassava processed products in Kenya; iv. to determine the status of the cassava market and marketability of cassava products in Kenya; v. to assess the comparative commercial potential of alternative product markets; and vi. to estimate the most important processed products sold by volume and value.

STUDY METHODOLOGY

The study was conducted in the coastal (Mombasa), eastern (Nairobi) and western (Busia) regions of Kenya where only the major markets were visited and cassava traders interviewed randomly. The classical theory of statistical inference based on the properties of samples and sampling distributions was used (Greene, 2000). In this kind of study it is not possible to predetermine the sample size. Randomly selected cassava traders were interviewed using a structured questionnaire. Data collected included information on traders’ characteristics, business activities, storage of cassava and cassava-based products, demand and supply characteristics. The data collected were analyzed using the Statistical Package for Social Sciences (SPSS).

RESULTS AND DISCUSSION

Market Characteristics

Market characteristics in the three study areas are shown in Tables 1 and 2. In Mombasa and Nairobi, marketing of cassava and cassava-based products was done on a daily basis Complimentary Contributor Copy 362 C. M. Githunguri, M. Gatheru and S. M. Ragwa

(Table 1). In Busia, daily marketing accounted for 22% while 78% of marketing was done through a local market that opens twice a week. Figures 1 and 2 shows the retailing of fresh cassava tuberous roots at Kongowea Market, Mombasa.

Figure 1. Farmers vend fresh cassava tubers at Kongowea Market, Mombasa.

Figure 2. A trader being interviewed by a market analysts about his business at Kongowea, Mombasa.

Table 1. Type of market by region

Market type Region Daily Other % respondents Mombasa 100 0 Nairobi 100 0 Busia 22.2 77.8

Complimentary Contributor Copy Trend in the Trade of Cassava Products … 363

Table 2. Market category for cassava and cassava based products

Market category Region Main market Secondary market Hotel % respondents Mombasa 100 0 0 Nairobi 93.8 0 6.2 Busia 50 50 0

In Mombasa, 100% of respondents reported that cassava and cassava-based products were sold at the main market (Kongowea). In Nairobi, 94% of respondents sold their products in local markets (Gikomba and Kibera) while about 6% sold their products to hotels. In Busia, 50% of respondents sold their products at the main market while 50% sold in secondary markets.

Figure 3. Processed cassava crisps at Mama Ngina Drive, Mombasa.

Figure 4. Cassava chips (fermented and sun-dried) ready to be mixed with other cereals for milling into flour.

Complimentary Contributor Copy 364 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Table 3. Characteristics of traders of cassava and cassava-based products

Region Characteristic Mombasa Nairobi Busia Mean Standard Mean Standard Mean Standard deviation deviation deviation Age of trader (yr) 42 17 43 13 44 13 Trading experience (yr) 19 13 7 6 12 15 Percent of respondents Gender of trader Male 58 19 22 Female 42 81 78 Origin of trader Native 75 25 67 Migrant 25 75 33 Education level None 25 25 44 Primary 50 25 33 Secondary 25 50 23 Post-secondary 0 0 0 Own a store Yes 8 73 33 No 92 27 67 Business category Wholesaler 8 19 0 Whole sale & retail 17 25 22 Retail 75 56 78 Own the business Yes 100 88 100 No 0 12 0 Sell other products Yes 25 100 78 No 75 0 22 Other products sold Grains (cereals etc) 0 87 85 Other root crops 33 0 0 Vegetables 0 0 15 Animal products 0 0 0 Others 67 13 0

CHARACTERISTICS OF CASSAVA TRADERS

Characteristics of traders of cassava and cassava-based products are shown in Table 3. Figure 3 shows sale of cassava crisps at Mama Ngina Drive, Mombasa. On the other hand Figure 4 shows cassava chips (fermented and sun-dried) ready to be mixed with other cereals for milling into flour in Busia in western Kenya. In Mombasa, cassava wholesalers constitute Complimentary Contributor Copy Trend in the Trade of Cassava Products … 365

8%; wholesalers/retailers 17% and retailers 75%. In Nairobi, 19% of respondents were wholesalers, 25% were wholesalers/retailers while 56% were retailers. In Busia 22% of respondents were both wholesalers and retailers while 78% were retailers. In Mombasa, the majority (58%) of those marketing cassava and cassava-based products were males while in Nairobi and Busia the business was dominated by females (Figure 5). The origin of cassava traders was as follows; in Mombasa (75%) were natives and 25% were migrants while in Busia (67%) were natives and 33% migrants. In Nairobi 75% of the traders were migrants. The average age of respondents was 42 years in Mombasa, 43 years in Nairobi and 44 years in Busia. The minimum (20 years) and maximum (85 years) age of respondents was recorded in Mombasa. The average number of years in cassava trading was highest (19 years) in Mombasa while Nairobi recorded the lowest (7 years). The average number of years in cassava trading was 12 years in Busia. Busia had the highest (44%) percentage of traders without formal education while Nairobi region recorded the highest (50%) percentage of traders that had attained secondary education. Ownership of storage facilities ranked highest (73%) in Nairobi and lowest (8%) in Mombasa. Busia had 33% of traders owning storage facilities. The low figure reported in Mombasa could be attributed to quick sales of fresh tubers that did not require storage facilities. Out of the number of cassava traders interviewed, only 25% in Mombasa were selling other agricultural products. In Nairobi, all traders interviewed were selling other agricultural products besides cassava while in Busia, 78% were dealing with other agricultural products. Other agricultural products sold in Mombasa included other root crops (33%), while in Nairobi and Busia cereals were dominant at 87% and 85% respectively.

80 Male 70 Female 60

% respondents 30

0 Mombasa Nairobi Busia

Region

Figure 5. Gender of cassava traders in Mombasa, Nairobi and Busia.

Complimentary Contributor Copy 366 C. M. Githunguri, M. Gatheru and S. M. Ragwa

BUSINESS ACTIVITIES

Figure 6 shows the overall trend in the trade of cassava and cassava-based products in the three study regions. Sale of cassava and cassava-based products in Mombasa, Busia and Nairobi dates as early as 1956, 1962 and 1987 respectively. In Mombasa, cassava crisps and fried fresh cassava constituted 8% and fresh roots 92% of cassava and cassava products sold. In Nairobi, boiled cassava constituted 6%, cassava flour 25%: dried chips 69% of cassava and cassava products being traded in. In Busia cassava flour constituted 33% and dried chips (for milling) 67% of the cassava processed products sold. In Mombasa, the minimum storage period for cassava and cassava-based products was one day with a maximum of four days. This was attributed to the fact that they sold fresh tuberous roots only. Though cassava was stored for a maximum 4 days, 75% of the traders experienced storage losses. In Nairobi the cassava products could be stored for up to 90 days because they were processed as dried cassava chips for milling. Although cassava was stored for a longer duration in Nairobi, storage losses were experienced by only 25% of the traders. In Busia, cassava-based products could be stored for up to 12 days but storage losses were reported by 25% of traders. Losses in storage were attributed mainly due to storage pests like rats and weevils and rotting.

20 Number of traders Number of

2005

2000 2003 2004

2002 2006

2001

1956 1957 1988 1997

1984 1987 1990 1992

1980 1989 1998

Year when cassava business started

Figure 6. Overall trend in cassava and cassava-based products in Busia, Nairobi and Mombasa for a period of 50 years.

Complimentary Contributor Copy Trend in the Trade of Cassava Products … 367

On treatment of cassava and cassava-based products during storage, none of the traders was applying any treatment in all the study regions. The reasons given for not applying any treatment to cassava and cassava-based products differed with region. In Mombasa, 75% of the respondents reported that the cassava sells quickly because of a ready market and hence does not need treatment while in storage. Similarly in Nairobi 50% of the respondents reported that cassava was sold quickly, while 29% of respondents reported that cassava products were not affected by pests. In Busia 75% of the respondents reported that cassava sold quickly due to a ready market while 13% reported that pests did not attack stored cassava products.

DEMAND CHARACTERISTICS

Demand characteristics for the sampled cassava and cassava-based products traders are shown in Table 4. The major customers of cassava and cassava-based products in Mombasa were final consumers (67%), retailers (25%) and processors (8%). In Nairobi 72% of customers were final consumers, 6% wholesalers, 12% retailers and 10 percent processors. In addition, the processors in Nairobi were mainly millers specializing in composite flour. In Busia 82% of customers were final consumers, 5% wholesalers, 5% retailers and 8% processors. Both males and females constituted 67% and 62% of main customers in Mombasa and Nairobi respectively. In Busia the major customers were females (56%). In the three study regions, main customers of cassava and cassava-based products were aged between 20 and 50 years. In Mombasa, the average price of a fresh root was 13 shillings during scarcity and 8 shillings during abundance. In Nairobi, the average price of a 2 kg tin (gorogoro) was 69 shillings during scarcity and 55 shillings during abundance. In Busia, the average price of a 2 kg tin was 35 shillings during scarcity and 31 shillings during abundance. In Mombasa, an average of 5 roots was sold during both scarcity and abundance period. In Nairobi, an average of 7 tins was sold during scarcity and 6 tins during abundance. In Busia, an average of 32 tins was sold during scarcity and 21 tins during abundance. The low volume of sales in Mombasa and Busia during abundance is attributed to the fact that most farmers have harvested enough cassava for their household use during this period. In Nairobi, the low volume of sales during the same period is attributed to the high volumes of cassava being brought to Nairobi from western Kenya.

SUPPLY CHARACTERISTICS OF CASSAVA TRADERS

Supply characteristics for the cassava traders sampled are shown in Figure 5. In Mombasa 67% of the principal suppliers of cassava and cassava-based products were both male and female. In Nairobi 53% of cassava and cassava-based products suppliers were women. In Busia 63% of cassava suppliers were both men and women. In Mombasa and Nairobi, 85% and 94% respectively, of principal suppliers were between 20 and 50 years old while in Busia, 100% of suppliers were in that age class. In Mombasa 67% of supplies came from Kilifi and 33% from Kongowea market. In Nairobi, 81% of

Complimentary Contributor Copy 368 C. M. Githunguri, M. Gatheru and S. M. Ragwa supplies came from Gikomba market, 13% from Kibera and 6% from Wakulima market. In Busia, 89% of supplies came from Busia market and 11% from Uganda. The supply prices of cassava and cassava-based products varied across the three study regions with Busia recording the lowest fluctuation throughout the year. In Mombasa, the average price for fresh roots per 90 kg was 900 shillings during scarcity and 400 shillings during abundance. In Nairobi, the average price of dried cassava chips was 43 shillings for a 2 kg tin (gorogoro) during scarcity and 31 shillings during abundance. In Busia the average price of dried cassava chips was 22 shillings for a 2 kg tin during scarcity and 19 shillings during abundance. Supply constraints were reported by 58% of respondents in Mombasa, 25% in Nairobi and 56% in western. The main supply constraints reported at the coast were lack of cassava during scarcity (43%), transport (29%), competition (14%) and lack of credit (14%). In Nairobi, the main supply constraints were competition from other related products like maize (50%) and lack of cassava during scarcity. In Busia, most important constraints recorded were transport (40%), lack of sorghum and finger millet for blending cassava (20%), lack of cassava during scarcity (20%) and unfavourable weather which makes the cassava chips not to dry well (20%).

Table 4. Characteristics of cassava and cassava-based products customers

Region Characteristic Coast Nairobi Western Percent respondents Major customers Final consumers 67 72 82 Wholesalers 0 6 5 Retailers 25 12 5 Processors 8 10 8 Gender of main customer Male 8 13 0 Female 25 25 56 Both 67 62 44 Age class of main customers 20-50 years 83 88 100 >50 years 0 6 0 All ages 17 6 0 Customers’ desirable attributes Sweet taste 75 0 56 White colour 25 50 44 Ease of milling 0 50 0 Mean Selling price during scarcity 13 69 35 Selling price during abundance 8 55 31 Daily quantity sold during scarcity 5 7 32 Daily quantity sold during abundance 5 6 21

Complimentary Contributor Copy Trend in the Trade of Cassava Products … 369

Table 5. Characteristics of cassava and cassava-based products suppliers

Region Characteristic Coast Nairobi Western Percent respondents Gender of principal supplier Male 25 14 0 Female 8 53 38 Both 67 33 62 Age class principal supplier 20-50 years 85 94 100 >50 years 0 6 0 All ages 15 0 0 Experience any supply constraints Yes 58 25 56 No 42 75 44 Most important constraints Competition 14 50 0 Lack of cassava during scarcity 43 50 20 Lack of credit 14 0 0 Transport 29 0 40 Bad weather 0 0 20 Lack of sorghum/millet 0 0 20 Mean Buying price during scarcity 900 43 22 Buying price during abundance 400 31 19

CONCLUSION

In Mombasa, cassava and cassava-based products were sold at the main market (Kongowea). In Nairobi, cassava and cassava-based products were sold in local markets (Gikomba and Kibera) and hotels. In Busia, cassava and cassava-based products were sold at the main and secondary markets. In Mombasa and Nairobi, cassava traders are mainly wholesalers, wholesalers/retailers and retailers while in Busia they were both wholesalers and retailers. In Mombasa, the majority of those marketing cassava and cassava-based products were males while in Nairobi and Busia the business was dominated by females. In Mombasa and Busia, the cassava business was dominated by the native people, while in Nairobi the business was dominated by migrants. The main cassava products sold in Mombasa were crisps and fried chips and fresh tuberous roots while in Nairobi the main products were boiled cassava, flour and dry chips. In Busia cassava flour and dried chips (for milling) were the main products. In Mombasa the major customers of cassava and cassava-based products were final consumers, retailers and processors while in Nairobi major customers were final consumers, wholesalers, retailers and millers. In Busia customers were final consumers, wholesalers, retailers and processors. In

Complimentary Contributor Copy 370 C. M. Githunguri, M. Gatheru and S. M. Ragwa

Mombasa the principal suppliers of cassava and cassava-based products were both male and female. In Nairobi the principal suppliers were women. In Busia principal cassava suppliers were both men and women. The main supply constraints reported at the coast were lack of cassava during scarcity, transport, competition and lack of credit. In Nairobi, the main supply constraints were competition from other related products like maize and lack of cassava during scarcity. In Busia, most important constraints recorded were transport, lack of sorghum and finger millet for blending cassava, lack of cassava during scarcity and unfavourable weather which makes the cassava chips not to dry well.

REFERENCES

FAO, (1990): Roots, Tubers, Plantains and Bananas in human nutrition. FAO, Rome, Italy. Goering, T.J. 1979. Tropical root crops and rural development. World Bank Staff working Paper No. 324. Washington, D.C., World Bank. Githunguri, C. M. 1995: Cassava food processing and utilization in Kenya. In: Cassava food processing. T. A. Egbe, A. Brauman, D. Griffon and S. Treche (Eds.) CTA, ORSTOM, pp119-132. Githunguri, C. M., I. J. Ekanayake, J. A. Chweya, A. G. O. Dixon and J. Imungi. (1998): The effect of different agro-ecological zones on the cyanogenic potential of six selected cassava clones. Post-harvest technology and commodity marketing, .IITA, 71-76pp. Githunguri, C.M., E. G. Karuri, J. M. Kinama, O. S. Omolo, J. N. Mburu, P. W. Ngunjiri, S. M. Ragwa, S. K. Kimani and D. M. Mkabili. (2006a) Sustainable Productivity of the Cassava Value Chain: An Emphasis on Challenges and Opportunities in Processing and Marketing Cassava in Kenya and Beyond. A project proposal present to the Kenya Agricultural Productivity Project (KAPP) Competitive Agricultural Research Grant Fund, Research Call Ref No.KAPP05/PRC- CLFFPS –03. KAPP Secretariat 106p. Githunguri, C.M., Karuri, E.G., Kinama, J.M., Omolo, O.S., Mburu, J.N., Ngunjiri, P.W., Ragwa, S.M. and Mkabili, D.M. (2006b): Sustainable Productivity of the Cassava Value Chain: An Emphasis on Challenges and Opportunities in Processing and Marketing Cassava in Kenya and Beyond. Githunguri, C. M., S.M. Ragwa and J. Abok. (2008a). Proceedings of the Cassava Value Chain Project Collaborators and Stakeholders’ in Kenya Inception Workshops. 2008 KARI Katumani Research Centre, Cassava Value Chain Project. 123pp. Githunguri, C.M., Kinama, J.M., Karuri, E.G., Gatheru M. and Ragwa, S.M. (2008b). Situational Analysis of Cassava Production, Processing and Marketing in Kenya. KARI Katumani Research Centre. Pp 51. Githunguri C. M, S. M Ragwa and S. Yatta. 2009a. Culinary Perceptions of Cassava-Based Products by Hoteliers’ Based in Kibwezi in Semi-Arid Eastern Kenya. In: C.M. Githunguri, Kizito Kwena, Erick Mungube and Mwangi Gatheru (eds). KARI Katumani Research Centre annual report 2008. Pp. 113. Githunguri C. M., S. M Ragwa and S. Yatta. 2009b. Popularization of Eight Cassava-Based Products and Recipes among Hoteliers and Consumers in Kibwezi in Semi-Arid Eastern

Complimentary Contributor Copy Trend in the Trade of Cassava Products … 371

Kenya. In: C.M. Githunguri, Kizito Kwena, Erick Mungube and Mwangi Gatheru (eds). KARI Katumani Research Centre annual report 2008. Pp. 113. GoK. 2010. Agricultural Sector Development Strategy (ASDS, 2010 - 2020). Pp. 101. Greene, W. H. (2000). Econometric Analysis. Upper Saddle River, New Jersey, Prentice-Hall, Inc. International Fertilizer Development Center (IFDC). (2012). Cassava+ Opportunities for Africa’s Smallholder Cassava Farmers. Cassava Project Report. IFDC Kenya, Duduville. Pp 6. Kadere, T.T. 2002. Marketing opportunities and quality requirements for cassava starch in Kenya. In Proceedings of regional Workshop on improving the cassava sub-sector, held in Nairobi, Kenya, April 2002, 8-18, pp. 81 – 86. KARI. 1995: Cassava Research Priorities at the Kenya Agricultural Research Institute, Cassava Priority Setting Working Group. Makokha, J. and Tunje, T.K. 2000: Study of Traditional Utilization and Processing of Cassava and Cassava Products in Kenya, First interim technical and financial report, JKUAT/EARRNET. Mbwika, J.M 2002. Cassava sub-sector analysis in the Eastern and Central African region. In Proceedings of regional Workshop on improving the cassava sub-sector, held in Nairobi, Kenya, April 2002, 8-18, pp. 8-18. Ministry of Agriculture. (2011): National Cassava Development Strategy 2012-2016. Ministry of Agriculture, 70pp. Ministry of Agriculture. (2009): Ministry of Agriculture Strategic Plan 2008 – 2012. Ministry of Agriculture. (1999): Provincial Annual Reports. Nweke, F. I., D. S. C. Spencer and J. K. Lynam. 2002. Cassava transformation. International Institute of Tropical Agriculture. 273p. Scott, G. J. (1995). Methods for Evaluating the Market Potential of Processed Products. Prices, Products, and People: Analyzing Agricultural Markets in Developing Countries. G. J. Scott. London, Lynne Rienner Publishers, Inc. Wambugu, S. M. and Mungai, J. N. 2000: The potential of cassava as an industrial/commercial crop for improved food security, employment generation and poverty reduction in Kenya, KIRDI.

Chapter 19

WILD RELATIVES OF CASSAVA: CONSERVATION AND USE

Márcio Lacerda Lopes Martins1,*, PhD Carlos Alberto da Silva Ledo2, PhD Paulo Cezar Lemos de Carvalho1, PhD André Márcio Amorim3,4, PhD and Dreid Cerqueira Silveira da Silva1, PhD 1Federal University of Recôncavo of Bahia, Center of Agronomical, Environmental and Biological Science, Bahia, Brazil 2Embrapa Cassava and Fruit, Bahia, Brazil 3State University of Santa Cruz, Department of Biological Science, Ilhéus, Bahia, Brazil 4Herbarium of Center of Research of Cocoa, CEPEC, Ilhéus, Bahia, Brazil

ABSTRACT

The genetic improvement of cassava is directly related to the increase of productivity of culture, this has an important role in feeding in developing countries. Therefore, knowledge about the biology, distribution and conservation status of their wild relatives is essential, because it allows the harvest and conservation efforts to be directed to those unfamiliar species of which there are more severe threats. These data become even more relevant since some of their wild relatives are resistant to common diseases, such as whitefly. This chapter discusses the closest conservation of the wild relatives of cassava from the evaluation of biological collection, as well as recent collections by authors in Brazil and their cultivation in Germplasm banks. This work is part of a program of study of wild species of Manihot developed in partnership with the Federal University of Bahia Recôncavo (UFRB) and Cassava and Fruits National Research Center (CNPMF) of the Brazilian Agricultural Research Corporation (EMBRAPA) both located in Cruz das Almas, Bahia, Brazil. The program, started in 2010 aims to harvest and cultivate wild

* Author for correspondence ([email protected], 55 75–3621-3176; 55 75–98127-1987). Complimentary Contributor Copy 374 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

species of the genus with taxonomic, conservation and agronomic purposes, especially with regard to improving the cassava (M. esculenta Crantz). Harvests were made during the first six years of the project in four Brazilian regions encompassing 14 states and over 150 municipalities mainly from the central and eastern South America region. About 60 of the 80 south American species of Manihot in various environments were seen and harvested. Thirteen species phylogenetically close to cassava were selected to discuss their conservation status based on their occupation Area (AOO), Occurrence Extension (EOO), and potential use for the improvement of this culture. According to the International Union for Conservation of Nature (IUCN) criteria, all species showed some degree of threat, two considered Critically Endangered and the other Endangered according to AOO. The EOO analysis showed different results with only three endangered species, which can indicate subsampling of natural populations of these species. In preliminary studies among the analyzed species only three presented suggest valuable features to cassava improvement as resistance to pests and diseases, such as African cassava mosaic virus, bacterial blight, anthracnose, green mite and caterpillar ‘mandarová,’ or high dry matter content and protein in roots. However, the fact that some species were not included in the analysis, because they do not appear in the same M. esculenta clade, which also presents important features for improvement, suggests that they may also be the subject of breeding programs due to the ease of hybridization verified gender. Regular expeditions of harvest of wild species of Manihot, that were conducted since 2010 have helped to increase the distribution of data and also to broaden the panorama of each species ‘in loco,’ allowing the verification of their habitat conservation status, the number of individuals of each population, etc. However, expeditions have not been made yet specifically aimed at the closest relatives of cassava, covered in this study. It is emphasized that maintaining wild relatives of cassava germplasm bank is a practice of fundamental importance for the improvement of this culture, because the programs rely on the introduction of alleles with valuable agronomic traits contained in these species to minimize the limitations found in culture as pests and diseases.

INTRODUCTION

The species of Manihot Mill. are exclusively Neotropical with majority distribution in South America, but with an important diversity center in Mexico (Nassar 1978a, Rogers & Appan 1973). The wide range of variables characters within the same species hinders the precise delimitation of various taxa and the number of accepted species is variable. Taking the example of Brazil, Rogers & Appan (1973) report 80 species, Allem (2002) estimates that the number of species varies between 47 and 50, while, Cordeiro et al. (2015) reports 76. The wild species of the genus for the improvement of cassava (Manihot esculenta Crantz) is important as it is considered the 4th main source of starch in the world which makes the definition of species of Manihot a relevant issue. At the same time the natural populations of these species are under constant threat due to the expansion of the agricultural frontiers and cattle raising, and are usually eliminated from rural areas due to the presence of hydrocyanic acid (HCN) (Allem 1999, Nassar 1978b). These actions accelerate the necessity to update the knowledge of the group with regard to the identity of their species, especially in relation to their conservation.

Complimentary Contributor Copy Wild Relatives of Cassava 375

CLASSIFICATION HISTORY

Originally described by Bauhin (1651), from cultivated samples in Brazil, Manihot has only been validated by Miller (1754) in the Gardener’s Dictionary. Crantz (1766) presented the first valid epithet for the genus (M. esculenta) from cultivated cassava specimens. From there several names have been published for this species (e.g., M. aipi Pohl, M. dulcis (J. F. Gmel) Pax, M. utilissima Pohl) (Rogers & Appan 1973). Pohl (1827), in Plantarum Brasiliae Icones et Descriptions, provided the first monograph about Manihot. In this work 48 species were presented, almost all accompanied by descriptions and boards. The author has been in Brazil and observed the material in the field, which made his descriptions and robust circumscriptions, with many species accepted today. Throughout the nineteenth century, Steudel (1840-41) combined various species originally described in Jatropha in Manihot, expanding and redefining the genus division. The second and third monographs of Manihot were published by Müller (1866, 1874). In his first work the author has listed 43 species, further divided into eight groups, in addition he presented the first identification key for species. Some species have expanded their area in relation to Pohl’s work (1827) (e.g., M. gracilis Pohl; M. palmata Pohl; M. tripartita (Spreng) Müll. Arg.). This paper presents descriptions that include characters related to flowers and inflorescences but lacks illustrations for species recognition. In Flora Brasiliensis (Müller 1874) 71 species were included which were divided into 10 groups, with few illustrations. In all Müller (1866, 1874) described 64 taxa between species and subspecies and added important distribution records, despite this he has not done any work in the field. Pax (1910) recognized 128 species in 11 sections. The author made a comprehensive review of the bibliography of existing collections and produced the first section in Manihot and the first hypothesis about the possible phenetics relations within the group. Ule (1907, 1914) described 13 species found in Bahia, with an emphasis on producing latex for rubber manufacturing, and discussed their relationship with other species of Manihot. Croizat (1943) assembled 17 species in South America and commented on the importance of establishing criteria to consider morphological diversity displayed by most species and their geographical distribution for the elaboration of a more consistent rating for the genus. Finally, Rogers & Appan (1973) presented in Flora Neotropica the latest monographic study of the genus. Many advances to the group’s taxonomy have been proposed by the authors who provided an important compilation of knowledge about Manihot, describing 13 taxa and synonymizing others to widely verify species morphologically (e.g., M. caerulescens Pohl; M. tripartita), which reduced the number of species to 98. The authors acknowledged 19 sections based on morphological phenetics analysis, some with a single species, (e.g., Sect. Grandibracteatae Pax emend D. J. Rogers & Appan, Tripartitae D. J. Rogers & Appan) and M. pauciflora (T. S. Brandegee) D. J. Rogers & Appan was recombined in genus Manihotoides D. J. Rogers & Appan, which is probably extinct in the wild (Nassar 2002). Unlike some of his predecessors, Allem (1977, 1978, 1979abc, 1980) discussed various aspects related to division of Brazilian species (e.g., M. anomala Pohl, M. caerulescens Pohl, M. carthagenensis Müll. Arg. and M. hilariana Baill.) based on his extensive field experience. In the late 1980s, this author published four new species (Allem 1989a) and the

Complimentary Contributor Copy 376 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al. revision of Section Quinquelobae Pax emend Rogers & Appan, based on morphological, palynological and cytogenetic (Allem 1989b). The author has also published new taxa and provided contributions to Glazioviannae Pax emend Rogers & Appan section (Allem 1999, 2001), and finally proposed a phenetic classification model, which includes about 50 species occurring in Brazil in 16 groups, and suggested probable phylogenetic relations among themselves (Allem 2002). Currently, some researchers have been devoted to genus study in South America, which has generated regional flora (Rodrigues 2007, Sátiro & Roque 2008, Mendoza 2010, Carmo- Jr et al. 2013) and several species have been described (Martins et al. 2011, 2014, Mendoza et al. 2013, Silva et al. 2013, Martins & Ledo 2015, Silva 2016).

MORPHOLOGICAL CHARACTERS

The Manihot genus can usually be recognized by lobed leaves with purplish hues and inflorescences racemose or paniculate with two basal pistillate flowers, accompanied by bracts and bracteoles usually evident (Rogers & Appan 1973). Furthermore, the formation of hydrocyanic acid (HCN) is common when the tissues suffer some injury (Dunstan et al. 1906). These characters, however, should be viewed with caution for the distinction of its kind (Allem 1977). For Léotard et al. (2009) Differentiation of the Manihot species based solely on morphological characters may lead to errors since some characteristics are highly variable due to environmental plasticity or developmental. Habit variation is observed for most species and seems to be directly related to the environment they occupy (Duputié et al. 2011). In general, the cerrado species have become shrubby or subshrubs, while species of the semiarid region tend to be woody and forest vine- like (Allem 1999; Nassar et al. 2008). The habit change according to the environment occurs with some species and can generate taxonomic doubts if the analysis is restricted to the herbarium materials with inaccurate notes (Allem 1999). According to Allem (1979b) the external morphology of the vegetative organs, especially leaves, is presented as a consistent characteristic for differentiation of the species closely related to Manihot, however, this proves to be fragile in related species. The author’s view, where the species have an extensive list of character states for certain structures (leaves, habit, pubescence, etc.) is supported in results found by Duputié et al. (2011). For these authors, the recent genus origin makes it difficult the species lines which are definitely isolated, so the delimitation of species and publication of new taxa should be surrounded by caution (Duputié pers. comm.). Pohl (1827) uses reproductive organs features to define the characters “essential” and “natural” within Manihot, however, in their diagnoses refers only to vegetative characters. In Manihot, the variation of the shape of the leaves is common, and one species may provide leaves 3, 4, 5, 6, 7, and 9-lobed, in addition several species are associated with whole leaves inflorescences (Allem 1979bc). This variation cannot generally be observed in herbarium materials and exclusive analysis of these materials may have contributed to the increase in the number of species of the genus recorded in Pax (1910). Some strains, such as M. caerulescens, M. pentaphylla Pohl and M. tripartita, are full of synonyms because of naming to the various forms that have their leaves (Rogers & Appan 1973). Another feature that

Complimentary Contributor Copy Wild Relatives of Cassava 377 should be noted is the nature of the leaves. Allem (1999) reports four species with leaves composed for Manihot, among them three from the Amazon. For Rogers & Appan (1973), these species have a strong constriction between leaf lobes of some species of the genus that resembles the leaflets, but its leaves are simple. The morphology of the stipules is important for the diagnosis of some taxa in Manihot (e.g., M. pusilla Pohl and M. stipularis Pax; Pax 1910), but they are usually caducous, and are used little in the delimitation of species of the genus (Rogers & Appan 1973). The inclusion of traits related to reproductive organs in the delimitation of Manihot species, as a kind of inflorescence size and margins of floral bracts and the coating of the fruits were introduced by Müller (1866, 1874). The shape of the bracts and bracteoles and inflorescence pattern are presented differentially which is useful in species distinction (Allem 1984). Some typically have bracts foliaceous (e.g., M. caerulescens, M. jacobinensis Müll. Arg. and M. tripartita), other setaceous (e.g., M. carthagenensis), while a few have variation in its form (e.g., M. anomala, Allem 1979b). The format of the bracts tends to remain stable in the same individual and the same population. The opposite is verified for the type of inflorescence (Allem 1977). Racemose inflorescences may occur solitary or in groups and some species may have panicles and racemes in the same individual (e.g., M. carthagenensis) (Allem 1979c). Nevertheless, these characteristics were used for the distinction of species of various sections which contributed to the inconsistency of the current rating. Rogers & Appan (1973) considered the shape of the flower bud staminate as a key character for the distinction of Manihot species, leaving aspects of fruit morphology in a lower plane. Flower buds, bi-fusiform, oval and orbicularis are diagnostic for species and may have subtle variations between pistillate and staminate flowers (Rogers & Appan 1973, Allem 1989a, 2001). Croizat (1943) highlights the importance of fruits for the genus. The fruits of Manihot are commonly found as capsules, but bacaceous fruit can be found in a small number of some related species (Allem 1999, Duputié et al. 2011). Barroso et al. (1999) includes the fruits bacaceous and drupe between the fruits of Euphorbiaceae, although some authors prefer to use the term indehiscent capsule (e.g., Oliveira & Oliveira 2009). Webster (1958) relates the kind of fruit to the size of the Phyllantaceae species. The same has not been tested in Euphorbiaceae but there is evidence that this relation does not exist in this group (Allem 1999). The indehiscence fruit in Manihot species apparently is related to the type of environment that they occupy which is directly linked to the type of dispersion presented by some species. The morphological diversity of Manihot fruit involves differences in their overall shape, size, and shape in the apex of the presence or absence of ribs on the septum. This last character has to be variable in certain species, in which the fruits can be smooth or ribbed, through several intermediate stages (e.g., M. caerulescens) (Allem 2001). Morphological variation in leaves, bracts, staminate buds, and fruits is shown in Figure 1. Unlike fruit, little taxonomic importance is given to the morphology of the seeds in Manihot. The species of the genus usually have oval seeds, ash brown, with dark spots, on the forehead, as well as usually have well-developed caruncles. Within Euphorbioideae, the morphology of the seed, especially the thickening of the integument and the presence of aryl, proved widely diverse (Tokuoka & Tobe 2002). Webster (1994) points out that caruncles presents considerable taxonomic and evolutionary interest within the Euphorbiaceae.

Complimentary Contributor Copy 378 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Figure 1. Morphologic diversity in Manihot species. (a)Leaves and stipules (note: entire and lobed leaves, peltate or not, setaceous and foliaceus stipules); (b)Staminate buds and bracts (note: bifusiform and ovoid buds, semifoliaceus and foliaceus bracts). (c)Fruits (note: the variation in the shape, colour and the presence of ribs) (Photo by authors).

The morphology of the pollen-grains presents homogenous Manihot (Allem 1993), though useful in differentiating some species of genera of Euphorbiaceae (Cruz-Barros et al. 2006, Cooke et al. 2010), within the subfamily Crotonoideae the-pollen-grains are typically inaperturates with exine formed by triangular elements connected to a network ‘muri’ with short columellae (Nowicke 1994), and those having exine periporate are considered “standard Manihot” (Punt 1962). Unlike macroscopic characters, the use of anatomical characters for Manihot species distinction is restricted, and most of the work has focused on cultivated species (Graciano- Ribeiro et al. 2008, 2009, Bomfim et al. 2011.). Hybrids tetraploid M. esculenta with M. oligantha Pax & K. Hoffm. showed a higher number of vessel elements and absence of growth rings in comparison with diploid hybrid, indicating a greater adaptation to xeric environments (Graciano-Ribeiro et al. 2008). The adaptation to this type of environment is one of the main characteristics of the species Manihot and, accordingly, the allotetraploid origin of the group may have been presented as of great adaptive value (Nassar 2000a). White latex and laticifers are commonly articulated in Manihot and Hevea Aubl., unlike the rest of Crotonoideae subfamily in which latex of different colors and unarticulated laticifers are more common (Rudall 1994). Few species of Manihot, however, have cream or yellow latex (Allem 1979b). Differences in the organization of tissues in the pericarp of fruits of M. caerulescens and M. tripartita are evidence of the importance of the morphology of fruits for their systematic, and that the use of these characters can prove useful (Oliveira & Oliveira 2009).

Complimentary Contributor Copy Wild Relatives of Cassava 379

Figure 2. Cross section of the mesophyll of the Manihot species of Chapada Diamantina, Bahia, Brasil, (Note: Epidermis variably papilose. (LP) – lacunose parenchyma, (PP) – palisade parenchyma, (st) – stomata. Scale bars: a, c, f = 60 μm; b, d, e, g = 30 μm)(Photo by I. Cunha-Neto and F. Martins, in Cunha-Neto et al. 2016, subm.).

The most comprehensive studies on the anatomy of wild species of the genus were developed by Allem (1984) evaluating the anatomy of the petiole, the type of venation, the ornamentation of the cuticle and the morphology of the epidermal cells. Epidermal waxes presented various forms, some even have been used to support the description of new species (e.g., M. xavantinensis D. J. Rogers & Appan) but have not enough stability for use with taxonomic purposes (Allem 1989b). The morphology of the leaf epidermal cells, however, proved to be an important character to be considered, especially in their abaxial surface (Allem 1984). Four groups were differentiated in the Quinquelobae section based on the absence of ornamentation on the leaf surface, or the presence thereof in the form of ridges, papillae or reticulated (Allem 1989b). Cunha-Neto et al. (2014) discuss the use of leaf anatomy in the differentiation of species of M. violacea Pohl complex group showing important characters as the cross section of the petiole, the shape of vascular bundles and the presence of epidermal papillae (Figure 2).

GENETIC AND MOLECULAR CHARACTERS

Some Euphorbiaceae groups have great stability in the number and morphology of chromosomes, while others show significant variation and may contribute to their taxonomy (Hassall 1976). Only the genus Euphorbia a diploid nucleus may have between 12 (Euphorbia

Complimentary Contributor Copy 380 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al. retusa Forssk) and 200 chromosomes (E. ferox Marloth) (Hans 1973; Hayirlioglu-Ayaz et al. 2002). The base chromosome number within the family is variable with the main values x = 7 and x = 13 and generally trend to reduce the size of the chromosomes as their number increases (Hans 1973). Among the genus near Manihot, several species of Cnidoscolus Pohl and Hevea have 2n = 36 which suggests that genus are cytologically uniform (Majumder 1964, Miller & Webster 1966, Leicth et al. 1998). A different situation was recorded for Jatropha L. presenting 2n = 20 or 22 (Hans 1973, Sasikala & Paramathma 2010). Jennings (1963) suggests that Manihot is an allotetraploid derivative of the basic number x = 9. The chromosome number found in the group is invariably 2n = 36, and their chromosomes are small, metacentric or submetacentric (Carvalho & Guerra 2002). The high frequency of regular meiotic pairing and large pollen viability observed in interspecific hybrids (Nassar & Freitas 1997) may be a result of this karyotype similarity, both in number and in size and shape (Carvalho & Guerra 2002). However, there are records of low pollen viability and deficiencies in the pairing of chromosomes (Nassar 2000a) as well as structural differences in the chromosomes and variable pollen viability in F1 generations originated from interspecific hybrids of Manihot (Bai et al. 1993). Nassar (2000b) believes that allopolyploidization is directly related to the rapid appearance of species lineages of Manihot, which led to weak reproductive barriers. Various reports of hybrids produced between each other wild species and the cultivated species confirm the absence of effective reproductive barriers for some groups (Nassar 1985, 2003, 2006, Nassar et al. 2008). In relation to the molecular data, regions that provide good results for the phylogeny of other groups, such as the spacer trnL-F useful in phylogenetic analyzes of Croton L., Macaranga Thouars and Mallotus Lour. (Berry et al. 2005, Kulju et al. 2007) does not exhibit phylogenetic signals that allow the distinction of most Manihot species (Chacon et al. 2008). This apparently is due to the fact that the DNA sequences being very well preserved in the Manihot species, thanks to its recent origin, and therefore uninformative (Leotard et al. 2009, 2011 Duputié et al.). Nevertheless, the results of these studies support the monophyly of the genus, which can be divided into the Central American and South American lineage, and the close relationship between biogeographic and the main lineages, and the need of infrageneric group taxonomic review. The definition of informative regions will allow them to be used as markers for introgression and classification of germplasm banks (Roa et al. 1997). Regions G3pdh nuclear gene has shown to be useful in phylogenetic and taxonomic analyzes involving the Amazon species M. esculenta subsp. flabellifolia (Pohl) Ciferi x M. surinamensis D. J. Rogers & Appan, and M. aff. quinquepartita Huber ex D. J. Rogers & Appan x M. brachyloba Müll. Arg. (Leotard et al. 2009), as well as most of the wild species of the genus (Chacon et al. 2008, Duputié et al. 2011). The relation between M. esculenta and their wild relatives, as well as the definition of its center of origin constitute the most common approaches inferred using molecular markers (Olsen & Schaal 1999, Allem 2002, Olsen 2002, Leotard et al. 2009). This species have been over investigated from the point of view of the genome which is actually a ‘flagship species’ in many evolutionary studies on larger scales (Daniell et al. 2008). And RAPD markers and AFLP have been effective in this matter, particularly in defining the positioning M. flabellifolia Pohl and M. peruviana Müll. Arg. (Roa et al. 1997, Colombo et al. 2000). These species were distinct and belonging to different sections by Rogers & Appan (1973), they had Complimentary Contributor Copy Wild Relatives of Cassava 381 their situation redefined as subspecies of M. esculenta (Allem 2002), but apparently are synonymous (Colombo et al. 2000). The genetic affinity between these wild species and M. esculenta was used for determination of gene pools within Manihot. Allem et al. (2001) consider as the primary gene pool subspecies of M. esculenta (M. esculenta subsp. esculenta, M. esculenta subsp. flabelifolia, M. esculenta subsp. peruviana) and M. pruinosa Pohl, and as pool other secondary gene 13 species from morphological evidence and reproduction tests. Other studies based on seed proteins proved useful for the evaluation of the similarity of 19 species of the genus, and showed the greatest similarity between geographically related species (Grattapaglia et al. 1987).

REPRODUCTIVE BIOLOGY

Manihot species are characterized by monoecious with pistillate and staminate flowers on the same inflorescence, except for some dioecious species of Stipularis Pax emend D. J. Rogers & Appan section (Rogers & Appan 1973). Its inflorescences are protogynous, so that the staminate flowers located at the top of inflorescence do not become anthesis when the pistillate have been fertilized or aborted in a range of approximately 20 days (Halsey et al. 2008). The selfing may occur as pistillate and staminate flowers of different branches can open simultaneously (Jennings & Iglesias 2002). Reproductive barriers are weak among the species of the genus (Jennings 1963). Cruz (1968) highlights that the Manihot species can interbreed causing natural hybrids as evidence in the cytogenetic group. Several interspecific hybrids between wild species and accessions of cultivated species were obtained, especially when the latter acts as a recipient of pollen grains (Nassar 1980) and even among wild species the process of hybridization seems to find great obstacles (Bolhuis 1953, Jennings 1959, Magoon et al. 1970, Nassar 1980). There are reports of natural hybrids between M. alutacea D. J. Rogers & Appan and M. reptans Pax, and between M. caerulescens and M. tripartita favored by low barriers and overlapping habitats (Nassar 1984, Nassar et al. 2008). Olsen & Schaal (2001) suggest the occurrence of inbreeding in natural populations of Manihot evidenced particularly by the low heterozygous rate, which may be due to the lack of genetic self-incompatibility systems within the genus and the seed dispersal mechanism (dehiscence explosive) that as a rule, does not reach great distances. Moreover, apomixis can influence this process. Nassar et al. (2008) showed that the polyploidy can offer the heterozygosity necessary for initiating the process of speciation, and apomixes could allow hybrid showing reproductive disorders which prevent and maintained seed source for fertilization. The apomixis rates in Manihot are variable and are related to the occurrence of apospory (development of embryo sacks for ovule cell mitosis), the main source apomixes mechanism in angiosperm (Nassar 2000b). Allem et al. (2001) evaluating M. dichotoma Ule and M. glaziovii Müll. Arg. concluded that the crossing is possible but gene transfer is difficult with the F1 tendency to be sterile, as well as chromosomes presenting weak pairing or being univalent. This scenario is shown different from that in Jatropha L. were the species presents variably isolated with different floral arrangements and phylogenetic relationships which may

Complimentary Contributor Copy 382 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al. inferred with from the reproductive success rates (Dehgann 1984). Some species of this genus occurring in the bush, however, have overlap in flowering period and aggregate distribution with predominantly autochoric dispersion, factors that favor the formation of natural hybrid (Neves et al. 2010). Phenological studies in Manihot are scarce. Data are directed generally to the study of M. esculenta (Reich et al. 2004, Rós et al. 2011) or, as in wild species, vegetative characters such as loss of leaves in caatinga environments with few data on the formation of flowers and fruits (Machado et al. 1997, Tannus et al. 2006).

BIOGEOGRAPHY

The distribution of Manihot is restricted to the Neotropics. Its species form two groups geographically isolated in Central America and South America, with only M. brachyloba and M. carthagenensis occurring in both regions (Rogers & Appan 1973, Allem 1979c). The monophyly of these two groups has been supported on molecular studies and apparently the disjunct distribution of these species was the result of human introduction (Rogers & Appan 1973, Chacon et al. 2008, Duputié et al. 2011). Croizat (1943) highlighted the important role of geographical isolation in the divergence of characters between populations of Manihot, this is essential in the process of speciation. This work may have influenced the review of Rogers & Appan (1973), which unlike their predecessors began their work based on geography, gender separating the groups of North/Central America and South America. Currently there are four recognized centers of diversity for the genus, distributed in descending order of number of species in central Brazil, Mexico, northeast Brazil and southern Mato Grosso do Sul and Bolivia (Nassar 1978a, 2000bc). Several microcenters of diversity can be recognized and its origin is probably related to frequent hybridization and the rugged topography of the environments where these species occur, which contributes to the isolation of small gene pools and consequent speciation (Nassar 1978ac, 1979ab, 1982). The cerrado is the richest ecosystem in the Manihot species and its origin seems to be corresponding with the largest group of diversification period (Duputié et al. 2011). Other environments such as caatinga, rain forest and dunes are less representative in number of species, but new records have been made (Allem 1989b, Martins et al. 2011). The Manihot species tend to be restricted or endemic to small areas (Rogers & Appan 1973). This pattern is observed for several species of central Bahia, in Chapada Diamantina and Chapada dos Veadeiros, that harbor together over three dozen species, some of which are recently described (Rogers & Appan 1973, Martins et al. 2014, Mendoza et al. 2015, Silva et al. 2015). Among the species to accepted in Brazil less than 20% have more than one distribution biome (Rogers & Appan 1973). Among these, there is M. caerulescens, considered the only species of Manihot recorded in the cerrado, caatinga, and Amazon rain forest, which are the three largest Brazilian ecosystems (Allem 2001). This situation, coupled with the small phenotypic alterations mentioned, generated many synonyms for this species. The same is observed for M. anomala, M. carthagenensis and M. tripartita (Rogers & Appan 1973, Allem 1979ac, 1989a).

Complimentary Contributor Copy Wild Relatives of Cassava 383

CONSERVATION OF WILD RELATIVES SPECIES OF CASSAVA

The Manihot species probably originated in the Mesoamericana region and subsequently distributed in South America, colonizing all types of environments (Duputié et al. 2011). The threat level that natural environments are subject to contributes to much of it being on lists of endangered species. The 1997 IUCN Red List of Threatened Plants (Walter & Gillett 1998), includes 65 taxa, between species and subspecies, under various degrees of threat. Recently, just to the northeast of Brazil, 14 species were considered threatened (Martins 2013). Still, the Red Book of Flora of Brazil (Martinelli & Moraes 2013) recognized only M. procumbens Müll. Arg. among the endangered species for the genus, as Vulnerable, highlighting the need for conservation studies for this group. Research on the closest wild relatives of cassava was increased by Duputié et al. (2011). Analysis of molecular phylogeny made from the genes Glyceraldehyde 3-phosphate (G3pdh) and Nitrate Reductase (NIA), showed a clade that includes the subspecies of M. esculenta and 12 other species, among their closest wild relatives. Much of these species needs further study related to their geographical distribution, conservation and taxonomy. Interest in the study of wild relatives of cassava is due to the characteristics of economic interest that these species may hold and can be transferred to cultivated species, this can solve serious problems considered for this culture. The gene pool of these species is threatened due to fragmentation and habitat destruction by deforestation being replaced by agriculture, extensive monoculture pastures and urbanization, and the introduction of exotic species and the influence of climate change (Oliveira 2011). Willis et al. (2007) highlighted several key conservation initiatives, including the identification of endangered species and predictions of species distribution towards future climate change. However, less than 1% of all species on the planet were evaluated to determine their conservation status, i.e., an assessment of the risk of extinction. With increasing loss of biodiversity, it is essential to carry out evaluations on the species conservation status of all groups, thus identifying which of them are at increased risk of genetic erosion, to guide conservation actions. According to Marchioretto et al. (2004), one of the priorities for conservation is obtaining and providing concrete and updated data on the geographical distribution of species. The geographical distribution of a species is a unit resulting from the interaction of factors that act at different intensities and scales as abiotic conditions, biotic interactions, regions that are accessible to the dispersion of species arising from another area and evolutionary capacity of populations to adapt to new environments (Brown 1995, Soberon & Peterson 2005). These characteristics reinforce the need for studies related to the geographical distribution of wild relatives of cassava in order to contribute to their conservation, making taxonomic decisions and direction of collection efforts. In the early twentieth century until World War II, some species were used for extraction of latex for rubber production (Ule 1907, 1914). Currently, however, the search for useful characters is related to improving resistance to pests, diseases and more severe weather conditions such as lack of water and temperature extremes. Such agronomic characteristics can be found among wild relatives of cassava and can be transferred to cultivated species, this can solve problems considered serious for this culture such as whitefly (Aleurotrachelus socialis Bondar), mandarová (Erinnyis ello L.), bacterial blight (Xanthomonas axonopodis pv.

Complimentary Contributor Copy 384 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Manihotis), anthracnose (Colletotrichum gloeosporioides) and African mosaic virus (Fukuda et al. 1999). However, the gene pool of these species is threatened due to fragmentation and destruction of habitat by deforestation process, being replaced by agriculture, extensive monoculture pastures and urbanization, and the introduction of exotic species and the influence of climate change (Oliveira 2011). So one of the priorities for conservation is obtaining and providing concrete and updated data on the geographical distribution of species and their conservation (Marchioretto et al. 2004). Thus, it can summarize the need to develop studies aimed at assessing the status of conservation of wild relatives of cassava due to:

1. Much of the species have been assessed as threatened in unofficial and official lists; 2. Little knowledge of the geographical distribution of most species; 3. Potential use of such species in cassava improvement.

DATA COLLECTION AND ANALYSIS

One way to identify the species conservation status is through analytical tools that are available to carry out such assessments. The GeoCAT tool (powered by Google® 2015) performs a geospatial analysis to facilitate the evaluation process, and taxa frame in red lists of endangered species drawn up criteria and categories defined by the IUCN (2014). Those considered the closest wild relatives of M. esculenta were selected primarily for the study of the conservation status of wild species of cassava by Duputié et al. (2011). The data on the occurrence of species distribution were compiled from:

1. Herbarium specimens

The herbariums ALCB, ASE, CEN, CEPEC, CPAP, CVRD, EAC, ESA, F, FLOR, FURB, HAS, HB, HPBR, HPUC, HRB, HST, HUEFS, HUFU, HURB, HVASF, IAN, IBGE, ICN, IMA, IPA, IPB, K, MBML, MG, NY, PEUFR, R, RB, SP, SPF, UEC, UFMT, UFP, UFRN, UNB, US, VIC e VIES (Table 1) were consulted and visited ‘in loco’ ‘between 2010 and 2013 or obtained through consultations with the pages of each herbarium in the world wide web based mostly on collections indexed.

2. Harvest data carried out by Embrapa Cassava and Fruit and UFRB.

In order to visit the populations referenced by the collections of herbaria and locate new populations, samples were taken in the period between March 2010 and March 2016, in the Brazilian states of Alagoas, Bahia, Espírito Santo, Goiás, Maranhão, Mato Grosso, Minas Gerais, Pará, Paraíba, Pernambuco, Piauí, Rio de Janeiro, Rio Grande do Norte, São Paulo, Sergipe and Tocantins, in areas with a predominance of caatinga, cerrado and tropical rainforest. The collected material also contained leaf samples for DNA extraction, stem segments (cuttings), seeds and seedlings for planting in Germplasm Banks of wild relatives of

Complimentary Contributor Copy Wild Relatives of Cassava 385

Manihot in EMBRAPA Cassava and Fruit (CNPMF) and Federal University of Bahia Recôncavo (UFRB) in Cruz das Almas, Bahia, Brazil. From the obtained material a pre-selection of herbarium specimens and examined materials was made, excluding those with bad location and duplicate records.

Table 1. Consulted herbaria list (acronyms second Thiers 2016)

Acronyms of Herbaria Nomenclature ALCB Herbário Alexandre Leal Costa ASE Herbário da Universidade Federal de Sergipe CEN Herbário da Embrapa Recursos Genéticos e Biotecnologia CEPEC Herbário do Centro de Pesquisas do Cacau CPAP Centro de Pesquisa Agropecuária do Pantanal CVRD Companhia Vale do Rio Doce EAC Herbário Prisco Bezerra ESA Herbário da Escola Superior de Agricultura Luiz de Queiroz F Field Museum of Natural History FLOR Flor Herbarium- Universidade Federal de Santa Catarina FURB Herbário Dr. Roberto Miguel Klein HAS Herbário Alarich Rudolf Holger Schultz HB Herbário Bradeanum HPBR Herbário Padre Balduíno Romba HPUC Herbário Pontifícia Universidade Católica de Minas Gerais HRB Herbário Radam Brasil HST Herbário Sérgio Tavares HUEFS Herbário da Universidade Estadual de Feira de Santana HUFU Herbário Uberlandense HURB Herbário do Recôncavo da Bahia HVASF Herbário Vale do Rio São Francisco IAN Herbário da Embrapa Amazônia Oriental IBGE Instituto Brasileiro de Geografia e Estatística ICN Herbário do Instituto de Biociências, UFRS IMA Instituto do Meio Ambiente de Alagoas IPA Herbário Dardano de Andrade Lima IPB Instituto Politécnico de Bragança K Royal Botanic Gardens MBML Museu de Biologia Professor Mello Leitão MG Museu Paraense Emilio Goeldi NY The New York Botanical Garden PEUFR Universidade Federal de Pernambuco R Museu Nacional RB Herbário Dimitri Sucre Benjamin Instituto de Pesquisas Jardim Botânico do Rio de Janeiro SP Herbário Maria Eneyda P. K. Fidalgo, Instituto de Botânica de São Paulo SPF Herbário da Universidade de São Paulo UEC Herbário da Universidade Estadual de Campinas UFMT Herbário da Universidade Federal de Mato Grosso UFP Herbário da Universidade Federal de Pernambuco UFPR Herbário da Universidade Federal do Paraná UFRN Herbário da Universidade Federal do Rio Grande do Norte UNB Herbário da Universidade de Brasília US Herbário da Universidade de Sevilla VIC Herbário da Universidade Federal de Viçosa VIES Herbário da Universidade Federal do Espírito Santo

We opted for the non-adoption of infraspecific taxa, as this preposition depends on phenotypic studies and population genotypic, which are in early stages of development. The

Complimentary Contributor Copy 386 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al. only subspecies accepted were those of M. esculenta (M. esculenta subsp. flabellifolia (Pohl) Ciferi and M. esculenta subsp. peruviana (Müll. Arg.) Allem). The primary distribution of 13 species are assessed in Table 2. The georeferencing was assigned by Google Earth 7 tool for herbarium specimens that did not have geographic coordinates to the collection site. Using a checkered mesh 0.5° × 0.5° longitude and latitude and the geographical distribution has been designed in the Neotropics which was divided into 6,818 cells. The ‘status’ of conservation of each species was evaluated from its occurrence Extension (EOO) and Occupation Area (AOO), understanding how EOO area contained within the shortest continuous boundary which can be drawn to encompass all points known, inferred or projected for the actual presence of a taxon, excluding cases of wandering and visitors and as AOO area or the sum of the areas occupied by a taxon within its extent of occurrence (IUCN 2014). Therefore, the distribution data were analyzed by GEOCAT tool (Geospatial Conservation Assessment Tool http://geocat.kew.org/?_ga=1.21973752.216021207. 1461090982) that provides distribution maps with the total area of occurrence and Extension occupation, and automatic categorizing according to the criteria of the International Union for Conservation of Nature (IUCN 2014). The species showed a predominantly central distribution in South America, mainly occupying areas of cerrado and the Amazon rainforest, the Brazilian Central Plateau and Amazon, respectively. Only M. pilosa Pohl showed a concentration in the southeastern region of Brazil, in areas occupied in mostly by deciduous or semideciduous forests and rainforests coastal forests (Figure 10). Conservation status of the species analyzed vary according to the criteria used. The criterion Occupation area (AOO) was less selective in the inclusion of the species most worrying threat levels. Eleven of the 13 analyzed species were considered as Endangered (EN) according to this criterion, with lower occurrence area of 500 km2. Two species (M. marajoara Chermonte de Miranda apud Huber and M. zehntneri Ule) were classified as Critically Endangered (CR) (Table 3).

Table 2. Wild relatives of cassava included in the assessment of the conservation status and geographical distribution

Espécies Distribuição M. brachyloba Müll. Arg. Brasil (AC, AM, PA); Caribe M. esculenta subsp. flabellifolia (Pohl) Ciferri Brasil (AC, AM, GO, MA, MT, TO) M. esculenta subsp. peruviana Müll. Arg. Brasil (MT, RO); Peru M. flemingiana D. J. Rogers & Appan Brasil (AC, GO, MT, RO, TO) M. fruticulosa (Pax) D. J. Rogers & Appan Brasil (DF, GO, MG, SP) M. guaranitica Chodat & Hassl. Brasil (MT); Argentina; Bolívia; Paraguai M. marajoara Chermonte de Miranda apud Huber Brasil (AP, PA) M. pilosa Pohl Brasil (ES, MG, RJ, SP) M. pruinosa Pohl Brasil (GO, MS, MT, TO) M. pusilla Pohl Brasil (DF, GO) M. surinamensis D. J. Rogers &Appan Guiana; Suriname; Venezuela Brasil (GO, MT, MG, RO, TO); Guiana; Suriname; M. tristis Müll. Arg. Venezuela M. zehntnery Ule Brasil (BA)

Complimentary Contributor Copy Wild Relatives of Cassava 387

Table 3. Evaluation of the ‘status’ of current conservation of the species based on the data of this study and comparison with other lists of threatened species (Critically Endangered (E typically five or fewer occurrences or 1,000 or fewer individuals); Danger (typically V- six to twenty occurrences or 1000-3000 individuals); Vulnerable (R-rare, typically 21-100 occurrences or 3,000 to 10,000 people); undetermined (undetermined I-); not in the list of endangered species (S) in Danger. (CR) Critically Endangered; (EN), Vulnerable (VU); Least Concern (LC). AOO (Occupation Area), EOO (Occurrence Extension)

Species IUCN (1997) Status Status EOO Status AOO Manihot brachyloba Müll. Arg. S EN LC Manihot esculenta subsp. flabellifolia (Pohl) Ciferri S EN LC Manihot esculenta subsp. peruviana Müll. Arg. R EN LC Manihot flemingiana D. J. Rogers & Appan E EN LC Manihot fruticulosa (Pax) D. J. Rogers & Appan V EN LC Manihot guaranitica Chodat & Hassl. R* EN LC Manihot marajoara Chermonte de Miranda apud Huber R CR CR Manihot pilosa Pohl S EN LC Manihot pruinosa Pohl S EN LC Manihot pusilla Pohl E EN EN Manihot surinamensis D. J. Rogers & Appan R EN LC Manihot tristis Müll. Arg. E**/R*** EN LC Manihot zehntneri Ule E CR CR *Manihot guaranitica subsp. boliviana (Pax & K. Hoffm.) D. J. Rogers & Appan. **Manihot tristis subsp. surumuensis (Ule) D. J. Rogers & Appan. ***Manihot tristis subsp. tristis Müll. Arg.

1. Manihot brachyloba Müll. Arg., Fl. Bras. 11(2): 451. 1874. Common name: Manioc bicha, maniva brava, maniva de veado, sacharumo, yuca cimarrona, yuca de indio, yuca silvestre. Manihot brachyloba is a species predominantly in the Amazon, occurring at the edge of the forest. It Presents an upright shrubby habit or more commonly vine-like, long roots, but not tuberous. There are few records of its occurrence in Central America (Costa Rica and Dominican Republic) that if proven may change their conservation status. Regarded as LC due to the large EOO and EN regarding the AOO, M. brachyloba had not yet been included in any list of endangered species (Figure 3).

Complimentary Contributor Copy 388 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Figure 3. Manihot brachyloba Müll. Arg.: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

Regarding criterion Extension Occurrence (EOO), 11 species were considered as LC, M. pusilla EN and the same two species (M. marajoara and M. zehntneri) CR. Unlike the results obtained by each of the criteria it took place probably because of the wide distribution of the species, together with the few records, increasing the potential area of occurrence and consequently its EOO, keeping low, however, the AOO values. Notable species considered Critically Endangered in both criteria, M. marajoara and M. zehntneri, which have complex taxonomic situation, which may have influenced the distribution of records and consequently their categorization front of the conservation status.

2. Manihot esculenta subsp. flabellifolia (Pohl) Ciferri, Arch. Bot. (Forlì) 18: 31. 1942. Common name: Mandioca-brava, mandioca-braba. Manihot esculenta subsp. flabellifolia occurs in the Amazon, in edges of forests and roads with tree vegetation. It has an upright shrubby habit or less rarely vine-like and tuberous roots. It offers potential for use in breeding programs with characteristics related to whitefly resistance, virus African mosaic, bacterial blight, anthracnose and mandarová, besides presenting high content of dry matter and protein (Carabali et al. 2010a, Akinbo et al. 2012). Regarded as LC due to the large EOO and EN regarding the AOO, M. esculenta subsp. flabellifolia has not yet been included in any list of endangered species (Figure 4).

3. Manihot esculenta subsp. peruviana (Müll. Arg.) Allem, Gen. Res. Cap. Ev. 41: 146. 1994. Common Name: Yuquilla. Manihot esculenta subsp. peruviana occurs in the Amazon region, in the northwest portion of South America, on edges of forests and roads with tree vegetation. It has vine-like habit and tuberous roots.

Complimentary Contributor Copy Wild Relatives of Cassava 389

Regarded as LC due to the large EOO and EN regarding AOO (Figure 5). It was included in the IUCN Red List (1997) as Vulnerable (R).

Figure 4. Manihot esculenta subsp. flabellifolia (Pohl) Ciferri: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

Figure 5. Manihot esculenta subsp. peruviana (Müll. Arg.) Allem: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

4. Manihot flemingiana D. J. Rogers & Appan, Fl. Neotrop. Monogr. 13: 143 1973. Manihot flemingiana occurs in open shrub cerrado areas in the Midwest region of Brazil and in neighboring areas of the Amazon rainforest. It has shrubby habit subshrub and there is no information about its root morphology. Regarded as LC due to the large EOO and EN regarding the AOO (Figure 6). It appears as EN in the IUCN Red List (Walter & Gillet 1998). Complimentary Contributor Copy 390 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

5. Manihot fruticulosa (Pax) D. J. Rogers & Appan, Fl. Neotrop. Monogr. 13: 149. 1973. Manihot fruticulosa occurs open shrub cerrado areas in the central-eastern region of Brazil. It presents subshrub habit, short roots and is not tuberous, but it is edible according to Nassar et al. (2008). Regarded as LC due to the large EOO and EN regarding the AOO (Figure 7). It appears as Endangered (VU) on the IUCN Red List (Walter & Gillet 1998) and as ‘Medium’ risk of extinction second Nassar et al. (2008).

Figure 6. Manihot flemingiana D. J. Rogers & Appan: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

Figure 7. Manihot fruticulosa (Pax) D. J. Rogers & Appan: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

Complimentary Contributor Copy Wild Relatives of Cassava 391

6. Manihot guaranitica Chodat & Hassl., Bull. Herb. Boissier II, 5: 671 1905. Common name: Higuerilla, Higuerita. Manihot guaranitica occurs in areas of open cerrado and shrub Chaco South-Central region of South America in Argentina, Bolivia, Brazil and Paraguay. It presents shrubby habit, short roots and it is not tuberous.

Figure 8. Manihot guaranitica Chodat & Hassl.: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

The IUCN Red List (Walter & Gillet 1998) includes M. guaranitica subsp. boliviana (Pax & K. Hoffman) D. J. Rogers & Appan as Vulnerable (R). The taxonomic status of this species and its subspecies is undefined and only after revision can its conservation status be evaluated with greater accuracy. At the time, it was decided not to consider the subspecies proposed by Rogers & Appan (1973). Thus, M. guaranitica was considered LC due to the large EOO and EN regarding the AOO (Figure 8).

7. Manihot marajoara Chermonte de Miranda apud Huber, Bol. Mus. Paraense Hist. Nat. 5: 120. 1908. Common name: Mandioca dos Índios, Maniva do Campo. Manihot marajoara occurs in the open areas of northern South America, and in the Brazilian states of Amapá and Pará. It features subshrub habit, short roots and has little tuberous. The 1997 IUCN Red List (Walter & Gillet 1998) includes M. marajoara as Vulnerable (R), however, limited occurrence records could support their inclusion as Critically Endangered (CR) (Figure 9). Gradually records are combined with the fact that their populations are few in number which highlights even more risks. Nevertheless, it is important to say that M. marajoara is closely related to M. surinamensis, with morphological characteristics that may lead to their synonymization, which would alter the status defined here.

Complimentary Contributor Copy 392 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Allem (1980) points out the difficulty of distinguishing M. marajoara and M. surinamensis, which has wider distribution, from Venezuela to Suriname. For the author the differences pointed out by Rogers & Appan (1973) are subtle and weak and may lead them to synonymization. If this occurs, the EOO M. marajoara (older binomial) will be extended by changing their classification.

Figure 9. Manihot marajoara Chermonte de Miranda apud Huber: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

8. Manihot pilosa Pohl, PI. Bras. Ic. et Descr. 1: 55. 1827. Common name: Mandioca Brava, Mandioca Braba, Maniva de Veado. Manihot pilosa is distinguished from other species belonging to the same cassava’s clade by having their distribution concentrated in the southeastern region of Brazil, in semideciduous and deciduous forests fragments. It presents shrubby habit tree, extended and tuberous roots like cassava, but more fibrous. Manihot pilosa was considered LC due to the large EOO and EN by AOO (Figure 10). However, it was Considered EN by Nassar et al. (2008).

9. Manihot pruinosa Pohl, PI. Bras. Ic. et Descr. 1: 28. t. 22. 1827. Nome vulgar: Mandioca Braba. Manihot pruinosa has concentrated distribution in the central region of South America, the Midwest of Brazil, and in areas of the woody cerrado. It presents shrubby habit scandent, tuberous roots like cassava, but more fibrous. It was considered by Allem (2002) as one of the closest species of cassava, the so-called ‘primary gene pool’ (GP1) (Figure 1) and therefore one of the most promising in interspecific crosses with cassava. Occurrence Extension data indicate LC while the AOO include it as EN (Figure 11). It can contribute in cassava breeding programs, increasing the nutritional value due to the high protein content that its roots have (Asiedu et al. 1994 Ojulong et al. 2008, Carabali et al. 2010b). Complimentary Contributor Copy Wild Relatives of Cassava 393

Figure 10. Manihot pilosa Pohl: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

Figure 11. Manihot pruinosa Pohl: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

10. Manihot pusilla Pohl, PI. Bras. Ic. et Descr. 1: 36. t. 26. 1827. Manihot pusilla has concentrated distribution in central Brazil, in areas of open shrub Cerrado in Goiás and the Federal District. It is among the smallest species of the genus, with about 30 cm. It has tuberous roots like cassava, but more fibrous. Manihot pusilla was considered EN in both criteria. According to the IUCN Red List (Walter & Gillet 1998) is among the species CR (Figure 12).

Complimentary Contributor Copy 394 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Figure 12. Manihot pusilla Pohl: Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

11. Manihot surinamensis D.J. Rogers & Appan, Fl. Neotrop. Monogr. 13: 80 1973. Manihot surinamensis is distributed in northern South America, Guyana, Suriname and Venezuela. It is an undergrowth species whose roots have not been investigated. Manihot surinamensis was considered LC due to the large EOO and EN regarding the AOO (Figure 13). According to the IUCN Red List (Walter & Gillet 1998) it was considered VU, but its status may change if synonymized to M. marajoara (see topic on the species). It is likely that the continuation of collecting expeditions in the northern region of South America can contribute to the discovery of more recorded occurrences of this species, contributing to raise the AOO values and consequently changing their conservation status.

Figure 13. Manihot surinamensis D. J. Rogers & Appan. Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

Complimentary Contributor Copy Wild Relatives of Cassava 395

12. Manihot tristis Mueller von Argau in Martius, Fl. Bras. 11(2): 449. 1874. Popular name: Boesi-ingi-kasabu, Maynoc, Wilde Cassava, Yuquilla. Manihot tristis has wide distribution in the north central region of South America. It is traditional distributed only to the north of the South American continent (Rogers & Appan 1973), but there are many records for the state of Goiás, and also to Mato Grosso, Minas Gerais, Rondônia and Tocantins, coming from herbarium material identifications made by Antonio Costa Allem, a specialist in the group taxonomy, therefore they were included in the sample. Manihot tristis is a species with complex taxonomy, with great morphological proximity to M. esculenta subsp. flabellifolia and M. leptopoda (Müll. Arg.) D. J. Rogers & Appan, including proposals synonymization the latter species (Allem 1978). It is a shrubby species whose roots have not been investigated. Was considered LC due to the large EOO and EN regarding the AOO (Figure 14). Manihot tristis subsp. surumuensis (Ule) D. J. Rogers & Appan and M. tristis subsp. tristis Müll. Arg. were included in the CR and EN categories respectively in the Red List of IUCN (Walter & Gillet 1998). It declined to consider these subspecies for categorizing effect on the conservation status because of the precariousness of distribution studies and taxonomy of this species. It has whitefly resistance source, the green mite and contains high content of dry matter (Asiedu et al. 1994, Ojulong et al. 2008, Carabali et al. 2010b). Bolhuis (1953) relates to its use as a protein source. These characteristics reinforce its importance in breeding programs, but it is important that it be accompanied by taxonomic studies.

Figure 14. Manihot tristis Müll. Arg. Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

13. Manihot zehntnery Ule, Bot. Jahrb. Syst. 114: 10. 1914. Manihot zehntneri Ule, referred to the state of Bahia (Rogers & Appan 1973, Nassar et al. 2008, Lamb et al. 2013) was not found in the field, or at least in herbarium collections, with the only record being the holotype. Collected by Zehntner in 1912 (Zehntner 598) in Riacho

Complimentary Contributor Copy 396 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al. de Santana, Bahia, described by Ule (1914), this species has similarities with the cultivated species M. esculenta Crantz. Rogers & Appan (1973), the holotype photo analysis pointed out this similarity but approximated the species of Glazioviannae Pax emend Rogers & Appan section due to the arboreal habit and its use for rubber extraction, common in the early XX century (Ule 1914). During an expedition held in the city of Riacho do Santana and region there were recorded only individual trees known as “Mandioca de Sete Anos” (Tree Cassava) (Araújo et al. 2001). The characteristics of this material fall on those described by Ule for M. zehntneri and the development of further studies can confirm its relationship to this species and indicate their synonymization M. esculenta. It was regarded as CR in both the analytical approach which supports the categorization of the IUCN Red List (Walter & Gillet 1998) (Figure 15).

Figure 15. Manihot zehntneri Ule. Distribution and Conservation Status according to Extent of Occurrence and Area of Occupancy (Image from Geocat, powered by Google® 2015).

CONCLUSION

Only three among the analyzed species presented preliminary studies that suggests valuable features to cassava improvement. They are all strains closely related to M. esculenta, according to latest phylogeny of the genus as M. esculenta subsp. flabellifolia, M. tristis and M. pruinosa, which tends to facilitate the crossing and, consequently, the share of those characteristics with the cultivated species. These characteristics are related to resistance to pests and diseases such as whitefly, African mosaic virus, bacterial blight, anthracnose, green mite and caterpillar ‘mandarová,’ as observed in M. esculenta subsp. flabellifolia and M. tristis. These species are still high dry matter content and the three mentioned species have high protein content in their roots. Some species not included in the analysis do not appear in the same clade M. esculenta also had important features to improve. According to Asiedu et al. (1994), Ojulong et al. (2008), Carabali et al. (2010b) M. reptans Pax has a high protein content, as well as M.

Complimentary Contributor Copy Wild Relatives of Cassava 397 alutacea D. J. Rogers & Appan, M. falcata D. J. Rogers & Appan, M. paviaefolia Pohl, and M. pentaphylla Pohl (Nassar 1978c). Other species also have potential use as M. glaziovii Müll. Arg. that is resistant to the virus African mosaic, bacterial blight resistance and drought tolerance (Hahn et al. 1980a, 1980b, Ambang et al. 2007, Nassar et al. 2010), M. tripartita (Spreng) Müll. Arg., drought tolerance (Nassar 1978a), M. oligantha Pax, rich in starch, protein and lutein and is resistant to drought, aluminum and cold (Nassar 1978a, 1986) and high content of essential amino acids such as methionine, lysine and tryptophan (Nassar & Sousa 2007), and further, M. dichotoma Ule, which shows high production of roots (Nassar et al. 2004). This fact suggests that these species can also be the subject of breeding programs due to the ease of hybridization seen in the genre. Regarding the conservation status of these species and the discrepancy observed between the occurrence Extension analysis (EOO) and Occupation Area (AOO) has probably given to the precariousness of the data. Regular expeditions of collection of wild species of Manihot, held since 2010 have helped to increase the distribution of data and also to broaden the outlook of each species ‘in loco,’ allowing the verification of their habitat conservation status, the number of individuals of each population, etc. However, expeditions have not yet been made expeditions specifically aimed at the closest relatives of cassava, addressed in this study. Associated with this, it is understood that the increase in studies on the taxonomy of these species can positively affect the categorization regarding the conservation status. Nevertheless, the data measured in this study contributes significantly to the Endangered Species List in Brazil, which so far includes only M. procumbens Müll. Arg., as inconsistent with the timely distribution presented by most species of Manihot and the level of degradation of their ecosystems. It is emphasized that maintaining wild relatives of cassava germplasm bank is a practice of fundamental importance for the improvement of this culture, because the programs rely on the introduction of alleles with valuable agronomic traits contained in these species to minimize the limitations found in culture as pests and diseases.

REFERENCES

Akinbo, O., Labuschagne, M. and Fregene, M. (2012). Introgression of whitefly (Aleurotrachelus socialis) resistance gene from F1 inter-specific hybrids into commercial cassava. Euphytica, 183(1): 19-26. Allem, A. C. (1977). Notas taxonômicas e novos sinônimos em espécies de Manihot - I (Euphorbiaceae). Revista Brasileira de Biologia, 37(4): 825–835. [Taxonomical and new notes synonyms in species Manihot - I (Euphorbiaceae). Brazilian Journal of Biology, 37 (4): 825-835.]. Allem, A. C. (1978). Notas taxonômicas e novos sinônimos em espécies de Manihot - II (Euphorbiaceae). Revista Brasileira de Biologia, 38(3): 721–726. [Taxonomical and new notes synonyms in species Manihot - II (Euphorbiaceae). Brazilian Journal of Biology, 38(3): 721–726.].

Complimentary Contributor Copy 398 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Allem, A. C. (1979a). Notas taxonômicas e novos sinônimos em espécies de Manihot - III (Euphorbiaceae). Revista Brasileira de Biologia, 39(3): 545–550. [Taxonomical and new notes synonyms in species Manihot - III (Euphorbiaceae). Brazilian Journal of Biology, 39(3): 545–550.]. Allem, A. C. (1979b). Notas taxonômicas e novos sinônimos em espécies de Manihot - IV (Euphorbiaceae). Revista Brasileira de Biologia, 39(4): 735–738. [Taxonomical and new notes synonyms in species Manihot - IV (Euphorbiaceae). Brazilian Journal of Biology, 39(4): 735–738.]. Allem, A. C. (1979c). Notas taxonômicas e novos sinônimos em espécies de Manihot - V (Euphorbiaceae). Revista Brasileira de Biologia, 39(4): 891–896. [Taxonomical and new notes synonyms in species Manihot - V (Euphorbiaceae). Brazilian Journal of Biology, 39(4): 891–896.]. Allem, A. C. (1980). Notas taxonômicas e novos sinônimos em espécies de Manihot - VI (Euphorbiaceae). Boletim do Museu Botânico Municipal. 40: 1–14. [Taxonomical and new notes synonyms in species Manihot - VI (Euphorbiaceae). Bulletin Municipal Botanical Museum, 40: 1–14.]. Allem, A. C. (1989a). Four new species of Manihot (Euphorbiaceae) from Brazil. Revista Brasileira de Biologia, 49(3): 649–662. Allem, A. C. (1989b). A Revision of Manihot section Quinquelobae (Euphorbiaceae). Revista Brasileira de Biologia, 49(1):1–26. Allem, A. C. (1999). A new species of Manihot (Euphorbiaceae) from the Brazilian Amazon. International Journal of Plant Sciences, 160: 181–187. Allem, A. C. (2001). Three New Infraspecific Taxa of Manihot (Euphorbiaceae) from the Brazilian Neotropics. Novon, 11(2): 157-165. Allem, A. C. (2002). Cassava: biology, product and utilization. In The origins and taxonomy of Cassava (Hillocks, R. J.; Thresh, J. M. and Bellotti, A. C., eds.). Natural Resources Institute, Greenwich. p. 1–16. Allem, A. C. (1999). The closest wild relatives of cassava (Manihot esculenta Crantz). Euphytica 107: 123–133. Asiedu, R., Hahn, S. K., Vijaya Bai, K. and Dixon, A. G. O. (1994). Interspecific hybridization in the genus Manihot-progress and prospects. Acta Horticulturae, 380: 110–113. Bai, K. V., Asiedu, R. and Dixon, A. G. O. (1993). Cytogenetics of Manihot Species and Interspecific Hybrids. In: W.M. Roca & A.M. Thro (Eds.), Proc First Intl Sci Meet 19 Cassava Biotechn Netw, pp. 51–55. Cartagena, Colombia, 25–28 August 1992. Centro Internacional de Agricultura Tropical, Cali, Colombia, 1993. CIAT Working Document n.123. Barroso, G. M., Amorim, M. P., Peixoto, A. L. and Ichaso, C. L. F. (1999). Frutos e sementes. Morfologia aplicada à sistemática de dicotiledôneas. Editora UFV, Universidade Federal de Viçosa. 443p. [Fruits and seeds. Morphology applied to the system of dicots. UFV, Viçosa Federal University. 443p.]. Bauhin, J. (1651). Historia plantarum universalis. 2: 794. Berry, P. E., Hipp, A. L., Wurdack, K. J., Van Ee, B. and Riina, R. (2005). Molecular phylogenetics of the giant genus Croton and tribe Crotoneae (Euphorbiaceae sensu stricto) using ITS and trnL-trnF DNA sequence data. American Journal of Botany. 92: 1520–1534. Complimentary Contributor Copy Wild Relatives of Cassava 399

Bolhuis, G. G. (1953). A survey of some attempts to breed cassava varieties with a high content of protein in the roots. Euphytica. 20: 107–112. Bomfim, N. N., Graciano-Ribeiro D. and Nassar N. M. (2011). Genetic diversity of root anatomy in wild and cultivated Manihot species. Genetic and Molecular Research. 10(2): 544–51. Carabalí, A., Bellotti, A. C., Montoya-Lerma, J. and Fregene, M. (2010a). Manihot flabellifolia Pohl, wild source of resistance to the whitefly Aleurotrachelus socialis Bondar (Hemiptera: Aleyrodidae). Crop Protection. 29(1): 34–38. Carabalí, A., Bellotti, A. C., Montoya-Lerma, J. and Fregene, M. (2010b). Resistance to whitefly, Aleurotrachelussocialis, in wild populations of cassava, Manihot tristis. Journal of Insect Science, 10(170): 1–10. Carmo-Jr, J. E., Sodre, R. C., Silva, M. J. da & Sales, M. F. (2013). Manihot (Euphorbiaceae s.s.) no Parque Estadual da Serra Dourada, Goiás, Brasil. Rodriguésia, 64: 727–746. [Manihot (Euphorbiaceae s.s.) in Golden Hill State Park, Goiás, Brazil. Rodriguésia, 64: 727–746.]. Carvalho, R. and Guerra, M. (2002). Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species. Hereditas. 136: 159–168. Chacon, J., Madriñan, S., Debouck, D., Rodriguez, F. and Tohme, J. (2008). Phylogenetic patterns in the genus Manihot (Euphorbiaceae) inferred from analyses of nuclear and chloroplast DNA regions. Molecular Phylogenetics and Evolution, 49: 260–267. Colombo, C., Second, G. and Charrier, A. (2000). Diversity within American cassava germoplasm based on RAPD markers. Genetics and Molecular Biology. 31(1): 189–199. Cordeiro, I., Secco, R., Silva, M. J. da, Sodré, R. C. and Martins, M. L. L. Manihot. Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. 2014 [Manihot. Species List of Flora of Brazil. Rio de Janeiro Botanical Garden]. (04 jul 2016). Available from URL: http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/ FB17591. Corrêa, A. M. S., Barros, M. A. V. C, Silvestre-Capelato, M. F. S., Pregun, M. A., Raso, P. G. and Cordeiro, I. (2010). Flora polínica da Reserva do Parque Estadual das Fontes do Ipiranga (São Paulo, Brasil). [Flora pollinic of Ipiranga Sources State Park] Hoehnea. 37(1): 53–69. Croizat, L. (1943). Preliminari per uno Studio Del genere Manihot nell' America Meridionale. Revista Argentina de Agronomia. 10(3): 213–226. [Preliminary study of genus Manihot of southern America. Argentina Journal of Agronomy. 10(3): 213–226.]. Cruz, N. D. (1968). Citologia no gênero Manihot Adans. I. Determinação do número de cromossomos em algumas espécies. Annais Academia Brasileira de Ciências. 40: 91-95. [Cytology in the genus Manihot Adans. I. Determination of the number of chromosomes in some species. Annals of Brazilian Academy of Sciences. 40: 91–95.]. Cruz-Barros, M. A. V., Corrêa, A. M. S. and Makino-Watanabe, H. (2006). Estudo polínico de Aquifoliaceae, Euphorbiaceae, Lecythidaceae, Malvaceae, Phytolaccaceae e Portulacaceae ocorrentes na restinga da Ilha do Cardoso (Cananéia, SP, Brasil). Revista Brasileira de Botânica. 29: 145-162. [Pollinic study by Aquifoliaceae, Euphorbiaceae, Lecythidaceae, Malvaceae, Phytolaccaceae and Portulacaceae occurring in the sandbank of Cardoso Island (Cananéia, SP, Brazil). Brazilian Journal of Botany. 29: 145–162.]. Cunha-Neto, I. L., Martins, F. M. Caiafa, A. N. and Martins, M. L. L. (2014). Leaf anatomy as subsidy to the taxonomy of wild Manihot species in Quinquelobae section (Euphorbiaceae). Brazilian Journal of Botany, 37: 481–494. Complimentary Contributor Copy 400 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Daniell, H., Wurdack, K. J., Kanagaraj, A., Lee, S. B., Saski, C. and Jansen, R. K. (2008). The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron. Theoretical Applied Genetics. 116: 723–737. Dehgann, B. (1984). Phylogenetic significance of interspecific hybridization in Jatropha (Euphorbiaceae). Systematic Botany, 9: 467–478. Dunstan, W. R., Henry, T. A. and Auld, S. J. M. (1906). Cyanogenesis in Plants. Part V. The Occurrence of Phaseolunatin in Cassava (Manihot aipi and Manihot utilissima). Proceedings of the Royal Society of London. B. 78: 152–158. Duputié, A., Salick, J. and McKey, D. (2011). Evolutionary biogeography of Manihot (Euphorbiaceae), a rapidly radiating Neotropical genus restricted to dry environments. Journal of Biogeography. 1: 1–11. FAO. Food and Agriculture Organization of the United Nations. 2015 (06 jan 2016). Available from: URL: http://faostat3.fao.org/faostat-gateway/go/to/download/Q/QC /E. Fukuda, W. M. G., Cavalcanti, J., Fukuda, C. and Costa, I. R. S. (1999). Variabilidade genética e melhoramento da mandioca (Manihot esculenta Crantz). In: Recursos Genéticos e Melhoramento de Plantas para o Nordeste Brasileiro. 1 ed. Petrolina. 14p. [Genetic variability and improvement of cassava (Manihot esculenta Crantz). In: Genetic Resources and Plant Breeding for the Brazilian Northeast. 1 ed. Petrolina. 14p.]. Graciano-Ribeiro, D., Hashimoto, D. Y., Nogueira, L. C & Teodoro, D. (2009). Internal phloem in an interspecific hybrid of cassava, an indicator of breeding value for drought resistance. Genetic and Molecular Research. 8: 1,139-1,146. Graciano-Ribeiro, D., Nassar, N. M. A., Hashimoto, D. Y. C & Miranda, S. F. (2008). Anatomy of polyploid cassava and its interspecific hybrids. Gene Conserve. 7: 620–635. Grattapaglia, D., Nassar, N. M. A. and Dianese, J. C. (1987). Biossistemática de espécies brasileiras do gênero Manihot baseada em padrões de proteína de semente. Ciência & Cultura. 39: 294–300. [Biosystematics of species of the genus Manihot based on seed protein patterns. Science & Culture. 39: 294-300.]. Halsey, M. E., Olsen, K. M., Taylor, N. J. and Chavarriaga-Aguirre, P. (2008). Reproductive Biology of Cassava (Manihot esculenta Crantz) and Isolation of Experimental Field Trals. Crop Science, 48: 49–58. Hans A. S. (1973). Chromosomal conspectus of the Euphorbiaceae. Taxon. 22: 591–636. Hassal, D. C. (1976). Numerical and Cytotaxonomic Evidence for Generic Delimitation in Australian Euphorbieae. Australian Journal of Botany. 24: 633–40. Hayirlioglu-Ayaz, S., Huseyin, I. and Beyazoglu, O. (2002). Cytotaxonomic studies on some Euphorbia L. (Euphorbiaceae) species in Turkey. Pakistan Journal of Botany. 34(1): 27– 31. IUCN International Union for Conservation of Nature (1997). Red List of Threatened Plants. Magnoliopsida. Compiled by the World Conservation Monitoring Center. IUCN - The World Conservation Union, Gland, Switzerland and Cambridge, UK. 862pp. IUCN International Union for Conservation of Nature. Guidelines for using the IUCN red list categories and criteria, ver. 1.1. 2014 (02 may 2016). Available from: URL: http:www.iucnredlist.org/documents/RedListGuidelines.pdf Jennings, D. L. (1959). Manihot melanobasis Müll. Arg. – A useful parent for cassava breeding. Euphytica 8: 157–162. Jennings, D. L. and Iglesias C. (2002). Breeding for crop improvement. In: Cassava biology, Complimentary Contributor Copy Wild Relatives of Cassava 401

production and utilization (Hillocks RJ, Tresh JM and Belloti AC, eds.). CAB International, Wallingford, Oxon, 149-166. Jennings, D. L. (1963). Variation in pollen and ovule fertility in varieties of cassava, and the effect of interspecific crossing on fertility. Euphytica. 12: 69–76. Kulju, K. K. M., Sierra, S. E. C. and Van Welzen, P. C. (2007). Reshaping Mallotus [Part 2]: inclusion of Neotrewia, Octospermum and Trewia in Mallotus s.s. (Euphorbiaceae s.s.). Blumea. 52: 115–136. Leitch, A. R., Lim, K.Y., Leitch, I. J., O.Neill, M., Chye, M. and Low, F. (1998). Molecular cytogenetic studies in rubber, Hevea brasiliensis Muell. Arg. (Euphorbiaceae). Genome. 41: 464–467. Léotard, G., Duputié, A., Kjellberg, F., Douzery, E., Debain, C., de Granville, J. J. and McKey, D. (2009). Phylogeography and the origin of cassava: new insights from the northern rom of the Amazonian basin. Molecular Phylogenetics and Evolution. 53: 329– 334. Machado, I. C. S., Barros, L. M. and Sampaio, E. V. S. B. (1997). Phenology of caatinga species at Serra Talhada, PE, Northeastern Brazil. Biotropica. 29: 57–68. Magoon, M. L., Krishnan, R. and Vijayabai, K. (1970). Cytogenetcs of the F. hybrid between cassava and ceara rubber and its back crosses. Genetica. 41: 3-12. Majumder, S. K. (1964). Studies on the germination pollen of Hevea brasiliensis in vitro and on artificial media. Journal of the Rubber Research Institute of Malaysia. 18: 185–193. Marchioretto, M. S, Windisch, P. G. and Siqueira, J. C de (2004). Padrões de distribuição geográfica das espécies de Froelichia Moench e Froelichiella R. E. Fries Amaranthaceae) no Brasil. Iheringia, Sér. Bot., Porto Alegre, 59(2): 149–159. Martinelli, G. and Moraes, M. A. (Orgs.). (2013). Livro Vermelho da Flora do Brasil. 1ªEd. Rio de Janeiro: Andrea Jacoksson: Instituto de Pesquisas Jardim Botânico do Rio de Janeiro. 1,100p. [Red Book of Flora of Brazil. 1ªEd. Rio de Janeiro: Andrea Jacoksson: Research Institute Botanical Garden of Rio de Janeiro. 1,100p.]. Martins, M. L. L. (2013). Avanços taxonômicos em Manihot Mill. para o Brasil. PhD Thesis, Universidade Estadual de Feira de Santana. [Taxonomic advances in Manihot Mill. to Brazil. PhD Thesis, University of Feira de Santana.]. Martins, M. L. L. and Ledo, C. A. S. (2015). Manihot cezarii (Euphorbiaceae), a new species from Central-Western of Brazil. Novon. 24: 179–181. Martins, M. L. L., Carvalho, P. C. L. and Amorim, A. M. A. (2011). A remarkable Manihot (Euphorbiaceae) from the coastal sand plains of Sergipe, Brazil. Phytotaxa. 32: 57–60. Martins, M. L. L., Carvalho, P. C. L., Ledo, C. A. S. and Amorim, A. M. A. (2014). What’s New in Manihot Mill. (Euphorbiaceae)? Systematic Botany. 39(2): 485–489. Mendoza, J. M. (2013). Manihot (Euphorbiaceae) en Bolivia, parte I: Tres especies nuevas y un nuevo registro. Brittonia. 38: 42–57. [Manihot (Euphorbiaceae) in Bolivia, Part I: Three new species and a new record. Brittonia. 38: 42-57.]. Mendoza, J. M. (2010). Los parientes silvestres del cultivo de la Yuca (casaba) en Bolivia: estado de conocimiento, grado de conservación y acciones de conservación propuestas. La Paz: Imprenta Sagitário. 142 p. [Wild relatives of cassava in Bolivia: state of knowledge, degree of conservation and proposed conservation actions. La Paz: Printing Sagittarius. 142 p.].

Complimentary Contributor Copy 402 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Mendoza, J. M., Simon, M. F. and Cavalcanti, T. B. (2015). Three new endemic species of Manihot (Euphorbiaceae) from the Chapada dos Veadeiros, Brazil. Arnaldoa. 22: 297– 312. Miller, K. I. and Webster, G. L. (1966). Chromosome Numbers in the Euphorbiaceae. Brittonia. 18(4): 372–379. Müller, A. (1873–1874). Euphorbiaceae. In.: Martius, C.F.P. and Eichler, A.G., Flora Brasiliensis. Monachii et Lipsiae. 11(2): 292. Müller, J. (1866). Euphorbiaceae exceto subordo Euphorbieae. In: A. P. De Candolle (ed.). Prodromus Systematics Universalis Regni Vegetabilis. 15: 189–1,286. Nassar, H. N. M. and Freitas, M. (1997). Prospects of polyploidizing cassava, Manihot esculenta Crantz, by unreduced microspores. Plant Breeding. 116: 195–197. Nassar, N. M. A. and Souza, M. (2007). Amino acid profile in cassava and its interspecific hybrid. Genetics and Molecular Research, 6: 92–197. Nassar, N. M. A. (1978a). Conservation of the genetic resources of cassava (Manihot esculenta): determination of wild species localities with emphasis on probable origin. Economic Botany. 32: 311–320. Nassar, N. M. A. (1978b). Hydrocyanic acid content in some wild Manihot (cassava) species. Canadian Journal of Plant Science. 58: 577–578. Nassar, N. M. A. (1978c). Microcenters of wild cassava Manihot spp. Diversity in Central Brazil. Turrialba. 28: 345–347. Nassar, N. M. A. (1979a). A study of the collection and maintenance of the germplasm of wild cassavas Manihot spp. Turrialba. 29: 221–221. Nassar, N. M. A. (1979b). Three Brazilian Manihot species with tolerance to stress conditions. Canadian Journal of Plant Science. 59: 533–555. Nassar, N. M. A. (1980). Attempts to hybridize wild Manihot species with cassava. Economic Botany. 34: 13–15. Nassar, N. M. A. (1982). Collecting wild cassavas in Brazil. Indian Journal of Genetic. 42: 405–411. Nassar, N. M. A. (1984). Natural hybrids between Manihot reptans Pax and M. alutacea Rogers & Appan. Canadian Journal of Plant Science. 64: 423–425. Nassar, N. M. A. (1985). Manihot neusana Nassar: a new species native to Paraná, Brazil. Canadian Journal of Plant Science. 65: 1,097–1,100. Nassar, N. M. A. (1986). Genetic variation of wild Manihot species native to Brazil and its potential for cassava improvement. Field Crops Research. 13: 177–184. Nassar, N. M. A. (2000a). Cassava, Manihot esculenta Crantz genetic resources: Their collection, evaluation, and manipulation. Advances in Agronomy. 69: 179–230. Nassar, N. M. A. (2000b). Cytogenetics and evolution of cassava (Manihot esculenta Crantz). Genetic and Molecular Biology. 23: 1,003–1,014. Nassar, N. M. A. (2000c). The transference of apomixis genes from Manihot neusana Nassar to cassava, Manihot esculenta Crantz. Hereditas 132: 167–170. Nassar, N. M. A. (2002). Keeping options alive and threat of extinction: a survey of wild cassava survival in its natural habitats. Gene Conserve. 1: 10. Nassar, N. M. A. (2003). Gene flow between cassava, Manihot esculenta Crantz, and wild relatives. Genetics Molecular Research. 4(2): 334–347.

Complimentary Contributor Copy Wild Relatives of Cassava 403

Nassar, N. M. A. (2006). Mandioca: Uma opção contra a fome estudos e lições do Brasil e do mundo. Ciência Hoje, 39(231): 31–34. [Cassava: An option against hunger studies and lessons from Brazil and the world. Science Today, 39 (231): 31-34.] Nassar, N. M. A. Hashimoto, D. Y. C. and Fernandes, S. D. C. (2008). Wild Manihot species: botanical aspects, geographic distribution and economic value. Genetics and Molecular Research. 7(1): 16–28. Nassar, N. M. A., Alves, J. and Souza, E. (2004). UnB 033: An interesting interspecific cassava hybrid. Revista Ceres. 51(296): 495–499. Nassar, N. M. A., Bomfim, N., Chaib, A., Abreu, L. F. A. and Gomes, P. T. C. (2010). Compatibility of interspecific Manihot crosses presaged by protein electrophoresis. Genetics and Molecular Research. 9(1): 107–112. Neves, E. L., Funch, L. S. and Viana, B. F. (2010). Comportamento fenológico de três espécies de Jatropha (Euphorbiaceae) da Caatinga, semi-árido do Brasil. Revista Brasileira de Botânica. 33: 155–166. [Phenological behaviour of three species of Jatropha (Euphorbiaceae) Caatinga, semi-arid region of Brazil. Brazilian Journal of Botany. 33: 155–166.]. Nowicke, J. W. (1994). A palynological study of Crotonoideae (Euphorbiaceae). Annals of the Missouri Botanical Garden 81: 245–269. Ojulong, H., Labuschangne, M., Herselman, L. and Fregene, M (2008). Introgression of genes for dry matter content from wild cassava species. Euphytica. 164(1): 163–172. Oliveira, M. M. de. Diversidade genética em espécies silvestres e híbridos interespecíficos de Manihot. 2011. 86 f. MSc. Dissertation (Agricultural Sciences) – Centro de Ciências Agrárias, Ambientais e Biológicas. Universidade Federal do Recôncavo da Bahia. 2011. [Genetic diversity of wild species and interspecific hybrids of Manihot. 2011. 86 f. MSc Dissertation (Agricultural Sciences) – Centre of Agricultural Sciences, Environmental and Biological. Federal University of Reconcavo of Bahia. 2011.]. Oliveira‚ J. H. G. and Oliveira‚ D. M. T. (2009). Morphology‚ anatomy and ontogeny of the pericarp of Manihot caerulescens Pohl and M. tripartita Müll. Arg. (Euphorbiaceae). Revista Brasileira de Botânica. 32(1): 117–129. Olsen, K. M. and Schaal, B. A. (1999). Evidence on the origin of cassava: Phylogeography of Manihot esculenta. Proceedings of the National Academy of Sciences. 96: 5,586–5,591. Olsen, K. M. (2002). Population history of Manihot esculenta (Euphorbiaceae) inferred from nuclear DNA sequences. Molecular Ecology. 11: 901–911. Olsen, K. M. and Schaal. B. A. (2001). Microsatellite variation in cassava (Manihot esculenta Euphorbiaceae) and its wild relatives: further evidence for a southern Amazonian origin of domestication. American Journal of Botany, Missouri, 88(1): 131–142. Orlandini, P. and Lima, L. R. (2014). Sinopse do gênero Manihot Mill. (Euphorbiaceae) no Estado de São Paulo, Brasil. Hoehnea. 41: 51–60. Pax, F. (1910). Manihot Adans. In:Engler, Pflanzenreich IV. 147(Heft 44): 21–111. Pohl, J. (1827). Plantarum Brasiliae Icones et Descriptiones. 1: 17–56. Punt, W. (1962). Pollen morphology of the Euphorbiaceae with special reference to taxonomy. Wentia. 7: 1–116. Reich, P. B., Uhl, C., Walters, M. B., Prugh, L. and Ellsworth, D. S. (2004). Leaf demography and phenology in Amazonian rain forest: A census of 40000 leaves of 23 tree species. Ecology Monograph. 74: 3–23.

Complimentary Contributor Copy 404 M. Lacerda Lopes Martins, C. A. da Silva Ledo, P. C. Lemos de Carvalho et al.

Roa, A. C., Maya, M. M., Duque, M. C., Tohme, J., Allem, A. C. and Bonierbale, M. W. (1997). AFLP analysis of relationships among cassava and other species. Theoretical and Applied Genetics. 95: 741–750. Rodrigues, A. S. (2007). As tribos Dalechampieae Müll. Arg. e Manihotae Melchior (Euphorbiaceae) no Distrito Federal, Brasil. Dissertation (Mestrado em Botânica), Universidade de Brasília. 104p. [The tribes Dalechampieae Müll. Arg. and Manihotae Melchior (Euphorbiaceae) in the Federal District, Brazil. Dissertation (MSc. Botany), University of Brasilia. 104p.]. Rogers, D. J. and Appan, S. G. (1973). Manihot and Manihotoides (Euphorbiaceae): A Computer Assisted Study. Flora Neotropica (Monograph No. 13). Hafner Press, New York. 272p. Rós, A. B., Hirata, A. C. S., Araújo, H. S. and Narita, N. (2011). Crescimento, fenologia e produtividade de cultivares de mandioca. Pesquisa Agropecuária Tropical. 41(4): 552– 558. [Growth, phenology and productivity of cassava cultivars. Journal of Tropical Agriculture. 41(4): 552–558.]. Rudall, P. (1994). Laticifers in Crotonoideae (Euphorbiaceae): Homology and Evolution. Annals of the Missouri Botanical Garden. 81(2): 270–282. Sasikala R. and Paramathma M. (2010). Chromosome studies in the genus Jatropha L. Electronic Journal of Plant Breeding. 1: 637–642. Satiro, L. N. and Roque, N. (2008). Levantamento da família Euphorbiaceae Juss. nas caatingas arenosas do Médio São Francisco. Acta Botanica Brasilica. 22(1): 99–118. [Family survey Euphorbiaceae Juss. in the sandy scrublands of the Middle São Francisco. Acta Botanica Brasilica. 22(1): 99–118.]. Silva, M. J. da (2016). Manihot gratiosa and M. lourdesii spp. nov. (Manihoteae, Euphorbiaceae) from the Brazilian Cerrado. Nordic Journal of Botany. 34: 66–74. Silva M., Sodré R. C. and Almeida L. C. S. (2013). A new endemic species of Manihot (Euphorbiaceae s. str.) from the Chapada dos Veadeiros, Goiás, Brazil. Phytotaxa. 131 (1): 53–57.\ Silva, M. J. (2015). Manihot appanii (Euphorbiaceae s.s.) a New Species from Brazil, and a Key to the Species with Unlobed or Very Shortly Lobed Leaves. Systematic Botany. 40: 168–173. Soberon, J. and Peterson, A. T. (2005). Interpretation of Models of fundamental ecological niches and species' distributional areas. Biodiversity Informatics. 2: 1–10. Steudel, E. T. (1840, 1841). Nomenclator Botanicus. Part I, 1,840, p. 779–800; Part II, 1,841, p. 99. Tannus, J. L. S., Assis, M. A. and Morellato, L. P. C. (2006). Fenologia reprodutiva em campo sujo e campo úmido numa área de Cerrado no sudeste do Brasil, Itirapina -SP. Biota Neotropica. 6(3): 1–27. [Phenology in dirty field and wet field in a Cerrado area in southeastern Brazil, Itirapina SP. Biota Neotropica. 6 (3): 1–27.]. Thiers, B. [continuously updated]. Index Herbariorum: A global directory of public herbaria and associated staff. New York Botanical Garden's Virtual Herbarium. Available from: URL: http://sweetgum.nybg.org/ih/. Tokuoka, T. and Tobe, H. (2002). Ovules and seeds in Euphorbioideae (Euphorbiaceae): structure and systematic implications. Journal of Plant Research. 115: 361–374. Ule, E. (1907). Vorläufi ge Mitteilung über drei noch unbeschriebene Kautschui liefernde Manihot-Arten in Bahia. Notizblatt Königl Botanischen Gartens und Museums zu Berlin- Complimentary Contributor Copy Wild Relatives of Cassava 405

Dahlem. 41: 119. [Preliminary Communication about three undescribed rubber supplying cassava species in Bahia. Notepad Royal Botanical Gardens and Museum in Berlin- Dahlem. 41: 119.]. Ule, E. (1914). Beitrage zur Kenntnis der brasilianischen Manihot-Arten. Beiblatt zu den Botanischen Jahrbuchern. 5: 1–12. [Contributions to the knowledge of Brazilian cassava species. Supplement to the Botanical year Bookers. 5: 1–12.]. Walter, K. S. and Gillett, H. J. 1997 IUCN Red List of Threatened Plants. Compiled by the World Conservation Monitoring Centre. IUCN - The World Conservation Union, Gland, Switzerland and Cambridge, UK. lxiv, 1998, 862p. Webster, G. L. (1958). A monographic study of the West Indian species of Phyllanthus. Journal of Arnold Arboretum. 39: 111–212. Webster, G. L. (1994). Synopsis of the genera and suprageneric taxa of Euphorbiaceae. Annals of Missouri Botanical Garden. 81: 33–144. Willis, K. J., Araujo, M. B., Bennett, K. D., Figueroa-Rangel, B., Froyd, C. A. and Myerl, N. (2007). How can a knowledge of the past help to conserve the future? Biodiversity conservation and the relevance of long-term ecological studies. Philosophicall Transactions of the Royal Society B-Biological Sciences. 362: 175–186.

Complimentary Contributor Copy Complimentary Contributor Copy

INDEX

bioproducts, xi, 22, 149, 150, 151, 156, 157, 158, # 159, 160, 161, 171, 174, 175, 192, 197, 269, 309 biosurfactants, xii, 158, 172, 173, 174, 175, 177, 16S rRNA gene, xiii, 231, 235 178, 192, 193, 194, 195, 196, 197 bitter cassava, 107, 108, 175, 263, 273, 298 A bivariate probit model, ix, 56, 57, 60, 61, 62, 66, 70, 73, 75, 81 agbelima, 321, 322, 330 amylopectin, 5, 30, 102, 118, 153, 275, 300, 303, C 338, 342, 343 amylose, 5, 30, 93, 94, 118, 153, 275, 300, 302, 303, calcium, vii, xi, 13, 15, 20, 29, 30, 90, 91, 106, 124, 307, 338, 342, 343 152, 153, 171, 176, 232, 241, 248, 253, 276, 304, ash, xiv, xv, 15, 20, 106, 110, 154, 271, 274, 275, 306 281, 286, 287, 298, 299, 336, 337, 339, 343, 347, Caldicellulosiruptor saccharolyticus, 137, 144 352, 377 Caloramator boliviensis, 146, 185, 197 carbohydrate, 5, 30, 91, 97, 99, 132, 169, 175, 180, B 193, 232, 252, 272, 274 carotenoids, xi, 107, 108, 149, 159, 161, 168, 169 bacterial universal primers, 235 cassava peel, xiii, xv, 114, 130, 132, 140, 143, 180, bacteriocin, xiii, 231, 237, 238, 239, 241, 242 181, 192, 231, 232, 233, 234, 236, 237, 244, 246, beta- glucosidase, xiii, 231 247, 248, 313, 318, 322, 324, 325, 326, 327, 328, bioethanol, 3, 4, 22, 23, 24, 25, 26, 27, 28, 131, 132, 329, 331, 332 135, 136, 138, 139, 140, 141, 143, 144, 145, 146, cassava processing waste, x, 129, 131, 132, 139, 145, 147, 160, 169, 173, 175, 182, 183, 184, 185, 197, 153, 167, 194, 199 252, 260, 266, 267, 268, 285, 286, 293, 331 cassava production, viii, ix, xiv, 35, 36, 41, 42, 43, bio-fertilizer, 131, 134, 135, 137, 139 44, 46, 55, 56, 57, 63, 72, 81, 82, 139, 152, 202, biofortified cassava, 108 203, 207, 209, 210, 211, 212, 213, 214, 232, 272, biofuels, vii, xi, 1, 5, 6, 16, 24, 25, 28, 130, 157, 158, 294, 313, 314, 315, 317, 328, 358 160, 171, 174, 182, 183, 184, 192, 194, 195, 256, cassava residue, xiv, 142, 176, 313, 324, 329 331, 353, 355 cassava wastewater, xi, xiii, 144, 152, 155, 156, 157, biogas, x, 129, 131, 133, 134, 135, 137, 138, 139, 158, 162, 171, 173, 174, 175, 176, 177, 178, 179, 140, 141, 142, 143, 144, 145, 146, 147, 175, 179, 180, 181, 182, 184, 185, 187, 188, 189, 190, 191, 180, 181, 182, 186, 187, 188, 189, 190, 194, 196, 192, 193, 194, 195, 196, 197, 199, 231, 233, 234, 198, 199, 260 236, 243, 245, 246, 247 bio-hydrogen, x, 129, 131, 133, 134, 136, 137, 144 chipping, 280, 318, 319, 323 bioprocesses, xi, 149, 152, 161, 174, 186, 190, 196 classification, 3, 107, 108, 175, 272, 347, 375, 376, 380, 392 Complimentary Contributor Copy 408 Index color, x, 95, 98, 100, 106, 107, 111, 116, 155, 264, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 280, 281, 282, 304 333 composition, x, xi, xiv, 5, 6, 11, 12, 16, 18, 20, 30, fibers, 106, 116, 172, 176, 184, 254, 255, 262, 266, 87, 88, 93, 94, 97, 100, 101, 103, 106, 109, 124, 267, 269, 284, 288, 304, 305, 307, 310 132, 133, 138, 141, 145, 150, 152, 153, 154, 156, films, xiv, 173, 254, 255, 262, 263, 264, 265, 267, 157, 159, 162, 171, 172, 173, 174, 175, 176, 191, 268, 269, 279, 297, 298, 300, 301, 302, 307, 308, 192, 199, 259, 271, 273, 275, 280, 281, 282, 283, 309, 311, 312, 355 284, 286, 291, 292, 293, 294, 295, 298, 324, 338, first and second-generation fuel ethanol, 2 339, 340, 342, 343, 347, 353, 354, 355 foams, xiv, 297, 298, 302, 303, 304, 305, 306, 307, cooking conditions, 132 308, 309, 310, 354 copper, 106, 152, 153, 166, 176, 196, 232, 246, 253, frying/drying/roasting, x, xvi, 34, 43, 105, 108, 109, 276 113, 114, 116, 118, 119, 123, 151, 162, 218, 280, culture dependent molecular method, 234 281, 300, 306, 318, 319, 320, 321, 322, 358 cyanogenic glycosides, 90, 107, 108, 155, 232, 233, 237, 272 G

D gari, xii, xiv, 130, 201, 202, 203, 204, 206, 207, 208, 209, 210, 211, 212, 220, 245, 248, 291, 293, 294, diseases, viii, xvi, 30, 32, 35, 36, 40, 41, 49, 88, 104, 313, 314, 320, 321, 322, 329, 360 131, 160, 272, 294, 317, 323, 373, 383, 396, 397 gelatinization, 8, 12, 92, 111, 114, 118, 122, 279, DNA-binding protein, 237, 238, 243, 244 300, 305, 309, 310 genomic DNA, 234 Ghana, vi, xiv, 36, 53, 248, 292, 294, 313, 314, 315, E 316, 317, 318, 319, 320, 321, 322, 328, 329, 330, 331, 332 eco-materials, 297 glutamate synthase, xiii, 231, 238, 239, 241 enzyme, 7, 9, 14, 27, 34, 36, 136, 141, 144, 147, 219, grating, 34, 107, 111, 114, 219, 318, 319, 320, 323 236, 237, 241, 245, 246, 248, 258, 261, 268, 277, 323, 325, 326, 327, 331 ethanol, v, 1, 2, 3, 8, 16, 17, 20, 23, 25, 27, 28, 34, H 135, 137, 139, 142, 162, 182, 244, 256, 257, 268, 285, 325, 326, 329, 330, 331, 332 harvesting, viii, 29, 31, 33, 34, 35, 44, 109, 110, 131, 137, 258, 265, 272, 275, 281, 314, 318, 327 hectares, 75, 157, 314 F HQCF, 35, 228, 322 Hydrocyanic Acid, 107 farinha d' água, 111, 112, 113, 116, 117, 118 hydroxynitrile lyase, xiii, 231, 237, 238 farm size categories, 56, 61, 68, 72, 73, 203, 207, 208, 211 fat, 93, 97, 99, 100, 106, 253, 298, 299 I fed-batch, 136, 139, 141, 144, 146, 158, 166, 168, 185, 325, 326, 329 improved cooking, 132 fermentation, x, xiv, 7, 8, 9, 10, 11, 13, 15, 16, 17, industrial cassava, 108 19, 20, 21, 24, 25, 26, 27, 28, 92, 105, 110, 112, iron, xi, 36, 91, 106, 108, 109, 124, 135, 143, 152, 113, 116, 118, 119, 120, 123, 129, 131, 135, 136, 153, 171, 232, 276, 320 137, 139, 140, 141, 142, 143, 144, 145, 146, 151, 152, 157, 163, 167, 168, 169, 173, 175, 179, 180, 181, 182, 183, 184, 185, 186, 188, 190, 191, 192, K 193, 194, 196, 197, 198, 199, 218, 229, 233, 237, 244, 245, 246, 247, 253, 257, 258, 260, 267, 268, kokonte, xiv, 313, 321, 322, 323, 329 271, 272, 275, 277, 278, 280, 281, 285, 288, 289, 290, 291, 292, 293, 294, 295, 318, 319, 320, 321,

Complimentary Contributor Copy Index 409

L P lactic acid, xii, xiv, xv, 116, 134, 163, 172, 179, 180, packaging, xiii, 93, 212, 243, 251, 254, 262, 263, 187, 195, 196, 198, 232, 233, 246, 259, 271, 272, 264, 266, 269, 297, 298, 303, 305, 306, 308, 337, 277, 288, 289, 290, 291, 294, 295, 303, 313, 324, 360 327, 328, 329, 330, 331, 333, 337 peel, 133, 152, 184, 232, 233, 236, 237, 255, 324, Lactobacillus plantarum, xiii, 179, 180, 181, 194, 326, 327, 336 199, 231, 233, 236, 237, 238, 242, 293, 323, 328, peeling, xv, 107, 108, 114, 118, 119, 151, 152, 175, 331, 332 273, 283, 284, 318, 320, 322, 323, 335, 337 linamarin, 34, 107, 114, 124, 155, 219, 237, 272, peplication protein, 238, 243 273, 298, 323 phosphorus, vii, 29, 32, 33, 90, 91, 106, 124, 135, lipid, 106, 158, 189, 193, 199, 253, 274, 284, 285 145, 152, 158, 189, 199, 232, 315 lotaustralin, 34, 107, 272, 273, 323 plasmid DNA, xiii, 231, 235 poly-glutamic acid, xiii, 231, 248 potassium, xi, 32, 33, 91, 106, 135, 152, 156, 171, M 232, 295 pretreatment step, vii, 1, 2, 8, 13, 18 magnesium, xi, 106, 152, 171, 232, 276 profitability, ix, 15, 36, 55, 56, 57, 58, 62, 72, 73, 74, manganese, 91, 106, 134, 135, 232 78, 81 maniçoba, 124 protein, vii, ix, xi, xiii, xiv, xv, xvii, 12, 20, 21, 22, Manihot glaziovii, 140, 141, 143, 144, 145, 146, 147, 29, 30, 34, 35, 36, 87, 89, 90, 91, 93, 94, 97, 99, 197, 273, 293 100, 106, 124, 130, 133, 139, 152, 154, 160, 171, manipueira, xi, 115, 123, 149, 150, 151, 155, 156, 176, 193, 218, 219, 229, 231, 232, 233, 236, 237, 158, 165, 166, 198 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, Manipueira, xi, 123, 149 248, 249, 253, 259, 264, 269, 271, 272, 274, 275, Maniva, 124, 391, 392 276, 280, 281, 282, 284, 285, 286, 287, 289, 291, metric tonnes, 4, 314, 328, 358 298, 299, 303, 304, 313, 323, 324, 329, 330, 374, microalgae, xi, 149, 150, 151, 158, 159, 160, 161, 388, 392, 395, 396, 397, 399, 400, 403 166, 168, 169, 189, 194, 199 modern technology adoption, ix, 56, 77, 81 moisture, x, 10, 16, 30, 51, 91, 97, 99, 105, 106, 110, R 111, 112, 116, 119, 121, 123, 151, 153, 156, 175, 191, 232, 250, 254, 255, 263, 266, 279, 280, 284, riboflavin, 106, 324 285, 286, 298, 299, 301, 303, 304, 305, 306, 319, RNA chaperones, 243 320, 324, 359 S N sieving, 12, 13, 151, 318, 320, 322 niacin, 106 site-specific recombinase, 240, 242, 243 Nigeria, v, vi, viii, ix, xi, xii, xiii, 29, 30, 31, 35, 36, sodium, 106, 136, 167, 232 37, 38, 39, 40, 53, 55, 56, 57, 58, 59, 61, 62, 67, spherical granules, 121 68, 72, 74, 75, 78, 81, 82, 83, 84, 85, 90, 104, sugarcane bagasse, vii, 1, 2, 7, 8, 11, 16, 17, 18, 19, 106, 130, 139, 140, 146, 150, 166, 171, 186, 201, 20, 21, 22, 24, 26, 27, 28, 145, 245, 304, 309, 202, 203, 207, 209, 211, 212, 213, 214, 220, 228, 310, 353 229, 231, 234, 245, 247, 249, 265, 274, 281, 283, sweet cassava, 104, 107 291, 330, 359 T O table cassava, 108 organic acids, xii, 152, 163, 172, 173, 175, 179, 180, tannase, xiii, 231, 236, 237, 238, 245, 246, 247, 248 181, 182, 187, 188, 193, 198, 233, 285

Complimentary Contributor Copy 410 Index tapioca, x, 34, 50, 91, 105, 108, 109, 110, 111, 112, 120, 121, 122, 123, 126, 127, 152, 162, 185, 186, W 198, 202, 220, 279, 298, 309, 314, 360 wastewater, v, vi, x, xi, 129, 143, 144, 149, 150, 151, tapioca starch, 108, 152, 185, 186, 198, 279, 309 152, 155, 156, 157, 158, 160, 161, 162, 163, 166, Thermotoga neapolitana, 137, 144 167, 168, 169, 171, 173, 174, 175, 176, 177, 179, thiamine, 106 180, 181, 182, 184, 185, 186, 188, 189, 190, 191, Thioredoxin, 243 192, 193, 194, 195, 196, 198, 199, 233, 234, 236, tucupi, x, 105, 106, 108, 115 245, 247, 248, 260

V Z vitamin A, 36, 40, 106, 107, 127, 324 zinc, xi, 106, 108, 135, 143, 171, 196, 232, 243, 249, vitamin B, 106 276 vitamin C, 90, 106, 124 vitamins, vii, xi, 29, 30, 35, 88, 106, 149, 152, 274, 324 volatile fatty acids, xii, 172, 190

Complimentary Contributor Copy