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Caladium Bicolor) for BIO-ETHANOL PRODUCTION USING INDIGENOUS FUNGAL ISOLATES

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Caladium Bicolor) for BIO-ETHANOL PRODUCTION USING INDIGENOUS FUNGAL ISOLATES i TITLE PAGE EVALUATING THE POTENTIAL OF WILD COCOYAM (Caladium bicolor) FOR BIO-ETHANOL PRODUCTION USING INDIGENOUS FUNGAL ISOLATES BY ONYENMA, NGOZI CHIZURUM PG/M.Sc./12/61544 A DISSERTATION SUBMITTED TO THE SCHOOL OF POST GRADUATE STUDIES, UNIVERSITY OF NIGERIA, NSUKKA, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE (M.Sc.) DEGREE IN INDUSTRIAL MICROBIOLOGY SUPERVISOR: PROF. A. N. MONEKE NOVEMBER, 2016 ii CERTIFICATION Onyenma, Ngozi Chizurum (Reg. No. PG/M.Sc./12/61544), a postgraduate student in the Department of Microbiology, majoring in Industrial Microbiology, has satisfactorily completed the requirements for course work and research for the degree of Masters in Science (M.Sc.) in Microbiology. This work embodied in her project is original and has not been submitted in part or full for either diploma or degree of this university or any other university. ………………………………… …………………………………… Prof. L.I. Ezeogu Prof. A.N. Moneke Head, Supervisor, Department of Microbiology, Department of Microbiology, University of Nigeria, Nsukka. University of Nigeria, Nsukka. iii DEDICATION This work is dedicated to the memory of my father Elder Johnson Onyenma, who passed on to me a love of reading and respect for education, and without whose caring support it would not have been possible. iv ACKNOWLEDGEMENTS I have made efforts in this project work. However, it would not have been possible without the kind support and help of many whom I would like to extend my sincere thanks to. I wish to thank most the Almighty God for giving me the determination to complete this project and to improve myself in a situation that I never imagined that I can surpass. My profound gratitude is extended to my HOD and my research supervisor, Prof. A. N. Moneke, who gave me a golden opportunity to do this wonderful project work, which helped me to learn about so many new things. I am also particularly grateful for the assistance given by Dr. Mrs. O. C. Amadi and Dr. Mrs. T. N. Nwagu. Their advice, assistance in keeping my progress on schedule and their willingness to give their time so generously has been very much appreciated. To my super mum and siblings, your prayers, unconditional support (both financially and emotionally) and your encouragement throughout my study saw me through. Finally, my thanks and appreciations go to my colleagues and friends who helped me a lot in developing and finalizing this project work with their abilities. v ABSTRACT Diminishing fossil fuel reserves and environmental pollutions associated with the use of fossil fuels prompts the search for alternative energy sources that are renewable, sustainable, cost effective and safe. This study evaluates the potential use of Caladium bicolor (fleshy part of corm, peel of corm, leaf and stalk) for ethanol production. Eighteen fungal strains were isolated from healthy and rotten corms of C. bicolor and also from fresh palm wine. They were screened for amylase activity, cellulase activity and only the yeast strains screened for ethanol fermentation ability. Hydrolysis was carried out in flasks containing mineral salts medium (MSM), at pH 5.0, 4% substrate concentration and 1.0 x 108 spores/ml inoculum size at 28 ± 2°C for 7 days and amount of reducing sugar produced determined. Effects of time, pH, substrate concentration, nitrogen source, and inoculum size on hydrolysis of the various substrates were studied. Optimal conditions were combined and the time course of the enzymatic hydrolysis obtained. Following hydrolysis, Saccharomyces sp. (4.0 x 107 cells/ml) isolated from fresh palm wine was used to ferment the sugar for 7 days. The fermented liquid was distilled and maximum ethanol yield determined. The results obtained from the screening showed that nine isolates (three organisms: Aspergillus spp., Penicillium spp. and Rhizopus sp.) showed enzymatic activity. Six showed multi-enzyme activity while three isolates showed just amylolytic activity. Aspergillus sp. (Org 2) showed the highest amylolytic and cellulolytic ability and thus used for further study. Maximum reducing sugar yield was achieved on day 5 when the Aspergillus sp. was cultured on the media containing fleshy part of corm (23.297g/L) and stalk (15.320g/L), while the maximum reducing sugar yield for peel of corm and leaf were 18.013g/L and 6.667g/L, respectively on day 4. Optimal reducing sugar yield was achieved with pH 5 for all the substrates. Optimal ethanol yield from fleshy part of corm, peel of corm and leaf were 0.485%, 0.210%, and 0.380%, respectively on day 5 while that of stalk and mixed substrate were 0.280% and 0.280% on day 3 and 6, respectively. This study therefore demonstrates the potential of utilizing fleshy part of corm, peel of corm, leaf and stalk of C. bicolor for glucose production which can be fermented for bio-ethanol production especially in areas where they are in abundance. vi TABLE OF CONTENTS Title page i Certification ii Dedication iii Acknowledgements iv Abstract v Table of Contents vi List of Tables xii List of Figures xiii List of Appendices xvi CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1.1 Introduction 1 1.1.1 Statement of Problem 2 1.1.2 Aim of Study 2 1.1.3 Objectives of the Study 3 1.2 Literature Review 3 1.2.1 Overview of Ethanol 3 1.2.2 Caladium bicolor (Wild Cocoyam) 5 1.2.3 Raw Materials for Ethanol Production 7 vii 1.2.3.1 Ethanol from Sugar 7 1.2.3.2 Ethanol from Starches 7 1.2.3.3 Ethanol from Lignocelluloses 8 1.2.4 Pretreatment of Lignocellulose Biomass 12 1.2.4.1 Physical Pretreatments 14 1.2.4.2 Chemical Pretreatments 17 1.2.4.3 Biological Pretreatments 18 1.2.4.4 Combined Pretreatments 19 1.2.5 Enzymatic Hydrolysis 19 1.2.5.1 Amylolytic Enzymes and their Producer Organisms 20 1.2.5.2 Cellulolytic Enzymes and their Producer Organisms 23 1.2.6 Yeast and Its Fermentative Ability 26 1.2.7 Hydrolysis and Fermentation Strategies 28 1.2.7.1 Separate Hydrolysis and Fermentation (SHF) 28 1.2.7.2 Simultaneous Saccharification and Fermentation (SSF) 28 CHAPTER TWO: MATERIALS AND METHODS 2.1.1 Collection of Sample (Substrate) 30 2.2 Identification of Sample 30 2.3 Proximate Analysis of Sample 30 2.4 Processing of Sample 30 viii 2.5 Isolation of Microorganisms 31 2.5.1 Samples 31 2.5.2 Media for Isolation 31 2.5.3 Isolation Procedure 31 2.6 Purification / Preservation of Isolates 32 2.7 Screening of the Isolated Microorganisms 32 2.7.1 Qualitative Screening of Isolates for Amylase Production 32 2.7.2 Qualitative Screening of Isolates for Cellulase Production 32 2.7.3 Quantitative Screening of Isolates for Ability to Generate Reducing Sugars from C. bicolor 33 2.7.4 Screening Yeast Isolates for Alcohol Production 33 2.8 Characterization/Identification of Selected Isolates 34 2.8.1 Characterization/Identification of Mould 34 2.8.1.1 Macroscopic Identification of Mould 34 2.8.1.2 Microscopic Identification of Mould 34 2.8.2 Characterization/Identification of Yeast Strains 35 2.8.2.1 Colonial Morphology of Yeast Isolates 35 2.8.2.2 Microscopy 35 2.8.2.3 Assimilation of Nitrogen Compound 36 2.8.2.4 Sugar Fermentation Test 36 ix 2.9 Preparation/Standardization of Inoculum 37 2.9.1 Preparation of Mould Inoculum 37 2.9.2 Preparation of Yeast Inoculum 37 2.9.3 Inoculum Standardization 37 2.10 Preparation of Standard Graphs 38 2.10.1 Preparation/Standardization of Glucose Concentration 38 2.10.2 Preparation/Standardization of Starch Concentration 39 2.10.3 Preparation/Standardization of Cellulose Concentration 39 2.11 Enzymatic Hydrolysis of Caladium bicolor 40 2.12 Analytical Methods in Enzymatic Hydrolysis of Caladium bicolor 40 2.12.1 Determination of Reducing Sugar Concentration 40 2.12.2 Determination of the Starch Content of Caladium bicolor (the fleshy part of corm) 41 2.12.3 Determination of the Cellulose Content of Caladium bicolor 41 2.13 Determination of Optimal Conditions on Hydrolysis of Caladium bicolor using the Selected Test Organism Aspergillus sp. (Org 2) 42 2.13.1 Effect of Incubation Time 42 2.13.2 Effect of Varying Initial pH of the Medium 42 2.13.3 Effect of Varying Substrate Concentration 42 2.13.4 Effect of Different Nitrogen Sources 43 x 2.13.5 Effect of Varying Inoculum Size 43 2.13.6 Time Course of Enzymatic Hydrolysis of Caladium bicolor 43 2.14 Fermentation 43 2.15 Measurement of Ethanol Concentration 44 2.16 Statistical Analysis 45 CHAPTER THREE: RESULTS 3.1 Proximate Analysis of Caladium bicolor (Wild Cocoyam) 46 3.2 Screening of the Isolated Microorganisms 48 3.2.1 Qualitative Screening of Isolates for Enzyme Production 48 3.2.2 Quantitative Screening of Isolates for Ability to Generate Reducing Sugar from C. bicolor 50 3.2.3 Screening Yeast Isolates for Alcohol Production 56 3.3 Characterization/Identification of Selected Isolates 58 3.3.1 Characterization/Identification of Mould Isolates 58 3.3.2 Characterization/Identification of Yeast Strains 58 3.4 Determination of Optimal Conditions on Hydrolysis of Various Parts of Caladium bicolor using the Selected Test Fungi Aspergillus (Org 2) 62 3.4.1 Effect of Incubation Time 62 3.4.2 Effect of Varying Initial pH of the Medium 68 3.4.3 Effect of Varying Substrate/Carbon Concentration 74 xi 3.4.4 Effect of Different Nitrogen Sources 80 3.4.5 Effect of Varying Inoculum Size (1.0 x 108 spores/ml) 86 3.4.6 Time Course of Enzymatic Hydrolysis of Various Parts of Caladium bicolor 92 3.5 Fermentation 98 CHAPTER FOUR: DISCUSSION AND CONCLUSION 4.1 Screening of Isolates for Enzyme Production 105 4.2 Optimal Production Studies (Hydrolysis of Starch and Cellulose) 106 4.3 Fermentation 110 4.4
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