Microbial Resources for Sustainable Energy Microbial Resources for Sustainable Energy This Is a FM Blank Page Basanta Kumara Behera • Ajit Varma

Basanta Kumara Behera · Ajit Varma

Microbial Resources for Sustainable Energy Microbial Resources for Sustainable Energy ThiS is a FM Blank Page Basanta Kumara Behera • Ajit Varma

Microbial Resources for Sustainable Energy Basanta Kumara Behera Ajit Varma Amity Institute of Microbial Technology Amity Institute of Microbial Technology Amity University Uttar Pradesh Amity University Uttar Pradesh Noida, Uttar Pradesh Noida, Uttar Pradesh India India

ISBN 978-3-319-33776-0 ISBN 978-3-319-33778-4 (eBook) DOI 10.1007/978-3-319-33778-4

Library of Congress Control Number: 2016940051

© Springer International Publishing Switzerland 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

Printed on acid-free paper

This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG Switzerland Foreword

“Nuclear power produces nearly 20 % of Germany’s energy, but in July 2011 (only 3 months after Fukushima) the German government vowed to shut down its nuclear capability within 10 years. Not just that, but to replace it with renewable energy, cut greenhouse-gas (GHG) emissions by 40 % by 2020 and 80 % by 2050, ensure renewables contribute 80 % of Germany’s energy by 2050, and ensure energy consumption drops 20 % by 2020 and 50 % by 2050.” In this connection, I believe that this book would have immense contribution to bring awareness about the potential of microbes to contribute green energy in the form of biogas, algal diesel, ethanol, hydrogen, and direct electricity to overcome the present energy crisis to a certain significant magnitude. This piece of book contributes a comprehensive review on the genesis of renewable green energy and its further development for commercialization. This book will not only be helpful to technology and policy worlds but will also appeal to a broader audience. Additionally, this work would be immensely inspiring to younger researchers in finding out issues and its solution to meet local energy demands through sustainable microbial resources available in and around a specific location. It is also a must-read piece of work for all youngsters who dream to find out solution for an eco-friendly sustainable environment by introducing better option for alternative source of energy. This book is systematized in such a way to give comprehensive and systematic knowledge on green energy to bring familiarity among readers on social acceptability of different options on biological renewable energy. The language is aimed at a Popular Science level of technical exposition and is relatively easy to understand although a wide spectrum of technologies are depicted. Each chapter includes an extensive list of references to assist the reader in finding sources and additional details of the referenced. The most vital part of this book is the way of dealing a highly debatable issue on energy crisis in a most conventional manner with the illustration of impressive figures and practicable data in tabular forms. Additionally, the book carefully addresses these complexities for each energy policy topic presented. The book intentionally avoids advocacy and tries best to be an honest broker to the readers to induce the sense of their own conclusions in spite of various pros and cons of the book. v vi Foreword

I am exceedingly proud the way the authors have worked out to draw international attention on sustainability of microbe origin green energy as an unavoidable option for alternate and clean energy to reduce the burden of greenhouse effect and also energy crisis.

Institute of Microbiology and Wine Research Prof. Dr. Helmut Koenig Johannes Gutenberg - University Mainz, Germany Preface

The security of global energy supplies is in great dilemma as oil and gas reserves are under the direct control of a small group of nations, most of which are considered politically unstable or have testy relationships with large consuming countries. About 80 % of the world’s oil reserves are located in just three regions: Africa, Russia and the Caspian Basin, and the Persian Gulf. More than half of the global gas reservoirs are restricted to only three countries: Russia, Iran, and Qatar. So, any serious energy security decision should be free from foreign energy sources. Due to uncertainty of Russian policy over natural gas supply in Europe, new coal-fired power stations are back on the political agenda. In the USA, home- grown biofuels have been promoted by successive administrations as an alternative to Middle Eastern oil imports, despite being more expensive. At present, we depend on about 80 % conventional energy for our immediate needs. However, this scenario may change a little in due course of time without drastic policy changes. On top of this, energy demand is expected to grow by almost half over the next two decades. Understandably, this is causing some fear that our energy resources are starting to run out, with devastating consequences for the global economy and global quality of life. The present energy crisis is not due to sharp downfall of natural energy resources as a result of drastic energy demand but because we are using it in the wrong way. The increase in global temperature is mainly due to heavy accumulation of CO2 in space as a result of burning fossil fuels for energy. The efficacy of energy industry used to be judged by two major criteria: its contribution to energy security and the cost of energy delivered to the consumer. However, now we are forced to include a third criteria related to emission of greenhouse gases, mainly CO2 into the atmosphere. So, now we feel the urgency of finding out suitable solution to overcome the present energy crisis. At first instant, we have to reduce the use of fossil fuels in order to have control over input of greenhouse gases into atmosphere. But coal is widely used to generate electricity, in most of the countries (especially the USA, China, and India) without carbon capture and storage (CCS) technology. It is right time that all these developing countries should make a common policy for reducing discharge of greenhouse gases to a marked quantity. However, at current rates of vii viii Preface population growth and with current technologies, this will be impossible without a global agreement to limit efflux of greenhouse gases into the atmosphere. Devel- oped countries must shoulder the initial burden with an agreement for immediate emission cuts. In return, the largest developing countries must agree to cut their own emissions in the future, but only after having achieved some recognizable level of economic development. The immediate solution to get partial relief from the present energy crisis is to find out effective strategies to encourage the use of alternate sources of renewable energy in order to lessen the burden of greenhouse gases. Besides various types of nonconventional energy (Solar power, Hydro-electric power (Dams in Rivers), Wind power, Tidal power, Ocean wave power, Geothermal power (heat from deep under the ground), Ocean thermal power, etc.), we have a lot of opportunities to use sustainable microbial resources for generating different energy carriers like hydrogen, ethanol, diesel directly from microalgae, macroalgae, Cyanobacteria, and fermentative bacteria. The most significant fact about microbial energy carriers is their less carbon dioxide emission property compared to conventional liquid fuels like petrol and diesel. In addition, abundant aquatic resources (both marine and freshwater) are sustainable storehouse of a variety of micro- and macroalgae which can be ultimate sources for generating green energy. Biodiesel is simple to use, biodegradable, nontoxic, and essentially free of sulfur and aromatics. It can be used in most diesel engines, especially newer ones, and emits less air pollutants and greenhouse gases other than nitrogen oxides. It’s safer to handle and has virtually the same energy efficiency as petroleum diesel. In addition, it has lubricity benefits that fossil fuels do not. Biodiesel blends as low as B2 have been found to significantly reduce the amount of toxic carbon-based emissions. Microor- ganisms like microalgae, bacillus, fungi and yeast are all available for biodiesel production. The present research has also revealed the possibility of obtaining hydrogen and electricity directly from microalgae and formative bacteria. When hydrogen is burned, the only emission it makes is water vapor, so a key advantage of hydrogen is that when burned, carbon dioxide (CO2) is not produced. Clearly, hydrogen is less of a pollutant in the air because it emits little tailpipe pollution. Hydrogen has the potential to run a fuel-cell engine with greater efficiency over an internal combustion engine. The same amount of hydrogen will take a fuel-cell car at least twice as far as a car running on gasoline. The concept of Microbial Fuel Cells (MFCs) has brought hope for using various types of microbes to generate electricity, directly. This piece of work is organized to bring attention of researchers, teachers, students and policymakers for considering microbial resources as sustainable storehouse of nature and pleads for its best use as alternate form of nonconventional energy. The first chapter narrates how green energy production begins with the photosynthetic fixation of CO2 into biomass and subsequent conversion of biomass by using microbes as biological catalyst to produce biofuels like ethanol, methane, hydrogen, and biodiesel, relatively free from hazardous gases like any oxide of carbon, nitrogen, and sulfur. In addition, it has also been explained how the policymakers boost the nonconventional biofuels by implementing special laws to Preface ix enforce them in auto industries. This has been nicely narrated with some historical facts and figures to convince readers the positive impact of biofuels in public life. In the second chapter, the authors intend to describe updated information on biogas production by the breakdown of organic matter in the absence of oxygen. Here, special attention has been paid to highlight commercialization of biogas production technology to meet the challenge in solving rural energy crisis under effective management program. In addition, technology on generation of electricity from biogas to solve localized energy problem has also been narrated in detail. Depletion of natural oil and diesel resources has created an enormous challenge in substituting suitable and economic fuels to meet the demand of locomotive engine and communication systems. In this regard, the authors have given special emphasis on explaining advanced process technology to obtain highly purified biogas to be used for commercial purpose and electricity generation. So, in the third chapter the authors have highlighted about the production of biodiesel from microbial biomass on commercial level to reduce the load on natural petroleum and gas resources. The most interesting part of this chapter is about direct production of ethanol from microalgae without any biomass extraction process. This has been nicely explained with well-illustrated figures and photographs to impress readers and make them realize the effective use of microalgae with the use of new technologies being developed by researchers from the field of industrial biotechnology. Unlike biofuels, microbial fuel cells (MFCs) are “Plug in and power” devices, which convert energy harvested from redox reactions directly into bioelectricity. MFCs can utilize low-grade organic carbons (fuels) in waste streams. The oxidation of the fuel molecules requires biofilm catalysis. In recent years, MFCs have also been used in the electrolysis mode to produce bioproducts in laboratory tests. MFC research has intensified in the past decade and the maximum MFC power density output has been increased greatly and many types of waste streams have been tested. The fourth chapter has been completely devoted to latest aspects of MFC technology and its possibility to commercialize. The last chapter has been organized to highlight the production of hydrogen from microalgae and formative bacteria. Hydrogen (H2) is being explored as a fuel for passenger vehicles. It can be used in fuel cell to power electric motors or burned in internal combustion engines (ICEs). It is an environment-friendly fuel that has the potential to dramatically reduce our dependence on imported oil, but several significant challenges must be overcome before it can be widely used. Hydrogen produces no air pollutants or greenhouse gases when used in fuel cells; it produces only nitrogen oxides (NOx) when burned in ICEs. The authors have taken maximum interest to draw attention of readers by illustrating impressive models on the production of hydrogen by using the vast marine ecosystem as green energy power station. Lastly, the authors want to extend thanks and are obliged to their respective family members for showing immense passion and extending cooperation while the manuscript was under preparation stage.

March 09, 2016 Basanta Kumara Behera Noida, India Ajit Varma ThiS is a FM Blank Page Contents

1 Microbial Energy Conversion Technology ...... 1 1.1 Microbes and Biofuels ...... 1 1.1.1 History of Ethanol as Transport fuel ...... 2 1.1.2 History of Biodiesel as Transport Fuel ...... 5 1.1.3 History of Biogas as Transport Fuel ...... 8 1.1.4 History of Miscellaneous Biofuels ...... 9 1.2 Renewable Energy Versus Fossil Fuels ...... 11 1.3 Salient Features of Microbial Biofuel ...... 15 1.4 Social Acceptability ...... 18 1.5 Global Partnership and Trading of Biofuels ...... 20 1.5.1 Algenol ...... 24 1.5.2 Solazyme ...... 24 1.5.3 Blue Marble ...... 25 1.5.4 Sapphire Energy ...... 26 1.5.5 Diversiﬁed Technologies Inc...... 27 1.5.6 OriginOil, Inc...... 27 1.5.7 Aquaviridis, Inc...... 27 1.5.8 Proviron ...... 28 References ...... 28 2 Biomethanization ...... 35 2.1 Introduction ...... 36 2.2 Anaerobic Digestion and Methane Production ...... 41 2.2.1 Methane-Producing Microbes ...... 46 2.2.2 General Mechanism of Methane Production ...... 47 2.3 Methane Emission and Organic Feedstocks ...... 51 2.3.1 Methane from Cattle Wastes ...... 54 2.3.2 Methane from Market Organic Wastes (MOW) ...... 55 2.3.3 Methane from Human Excreta ...... 60 2.3.4 Methane from Whey ...... 67 2.3.5 Methane from Algae ...... 68

xi xii Contents

2.3.6 Methane from CO2,H2, and CO by Anaerobic Fermentation ...... 70 2.3.7 Methane from Slaughterhouse Wastes ...... 71 2.3.8 Sewage Sludge ...... 75 2.3.9 Methane from High Solid Agro-wastes ...... 78 2.4 Raw Biogas Upgradation Process ...... 79 2.4.1 Removal of Water ...... 80 2.4.2 Removal of Nitrogen ...... 80 2.4.3 Removal of Ammonia ...... 80 2.4.4 Removal of Siloxanes ...... 80 2.4.5 Removal of Particulates ...... 81 2.4.6 Removal of Hydrogen Sulﬁde ...... 81 2.4.7 In Situ Biogas Upgradation ...... 84 2.5 Full-Scale Technology for Biogas Upgrading ...... 85 2.5.1 Water Scrubber ...... 85 2.5.2 Chemical Scrubbing ...... 86 2.5.3 Membrane Technology (Gas Permeation) ...... 87 2.5.4 Cryogenic Upgrading ...... 88 2.5.5 Pressure Swing Adsorption (PSA) ...... 89 2.5.6 Bioﬁltration ...... 90 2.5.7 Organic Physical Scrubbing ...... 91 2.6 Factors Affecting the Biogas Generation ...... 92 2.6.1 Temperature and Pressure ...... 92 2.6.2 Organic Loading Rate (ORL) ...... 93 2.6.3 Hydraulic Retention Time (HRT) ...... 93 2.6.4 pH Value or Hydrogen Ion Concentration ...... 93 2.6.5 Carbon to Nitrogen Ratio ...... 94 2.6.6 Nutrients Concentration ...... 94 2.6.7 Reaction Period ...... 95 2.6.8 Stirring or Agitation of the Content of Digester ...... 95 2.6.9 Inoculation and Start-Up ...... 95 2.6.10 Inhibition ...... 96 2.7 Pretreatment for Enhanced Biogas Production ...... 97 2.7.1 Mechanical Pretreatment ...... 97 2.7.2 Thermal Pretreatment ...... 97 2.7.3 Chemical Pretreatment ...... 98 2.7.4 Alkali Pretreatment ...... 98 2.7.5 Oxidative Pretreatment ...... 99 2.7.6 Ionic Liquid Pretreatment ...... 99 2.7.7 Biological Pretreatment ...... 99 2.8 Type of Biogas Digester ...... 100 2.8.1 Fixed Dome Digesters ...... 101 2.8.2 Floating Drum Digester ...... 102 2.8.3 Plug Flow Digesters ...... 102 Contents xiii

2.8.4 Biogas Digester ...... 103 2.8.5 The Batch Reactor ...... 103 2.8.6 Continuous Biogas Digester ...... 104 2.8.7 Anaerobic Baffle Reactor ...... 104 2.8.8 Anaerobic Filter-Type Reactor ...... 104 2.9 Biogas Utilization and Problems ...... 105 2.9.1 Biogas for Cooking and Lighting ...... 105 2.9.2 Biogas-Based Diesel Engine ...... 105 2.9.3 Electricity Generation from Biogas ...... 106 2.9.4 Biogas for Automobile ...... 106 2.9.5 Optimization of Process for Biogas Production ...... 107 2.10 Biogas Utilization Problems ...... 107 2.10.1 Choice of Digester ...... 107 2.10.2 Supply of Feed ...... 107 2.10.3 Waste Collection Transport and Storage ...... 108 2.10.4 Nature of Biogas ...... 108 2.10.5 Gas Flammability ...... 109 2.10.6 Gas Compressibility ...... 109 2.10.7 Biogas Storage ...... 109 2.10.8 Social Problem ...... 111 References ...... 112 3 From Algae to Liquid Fuels ...... 123 3.1 Biodiesel from Microalgae ...... 123 3.1.1 What Is Microalgae? ...... 124 3.1.2 Benefit of Using Microalgae for Biodiesel Production ...... 124 3.1.3 Algae for Value-Added Products ...... 126 3.1.4 Viability of Microalgae for Biodiesel ...... 129 3.1.5 Overview of Algal Biodiesel Supply Chain ...... 131 3.2 Large-Scale Microalgae Production ...... 132 3.2.1 Microalgae Cultivation ...... 132 3.2.2 Algae Cultivation Methods ...... 135 3.3 Harvesting and Biomass Concentration ...... 147 3.3.1 Concept ...... 147 3.3.2 Harvesting Methods ...... 148 3.3.3 Dehydration Processes ...... 153 3.4 Extraction and Purification of Biofuels ...... 154 3.4.1 Lipid Contents of Algal Biomass ...... 154 3.4.2 Algae Lipid Extraction ...... 157 3.4.3 Transesterification for Biodiesel Production ...... 158 3.5 Bioethanol from Algae ...... 160 3.5.1 Introduction ...... 160 3.5.2 Microalgae as a Potential Source of Bioethanol ...... 163 3.5.3 Ethanol Production from Macroalgae ...... 166 xiv Contents

3.5.4 Algae Ethanol Commercialization ...... 166 3.5.5 Processing Ethanol from Algae ...... 170 References ...... 171 4 Microbial Fuel Cell (MFC) ...... 181 4.1 Introduction ...... 181 4.2 Fuel Cell Versus MFC ...... 184 4.3 Generation Energetic Molecule and Ions Gradient in MFCs .... 190 4.3.1 History of the MFC ...... 190 4.4 Microbiology of Fuel Cell ...... 191 4.4.1 Types of Microbes in FMCs ...... 191 4.4.2 Bacterial Metabolism and Electron Transfer in MFC . . . 193 4.4.3 Factors Affecting Performance of MFCs ...... 196 4.4.4 Power Generation in MFCs ...... 198 4.5 Types of Microbial Fuel Cells and Components ...... 198 4.5.1 Two-Chambered Microbial Fuel Cell ...... 199 4.5.2 One-Chambered Microbial Fuel Cell ...... 200 4.5.3 Upﬂow Microbial Fuel Cell ...... 201 4.5.4 Stacked Microbial Fuel Cell ...... 202 4.5.5 The Plant Microbial Fuel Cell (PMFC) ...... 203 4.5.6 Tubular MFC ...... 205 4.6 Components and Architecture of Microbial Fuel Cell ...... 206 4.6.1 Anode ...... 206 4.6.2 Cathode ...... 208 4.6.3 Ion Exchange Membrane ...... 210 4.7 Applications of Microbial Fuel Cell Technology ...... 210 4.7.1 Wastewater Treatment ...... 211 4.7.2 MFC for Landﬁll ...... 212 4.7.3 Generation of Electricity Directly from PMFC ...... 213 4.7.4 Microbial Fuel Cell for Health Care ...... 213 4.7.5 Power Supply to Microelectronic Devices ...... 214 4.7.6 Hydrogen Production ...... 215 References ...... 216 5 The Microbiological Production of Hydrogen ...... 223 5.1 Introduction ...... 226 5.2 History and Milestone of Microbial Hydrogen Production . . . . . 228 5.3 Hydrogen Production Process ...... 230 5.3.1 Sustainable Hydrogen from Water ...... 230 5.3.2 Hydrogen from Fossil Fuel ...... 233 5.3.3 Hydrogen from Cellulose Biomass ...... 234 5.3.4 Hydrogen Production by Bioethanol Reforming ...... 234 5.3.5 Hydrogen Production by Water Biophotolysis ...... 235 5.4 Microbial Resources for Hydrogen ...... 235 5.4.1 Fermentative Hydrogen Production ...... 235 5.4.2 Hydrogen from Rhodopseudomonas palustris ...... 237 Contents xv

5.4.3 Hydrogen from MEC ...... 238 5.4.4 Hydrogen Production in Extreme Bacterium ...... 239 5.4.5 Hydrogen Production by Clostridium sp...... 240 5.4.6 Hydrogen Production by Acetogenic Bacteria ...... 240 5.4.7 Hydrogen Production by Desulfurizing Bacterium (Desulfovibrio vulgaris)...... 242 5.4.8 Hydrogen Production by Cyanobacteria ...... 243 5.4.9 Hydrogen Production by Green Algae ...... 249 5.5 Bioreactors for Hydrogen Production ...... 253 5.6 Future Prospects of Microbial Hydrogen Production ...... 258 References ...... 261

Index ...... 273 ThiS is a FM Blank Page Abbreviations

AGATE Acid, Gas, and Ammonia Targeted Extraction AFPRO Action for Food Production AMIMCI 1-Allyl-3-methylimidazolium chloride AC After Christ AD Anaerobic Digester ATP Adenine Triphosphate ASP Aquatic Species Program BOD Biological Oxygen Demand BC Before Christ BVS Biodegradable VS BSA Bovine Serum Albumin BETO Department’s Bioenergy Technologies Office BDTACI 1-N-butyl-3-methyl (tetradecyl) ammonium chloride CO2 Carbon Dioxide CEO Chief Executive Officer CNG Compressed Natural Gas CBM Compressed Biomethane CV Calorific Value CoTMPP Cobalttetramethoxyphenylporphyrin COD Chemical Oxygen Demand DAF Dissolved Air Flotation DMDO Dimethyl Dioxirane DOE Department of Energy, Government of USA DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea DEA Diethanolamine EPA Environmental Protection Agency FFV Flexible-Fuel Vehicles EPA Energy Policy Act EDA Electron Donor–electron Acceptor EDTA Ethylenediaminetetraacetic Acid FAME Fatty Acid Methyl Esters xvii xviii Abbreviations

FECVs Fuel-Cell Electric Vehicles GHG Greenhouse Gas HRT Hydraulic Retention Time HE Human Excreta HDP High-Density Polyethylene HNQ 2-Hydroxy-1,4-naphthoquinone HTS Interspecies Hydrogen Transfer IABR Integrated Algal BioRefinery KVIC Khadi and Village Industry Commission LPG Liquid Petroleum Gas LDPE Low-Density Polyethylene LLDPE Linear Low-Density Polyethylene LBM Liquefied Biomethane LCFA Long Chain Fatty Acids MDGs Millennium Development Goals MFC Microbial Fuel Cell MOW Market Organic Wastes MDEA Methyldiethanolamine MEA Monoethanolamine MGDG Monogalactosyldiacylglycerol MEC Microbial Electrolysis Cell NGO Nongovernmental Organization NMMO N-methylmorpholine-N-oxide monohydrate NAD Nicotinamide Adenine Dinucleotide NADP Nicotinamide Adenine Dinucleotide Phosphate NREL National Renewable Energy Laboratory NREL National Renewable Energy Laboratory OLR Organic Loading Rate PS I Photo-System I PS II Photo-System II PNAS Proceedings of the National Academy of Sciences PEF Pulsed Electric Field POMS PeroxyMonoSulfate PUFA Polyunsaturated Fatty Acids P-MFC Plant-Microbial Fuel Cell PMFC Plant Microbial Fuel Cell PBR Photo Bio-Reactor PAA Peracetic Acid PEM Proton Exchange MembraneRED PEC Photo-Electrochemical PNSB Purple Non-Sulfur Bacterium RED Renewable Energy Directive SCP Single Cell Protein SzIBR Solazyme Integrated Biorefinery Abbreviations xix

SRT Solids Retention Time SAO Syntrophic Acetate Oxidizers SOD Super Oxide Dismutase SMFC Sediment soil MFC TOS Total Organic Solid TITE Research Institute of Innovative Technology for the Earth TAGs Triacylglycerides US United States USDE United States Department of Energy VFAs Volatile Fatty Acids VMOW Vegetable Market Organic Wastes VFA Volatile Fatty Acids VS Volatile Solids WGS Water-Gas Shift Reaction Chapter 1 Microbial Energy Conversion Technology

1.1 Microbes and Biofuels

Mainly, three types of biofuels, i.e., ethanol, biodiesel, and biogas, in the form of methane and hydrogen are available as alternative renewable sources for green energy production [1]. The most commonly used biofuel for transport is ethanol being produced by microbial catalytic reaction (Fig. 1.1) by using feedstocks that contain signiﬁcant amounts of carbohydrate, such as sugarcane, sugar beet, maize, and wheat (Fig. 1.2) In addition, methane is being used as cooking gas and also to generate electricity to meet local energy demand. Fortunately, the last two decades have witnessed biodiesel as transport biofuel being processed from plant materials or microbial biomass. But, the present discussion is restricted to microbial- catalyzed liquid biofuels like ethanol, biodiesel from biomass of microbes being processed by chemical technology, and biogas like methane (based on microbial- catalyzed reaction) and hydrogen directly from microbes. Burning of wood directly or using biomass-derived liquid fuels like biodiesel and ethanol for transport purpose emits CO2 as in the case of using fossil fuels as energy carrier. However, direct burning of raw biomass emits more carbon dioxide as compared to fossil fuels to generate equal amount of energy. Still, it has been claimed that use of biofuel reduces the greenhouse effect as compared to using fossil fuel. This is because autotrophic plants absorb CO2 during their growth and development. However, from transport point of view, both fossil fuels and biofuels are suitable (Table 1.1), and moreover biofuels are having some better advantages compared to nonrenewable fuels.

© Springer International Publishing Switzerland 2016 1 B. Kumara Behera, A. Varma, Microbial Resources for Sustainable Energy, DOI 10.1007/978-3-319-33778-4_1 2 1 Microbial Energy Conversion Technology

Starch bearing Sugar bearing Cellulose biomass biomass biomass

Hydrolysis Separation by enzymatic Extraction Or Thermal acid hydrolysis

Fermentable sugar Sugar crystal

Sugar solution molasses

Carbon Based on microbial Fermentation dioxide catalysts

Rectification Stilage by products Of distilation

Ethanol 95%

Purification

Anhydrous ethanol

Fig. 1.1 Process ﬂow of ethanol production from carbohydrate-rich biomass being catalyzed by microbes

1.1.1 History of Ethanol as Transport fuel

World archaeological evidences nicely illustrated the conceptual development of the use of alcoholic beverage since early 10,000 BC. The reminiscence of late Stone Age jugs located at Neolithic village indicates the practice of making beverage through fermentation technology existed in 10,000 BC [2]. The Proceedings of the National Academy of Sciences (PNAS) reports that a fermented drink made of grapes, hawthorn berries, honey, and rice was being produced in 7000–6600 BC 1.1 Microbes and Biofuels 3

Feed Algal biomass, Agriculture wastes Petrocrop biomass Municipal Grain crops, Sugar crops & Other wastes stock starch crops Lignocellulosic Hyrdrocarbon Residue from Oil industry

Biological Chemical Biological Direct Gasification Process conversion hydrolysis Cracking & liquefaction chemical pyrolysis cracking

Products Biogas Ethanol Petroleum Methanol Like fuel

Fig. 1.2 Feedstock production systems for biofuels

Table 1.1 Fuel characteristic Property Petrodiesel Biodiesel of renewable fuels and Speciﬁc gravity 0.85 0.88 nonrenewable fuels Viscosity mm2 at 40 C 2–3 4–5 C 80.80 76.20 H 13.20 12.60 O 0.0 11.20 Energy content (LHO) 43 39

[3–5]. This is approximately the time when barley beer and grape wine manufacturing process began in the Middle East and Georgia [6]. Reports on alcoholic beverage have also been found dating from 3400 BC in Egyptian, 3150 BC in ancient Egypt [7], 3000 BC in Babylon [8], 3000 BC to 2000 BC in India as “Sura” [9], 2000 BC in pre-Hispanic Mexico [10], 2000 BC to 1750 BC in Babylonians and Greece [10], and 1500 BC in Sudan [10]. The medieval era witnessed the discovery of distillation process being practiced by Greek alchemists working in Alexandria in the first century AD [11]. However, Alexander of Aphrodisiac for the first time highlighted the process of distillation in 200 AD [12]. Subsequently, the basic knowledge on distillation process leads to alcoholic beverage production from raw materials like fermentable fruits, plant juice, and starchy materials like various grains and organic compounds rich in carbohydrates. The early modern period (1500–1800) was well known for use of alcoholic beverage as God’s gift for modernizing social pleasure, enjoyment, and health care [13]. It was only in the year 1824–1826 that the US inventor Samuel Morey was able to show for the first time a special designed internal combustion engine that runs on ethanol and turpentine [14]. In 1860, German inventor Nikolaus Otto uses ethanol as fuel in one of his engines [15–17]. For the first time in the world history in the USA, a special tax is placed on industrial alcohol by the Union Congress to help pay for the Civil War. The tax is $2 per gallon, and this makes ethanol fall out 4 1 Microbial Energy Conversion Technology of favor as a fuel in the USA. Prior to 1962, ethanol was commonly used in lamps. Fortunately, in the same year, Henry Ford builds his first automobile (the Quadricycle), and the engine was designed to run on pure ethanol. This was a noble achievement in ethanol car industry. In the year 1906, the US Congress replaced the earlier tax imposed in the year 1862. In the year 1908, for the first time Ford manufactured “Ford Model T” engine which was a flexible hybrid engine capable of using ethanol, gasoline, or kerosene [18, 19]. This car was produced until 1927. In 1914, with the outbreak of the First World War, Henry Ford started the campaign for production of alcohol from grains to meet the problem of gasoline shortage. After the First World War, in 1920, the gasoline, not ethanol, becomes the most popular fuel in the USA as well as in many other parts of the world. In the year 1922–1923, the Standard Oil Company started marketing a blend of 25 % alcohol and 75 % gasoline in Baltimore when a period of high gasoline and low alcohol prices existed. The alcohol used by Standard Oil had been produced during the First World War for ammunition production, but after the war it was not needed and made available by the military at low prices in the Baltimore area. After the Second World War, in 1945, gasoline became cheap and easily accessible. As a result, the interest in ethanol cars was reduced. From the US and European perspective, the following decades were not a very productive part of ethanol car history. However, in other parts of the world, such as Brazil, the interest in ethanol car continued. In 1945, oil embargoes and higher oil prices once again increased the use of ethanol car. Since then, continuous efforts have been made by US scientists and technol- ogists to use alcohol as partial substitute in petrol- and alcohol-branded fuel. In 1979, Ma´rio Garnero, President of the National Association of Automotive Vehicle Manufacturers, convinced the four major automobile producers in the country— Ford, Volkswagen, General Motors, and Fiat—to establish the dauntless goal of producing 1 million ethanol cars. This is equal to the entire automobile production of 1978. Fiat 147, the first modern car running on ethanol only, was launched on the Brazilian market the same year. Three years later, 90 % of Brazil’s new automobiles were ethanol cars. Garnero is dubbed “The Father of the Ethanol Car” for his role in ethanol car history [20]. In the same year, the Amoco Oil Company started marketing alcohol-blended fuels and soon followed by others, such as Texaco, Beacon, Ashland, and Chevron. The year 1992 is an important year in US ethanol car history, because this is when the Energy Policy Act of 1992 is enacted. The Act makes it mandatory for certain car fleets to start buying vehicles capable of running on alternative fuels. The Act defines ethanol blends with at least 85 % ethanol as alternative fuel. The Act also gives tax deductions to those who purchase a vehicle capable of running on alternative fuel or convert an old vehicle for the same purpose. E85 is a fuel made from a blend of 85 % ethanol and 15 % unleaded gasoline. E85 was created for flexible-fuel vehicles or vehicles that can run on any blend of ethanol up to 85 %. In the USA, the ethanol used in E85 is normally derived from corn. Till 2005, over 4 million flexible-fuel vehicles now exist in the USA. They 1.1 Microbes and Biofuels 5 can run on gasoline as well as on E85. Roughly 400 gas stations sell E85, and most of them are located in the Midwest, where a lot of corn is grown. The Midwest has always been an important region in the E85 ethanol history. The fleet of flexible-fuel vehicles (FFVs) in Brazil is the largest in the world, and since their inception in 2003, a total of 20 million flex-fuel cars and light trucks has been manufactured in the country by June 2013 [21] and over 3 million flexible-fuel motorcycles by October 2013. This is followed by the USA where about 11 million flex-fuel cars and light trucks were in operation in early 2013 [22, 23]. Brazilian flexible-fuel vehicle are optimized to run on any mix of E20 to E25 gasoline and up to 100 % hydrous ethanol fuel (E100). Flex vehicles in Brazil are built-in with a small gasoline. Registrations of flex-fuel autos and light trucks represented 87.0 % of all passenger and light-duty vehicles sold in 2012 [24], while flexible-fuel motorcycles represented 48.2 % of the domestic motorcycle production in 2012 [25]. There are over 80 flex car and light truck models available in the market manufactured by 14 major carmakers [26] and five flex-fuel motorcycles models available as of December 2012 [24]. In Europe, Sweden now has 250,000 flex-fuel vehicles on the road, about 70 % of the European total. The growth is the result of Sweden’s National Climate in Global Cooperation Bill, which signed the Kyoto Protocol and vowed to free Sweden from fossil fuels by 2020. Sweden also has the largest number of flex- fuel filling stations in Europe: 1750. Ethanol-based FFVs are supposed to be the most common in the world market having the record of about 39 million automobile, motorcycles, and light-duty trucks manufactured and sold worldwide through October 2013 and concentrated in four markets [27–31]: Brazil (23.0 million), the USA (10 million), Canada (more than 600,000), and Europe, led by Sweden (229,400).

1.1.2 History of Biodiesel as Transport Fuel

The bitter experience in operating steam engines in the late 1800s resulted in the development of diesel engine by Rudolph Diesel in the 1890s [32–34]. The diesel engine has become the engine of choice due to its reliability, consistency in power generation, and high fuel economy, worldwide. The most advantage of use of vegetable oil in running diesel engine is its use in running diesel engine in most remote area and cultivated field for irrigation, where availability of electricity is extremely poor. In order to increase the efficiency of diesel engines, the modern biodiesel fuel is made by converting vegetable oil into compounds called fatty acid methyl ester (FAME), in the year 1930 in Belgium [35]. The diesel engine works on the principle of compression ignition, in which fuel is injected into the engine’s cylinder after air has been compressed to a high pressure and temperature. As the fuel enters the cylinder, it self-ignites and burns rapidly, forcing the piston back down and converting the chemical energy in the fuel into mechanical energy. In the early 1990s, Martin Mittelbach with the US [36] industry further developed 6 1 Microbial Energy Conversion Technology biodiesel engine’s working quality. Pacific Biodiesel became one of the first biodiesel plants in the USA in 1996. In 2001, the biodiesel engine manufacturing became most popular, even in small industry level in the USA after the terrorist attack in 2001 which resulted in historically high oil price and an increase awareness of energy security [37]. Rapeseed and soybean oil, palm oil, and sunflower seed oil are commonly being used as alternative substitutes for diesel, after transesterification process. In addition, yellow grease, waste vegetable oil, waste cooking oil, and animal fat are also used for biodiesel purpose, mostly to meet local demand. Under the present energy crisis, biodiesel demand is gaining popularity, all over the world. It is mainly due to its clean emission profile, ease of use, and many other benefits. With minimum subsidy, biodiesel is cost competitive with petrodiesel. Meanwhile, biodiesel from algal biomass has shown the hope of commercialization for transport purpose. Harder and von Witsch, in the year 1942, had taken the credit of discovering microalgae as rich source of lipid which can be used both as food additive and fuel [38, 39]. With this noble information, after the Second World War, scientists from the USA [40–42], Germany [43], Japan [44], England [45], and Israel [46] remain busy in blue-green algae research to produce high-quality lipids suitable for use as biofuel [47]. Mainly, species in genus Chlorella are being used as target materials to improve large-scale biomass production, both in closed and open systems. During this period H.G. Aach findings were disclosed that lipid biosynthesis induction to significant level by nitrogen starvation technique [48]. Unfortunately, after the Second World War, the need for alternative fuels was subsided, and more focus was given to use algae as food and wastewater treatment [49]. In the 1970s, oil embargo and oil price surges have drawn the attention of the US Department of Energy to initiate the Aquatic Species Program in 1978 [49]. In this program, it was targeted to develop liquid transport fuel within a period of 18 years amounting to a budget of about $25 million [50]. About 3000 algal species were collected from all over the world to understand the growth and development under various stress conditions to increase the lipid contents. Almost all experimental trials were conducted in open outdoor pond under variable environmental conditions. The most significant results obtained from such huge project were that the enhancement of lipid production could be possible only by limited supply of nutrients [50], where as high growth was monitored by enrich culture technique. It was also realized that algal strain improvement for high lipid production could be possible by genetic engineering means. Although the project was successful in outdoor open ponds, the cost of unrestricted algal oil was about $59–186 per barrel [50], while the petroleum cost was less than $20 per barrel in 1995 [49]. Therefore, under constrain budget in 1996, the Aquatic Species Program was abandoned [49]. Fortunately, in the 1990s, the finding of Japan’s Research Institute of Innovative Technology for the Earth (RITE) on fixation of CO2 by algae was given an important clue to algal biofuel research [49]. At the same time other research project on harvesting hydrogen gas, methane, or ethanol from algae, as well as nutritional supplements and 1.1 Microbes and Biofuels 7 pharmaceutical compounds, has also helped inform research on biofuel production from algae [49]. Fortunately, algae are widely distributed, all over the world. In the USA, several companies are involved in the production of algal biomass through mass cultivation process, both in closed and open systems [51] for the purpose of algae–fuel. The present energy shortage and the world food problem have created interest in mass culture of blue-green algae for biodiesel production using arboreal land unsuitable for agriculture. The most advantage in algae cultivation is that they can be easily grown with minimum impact on freshwater system [52, 53]. They can also be cultivated well in saline water system with minimum management [54]. Interest- ingly, it has been observed that the algal biomass production cost is comparatively more than the production of second-generation fuel crops; however, the yield of biofuel is about 10–100-fold more per unit area [55]. The US Department of Energy reports that the algae–fuel can meet the petroleum fuel demand of the entire USA if only 0.42 % of US land amounting about 15,000 square miles (39,000 km2) would be used for blue-green algae cultivation to get biodiesel [56]. Surprisingly, this area is equivalent to 14 % of land used for corn cultivation in the USA [57]. The USA is known to consume the highest quantity of petrol and is about 25 % of the world petroleum consumption per year. It has been hypothetically calculated that about 1200 billion liters of petrol per year can be obtained from about 30 million hectares of land equivalent to 4 % land area of the continental USA [58, 59]. Algal biomass can be used to derived various types of energy for transportation, including biodiesel, jet fuel, electric power, and ethanol. The main advantages of algae-based biofuel over petro crop cultivation include higher biomass yields per acre of cultivation, little to no competition for arable land, use of a wide variety of water sources, the opportunity to reuse carbon dioxide from stationary sources, and the potential to produce “drop-in” ready-to-use fuels. The main drawback in the practice of algae cultivation is the anticipated cost of production, availability of land, and water resource. Fortunately, as earlier stated even saline water and arboreal land can well be used for commercial level of algae cultivation. Algae- based biofuel research and development is in their infancy, although work has been conducted in this area for decades to explore the possibility of using closed systems to cultivate blue-green algae by using solar energy, wastewater, and carbon dioxide emitted from industries. Algae Biomass Organization has the opinion that in 2018 algae–fuel can reach price parity with petrodiesel if granted production tax credits [60]. Unfortunately, in 1913, the mobile giant ExxonMobil Chairman and CEO Rex Tillerson had a joint venture project with J. Craig Venter’s Synthetic Genomics with the commandment of spending $600 over 10 years but with the lapse of 4 years declined from the project with an opinion that algae–fuel is “probably further” than 25 years away from commercial viability, even after spending 4 years with the project at the expense of about $100 million [61]. Fortunately, on the other hand, three American companies Solazyme [62], Sapphire Energy [63], and Algenol [64] were able to commercialize algae–fuel in 2012, 2013, and 2014, respectively. 8 1 Microbial Energy Conversion Technology

Green diesel processing from algal biomass can be further integrated to obtain other value-added products like natural dyes and pigments, antioxidants, amino acids, and vitamins [65] as well as pigments that may be beneﬁcial, such as beta- carotene and chlorophyll [66] and other high-value bioactive compounds [67–68].

1.1.3 History of Biogas as Transport Fuel

Biogas, otherwise known as landfill gas, is a mixture of methane, carbon dioxide, nitrogen, hydrogen, hydrogen sulfide, and oxygen. The quantity of methane can be varied from 50 to 80 % by using advanced process technology. It is guessed that during the tenth century BC and in Persia in 16th AD, the biogas was supposed to be used for heating bathwater [69]; however, not much evidence is available regarding its production and social use. Historical evidences are available on casual citation of biogas in 1630 by van Helmont and in 1667 by Shirley. In the scientific world, Volta noted as early as 1776 that biogas production is a function of the amount of decaying plant material and that the biogas is flammable under certain conditions. By 1884, a student of Pasteur in France, Gauon, had anaerobically produced biogas by suspending cattle manure in a water solution at 35 C. At that time he was able to obtain 100 L of biogas per meter. In 1808 H. Davy demonstrated decomposition of straw manure and collected biogas under vacuum condition without showing any interest on biogas. In the seventeenth century, Jan Helmont demonstrated the evolution of flammable gas from decomposed organic materials. In 1808, Sir Humphrey Davy determined that methane was present in the gases produced during the anaerobic digestion of cattle manure. The first digestion plant was built at a leper colony in Mumbai, India, in 1859 [70] to generate biogas for street lamp lighting purpose. Anaerobic digestion reached England in 1895 when biogas was recovered from a “carefully designed” treatment facility and used to fuel street lamps in Exeter [69]. The development of microbiology as a science led to research by Buswell and others in the 1930s to identify anaerobic bacteria and the conditions that promote methane production. In the 1890s [71], England for the first time realized the use of biogas in lighting lamps. In the 1930s, Buswell and others isolated and cultured anaerobic bacteria for biogas production. India, as one country with many biogas reactors installed, has a quite long history of biogas development. During the same time, many other parts of the world simultaneously started using methane for household works. They used to wonder how such small-scale methane production unit could be installed at their farms to convert waste into something more valuable [72]. Today, million-microlevel biogas plants are available in developing countries like India, Nepal, and Pakistan [73]. While developed countries are in progress in the process of generating significant amount of electricity, in Germany, biogas is used to generate electricity in megawatt quantity [74]. 1.1 Microbes and Biofuels 9

At present, compressed biogas is widely being used in Sweden, Switzerland, and Germany. Interestingly, biogas-empowered train “Biogasta˚get Amanda” has been in service in Sweden since 2005 [75, 76]. Even in Sweden, pig manure is being used to run customer-adapted combustion engine [77, 78]. In 2007, an estimated 12,000 vehicles were being fueled with upgraded biogas worldwide, mostly in Europe [79].

1.1.4 History of Miscellaneous Biofuels

1.1.4.1 Butanol

It is more or less similar to gasoline and less fuel value with reference to ethanol. It can be used as transport fuel. The green waste left over from the algae oil extraction can be used to produce butanol. In addition, it has been shown that macroalgae (seaweeds) can be fermented by Clostridia to butanol and other solvents [80]. Carbohydrates by anaerobic digestion by strain of Clostridium, called “TU- 103,” can be converted to butanol, acetone, and ethanol. However, cost issues, the relatively low yield, and sluggish fermentations, as well as problems caused by end product inhibition and phage infections, meant that acetone–butanol–ethanol (ABE) could not compete on a commercial scale with butanol produced synthetically, and almost all ABE production ceased as the petrochemical industry evolved. However, there is now an increasing interest in the use of biobutanol as a transport fuel. Eighty-ﬁve percent butanol/gasoline blends can be used in unmodiﬁed petrol engines. It can be transported in existing gasoline pipelines and produces more power per liter than ethanol. Biobutanol can be produced from cereal crops, sugarcane, sugar beet, etc., but can also be produced from cellulosic raw materials.

1.1.4.2 Hydrogen

Since the pioneering discovery by Gaffron and coworkers over 60 years ago, the ability of unicellular green algae, Chlamydomonas reinhardtii, to produce H2 gas upon illumination has been mostly a biological curiosity [81, 82]. He was one of the earlier researchers trying to elucidate the mechanistic and biochemical details of photosynthesis and plant metabolism. His most famous ﬁnding was that unicellular green algae can produce molecular hydrogen (H2) in the presence of light [83]. Sub- sequently, his noble ﬁnding has led to many efforts to develop H2 as a renewable biofuel [84]. In the late 1990s, Professor Anastasios Melis a researcher at the University of California at Berkeley discovered that if the algal culture medium is deprived of sulfur, it will switch from the normal photosynthesis to the production of hydrogen. At present, scientists at the US Department of Energy’s Argonne National Laboratory are working on the physiology and biochemistry of Chlamydomonas moewusii of hydrogen production and its monitoring [85, 86]. 10 1 Microbial Energy Conversion Technology

It was in the year 1911 M. Potter conceived the idea of using microbial cells, E. coli, for production of electricity, but the work did not receive any major coverage [87]. In 1931, however, Barnet Cohen drew more attention to the area when he created a number of microbial half fuel cells that, when connected in series, were capable of producing over 35 V, though only with a current of 2 milliamps [88]. Del Duca et al. (1963) demonstrated electricity production through fermentation of glucose by using Clostridium butyricum. Though the cell functioned, it was found to be unreliable, owing to the unstable nature of hydrogen production by the microorganisms [89]. Although this issue was later resolved in work by Suzuki et al. in 1976 [90], the current design concept of an MFC came into existence a year later with work once again by Suzuki [91]. But he could not clear the detail of functional aspect of microbial fuel cell. However, in the early 1980s, the works of MJ Allen and H. Peter Bennetto, from King’s College London, reported, individually, the detail about the functional part of microbial fuel cell. It is now known that electricity can be produced directly from the degradation of organic matter in a microbial fuel cell. Like a normal fuel cell, an MFC has both an anode and a cathode chamber. The anode chamber is connected internally to the cathode chamber via an ion exchange membrane with the circuit completed by an external wire. In May 2007, the University of Queensland, Australia completed its prototype MFC as a cooperative effort with Foster’s Brewing. The prototype, a 10 L design, converts brewery wastewater into carbon dioxide, clean water, and electricity. With the prototype proven successful [11], plans are in effect to produce a 660 gallon version for the brewery, which is estimated to produce 2 kW of power [92]. In 2011, biofuels were approved for aviation purpose [93]. Since then, some airlines have taken trials by using biofuels on commercial flights [94]. Currently, aviation represents 2 % of global emissions but is expected to grow to 3 % by 2050 [95]. Mainly, aviation biofuels are made from vegetable oils, waste cooking oil, and waste animal fats. In 2014, Boeing (NYSE:BA) has completed the world’s first flight using biodiesel, a blend of 15 % green diesel and 85 % petroleum jet fuel [96]. Rapid development in mass culture of algae has favored strongly in further use of algal biofuel in aviation purpose. Various companies working on algae jet fuel are Solazyme, Honeywell UOP, Sapphire Energy, Imperium Renewables, and Aquaflow Bionomic Corporation. In addition, universities like Arizona State Uni- versity and Cranfield University are also busy in algae jet fuel research. The long-chain hydrocarbons derived from algal biomass can be made to shorter form equal to petrodiesel through hydrocracking refinery process [96–98]. It has the same chemical properties as petrodiesel, meaning that it does not require new engines, pipelines, or infrastructure to distribute and use. It has yet to be produced at a cost that is competitive with petroleum [97]. 1.2 Renewable Energy Versus Fossil Fuels 11

1.2 Renewable Energy Versus Fossil Fuels

The chemistry of biofuels is substantially more complex than the chemistry of petroleum. Biofuels contain oxygen. By adding just that one atom, the complexity of these molecules begins to rise dramatically. Whereas the major difference between fossil fuels revolves around whether they have single or double bonds and how long they are, the differences between biofuels are far more complex. Because of the oxygen, biofuels can contain alcohols, esters, ethers, and acid groups. Each of these groups is a whole subset of organic chemistry, and each has special reaction characteristics. Alcohols are really nothing like ester, which are not like acids at all. The net result of adding oxygen is a huge jump in complexity [98]. Biofuels, like fossil fuels, come in a number of forms and meet a number of different energy needs. The class of biofuels is subdivided into two generations, each of which contains a number of different fuels. First-generation biofuels are made from sugar, starch, or vegetable oil. They differ from “second-generation biofuels” in that their feedstock (the plant or algal material from which they are generated) is not sustainable/green or, if used in large quantity, would have a large impact on the food supply. First-generation biofuels are the “original” biofuels and constitute the majority of biofuels currently in use. Second-generation biofuels are “greener” in that they are made from sustainable feedstock. In this use, the term sustainable is deﬁned by the availability of the feedstock, the impact of its use on greenhouse gas emissions, its impact on biodiversity, and its impact on land use (water, food supply, etc.). At this point, most second-generation fuels are under development and not widely available for use. Diesel and biodiesel are two products that can perform the same function but come from very different sources. Both diesel and biodiesel can be used to fuel diesel vehicles, such as cars, trucks, tractors, and powered lawn mowers. The major difference between these two fuel sources is that diesel comes from petroleum, a nonrenewable fossil fuel by-product (Fig. 1.3), whereas biodiesel is extracted from plant, seed, and animal oils (Fig. 1.4).

Fig. 1.3 Petrodiesel with 16 carbon molecules

Fig. 1.4 Biodiesel with 17 carbons (also called 16 carbons with an ester group) 12 1 Microbial Energy Conversion Technology

Biodiesel is a renewable form of diesel made out of biodegradable oils, such as soybean or peanut oil. When combined with certain alcohols, the fat in these oils create long chains of a chemical substance known as esters, which make the oil usable as a fuel. Biodiesel can be used in nearly any diesel engine with few modiﬁcations and no damage to the engine. In addition to coming from a renewable source, biodiesel releases extremely marginal levels of pollutants into the air. Compare the petrodiesel molecule above with a typical biodiesel molecule as shown here. In many ways, the biodiesel and petrodiesel molecules are similar. In fact, the only real difference is on the right side of the molecule where the biodiesel has two oxygen atoms compared to the petrodiesel molecule. These oxygen atoms are what make all the difference in biofuels like biodiesel, when they are burned. Oxygen is present in biodiesel because of the way it is produced. Petrodiesel is produced under anaerobic conditions over very long periods of time. These conditions result in the removal of oxygen from dead plant and animal matter, leaving only hydrogen and carbon to form petroleum and other fossil fuels. Biofuels, on the other hand, are produced through a process known as transesteriﬁcation. In this process, fats and oils from living organisms are broken apart to yield very long molecules that contain the oxygen we see above. Here is how the process works (Fig. 1.5). Oxygen makes all the difference in these molecules. The chart below lists some of the physical characteristics of biodiesel and compares them to petrodiesel (Table 1.2).

Fig. 1.5 Transesteriﬁcation of fatty acid (glycerol shown in red, methanol shown in blue)

Table 1.2 Difference Property Petrodiesel Biodiesel between biodiesel and Cetane 40–50 50–65 petrodiesel Energy content (BTU/gal) 128 K 118 K Energy density (MJ/kg) 43 38 Nitric oxide emission Baseline +10 Sulfur content <10 ppm <5 ppm Cloud point (C) À520 Cold ﬂow property Baseline Poor Lubricity Baseline Poor 1.2 Renewable Energy Versus Fossil Fuels 13

The second chemical difference between petro- and biodiesel deals with degradation. Biodiesel is biodegradable. The ester group makes it possible for biodiesel to be taken up by microorganisms and degraded. It also allows biodiesel to degrade through processes such as exposure to light, and it means that biodiesel spontaneously degrades at a set rate over time. This is good when the fuel is spilled but can cause problems during long-term storage. The third difference, and one that is a problem with biodiesel, is that the oxygen atoms make the molecule polar. That simply means that the end of the molecule near the oxygen has a net negative change and the end farthest from it has a net positive charge. In petrodiesel, there is no charge and the entire molecule is neutral. The problem with having polarity is that it makes it easier for the molecule to form into a crystal structure. In other words, it makes it easier for the molecule to freeze. Biodiesel freezes at temperature as much as 25 C above that of petrodiesel. Thus, biodiesel is less suitable for use in cold climates. The fourth chemical difference between petro- and biodiesel has to do with corrosion. The polarity of biodiesel makes it more prone to react with materials around it. This is particularly true of metals like zinc, copper, tin, and lead. Thus, biodiesel can speed up the process of corrosion (rusting) of these metals and decrease the life span of some engine components. In the same way, biodiesel is more reactive with molecules called elastomers, which give rubber its ﬂexibility and durability. When biodiesel damages elastomers, it makes rubber more brittle and causes it to crack. The result is damage to gaskets, fuel lines, and other engine connections that contain rubber. As a result of the last two differences, engines often have to be modiﬁed or built differently to handle biodiesel. This means that older diesel engines cannot handle biodiesel at all. Modern engines are often rated using terms like B2, B5, B20, and B100, which refer to the percent of biodiesel that they can handle. A B5 engine can tolerate fuels that are up to 5 % biodiesel without damage to engine components. A B100 engine can run on 100 % biodiesel. The biggest difference between these biofuels (ethanol and butanol) and biodiesel is that organisms can produce ethanol and butanol on their own. This means there is no need to harvest an organism to obtain a raw product that is converted to biofuel. The advantage of such an approach is that fewer nutrients and less water are required. The molecular structure of ethanol, butanol, and gasoline are diagramed below. Note that the only real difference, structurally, between ethanol and butanol is the number of carbon atoms they contain. Note that octane is used to represent gasoline even though gasoline is actually a mixture of hydrocarbon molecules ranging in size from four to twelve carbon atoms long (Fig. 1.6). Ethanol (Fig. 1.7) and butanol (Fig. 1.8) are both considered biofuels because they can be produced from living or recently living organisms. Like the differences between petrodiesel and biodiesel, the big difference between ethanol/butanol and gasoline is the presence of oxygen. In the case of alcohols (ethanol and butanol are alcohols), there is no ester group, but there is an alcohol group (just an oxygen with a hydrogen attached). This 14 1 Microbial Energy Conversion Technology

Fig. 1.6 Octane, one of several molecules in gasoline

Fig. 1.7 Ethanol (blue represents oxygen)

Fig. 1.8 Butanol (blue represents oxygen) difference means that alcohols behave differently from the esters that make up biodiesel. The chart below compares gasoline to ethanol, butanol, and biodiesel (Table 1.3). The thing to note is that ethanol has a very low energy density compared to gasoline, but butanol is pretty comparable. It requires about 1.5 times as much ethanol as it does gasoline to generate the same power. In a car, that means you need 1.5 gal of ethanol to travel the same distance as you would on one gallon of gasoline. An understanding biology is assisting in the production of biofuels. By genetically modifying organisms, some companies have been able to induce things like 1.3 Salient Features of Microbial Biofuel 15

Table 1.3 Difference between gasoline, ethanol, butanol, and biodiesel Property Gasoline Ethanol Butanol Biodesel Octane (average) 85–96 99.5 97 (Cetane) 50–65 Energy density 33 20 30 38 algae to produce fuel that is so close to gasoline that one can hardly tell the difference. If it is possible to complete this process without investing a great deal of energy from standard fuels and thus obtain most energy from sunlight, then the production of fuel in this manner can actually reduce carbon emissions. This occurs because more carbon is taken up to produce the fuel than is released when it is burned. This holy grail of biofuel production has yet to be obtained, but it is theoretically possible.

1.3 Salient Features of Microbial Biofuel

Basically energy resources can be broadly divided into three types: (1) fossil fuels, (2) fissionable nuclear fuels, and (3) non-fossil and nonnuclear energy. In spite of their significant use, fossil fuels have two major problems. Firstly, these are nonrenewable, and thus supply of such furls is either approaching exhaustion or getting more difficult to procure due to transport bottlenecks and steep hike in their price level; secondly, their continuous consumption may cause extreme environmental problems leading to ecological imbalance. Like fossil fuels, fissionable nuclear fuels also suffer from two serious drawbacks. Their supply from relatively cheap sources is drying up even for the developed countries. Moreover, the production and use of these sources cause plethora of hazards to this habitable world. The non-fossil and nonnuclear energy possibilities fall in three groups: (1) on solar such as geothermal and tides, (2) indirectly solar such as winds and ocean thermal gradients, and (3) directly solar which among other options includes photosynthesis. All other alternative sources under this category have one or the other practical hurdles in the way of their harnessing energy. Viewed from economic, technological, social, and material factors, they lack the capacity of meeting our future energy needs. Photosynthesis helps to remove carbon dioxide from the atmosphere and generate oxygen, the life-sustaining gas. It thus helps to control environmental pollution. It is believed that during the last hundred years, the concentration of carbon dioxide has significantly increased due to an ever-increasing use of fossil fuels. In the last decades alone, it has increased by about 100 ppm. This is likely to warm up the upper layer of the ocean and cause a raise in the sea level. Since plants use carbon dioxide for their growth, greater stress on biomass production may help to restore its level to the normal. Biomass energy is, thus, environmentally a very acceptable resource. The wide use of biomass for development produces minimal ecological imbalance and provides means to recycle nutrients and carbon dioxide from the atmosphere. 16 1 Microbial Energy Conversion Technology

An analysis of the energy scenario on the earth reveals that the only alternative appropriate to the socioeconomic conditions prevailing in the world is the photosynthetic way of trapping solar energy and preserving the same as various types of energetic biopolymers in the form of biomass. It has been the source of an old, reliable, and renewable form of energy, referred to under a new name “biomass,” presently. The earth receives 176,000 terawatts of incident primary power, while the rate of human world energy consumption is about 13.5 terawatts or less than 0.01 % of the solar resource. While solar energy is dispersed, nevertheless it represents a daily average energy equivalent of 20 kt of TNT per 1.5 square miles of the earth’s surface. Nature has provided a very useful storage process for capturing this light energy, namely, photosynthesis. In fact photosynthesis on earth stores over ten times the energy each year than that used currently by all humanity. Photosynthesis is the only process which produces enormous quantities of organic matter for sustaining the life on this globe. Coal, petroleum, and natural gas represent the photosynthetic capital of the past geological ages. They have been formed by the application of heat and compression over the plant and animal bodies in the deeper layers of earth. Along with wood, they provide a sufficient portion of energy required for domestic, industrial, and transport needs. Oxygen is being constantly consumed, and carbon dioxide evolved by the respiration of animals and plants and by the burning of wood, coal, petroleum, or natural gas. High carbon dioxide content of the atmosphere is toxic. Lower concentrations of oxygen are equally harmful. Luckily, green plants keep the concentration of the two gases almost constant by absorbing carbon dioxide and evolving oxygen during photosynthesis. This means the significance of biomass in sustainable ecosystem is unavoidable. Therefore, terrestrial and aquatic biomass merits a great deal of consideration in any energy development program, particularly for developing country. Renewable energy, presently being used, is mainly of three forms, i.e., ethanol, biodiesel, and biogas, and is ultimately derived from terrestrial biomass or aquatic micro- and macroalgae. All types of biological-origin green fuels are having properties like petrol and petrodiesel. Due to the following special features, biofuels are considered as better option for alternative energy: (a) Biodiesel is compatible with any diesel engine. This eliminates the need for converting the engine to support biodiesel. Biodiesel operates in compression ignition engine, which essentially requires very little or no engine modifications because biodiesel has properties similar to petrodiesel fuels. (b) As compared to petrol and petrodiesel, both ethanol and biodiesel are nontoxic and environmentally friendly as they produce substantially less carbon monoxide and 100 % less sulfur dioxide emissions with no unborn hydrocarbons. 1.3 Salient Features of Microbial Biofuel 17

(c) Biodiesel and ethanol are renewable energy sources unlike other petroleum products that will vanish in the years to come. Since it is made from animal and vegetable fat, it can be produced on demand. (d) Biodiesel can be used in pure form (B100) or can be blended with petrodiesel in the form of B2 (20 %, 80 % petrodiesel), B5 (5 % biodiesel, 95 % petrodiesel), or B20 (20 % biodiesel, 80 % petrodiesel). Like this ethanol- blended fuel, E10 (10 % ethanol and 90 % gasoline) reduces greenhouse gases by up to 3.9 %. (e) Fossil fuels are limited and may not be able to fulfill our demand for coal, oil, and natural gas after a certain period. But, ethanol and biodiesel can work as alternative forms of fuels and can reduce our dependence on foreign suppliers of oil as are produced from domestic energy crops and algal biomass. These fuels are produced in local refineries which reduce the need to import expensive finished product from other countries. (f) Biofuels are having less greenhouse gas emissions (e.g., B20 reduces CO2 by 15 %) as compared to fossil fuels and when burnt release greenhouse gases like carbon dioxide in the atmosphere that raises the temperature and causes global warming. To protect the environment from further heating up, many people have adopted the use of biofuels. Experts believe that using biodiesel instead of petrodiesel can reduce greenhouse gases up to 78 %. (g) Biofuels can be produced and distributed locally, whereas, fossil fuels are limited and may not be able to fulfill our demand for coal, oil, and natural gas after a certain period. Biodiesel can work as an alternative form of fuel and can reduce our dependence on foreign suppliers of oil as it is produced from domestic energy crops. (h) When fossil fuel is extracted from underground, it has to be refined to run diesel engines. It cannot be used straight away in the crude form. When it is refined, it releases many chemical compounds including benzene and butadi- ene in the environment which are harmful for animals, plants, and human life. Biofuel refineries, which mainly use vegetable and animal fat into biofuel, release less toxic chemicals, if spilled or released to the environment. (i) Vehicles that run on biodiesel achieve 30 % fuel economy than petrodiesel engines which means it makes fewer trips to gas stations and run more miles per gallon. (j) Ethanol is considered a renewable energy resource because it is primarily the result of conversion of the sun’s energy into usable energy. Creation of ethanol starts with photosynthesis, which causes feedstocks, such as sugarcane, maize, and other carbohydrate-rich annual crops to grow. These particular feedstocks are processed into ethanol. (k) Ethanol is carbon neutral, i.e., the carbon dioxide released in the bioethanol production process is the same amount as the one the crops previously absorbed during photosynthesis. (l) Ethanol has a lower heat of combustion (per mole, per unit of volume, and per unit of mass) than petrol. 18 1 Microbial Energy Conversion Technology

(m) Biogas systems convert organic household waste and manure organic market wastes into gas for cooking and lighting to meet household and local energy demand. Besides this, it helps to manage organic wastes and contributes to improved hygiene. (n) The biogas-slurry that comes out of biogas systems is rich in nutrients and can be applied directly to plants and vegetables to help them grow. Biogas-slurry replaces chemical fertilizers. This saves money and is better for the environment.

1.4 Social Acceptability

Complete success of any events or activities needs social acceptability in totality. Without it sustainability of any decision on project implementation is not feasible, in its true sense [99]. Hence, public acceptability is a complementary factor while thinking for any renewable energy project implementation to minimize logistic expenditures and to secure energy crisis [100]. By this means only, biofuel project can be timely successful. In this regard, the NGOs, policymakers, and politician as representatives of the public and press play a signiﬁcant role for the convenience and mobilization of the public. In addition, healthy interaction with certain geographical factors, state-of-the-art technology development, and social factors helps in accelerating the development of biofuel-based projects [101, 102]. Sometimes, opposition to renewable energy at micro-social level may play an important role in the success of the workout of biofuel-based projects [103]. Biofuels are noticed to be the most promising alternative sources of energy which can be helpful in bringing down the problems caused due to greenhouse effect as a result of extensive use of fossil fuels. As the biofuels are blended with ethanol or biodiesel, the chances of more emission of various noxious gases along carbon dioxide get reduced to a signiﬁcant level. In addition, a variety of feedstocks available on earth are autotrophs and are sustainable sources of energy. The factors responsible for social acceptance of biofuel utilization [104] depend on the geographical location of the areas where biofuel-based project is to be implemented to reduce the cost of logistics and to relieve people, especially city life based, from environmental pollution and greenhouse effects. Delshed et al. [105] investigated on public opinion on biofuel technology development and suggested some policy measures to use renewable energy, effectively [105]. In the year 2007, Wilstahagen et al. [106] suggested triangular relationship model (Fig. 1.9) on biofuel utilization on the basis of social, community, and market acceptance. The global emissions of carbon dioxide increased by 1.4 % over 2011, reaching a total of 34.5 billion tonnes in 2012. After a correction for the leap year 2012, this increase was reduced to only 1.1 %, compared with an average annual increase of 2.9 % since 2000. Interestingly, after 2012–2013 carbon dioxide emission has been reduced to about 1 %. The growth in global CO2 emissions almost stalled, 1.4 Social Acceptability 19

Social-political acceptance • Technologies & policies • By the public • By key stakeholders • By policy makers

Community Market acceptability • Acceptance • Consumers • Procedural • Investors justice • Intra firm • Distributiona

Fig. 1.9 Diagram of triangle dimension model of social acceptance increasing by only 0.5 % in 2014 compared to 2013, while the world’s economy grew by 3 %. In that year, emissions from fossil fuel combustion and industrial processes were about 35.7 billion tonnes CO2. China and the USA increased their emissions by 0.9 %. In the European Union, CO2 emissions decreased by an unprecedented 5.4 %, while India’s emissions continued to increase strongly, by 7.8 %. The CO2 emission trend mainly reflects energy-related human activities over the past decade. Transportation sector is the second largest carbon contributor next to industry sector [107]. In general, burning fossil fuel will produce methane (CH4) together with emissions of CO2, CO, NOX, SOX, and moisture water. Among them, carbon emission is the major concern as it is rapidly increasing, and this gas is the major contribution for the greenhouse effects [108]. Fortunately, over the last few years, the role of biofuels has been realized both by public and government sectors, and maximum efforts have been made to make the people realize the urgency of using renewable biofuels in transport and industrial sectors. On the basis of the International Energy Agency, an appreciable rise in global biofuel production has been observed and may be more till the end of 2020 [109]. In 2013 the global biofuel production was about more than 115 billion liters, which is about 7 % of the value over the earlier year. It was mainly due to high recorded amount of sugarcane production, in Brazil, resulting in two billion L additional ethanol production compared to the earlier forecast data. In the same year in the USA, ethanol production increased due to elevated corn prices resulting from intensified drought in the previous year. The US policy in regard to biofuels, such as ethanol, biofuel, and biodiesel, began in the early 1990s as the government began looking more intensely at 20 1 Microbial Energy Conversion Technology biofuels as a way to reduce dependence on foreign oil and increase the nation’s overall sustainability. Since then, biofuel policies have been refined, focused on getting the most efficient fuels commercially available, creating fuels that can compete with petroleum-based fuels, and ensuring that the agricultural industry can support and sustain the use of biofuels. Ethanol production from maize currently dominates the US biofuel production, with production of 14.30 billion gallons of ethanol from maize. The USA produced 60 % of global ethanol in 2014. Continuously, since 2000, the USA has been implementing series of policies to use to increase the efficiency of using biofuel in transport sectors. Among such policies are the Biomass Research and Development Act of 2000, the Farm Bill of 2002, the Energy Policy Act 2005, the Energy Independence Security Act of 2007, and the American Recovery and Reinvestment Act 2009. In most developed countries, the biofuel industry has developed through regulations to ensure sustainable market trading policy. This could be possible due to developing facilities by implementing tariffs on imports of raw materials and government subsidies to production factors, agricultural subsidies, environmental subsidies, tariffs, mandated purchases, and national goals for blending biofuels (ethanol and biodiesel) with fossil fuels (regular gasoline and diesel). The last factor is supposed to be unavoidable and a key regulation that would be helpful in consumption of biofuels in the fuel market. These goals allow new investment projects, and the industry can be sure that their product will be demanded in the domestic market of the country that imposes these goals through mandates or laws. Table 1.1 shows the countries considered as the main producers and exporters of biofuels have set national goals for blending. Some of the states of the USA expend E5 or E10 (gasoline blended with ethanol 5–10 %) in all stations, that is, even if the consumer does not have complete information on the existence or production of biofuels, these individuals in those states are consuming this alternative fuel. Additionally, the USA has ruled that government vehicles use E85 (85 % ethanol); they have also established incentives for school vehicles to use E85 or biodiesel. Promising results have been seen in imposing rule in using blended flexible fuels for school transport in the USA [110]. It has been noticed that after imposing Energy Policy Act 2005, there has been a rapid growth in biofuel production and consumption in the USA, and the European Union and Brazil appear to be shifting gears. The global biofuel production is seen reaching 139 billion liters in 2020. With a less optimistic outlook for the USA and Brazil, world ethanol output is now forecast to reach 104 billion L in 2020. For 2018, the ethanol forecast has been cut by almost 4 billion L from levels projected last year [111].

1.5 Global Partnership and Trading of Biofuels

The large-scale production of biofuels began in the early 1970s. In 1975, Brazil launched a pro-alcohol program for biofuel production. Since then world attention has been keen in enhancing biofuel use as alternative to fossil fuels [112]. This is 1.5 Global Partnership and Trading of Biofuels 21 due to their noble properties as clean fuels compared to fossil fuels responsible for greenhouse effect on which Kyoto protocol meeting was organized. The main reason for the increasing interest in biofuels is mainly due to energy security, currency savings through a reduced oil bill, rural development, and poverty reduction. Developed countries are trying desperately to establish perfect coordination between agriculture, land, and green energy utilization. The Millennium Summit held on 2000 aimed to have a new global partnership to reduce extreme poverty by 2015 in line with a series of targets that have become known as the Millennium Development Goals (MDGs). The MDGs are crafted around eight themes, i.e., (1) values and principles; (2) peace, security, and disar- mament; (3) development and poverty eradication; (4) protecting our common environment; (5) human right, democracy, and good governance; (6) protecting the vulnerable; (7) meeting the special needs of Africa; and (8) strengthening the United Nations. Of particular relevance here is the fourth one, which calls for environmental sustainability. One of its targets is to addition; widespread biofuel production may result in, or enhance, poor labor practices [112, 113]. Recently, biofuel market has gained speed to meet global demand for alcohol and biodiesel. In the future, the scope of biofuel trading seems to be bright but needs strong infrastructure—compatible with biofuel promotion as the volume and direction of biofuel trade depend on many factors, including policies, tariffs, and crop yields. Ethanol global market has been in better demand compared to biodiesel, as international policies and incentives in ethanol marketing in the European Union are more practicable. The current major participants in the liquid biofuel trade are the USA, EU, Brazil, and Argentina. Basically, effective utilization of biofuels depends on three major factors, i.e., (a) domestic production, (b) domestic consumption, and (c) trade-related measures [114]. The ﬁrst factor includes production mandates, investment support such as loan guarantees and tax credits for biofuel facilities, and research and development for feedstock improvement. In the second factor issues related to consumption mandates, incentives such as tax exemptions for biofuels or incentives promoting ﬂex-fuel vehicles are incorporated. The third factor covers protective actions, such as import tariffs, and measures to prevent exports, such as export tariffs. All the policies so far that have been framed by the USA and European countries are based on these factors. Through government incentives, Brazil’s ethanol industry became a major source of bio-based motor fuel in the 1990s and in recent years has been a major ethanol exporter. Brazilian ethanol is produced from sugar cane, a low-cost feedstock that also allows processors to use the cane stalks as fuel for processing plants after the juice has been removed for ethanol or cane sugar production. Sugarcane is a perennial crop that needs to be replanted only every 6–8 years. The sugar marketing year [104] in Brazil’s major center-south sugar-producing region runs from May through April, although crushing of sugarcane has sometimes started as early as late March. In the northeastern Brazil’s sugar area, the marketing year runs from September through August. Brazil sources indicate that 90 % of the crop is produced in the center-south region. 22 1 Microbial Energy Conversion Technology

With large ethanol production, Brazil’s government has been able to mandate ethanol–gasoline average blends of 18–25 %. The percentage blend can vary from time to time, depending on the available supply of ethanol. In October 2011, it was lowered from 25 to 20 %, due to tight sugar and ethanol supplies. In August 2012, it was expected to remain at that level until at least the start of the 2013–2014 sugar marketing year [115]. In recent years before June 2012, Brazil had a federal tax on gasoline but not on ethanol, thus helping to provide an economic advantage for ethanol. In June 2012, the gasoline tax was removed, thus tending to reduce the advantage of ethanol relative to gasoline. The number of flex-fuel vehicles in Brazil’s automobile fleet has increased rapidly in the last few years and now accounts for most new car sales. As a result, Brazil has a large fleet of vehicles that can be powered by hydrous ethanol, a mixture that includes a small amount of water in ethanol and no gasoline. Alternatively, anhydrous ethanol can be blended with gasoline to create the 20 or 25 % ethanol–gasoline blends. Motorists in Brazil have indicated to us that E100 needs about a 30 % price discount per liter to offset its lower fuel mileage than gasoline. Ethanol production grew consistently from around 800 million gallons per month in the beginning of 2009 to historically high levels of more than 1100 million gallons per month from January 2011 through July 2012. Ethanol production stopped growing at the rates of prior years because of the saturation of the US gasoline market with E10 coupled with less-favorable export markets. Neverthe- less, ethanol production in 2011 was 13.9 billion gallons, compared to 13.3 billion gallons in 2010. Ethanol grew from 8 % of US gasoline consumption by volume in 2009 to nearly 10 % in 2011 and in the first eight months of 2012. With almost all gasoline in the USA already blended with 10 % ethanol (E10), the maximum level approved for use in all cars and light trucks, significant increases in domestic consumption of ethanol face a blend wall, unless higher percentage of ethanol blends can achieve significant market penetration. While the US Environmental Protection Agency (EPA) has approved the use of a 15 % ethanol blend (E15) for model year 2001 and newer cars and light trucks, concerns related to automobile warranties, potential liability for misfueling, and infrastructure costs are likely to limit E15 use to low volumes in the near term. Exports of ethanol increased substantially as producers looked abroad for new markets and Brazil experienced a poor sugar harvest during 2011–2012. The EU has been a major consumer of both biodiesel and ethanol since 2003, and the acceptance of Renewable Energy Directive (RED) in 2009 has only increased the production and consumption of biofuels. RED requires a 10 % renewable energy share in the transportation sector by 2020 and outlines sustainability criteria, which include land use change and greenhouse gas (GHG) savings. In addition to RED, the EU’s Fuel Quality Directive also created requirements for renewable fuel use—including a 6 % reduction in transportation-related GHG emissions—and dictated fuel quality standards. The technical standards from the Fuel Quality Directive in conjunction with the sustainability requirements may increase barriers to trade but at the same time increase consistency in fuel quality and provide certainty for producers and consumers. Currently, only direct land use change is 1.5 Global Partnership and Trading of Biofuels 23 considered in both regulations, but future revisions may include indirect land use change [114]. It has been noticed that the use of biofuels has reduced the greenhouse effect to a remarkable extension. This impact mainly depends on the type of feedstock, cultivation methods, conversion technologies, and energy efficiency assumptions. The practice of sugarcane cultivation helps in reduction of greenhouse effect better way compared to the other crops. One issue is the “energy balance” of biofuels, i.e., the amount of energy required to produce one unit of biofuel compared to the energy contained in the same unit of biofuel. In some cases, notably where fossil fuels are used in the production process, biofuels may not fare better than conventional fuels. However, emission from biofuel is noticed to be free from lethal gases compared to fossil fuel use. The only problem with burning sugarcane field after harvest resulted in emission of greenhouse gas and nitrogen oxide. The practice of energy crop cultivation causes environmental problems associated with agricultural commodity production such as deforestation, monocropping, water usage, land degradation, and water pollution. The practice of soy production by replacing forest area may create environmental problem leading to ecosystem imbalance in semidry land like north-central Brazil (the cerrado) and in the Amazon forests. Unlike Brazil, Argentina has primarily focused on exporting biodiesel. In 2013, Argentina exported 1.5 million tonnes of biodiesel to EU. The main exporters are Cargill, Bunge Ltd and Louis Dreyfus. Argentina’s policies have included tax credits for production as well as much lower export taxes than other vegetable oils (Lamers et al.) [114]. The result has been an industry focused on export primarily to the EU often via the USA (Junginger 2008) [116]. The RFS is not expected to greatly increase the demand for biodiesel in the USA, so most Argen- tinean biodiesel will likely continue to go to the EU. Argentina has implemented some policies encouraging domestic consumption, but production volumes are expected to remain high enough to continue its focus on exports (Lamers et al.) [114]. About 50 companies ((1) Solazyme, (2) KiOR, (3) LanzaTech, (4) Novozymes, (5) POET, (6) DuPont Industrial Biosciences, (7) Gevo, (8) Sapphire Energy, (9) Joule Unlimited, (10) ZeaChem, (11) Honeywell’s UOP, (12) BP Biofuels, (13) LS9, (14) Chemtex/Beta Renewables, (15) Amyris, (16) Virent, (17) INEOS Bio, (18) Enerkem, (19) Abengoa Bioenergy, (20) Ceres, (21) Neste Oil, (22) Renmatix, (23) Coskata, (24) DSM, (25) Mascoma, (26) Virdia, (27) Codexis, (28) Butamax, (29) Waste Management, (30) Petrobras, (31) Elevance Renewable Sciences, (32) Cobalt, (33) Inbicon, (34) Dynamic Fuels, (35) Algenol, (36) Ful- crum BioEnergy, (37) Valero, (38) EdeniQ, (39) Rentech, (40) Boeing, (41) OriginOil, (42) SG Biofuels, (43) Cosan, (44) Ensyn, (45) Renewable Energy Group, (46) Dyadic, (47) OPX Biotechnologies, (48) BlueFire Renewables, (49) Catchlight Energy, (50) Fiberight) all over the world are working in algae– fuels for commercialization. The primary products from algae-based technology platform are ethanol, diesel, and gasoline and eventually jet fuel primarily intended for transportation fuel. Following are few emerging companies actively engaged in algae–fuel marketing along or in joint venture with other foreign companies. 24 1 Microbial Energy Conversion Technology

1.5.1 Algenol

Since 2006, Algenol is a global, industrial biotechnology company that is commer- cializing its patented algae technology platform for production of ethanol and other biofuels. Based in Southwest Florida, Algenol’s patented technology enables the production of the four most important fuels (ethanol, gasoline, jet, and diesel fuel) using proprietary algae, sunlight, carbon dioxide, and salt water for around $1.27 per gallon and at production levels of 8000 total gallons of liquid fuel per acre per year. Algenol’s technology produces high yields and relies on Algenol’s patented photo bioreactors and proprietary downstream separation techniques for low-cost fuel production. These novel, low-cost techniques have the added benefit of consuming carbon dioxide from industrial sources, not using farmland or food crops and being able to provide freshwater. The company’s technology is a unique two-step process that first produces ethanol directly from the algae and then converts the spent algae biomass to biodiesel, gasoline, and jet fuel. It is the only renewable fuel production process that can convert more than 85 % of its CO2 feedstock into the four most important fuels. Algenol’s goal is to produce 20 billion gallons per year of low-cost ethanol by 2033 [117]. The second large partner to join Algenol is Reliance Industries Ltd. based in Mumbai, India (BioField). Reliance operates the world’s largest refinery among many business interests and has a demonstrated commitment toward developing sustainable and renewable energy sources. Other dedicated partners include Uni-Systems that is working on developing co-location strategies with existing sugarcane ethanol facilities and Mizrahi, working to evaluate a land site in Israel next to a large power plant.

1.5.2 Solazyme

With a breakthrough production process, Solazyme is revolutionizing the way the world makes oil. This company works on the basis of today’s biggest issues—such as sustainably harvested oil and resource scarcity with microalgae. Solazyme’s manufacturing process can produce a wide range of products from a variety of sugar feedstocks at a single manufacturing facility using standard industrial equipment, thus providing exceptional input, output, and capital flexibility. A 100 % algae-derived biodiesel, Soladiesel BDR, can be used with factory- standard diesel engines without modification. The fuel is fully compliant with the ASTM D 6751 specifications for fatty acid methyl ester (FAME)-based fuel that meets ASTM D 975 and significantly outperforms ultra-low sulfur diesel in total THC, carbon monoxide, and particulate matter tailpipe emissions. Soladiesel BD also demonstrates better cold temperature properties than any commercially available biodiesel. 1.5 Global Partnership and Trading of Biofuels 25

Solazyme is one of a handful of companies which is supported by oil companies such as Chevron. Additionally, this company is also backed by Imperium Renew- ables, BlueCrest Capital Finance, and The Roda Group. Solazyme has developed a way to use up to 80 % percent of dry algae as oil [118]. This process requires the algae to grow in a dark fermentation vessel and be fed by carbon substrates within their growth media. The effect is the production of triglycerides that are almost identical to vegetable oil. Solazyme’s production method is said to produce more oil than those algae cultivated photosynthetically or made to produce ethanol. Oil refineries can then take this algal oil and turn it into biodiesel, renewable diesel, or jet fuels. Part of Solazyme’s testing, in collaboration with Maersk Line and the US Navy, placed 30 tonnes of Soladiesel (RD) algae fuel into the 98,000 tonnes, 300 m container ship Maersk Kalmar. This fuel was used at blends from 70 to 100 % in an auxiliary engine on a month-long trip from Bremerhaven, Germany, to Pipavav, India, in December 2011. In July 2012, the US Navy used 700,000 gal of HRD76 biodiesel in three ships of the USS Nimitz “Green Strike Group” during the 2012 RIMPAC exercise in Hawaii. The Nimitz also used 200,000 gal of HRJ5 jet biofuel. The 50/50 biofuel blends were provided by Solazyme and Dynamic Fuels [117– 119]. Since 2008, Solazyme has partnered with the US Navy and the Department of Defense to develop, test, and certify advanced drop-in renewable fuels that meet their strictest standards. Specifically, Solazyme has developed jet fuel, marine diesel, and on-road diesel that have been rigorously tested by the US Navy and shown to meet its HRD76 and HRJ5 military specifications. We are proud that Solazyme fuels were used as the reference fuels during the Navy’s successful multi- year certification process for renewable marine diesel fuel.

1.5.3 Blue Marble

Blue Marble Energy is a Seattle-based company utilizing unique bacterial consortia to produce specialty biochemicals and renewable natural gas. BME’s mission is to produce drop-in replacements to petrochemicals which are safe, carbon-neutral, and fully sustainable. BME’s proprietary AGATE (acid, gas, and ammonia-targeted extraction) system processes nearly any organic feedstock, utilizing cultured strains of bacteria to perform fermentation (like brewing beer) to produce a wide variety of biochemicals. This solution will allow manufacturers to replace petroleum with renewable feedstocks and produce some of the chemical industry’s highest value products, including esters, amides, and anhydrous ammonia. In addition to these biochemicals, the AGATE system generates renewable natural gas as a by-product [120]. Bionavitas is a bioscience company based in Redmond, Washington, committed to revolutionizing the clean technology industry. Bionavitas harnesses the power of algae for biofuel production, health and nutraceutical products, and environmental 26 1 Microbial Energy Conversion Technology remediation. The company’s innovative, patent-pending Light Immersion Technol- ogy™ addresses the problem of algae “self-shading” by allowing more light to penetrate the algae biomass. This permits the algae to grow deeper and thicker, resulting in the more efﬁcient and expansive algae production necessary for cost- effective commercial application.

1.5.4 Sapphire Energy

Sapphire Energy was founded in 2007 with the mission to change the world by developing a domestic, renewable source of energy that benefits the environment and hastens America’s energy independence [121]. The company’s founders— backed by a team of the nation’s leading researchers, scientists, and blue-ribbon investors in early-stage companies—set out to become the world’s leading producer of commercial-scale, renewable, drop-in replacement fuels for gasoline, diesel, and jet fuel with its algae-derived biofuel solution. To accomplish this, they brought together a team of people who, as entrepreneurs, investors, scientists, and concerned citizens, shared the belief that climate change is a threat to the environment and that dependence on imported oil is a threat to national security and the security of future generations. As of February 2014, Sapphire Energy employs over 150 employees. The company is headquartered in San Diego, California and has an engineering office in Orange County, California, and a research and development facility in Las Cruces, NM. In addition, the company’s green crude farm, the world’s first commercial demonstration algae-to-energy facility (also known as the Integrated Algal BioRefinery or IABR), for which construction began in June 2011, is now operating in Luna County, near Columbus, New Mexico. Sapphire’s green crude farm integrates the entire value chain of algae-based fuel, from cultivation, to harvest, to conversion of ready-to-refine green crude, representing the convergence of biotechnology, agriculture, and energy [4]. The Farm is now producing barrels of crude oil year-round, and the company expects to be producing 100 barrels of green crude per day in 2015 and at commercial-scale production in 2018. Sapphire Energy produces “green crude” from algae, creating a crude oil containing many of the properties found in fossil crude oil. Green crude is a low-carbon, 100 % renewable crude oil offering a reduction in carbon emissions compared to petroleum-based equivalents. Its production does not impact agricultural crops, land, or water, since algae are grown in salty, non-potable water, using lands not suitable for agriculture, and require only sunlight and CO2 to grow. It also drops right into the current infrastructure of today’s cars, trucks, and aircraft without modifications. Sapphire Energy’s green crude can be refined in a typical refinery and scaled to meet the growing demand for new sources of energy which are renewable and environmentally friendly and reduce dependence on foreign oil. For the first time, in 2013, Sapphire sold algal biofuel to Tesoro [119]. 1.5 Global Partnership and Trading of Biofuels 27

1.5.5 Diversiﬁed Technologies Inc.

Diversified Technologies Inc. has created a patent-pending pretreatment option to reduce costs of oil extraction from algae. Pulsed electric field (PEF) technology is a low-cost, low-energy process that applies high-voltage electric pulses to an algal slurry [118]. These pulses rupture the algal cell walls, increasing the availability of intracellular materials for downstream separation and extraction. The process is in-line and scalable to high volumes. Using PEF treatment would help biorefiners simplify the processing of algae for biofuels and accelerate downstream extraction processes. It has been calculated that PEF treatment would account for about $0.10 per gallon compared to about $1.75 per gallon for conventional drying process.

1.5.6 OriginOil, Inc.

OriginOil helps algae growers extract oil from algae for use as a feedstock for the commercial production of transportation fuels, chemicals, and foods by using a noble method known as Helix BioReactor [120]. This reactor is fitted with special type of light device which can supply specific wavelength of light, as full spectrum of light is not required for algae growth [121]. In a single step, our breakthrough technology efficiently dewaters and breaks down algae for its useful products, overcoming one of the greatest challenges in making algae a viable replacement for petroleum. As a pioneer and the emerging leader in the global algae oil service field, OriginOil supports its core algae extraction technology with an array of process innovations for some of the world’s most successful algae growers and refiners, just as pioneers like Schlumberger and Halliburton have done in the oil field service industry.

1.5.7 Aquaviridis, Inc.

Aquaviridis is a commercial algae grower headquartered in Preston, Minnesota, with production facilities in the Mexicali Valley of Northern Mexico. Its focus is on the production of algae feedstocks for high-value markets such as nutraceuticals, human food, animal/aquaculture feed, and renewable biofuels. Aquaviridis intends to partner with leading algae industry researchers, equipment vendors, and government and local organizations to develop best-in-class products and solutions for these markets. 28 1 Microbial Energy Conversion Technology

1.5.8 Proviron

This company has been working on a new type of reactor (using flat plates) which reduces the cost of algae cultivation [122]. Proviron is a Flemish chemical business that is still a 100 % family company, which is known for its brake fluids and anti- frost products for the aircraft industry. This company has developed an alternative system for mass-scale algae cultivation by using flat-plate reactor surrounded by a water buffer, manufactured from thin plastic film. This system is around 7 meters long and can be rolled out like an air mattress. The entire structure is held up by a surrounding water buffer. The water also ensures that the temperature does not get too high during bright sunshine, as this would kill the algae. The ideal growth temperature lies between 20 and 25 C. Enclosed pipe systems must be cooled in high summer with sprayers in order to ensure that 25 is not exceeded. The current trial installation is located at the Hooge Maey, known to many Antwerp residents as the former landfill site. During the winter months, Proviron uses residual heat from converted diesel generators that run on methane that is captured from the landfill site.

References

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In recent year, there has been significant interest in biomethanization technology and its application for biogas production [1–10]. The increasing demand for biogas is mainly due to a number of factors. Firstly, the escalating cost of fossil fuels, and the decreasing availability of renewable sources of fuel, i.e., firewood, has forced many developing countries to look more at renewable energy technologies, i.e., solar, wind, and biofuels such biogas, alcohol, and biodiesel. Among these techniques, biogas is one of the lowest financial inputs per kWh of output. Secondly, since biogas mimics natural environmental cycle, nutrients such as nitrogen, phos- phorous, and potassium are conserved in the process and can be recycled back to the land in the form of slurry. This is in contrast to the burning of biomass where most of the nutrients are perished. Thirdly, through the digestion of animal manures and night soil, biogas has the potential of considerably reducing the plant, animal, and human pathogens. This breaks the cycle of reinjection and leads to a considerable improvement in public health [11, 12]. Finally, since biogas is a clean burning fuel, its use in domestic applications can reduce the incident of eye and lung problem commonly encountered with the use of smoky fuel, like firewood, agricultural residues, or coal. Microbial-catalyzed biogas production resulting from anaerobic decomposition of waste biomass is gaining interest as a versatile source of energy having high potential to meet growing demand within the power, heat, fuel, and biochemical markets. Pike Research, a market research and consulting firm that provides in-depth analysis of global clean technology markets, says that the biogas market gained $17.3 billion in global revenue in 2011 and likely to reach about $33.1 billion in the year 2022 [13]. In fact, biofuel provides around 3 % of the world’s fuel for transport. Biomass energy resources are readily available in rural and urban areas of all countries. Biomass-based industries can provide appreciable employment opportunities and promote biomass regrowth through sustainable land management practices. The negative aspects of traditional biomass utilization in developing countries can be mitigated by promotion of modern waste-to-energy technologies which

© Springer International Publishing Switzerland 2016 35 B. Kumara Behera, A. Varma, Microbial Resources for Sustainable Energy, DOI 10.1007/978-3-319-33778-4_2 36 2 Biomethanization provide solid, liquid, and gaseous fuels as well as electricity. Bioenergy systems offer signiﬁcant possibilities for reducing greenhouse gas emissions due to their immense potential to replace fossil fuels in energy production. Bioenergy usually provides an irreversible mitigation effect by reducing carbon dioxide at source, but it may emit more carbon per unit of energy than fossil fuels unless biomass fuels are produced unsustainably. Biomass can play a major role in reducing the reliance on fossil fuels by making use of thermochemical conversion technologies. In addition, the increased utilization of biomass-based fuels will be instrumental in safeguarding the environment, generation of new job opportunities, sustainable development, and health improvements in rural areas. The biogas production technology is an extremely versatile technology and can utilize a wide variety of organic feedstocks (animal manures, night soil, agricultural residues, aquatic plants, and organic industrial wastes) in the presence of methanogens [14]. These microbes are a group of archaea named for the unique way they obtain energy. They use carbon dioxide to oxidize hydrogen-releasing methane as a waste product. They are poisoned by oxygen. Methanogens live in extreme environments and swamp and marshes where the oxygen has been consumed by other microorganisms. Some species inhabit the anaerobic environment within the guts of cattle, termites, and other herbivores, playing an essential role in the nutrition of these animals. Methanogens also act as decomposers in sewage treatment facilities.

2.1 Introduction

Essentially, biomass is stored solar energy that man can convert to electricity, fuel, and heat. Through photosynthesis the energy from the sun is stored in the chemical bonds of the plant material. Typically biomass energy comes from three sources: agricultural crop residues, municipal and industrial waste, and energy plantations. Numerous processes, such as cogeneration, gasification, and fermentation, can tap into this energy source to produce energy available for human use. Biomass has been used for many thousand years as a source of heat energy since man first discovered fire. Many people around the world still burn wood as their primary source of heat during the winter. Today, there are 1.8 billion rural people in the developing countries of the world still using straw as their domestic fuel source. Biomass has a limited value if it can only be used as a solid fuel for cooking or for heating to produce steam. However, converting into combustible gases by biotech- nical means [15–19], it has much wider application and can be used to drive locomotive engine or as an efficient fuel in transport sector. So, many direct and indirect techniques are presently being operated on a large scale or at pilot plant stage. These biomass conversion techniques, mainly of three fundamental types (combustion, dry chemical process, and aqueous process), are chiefly based on the biomass water contents. The principal routes and product involved in these techniques are given in Fig. 2.1. This chapter investigates biomass feedstock opportunities and provides a technical overview and assessment of production techniques 2.1 Introduction 37

Combustion Biomas Aqueous process

Chemical Alcohol Anaerobic Production Treatment Digestion Electricity High pressure Wood, crop Cellulosic Night soil, cow steam Residue Waste, starch Dung, cattle Grass MSW Bearing Litter, crop plants residue

Dry Oil Ethanol Methane Chemical

Pyrolysis Gasification Hydrogasification

Dry wood dry litter Dry wood dry litter Methane Straw Straw Ethanol Baggasse Baggasse Char Dry lignocellulosic Dry lignocellulosic matters Matters MSW

Oil Low Gas Medium Char Energy Gas

Methane Methanol Ammonia Electricity

Fig. 2.1 The principal routes in biogas production process from waste biomass for converting biomass into transport fuel, either in the form of liquid fuel or gaseous fuel by means of suitable microbes. Microbial decomposition of organic biomass under anaerobic condition generates a flammable gas popularly known as biogas (Table 2.1). However, this gas is a mixture of various other gases being dominated by methane. Different sources of production lead to different specific compositions (Tables 2.2 and 2.3). The presence of H2S, CO2, and water makes biogas very corrosive and requires the use of adapted materials. The composition of a gas issued from a digester depends on the substrate, of its organic matter load, and the feeding rate of the digester. Combus- tion of methane, hydrogen, and carbon monoxide present in biogas with oxygen generates energy which can be used for generating heat and electricity. A great variety of organic material can be used in anaerobic digesters as a feedstock for generating biogas [37–39]. However, there are scientific, engineering, and legal limits to what can be added successfully to a digester. In addition, the feedstock needs to be a liquid mixture with appropriate moisture content. The energy production potential of feedstocks varies depending on the type, level of processing/ pretreatment, and concentration of biodegradable material. Before obtaining clean 38 2 Biomethanization

Table 2.1 Chemical characteristics of biogas from different sources of production Wastewater Waste from Household treatment plants Agricultural agri-food Component waste sludge waste industry

CH4 % vol 50–60 60–70 60–75 68

CO2 % vol 30–34 33–19 33–19 26

N2 % vol 5–0 1–0 1–0 –

O2 % vol 1–0 <0.5 <0.5 – H2O % vol 6 (@ 40 C) 6 (@ 40 C) 6 (@ 40 C) 6 (@ 40 C) Total % vol 100 10 100 100 3 H2S mg/m 0–200 1000–4000 3000–10,000 400 3 NH3 mg/m – 50–100 – Aromatic mg/m3 0–200 – – – Organochlorinated or 100–800 – – – organoﬂuorated mg/m3

Table 2.2 Physical characteristics of biogas from different sources of production Type of gas Household waste Agri-food industry Natural gas

Composition 60 % CH4 68 % CH4 97.0 % CH4

33 % CO2 26 % CO2 2.2 % C2

1%N2 1%N2 0.3 % C3

0%O2 0%O2 0.1 % C4+

6%H2O5%H2O 0.4 % N2 PCS (kWh/m3) 6.6 7.5 11.3 PCI (kWh/m3) 6.0 6.8 10.3 Density 0.93 0.85 0.57 Mass (kg/m3) 1-21 1.11 0.73 Index of Wobbe 6.9 8.1 14.9 biogas, three major steps are to be followed (Fig. 2.2). Listed below are feedstocks that can be commonly used in anaerobic digesters: • Livestock manures • Waste feed • Food-processing wastes • Slaughterhouse wastes • Farm mortality • Corn silage (energy crop) • Ethanol spillage • Glycerin as the product from biodiesel production • Milk house wash water • Fresh produce wastes • Industrial wastes • Food cafeteria wastes • Sewage sludge 2.1 Introduction 39

Unlike other renewable fuels such as biodiesel and bioethanol, biogas production is relatively simple and can operate under any conditions and is not monopo- listic [40–42]. Dung is a potential substrate for biogas production, seen only as a floor polish and fertilizer in the garden for hundreds of years. Biogas for rural energy is sustainable and affordable and has no negative effect on people’s health or the environment, if handled properly [43, 44]. Complicated construction, difficult operation of the systems, high investment, and maintenance costs have pushed farmers to adopt cheaper and simpler anaerobic systems [45]. There are currently more than 30 million household digesters in China, followed by India with 3.8 million, 0.2 million in Nepal, and 60,000 in Bangladesh [46–48]. China has increased its investments in biogas infrastructure very rapidly. By 2020, 80 million households in China are expected to have biogas digesters serving 300 million people [49]. India is implementing one of the world’s largest renewable energy programs with different scales of technologies. One of the strategies is to promote biogas plants [50, 51]. India began the project half a century ago and was further supported by the National Project on Biogas Development in 1982. Similar trends were more or less observed in other Asian countries. For instance, SNV, a nongovernmental organization (NGO) from the Netherlands, has installed 23,300 plants in Vietnam [52]. At present, the USA has over 2000 sites producing biogas: 239 anaerobic digesters on farms, 1241 wastewater treatment plants using an anaerobic digester (~860 currently use the biogas they produce), and 636 landfill gas projects [53–60]. The number of farm-scale digesters in Europe has increased drastically. At the end of 2011, the number of these digesters was more than 4000 in Germany, 350 in Austria, 72 in Switzerland, and 65 in the UK followed by Denmark with 20 community-type and 35 farm-scale plants, and Sweden had 12 plants [22, 23, 61, 62]. The level of biogas technology for household purposes is very low in many African countries [63]. Kenya has 1884 household biogas plants and Ethiopia has more than 1140 plants [64]. Small-scale biogas plants are located throughout Africa, but only a few are working. Poor technical quality of construction and material used, inexperienced contractors, insufficient knowledge on the system in practice, as well as in research institutes and universities are some of the reasons responsible for the failure [62]. Although the potential need is very high in Africa, the technology is at embryonic stage with the countries struggling to meet their energy demands [2, 24, 65]. At the end of 2013, there were about 130 non-sewage biogas plants in the UK. Most are on farm, and some larger facilities exist off farm, which are taking food and consumer wastes. On October 5, 2010, biogas was injected into the UK gas grid for the first time. Sewage from over 30,000 Oxfordshire homes is sent to Didcot Sewage treatment works, where it is treated in an anaerobic digester to produce biogas, which is then cleaned to provide gas for approximately 200 homes (Anaerobic Digestion and Bioresources 2014). 40 2 Biomethanization

Table 2.3 Production of biogas from various organic wastes Name of organic substances Methane production References Biomass (Calotropis procera) 2.9 to 3.6 L biogas per day (50–90 % [20] methane) Napier gas (in presence of 0.7 L/day [21] micronutrients) Rabbit waste 127.00 L/kg VS [22] Rabbit waste + tomato plant wastes 0.115 m3/kg VS [20] Cow paunch manure 442 L/mg TVS [23] Pea shell 220 L/kg TS [24] Cattle dung 129 L/kg TS (40-hrt) [21] Night soil 69.72 L/kg VS 73 % methane [25] Tomato wastes 0.42 m3/kg/VS [26]

Hydrogen + CO2-based gas transfer 470 L/day [27] reactor

Efﬂuent gas contents (CO2 and H2) 286 L/day [28]

CO2,H2, and CO (coal gasiﬁcation 33.7 mm/L/h (cell concentration/ [29] product) L ¼ 2.98) Vegetable-processing wastes 0.55 m3/kg VS [26]

Market organic wastes 4.8 L/day (CH4 ¼ 60 % hrt 10 days. TS [30] % ¼ 0.7) Apple pomace 155.6 L/kg TS [24] Cauliflower and radish wastes 150.7 L/kg TS Rotten cabbage 217.6 L/kg TS Wheat straw 360 L/kg TS Rice straw 487 L/kg TS Mirabilis leaves 418/kg TS [31] Cauliflower leaves 520/kg TS Ipomeas fistula leaves 507/kg TS Dhub grass 282 L/kg TS Banana peeling 460/kg TS Boiled rice 0.294/kg VS added Cooked meat 0.482/kg VS added Fresh cabbage 0.277/kg VS added [32] Cellulose 0.356/kg VS added m.f.w (mixed food wastes) 0.472 m3 MSW (municipal solid wastes) 0.186–0.222 m3/kg VS added Yard wastes 0.134–0.209 m3/kg VS added [33] Paper 0.084–0.349 m3/kg VS added Food packaging 0.318–0.349 m3/kg VS added Sorghum cultivars 0.28–0.40 m3/kg VS added [34] Lingo-cellulose 0.28–0.40 m3/kg VS added Freshwater aquatic 0.07–0.43 m3/kg VS added Forage and grasses 0.07–0.41 m3/kg VS added Root and tuber 0.19–0.43 m3/kg VS added [35] (continued) 2.2 Anaerobic Digestion and Methane Production 41

Table 2.3 (continued) Name of organic substances Methane production References Marine 0.08–0.38 m3 /kg VS added Crop residue 0.08–0.53 m3/kg VS added Vegetable oil 0.94 m3/kg VS added Primary sludge 0.59–0.54 m3/kg VS added Food wastes 0.014 m3/kg VS added Eucalyptus 0.014 m3/kg VS added [36] Pine 0.059 m3/kg VS added Bamboo 0.0163 m3/kg VS added Cellulose biomass 0.7 m3/kg VS added

2.2 Anaerobic Digestion and Methane Production

Anaerobic digestion is a series of biological processes in which microorganisms break down biodegradable material in the absence of oxygen. One of the end products is biogas, which is combusted to generate electricity and heat or can be processed into renewable natural gas and transportation fuels. A range of anaerobic digestion technologies are converting livestock manure, municipal wastewater solids, food waste, high strength industrial wastewater and residuals, fats, oils and grease, and various other organic waste streams into biogas (Table 2.3). Separated digested solids can be composted, utilized for dairy bedding, directly applied to cropland, or converted into other products. Nutrients in the liquid stream are used in agriculture as fertilizer. Figure 2.3a shows carbon flow diagram of the biogas process, and detail sequential steps are described below: Hydrolysis In general, hydrolysis is a chemical reaction in which the breakdown of water occurs to form H+ cations and OHÀ anions. Hydrolysis is often used to break down larger polymers, often in the presence of an acidic catalyst. In waste biomass hydrolysis (Fig. 2.3b), organic polymers are degraded into soluble monomers, e.g., cellulose is hydrolyzed to glucose units in enzymatic reactions [21]. Efficient hydrolysis of cellulose involves at least three groups of enzymes, namely, endoglucanase, exoglucanase, and glucosidase. More enzymes are required for complete degradation of hemicellulose because of its greater complexity compared to cellulose. Of these, xylanase is the best studied. Anaerobic bacterial species, especially Clostridium spp. (e.g., C. cellobioparum, C. acetobutylicum), contain complexed cellulase systems which enables high hydrolysis efficiency [21, 29]. Other anaerobic bacteria with cellulolytic activity are, e.g., Acetivibrio cellulolyticus, Ruminococcus albus, and Eubacterium cellulosolvens [29]. The enzymatic hydrolysis of lignin is limited due to its irregular shape [21], and lignin is hardly degraded in anaerobic conditions [30]. Through hydrolysis, these large polymers, namely, proteins, fats, and carbohydrates, are broken down into smaller molecules such as amino acids, fatty acids, and simple sugars. 42 2 Biomethanization

Biogas Removal of moister content digester Dryer Water removal process Buster Heat exchanger/Blower/ Raw biogas Chiller/Dessicant

Activated Silica gel Moister free biogas charcoal

Removal of impuries Sulfa Iron sponge Bio- treatment filtration

Medium For electricity BTU generation Biogas

CO2 Removal process

Membrane Water wash Or Or Cryogenic Amine scrubber Or PSA

High BTU Biomethane 100-500 psig to meet Pipe line specification

COMPRESSION

USE

On-site Tube trailers Gas grid Vehicle Generation or 4000-psig injection 3500-psig Direct use 50-1000 psig

Fig. 2.2 Raw biogas process technology to generate clean biomethane. Adapted from American Biogas Council, promoting the anaerobic digestion and biogas industry 2.2 Anaerobic Digestion and Methane Production 43

a WASTE BIOMASS 1st step Proteins Carbohydrates Fats Hydrolysis

nd 2 step Amino acid & Sugar Fatty acids

Acidogenesis Intermediates 3rd step Propreonic acid Butyric acid

Acetogenesis

Acetic acid Hydrogen

4th step Methanogenesis METHANE

b ORGANIC MATERIALS

Carbohydrates Proteins Lipids

30% Fermentative bacteria

51 19% Organic acids Alcohols Lactate

Acetogenic baceria

Acetate oxdising bacteria Acetate H2 CO2

Homoacetogenic bacteria 70% 30%

Aceticclastic methanogensCH4 CO2 Hydrogenotrophic methanogens

Fig. 2.3 (a) Carbon ﬂow diagram of the biogas process. Adapted from Angelidaki et al. [27]. (b) Schematic diagram of four major steps involve in waste biomass hydrolysis and biogas production 44 2 Biomethanization

Proteins are generally hydrolyzed to amino acids by proteases, secreted by Bacteroides, Butyrivibrio, Clostridium, Fusobacterium, Selenomonas, and Strepto- coccus. The amino acids produced are then degraded to fatty acids, such as acetate, propionate, and butyrate, and to ammonia as found in Clostridium, Peptococcus, Selenomonas, Campylobacter, and Bacteroides. Lipases convert lipids to long-chain fatty acids. A population density of 104–105 lipolytic bacteria per ml of digester ﬂuid has been reported. Clostridia and the Micrococci appear to be responsible for most of the extracellular lipase producers. The long-chain fatty acids produced are further degraded by p-oxidation to produce acetyl-CoA. Acidogenesis Acidogenesis is the next step (Fig. 2.3b) of anaerobic digestion in which acidogenic microorganisms further break down the biomass products after hydrolysis. The soluble compounds formed in the hydrolysis (e.g., glucose, xylose) are further oxidized to, e.g., VFAs (acetic, propionic, butyric, etc.), H2, and CO2 in the acidogenesis step (also referred to as fermentation) by fermentative bacteria. These fermentative bacteria produce an acidic environment in the digestive tank while creating ammonia, H2,CO2,H2S[51–54], shorter volatile fatty acids, car- bonic acids, alcohols, as well as trace amounts of other by-products. The sugars obtained from the hydrolysis step can easily be degraded by fermentation process, while long-chain fatty acids (LCFA) must be obligately oxidized by an external electron acceptor [31]. Amino acids can either be degraded through Stickland reactions, where one amino acid acts as an electron donor and another acts as an electron acceptor, or be oxidized with an external electron acceptor [20]. Glucose fermentative microbes have branched metabolisms, which means they are able to metabolize the substrate in different pathways which give different amount of energy and produce different fermentation products [25]. The fermentative bacteria can function at high concentrations of hydrogen and formate. However, under this condition, the bacteria will use a metabolic pathway in which more reduced metabolites are produced, such as VFA, lactate, and ethanol [27]. Examples of different products from glucose fermentation are shown in Table 2.4. Acetogenesis In acetogenesis, VFAs (e.g., propionic and butyric acids) are oxidized by acetogenic bacteria to acetic acid and H2, which are used as substrates in methanogenesis (Fig. 2.3b). In general, acetogenesis is the creation of acetate, a derivative of acetic acid, from carbon and energy sources by acetogens. These microorganisms catabolize many of the products created in acidogenesis into acetic acid, CO2, and H2. Most acetogenic bacteria that produce acetate are gram-positive bacteria, and many are species of the spore-forming Clostridium (e.g., C. aceticum) or the nonspore-forming Acetobacterium [26]. Acetogenic bacteria typically grow more slowly when compared to acidogenic bacteria. Most acetogenic bacteria can grow heterotrophically by fermenting sugars [26]. Homoacetogens consume CO2- and H2-producing acetate according to the following equation: 2.2 Anaerobic Digestion and Methane Production 45

Table 2.4 Methane emission Nature of resource Methane emission in kg from biological resources Hybrid cattle 120 Local cattle 60 Sheep 8 Pig 1.5 Human being 0.12 Source: NASA Institute for Space Science

À þ À 4H2 þ 2HCO3 þ H ! CH3COO þ 4H2O ð2:1Þ

Methanogenesis Methanogenesis constitutes the final stage (Fig. 2.3b) of anaerobic digestion in which methanogens create methane from the final products of acetogenesis as well as from some of the intermediate products from hydrolysis and acidogenesis. Methanogens are obligate anaerobic Archaea [26], divided into five phylogenetic orders, namely, Methanosarcinales, Methanobacteriales, Methanomicrobiales, Methanococcales, and Methanopyrales showing diverse cell morphology and optimal growth conditions [66]. Three classes of methanogenic substrates are known, i.e., CO2-type substrates (CO2, CO, formate), methyl substrates (methanol, methylamine, dimethylamine, trimethylamine), and acetotrophic substrates (acetate) [26]. There are two general pathways involving the use of acetic acid and carbon dioxide, the two main products of the first three steps of anaerobic digestion, to create methane in methanogenesis:

À CH3COO þ H2O ! CH4 þ HCO3 ð2:2Þ

CO2 þ 4H2 ! CH4 þ 2H2O ð2:3Þ

While CO2 can be converted into methane and water through the reaction, the main mechanism to create methane in methanogenesis is the path involving acetic acid. This path creates methane and CO2, the two main products of anaerobic digestion. Anaerobic reactions will ultimately lead to production of CH4 and CO2; thus, in traditional AD process, the biogas is mainly composed of methane (50–70 %) and carbon dioxide [26]. Landfill gas is produced during anaerobic digestion of organic materials in landfills and is very similar to biogas. Its methane content is generally lower than that of biogas, and landfill gas usually also contains nitrogen from air that seeps into the landfill gas during recovery. Landfill gas can also, in contrast to, e.g., biogas from farms, contain a great number of trace gases. 46 2 Biomethanization

2.2.1 Methane-Producing Microbes

Methanogenic archaea, or methanogens, are an important group of microorganisms that produce methane as a metabolic by-product under anaerobic conditions. Methanogens belong to the domain archaea, which is distinct from bacteria. Methanogens are commonly found in the guts of animals, deep layers of marine sediment, hydrothermal vents, and wetlands. They are responsible for the methane in the belches of ruminants, as in the ﬂatulence in humans, and the marsh gas of wetlands. Methanogens should not be confused with methanotrophs, which consume methane rather than produce it. Methanogens, differ from other bacteria not only by their types of metabolism but also by a number of characteristic features in the composition of their cell constituents. They lack a typical peptidoglycan skeleton. Methanococcus has only a protein envelope; a peptide sheath is found in Methanospirillum, while the cell wall of Methanosarcina barkeri consists of a polysaccharide composed of uronic acid, neutral sugars, and amino sugars. The methanogenic bacteria are not subject to growth inhibition by penicillin. Almost all the shapes known in the Eubacteria can be found in the methanogens: Methanospirillum hungatei Spirilla Methanosarcina barkeri Coccal packets Methanothrix soehngenii Filamentous Methanoplanus limicola Square type

Methanobacterium bryantii Long rod Methanobacterium formicum Rods Methanobrevibacter smithii Shortrods and chains

Thermolithotrophicus Cocci Cocci Methanomicrobium mobile Short rod Methanosarcina acetivorans Irregular cocci

Besides these, there are mesophilic and thermophilic species (Methanothermobacter thermautotrophicus (Methanobacterium thermoautotrophicum), Methanothermobacter thermoﬂexus (Methanobacterium thermoﬂexum), Methanosaeta thermophila, Methanothermus fervidus). Six families have been distinguished already, and the names of known species and genera are constantly increasing. 2.2 Anaerobic Digestion and Methane Production 47

Due to strict anaerobic nature, methane-producing bacteria are very lethal to air. These bacteria are devoid of any catalase and/or superoxide dismutase enzyme. However, Methanosarcina barkeri is exceptional in possessing a superoxide dismutase (SOD) enzyme and may survive longer than the others in the presence of O2 [32]. Some methanogens, called hydrogenotrophic, use carbon dioxide (CO2) as a source of carbon and hydrogen as a reducing agent. Methanogens play a vital ecological role in anaerobic environments by removing excess hydrogen and fermentation products produced by other forms of anaerobic respiration. Because of this, methanogens thrive in environments in which all electron acceptors other than CO2 (such as oxygen, nitrate, trivalent iron, and sulfate) have been depleted. In the human gut, accumulation of hydrogen reduces the efficiency of microbial processes, reducing energy yield. Methanogens such as M. smithii are pivotal in the removal of this excess hydrogen from the gut and may be useful therapeutic targets for reducing energy harvest in obese humans. In marine sediments, biomethanation is generally confined to where sulfates are depleted, below the top layers. Methanogens play a key role in the demineralization of organic carbon in continental margin sediments and other aquatic sediments with high rates of sedimentation and organic matter. Under the correct temperatures and pressure, biogenic methane can accumulate in massive deposits, which account for significant fractions of organic carbon and key reservoirs of a potent greenhouse gas. Some methanogens, called extremophiles, can thrive in extreme environments such as hot springs, submarine hydrothermal vents, and hot, dry deserts. Methanogens have been found buried under kilometers of ice in Greenland, as well as in the “solid” rock of the earth’s crust, kilometers below the surface. Generally, organic compounds like succinate, propionate, butyrate, lactate, acetate, alcohols, and gaseous substances like CO2 and H2 are the main products during the anaerobic digestion of organic substances. These organic products ultimately are the substances for the methanogenesis bacteria. This means the methanogens are the last link in an anaerobic food chain (Fig. 2.4).

2.2.2 General Mechanism of Methane Production

Generally, it has been noticed that the methanogens are in close association with hydrogen-producing bacteria. During such association, the hydrogen is rarely liberated in gaseous forms. Rather, the excreted hydrogen dissolved in the aquatic medium is directly used by methanogens for producing methane. This sort of mutual symbiotic process for generation of methane is commonly noticed in anaerobic area, in sediment water or soil which are rich in cellulose, or in such other detritus organic matters. Methane bacteria are able to activate on hydrogen and to couple its oxidation to the reduction of carbon dioxide. Since they can synthesize all their cell substances 48 2 Biomethanization

Succinite Fermenting bacteria Propionate H2O CO2 Butyrate Acetate Lactate. . .

Polysaccarides BIOMASS Cellulose, starch, Proteins lipids

CH2 Acetate CO2 Anaerobic Nutritional Chain Formate CO2 H2

Fig. 2.4 Role of methanogenesis bacteria in anaerobic food chain using carbon dioxide as sole carbon source, their mode of life is evidently to be regarded as chemoautotrophic. The microbial synthesis of methane, sometimes, is regarded analogous to carbonate respiration. The overall reaction of biomethane production by using hydrogen and carbon dioxide is as follows:

4H2 þ CO2 ! CH4 þ 2H2O; ΔG0 À n131 kj=mol

Theoretical maximum of 12 moles of H2 from one mole of glucose is not thermodynamically possible reaction. Instead, the highest H2 production (4 moles) can be achieved, when one mole of glucose (C6H12O6) is degraded to 2 moles of acetate (Eq. 2.4), resulting in COD reduction of 33 % in the form of H2 [28]. When glucose is degraded to butyrate, in theory 2 moles of H2 per one mole of glucose (Eq. 2.5) can be produced [34]. Theoretical maximum from one mole of xylose (C5H10O5) is 3.33 moles H2 with acetate as the sole end product [33], while theoretical maximum from one mole of sucrose (C12H22O11) is 8 moles of H2 [35].

C6H12O6 þ 4H2O ! 2acetate þ 2CO2 þ 4H2 ð2:4Þ

C6H12O6 þ 4H2O ! butyrate þ 2CO2 þ 2H2 ð2:5Þ

Among a large number of microbial species, strict anaerobes, such as clostridia (e.g., C. pasteurianum, C. butyricum, C. beijerinckii), are efﬁcient producers of hydrogen, with theoretical H2 yield of 4 moles per mole of glucose. Practical yields from these fermentations are near 2 or slightly above [34, 36, 67] as some of the substrate is used as energy and carbon source for bacteria [68] other degradation products than acetic acid can be produced, and H2-consuming reactions can occur [69]. Hydrogen is produced during the exponential growth phase of clostridia [28], and in addition to acetate, fermentation can yield ethanol, butyrate, butanol, and acetone [34, 70]. Clostridia form spores under unfavorable conditions, such as lack 2.2 Anaerobic Digestion and Methane Production 49 of nutrients or heat treatment, and are highly sensitive to oxygen [28]. In addition, facultative anaerobic bacteria, such as enteric bacteria, can produce at most 2 moles of H2 per mole of glucose [36], while in practice about one half of this theoretical H2 yield is observed [34]. Enterobacter sp. can tolerate oxygen [69] and carry out a mixed-acid fermentation producing lactate, ethanol, acetate, formate, H2,CO2, succinate, and butanediol. In mixed cultures, mesophilic Clostridium sp. and thermophilic Thermoanaerobacterium sp. are the species most often indicated [28]. Moreover, extreme-thermophilic (70 C) hydrogen (ethanol) producers (from glucose) have been found, e.g., Thermoanaerobacter, Thermoanaerobacterium, and Caldanaerobacter [71]. In addition to hexose or pentose utilizing H2 producers, some bacteria, e.g., C. cellulolyticum, C. acetobutylicum X9, C. cellobioparum, and C. thermocellum 14, are capable of H2 production from cellulose [61] with, e.g., acetic acid, lactic acid, succinic acid, and ethanol as fermentation products. More- over, species such as C. butyricum, C. acetobutylicum, C. cellobioparum, C. pasteurianum, and C. perfringens are capable of fermentation of starch and pectin in addition to sugars, with fermentation products of acetone, butanol, ethanol, isopropanol, butyric acid, acetic acid, propionic acid, and lactic acid. H2 yields from cellulose are typically between 1 and 2 mol/(mol hexose) [70]. Recently, H2 yield of 10.1 mmol/(g cellulose) has been reported [72], while 2.32 mol/(mol glucose) has been obtained from starch in CSTR [73]. Factors affecting cellulose degradation are among others initial and ﬁnal pH, substrate type, which are rich in cellulose, and concentration as well as inhibition by degradation products [74]. Limited literature is available on the enzyme(s) involved during the biochemical conversion of H2 and CO2 to methane and acetate to methane and carbon dioxide. However, a number of coenzyme and prosthetic groups are noticed in such process (Fig. 2.5). The mechanism of ATP generation during such reaction [4] is not clearly understood. It has also been noticed that some methanogens utilize carbon monoxide for methane biosynthesis. Carbon monoxide and hydrogen are the main intermediary products during such process:

4CO þ 4H2O ! 4CO þ 4H2 CO2 þ 4H2 ! CH4 þ 2H2O 4COþ 2H2O ! CH4 þ 3CO2

Methane can also be produced from methanol and hydrogen by Methanosarcina barkeri. In such process, the enzyme methyl transferase convert methanol directly to methyl-coenzyme, which is reduced with the liberation of methane (Fig. 2.6). In contrast to phototrophic microorganisms, anaerobic bacteria, in dark, convert a large variety of carbohydrate sources to hydrogen, CO2, volatile fatty acids, and ethyl alcohol. In natural environments (compost, anaerobic digester sludge, soil, etc.), hydrogen-producing bacteria coexist with methane-forming anaerobes which consume the above-listed metabolites including hydrogen and produce their own end products like methane and CO2. Two-stage AD producing both H2 and CH4 has 50 2 Biomethanization

2Ethano

CO2 2NAD 2Fd 2H 2NADH2 2FdH2

2 Acetaldehyde 4H

2Fd ATP ATP 2H2 2FdH2 CH4 2Acetate

2 Acetate

Fig. 2.5 Biochemical conversion of hydrogen to methane by methanogens

ATP?

H2 H2

Methyl Co-Enzyme

Methyl CH2 S-CoM MS-Co-+H2O+ Methanol Methanol CH4 CH4 CH3OH Transferase

Methanosarcina barkeri

Fig. 2.6 Biological production of methane from hydrogen and methanol by M. barkeri been suggested as a feasible technology to improve the overall energy conversion efﬁciency [75, 76]. The growth rates and pH optima are different for acidogens and methanogens [77], and thus, in a two-stage AD system, faster-growing acidogens are developed in the ﬁrst-stage hydrogenic reactor and are involved in the production of VFAs and H2. On the other hand, slow-growing acetogens and methanogens are developed in the second-stage methanogenic reactor, in which the produced VFAs are further converted to CH4 and CO2. In addition, the optimal temperature for hydrolysis/acidogenesis can differ from optimal temperature for methanogenesis [78]. Two-phase processes thus allow the selection and enrichment of different microorganisms in each phase and can increase the stability of the process [79]. The application of two-stage AD process for sequential H2 and CH4 production has been proposed as a promising technology for better process performance and 2.3 Methane Emission and Organic Feedstocks 51

higher energy yields as compared to the traditional one-stage CH4 production process [80, 81]. Two-stage H2 +CH4 system has been shown to improve CH4 yield when compared to traditional one-stage methane process, as, e.g., 21 % more CH4 was obtained in a two-stage system from household solid waste [82] and 22 % more from lipid-extracted microalgae [83]. The reducing enzyme, methyl-coenzyme M methyl reductase (MCR), is a multienzyme complex which contains, besides other protein, F420 F430 and hydrogenase. Most probably, the reaction is accompanied by the extrusion of protons from the cell, and the resulting protein potential can then drive the regeneration of ATP. This ATP generation by electron transport phosphorylation under anaerobic conditions is in contrast with the substrate phosphorylation under anaerobic conditions noticed in other lower plants.

2.3 Methane Emission and Organic Feedstocks

Globally, over 60 % of total CH4 emissions come from human activities. In 2013, CH4 accounted for about 10 % of all US greenhouse gas emissions from human activities. Methane is emitted by natural sources such as wetlands, as well as human activities such as leakage from natural gas systems and the raising of livestock. Domestic livestock such as cattle, buffalo, sheep, goats, and camels [1–4] produce large amounts of CH4 as part of their normal digestive process (Table 2.4). Also, when animal manure is stored or managed in lagoons or holding tanks, CH4 is produced. Because humans raise these animals for food, the emissions are considered human related. Globally, the agriculture sector is the primary source of CH4 emissions. Natural processes in soil and chemical reactions in the atmosphere help remove CH4 from the atmosphere. Methane’s lifetime in the atmosphere is much shorter than carbon dioxide (CO2), but CH4 is more efficient at trapping radiation than CO2. If the methane emission from human activities is regulated, then it would help a lot in reducing the global greenhouse effect phenomenon. Proper management of these agriculture wastes and livestock may significantly help in generating biogas through anaerobic digestion process. The material that is used in anaerobic digestion is called feedstock. A great variety of organic material can be used in anaerobic digesters as a feedstock for generating biogas (Table 2.5). However, there are scientific, engineering, and legal limits to what can be added successfully to a digester. In addition, the feedstock needs to be a liquid mixture with an appropriate moisture content. For example, mesophilic complete-mix tank digesters (the type most commonly used today) typically operate best with a mixture of 4–8 % solids in water. Digesters require various moisture contents, depending on the design and operation of the system. The feedstock does not have to be waste; any biodegradable plant or animal matter that is not woody is a suitable feedstock for a digester. However, anaerobic microorganisms cannot break down lignin, the complex polymer that gives plants their strength, which means that wood products, paper, and straw will slow the 52 2 Biomethanization

Table 2.5 Biogas production from various types of plant biomass and organic wastes Type of biomass/organic waste Biogas yield Marine sea grass Thalassia testudinum shoot 10.4 L/kg wet weight Thalassia testudinum root 7.4 L/kg wet weight Syringodium filiforme 7.377 L/kg wet weight Algae Gracilaria tikvahiae 22.2–32.4 L/kg wet weight Ulva spp. 32–43.0 L/kg wet weight Hypnea musciformis Sargassum fluitans 8.5 L/kg wet weight Aquatic Biomass Eichhornia crassipes (water hyacinth) 7.05 L/kg wet weight Freshwater aquatic 0.07–0.43 m3 kg1 VS Waste biomass Sugar beet pulp 0.45 m3 kg1 VS Lignocelluloses 0.02–0.27 m3 kg1 VS Crop residue 0.08–0.53 m3/kg VS Wheat straw 162–249 L/kg VS added Rice raw 241–367 L/kg VS added Mirabilis lees 290–341 L/kg VS added Cauliflower leaves 350 L/kg to 423 kg VS added Ipomoea fistulosa 387–429 L/kg VS added Dhub grass 137–228 L/ kg VS added Banana peeling 271–409 L/kg VS added Market organic waste 0.478 m3/kg VS added Mixed vegetable waste 0.6 m3/kg VS added Wheat straw 0.36 m3/kg VS added Potato tops 0.61/kg VS added Maize top 0.52 m3/kg VS added Beet leaves 0.45 kg/VS added Grass 0.56 kg VS added Brassica (leafy vegetable) 22.6 kg wet biomass Solanum tuberosum (potato) 79.0 kg wet biomass Calotropis procera 1.6–2.4 L/day Municipal sewage, sludge 0.60 m3/kg VS added Municipal sewage skimmings 0.57 m3/kg VS added Municipal garbage 0.63/kg VS added Waste paper 0.26 m3/kg VS added Municipal refuse 0.31 m3/kg VS added Abattoirs waste Cattle paunch contents 0.53 m3 kg VS added Intestines 0.09 m3 kg VS added Cattle blood 0.16 m3 kg VS added (continued) 2.3 Methane Emission and Organic Feedstocks 53

Table 2.5 (continued) Type of biomass/organic waste Biogas yield Dairy waste, sludge 1.03 m3 kg VS added Yeast waste, sludge 0.80 m3 kg VS added Paper waste, sludge 0.25 m3 kg VS added Horse manure 0.44 m3 kg VS added Cattle manure 0.32 m3 kg VS added Pig manure 0.42 m3 kg VS added Brewery waste 0.45 m3 kg VS added Potatoes 276–400 m3 tonne Rye grain 283–292 m3 tonne Clover grass 290–390 m3 tonne Sorghum 295–372 m3 tonne Grass 298–467 m3 tonne Red clover 300–350 m3 tonne Jerusalem artichoke 300–370 m3 tonne Turnip 314 m3 tonne Rhubarb 320–490 m3 tonne Triticale 337–555 m3 tonne Oilseed rape 340 m3 tonne Reed canary grass 340–430 m3 tonne Alfalfa 340–500 m3 tonne Clover 345–350 m3 tonne Barley 353–658 m3 tonne Hemp 355–409 m3 tonne Wheat grain 384–426 m3 tonne Peas 390 m3 tonne Ryegrass 300–410 m3 tonne Leaves 417–453 m3 tonne Fodder beet 160–180 m3 tonne Cattle slurry 15–25 m3 tonne Pig slurry 15–25 m3 tonne Poultry 30–100 m3 tonne Grass silage 160–200 m3 tonne Whole wheat crop 185 m3 tonne Maize silage 200–220 m3 tonne Maize grain 560 m3 tonne Crude glycerin 580–1000 m3 tonne Wheat grain 610 m3 tonne Rape meal 620 m3 tonne Fats Up-1200 m3 tonne Nettle 120–420 m3 tonne Sunﬂower 154–400 m3 tonne Miscanthus 179–218 m3 tonne (continued) 54 2 Biomethanization

Table 2.5 (continued) Type of biomass/organic waste Biogas yield Whole maize crop 205–450 m3 tonne Flax 212 m3 tonne Sudan grass 213–303 m3 tonne Sugar beet 236–381 m3 tonne Kale 240–334 m3 tonne Straw 242–324 m3 tonne Oats grain 250–295 m3 tonne Chaff 270–316 m3 tonne digester. Some examples of feedstock streams are cow dung, urban organic wastes, algae, vegetable wastes, human excreta, etc.

2.3.1 Methane from Cattle Wastes

For ages, cow’s excreta traditionally have been used as manure in the ﬁelds and dry cow dung as source of heat energy for rural people. But it is also a source of viable energy biogas, which is capable of providing electricity for domestic purposes. One cow can produce over 30 gallons of manure a day. One hundred cows can produce about 30,000 gallons of manure. All that manure can be turned into serious energy generation process for powering electrical equipments for various domestic purposes. Many developing countries have been using biogas for generating energy [59, 83–86] to meet local demand for electricity. It has a lot of potential to reduce carbon footprint. By capturing and storing CO2 from biogas into the ground, the biogas becomes carbon negative and scrubs our past CO2 emissions out of the atmosphere. The general characteristic of cow dung available in cattle house is given in the Table 2.6. In India, a buffalo/cow gives 20–30 kg, by dry weight, dung per day. The biogas production is about 0.18 m3 g/kg of manure. About 1.7 m3 of biogas equals 1 L of gasoline. The manure produced by one cow in one year can be converted to methane, which is the equivalent of over 200 L of gasoline. Cow Dung Processing India has more cattle than any other country (450 million head, 19 % of the world population), and cow is worshipped for auspicious purpose. Besides this, cow dung and urine are used as purifying agents. Cow dung is also widely used in India as composted fertilizer and as a cooking fuel (dung cakes). Dung accounts for over 21 % of total rural energy use in India and as much as 40 % in certain states of the country. The cow dung, water, and various organic residues from agricultural waste are supplied as feed to the digester. The proportions recommended are cow dung + solid waste 1:1 by weight and forming to about 10 % of solid content and 90 % of water. 2.3 Methane Emission and Organic Feedstocks 55

Table 2.6 General Parameters Contents characteristic of cow dung TS (mg/L) 156 available in cattle farm house VS (mg/L) 32.5 COD (mg/L) 2.200

NH3–N (mg/L) 680 pH 7.1–7.4 Moister 41.2 % Methane 55–56 %

CO2 30–35 % Nitrogen 1.8–2.4 % Phosphorus 1.0–1.2 Potassium 0.6–0.8 Burning efﬁciency 60 % Caloriﬁc value 540–600 btu/ft3

The amount of feed supply per day to the digester is called loading rate. It is dependent on the size of the plant. The recommended loading rate is about 0.2 kg/m 3 of digester capacity. The under loading and overloading reduces the biogas production. The loading of feed must be carried out every day at the same time as to keep the solid concentration ratio constant in the digester. Cow dung and waste materials of other cattle mainly consist of undigested cellulosic material or degraded cellulosic materials. Besides these, cow dung and cattle waste carry residual carbohydrate in the form of xylans, fructosans, and residual fatty acid and various type of bacteria (Table 2.7). Various types of reactors starting from simple portable to more modern type are being used for biogas production at present. Despite their differences in design and operational characteristics, the biology of methane production is essentially same. The main concern in operating these systems is to ensure that various bacterial species (Table 2.7) function in a balanced and sequential way. The overall process of microbial transfer of cow dung to methane is mainly in three sequences. The fermenting bacteria, in the ﬁrst phase of digestion, convert the cellulosic and other organic wastes of cow dung to simple organic compounds. The products of cellulosic materials are ethanol, acetate, formate, molecular hydrogen, and carbon dioxide. In the second phase, the acetogenic bacteria transfer these primary products to acetate, fermate, butyrate carbon dioxide, and hydrogen. In the ﬁnal stage of this anaerobic digestion, these products are the substrate for the methanogenesis, and subsequently methane is produced along with CO2 and H2 (Fig. 2.7).

2.3.2 Methane from Market Organic Wastes (MOW)

Rapid urbanization all over the world has created a serious problem of solid waste disposal. Large vegetable and fruit market in the big cities [87–91] contribute much 56 2 Biomethanization

Table 2.7 Microbes involve Gram-negative cocci in anaerobic digestion of Ruminococcus albus cow dung Ruminococcus ﬂavefaciens Gram negative non motile bacteria Bacteroides succinogenes Butyrivibrio ﬁbrisolvens Clostridium cellobioparum Veillonella opalescens Desulfotomaculum ruminis Selenomonas ruminantium Methanobacterium ruminantium

to the accumulation of this waste which is disposed of by composting, spreading on the land or sometimes as an animal feed. It has been observed that of the enormous supply of food for human consumption, about one third gets wasted globally [92, 93]. India stands second in the production of fruits and vegetables in the world. It contributes about 10 and 14 % of fruits and vegetables in the world production [90]. As per National Horticulture Database published by the National Horticulture Board, during 2012–2013, India produced 81.285 million metric tonnes of fruits and 162.19 million metric tonnes of vegetables (National Horticulture Database published by the National Horticulture Board, during 2012–2013). Only 1 % of total vegetable and fruit production is used in processing industries. The solid wastes normally account for 40–50 % of the raw material processed and have a total solid concentration of 10–15 %. Even in a developed country, the problem of proper disposal of waste from vegetable market is still unsettled [94, 95]. These vegetable wastes are rich in organic matter (Table 2.8) and can act as good source of nonconventional energy, although these wastes cause environmental pollution. These wastes are presently sent to sanitary landﬁll by taking it for granted that the same process is relatively less expensive. Dumping of these vegetable wastes on unused land creates tremendous problems like foul smell and dangerous aerobic problem. Besides this, methane generated during decomposition of organic wastes diffuses through underground, to a far site from its production point, and causes explosion. There have been a number of reports on the utilization of fruit- and vegetable-processing wastes individually as feedstock for biogas production. Composition of Market Organic Wastes The market organic wastes (MOW) consist of mainly seasonal vegetable waste, waste from fruit processing and fruit storage house, various packing materials, plastic, etc. (Fig. 2.8). Composition of a typical organic waste from a vegetable market is given in Tables 2.9 and 2.10. On the basis of physic chemical nature, the MOW can be differently fractioned (Fig. 2.9). 2.3 Methane Emission and Organic Feedstocks 57

1 st Stage Fermentive bacteria Cow dung and cattle wastes

Hydrolyze and Ferments Organic Substance

Cellulose degrading bacteria Protein degrading bacteria Fat degrading bacteria

Fatty acids Saccharides Aminoacids

Volatile Acids

2nd step Hydrogen producing acetogenic H2 & CO2 bacteria

Acetogenesis CO2 H2 Acetic acid Organic electron donor

rd 3 Stage Hydrogenophilic Acetophilic Methanogenesis Methanogens Methanogens

CH4 CH4 H2+ +CO2 BIOGAS

Fig. 2.7 Anaerobic digestion and biogas production mechanism in cow dung and cattle wastes

Generally, the production of biogas depends on the composition of seasonal vegetable and other organic wastes present in the VMOW. Most vegetable and fruits are processed on a seasonal basis, only for a period of 1–3 months, and the wastes that emanate during these process vary considerably in their physicochemical characteristics (Tables 2.11 and 2.12). Some of them are rich in toxic constituents such as limonene in citrus wastes, and most of them are deﬁcient in nitrogen (such as mango- and pineapple-processing wastes). It has been noted that the total carbon content in market organic waste approached almost 40 %. The content of total nitrogen was noticed to be in wide range of 21.32 to 76.5. Under deﬁcit of nitrogen, it is advised to use synthetic fertilizer such as urea and ammonium sulfate. However, the use of synthetic fertilizer is uneconomical. Recently, it has been 58 2 Biomethanization

Table 2.8 A typical data on vegetable market organic waste Potato Salad Green peas and Mixture of Mixture of Wastes (g/kg) peelings waste carrots FVW FVW Total solid 119.2 79.4 179.4 90.4 85 Volatile solid 105.5 72.1 176 82.9 77.4 Total COD 126 97.8 185 104.5 – Particulate COD 30.6 39.3 123.9 – – Total suspended 80 39 14.5 – 58.6 solid Total Kjeldhal – – – 2 2.7 nitrogen Cellulose 12.4 13.5 16.1 9.2 – Sugar and ––– 62– hemicelluloses Lignin – – – 4.5 –

Vegetable Market Organic Wastes (VMOW)

Organic fraction Fuel fraction Plastic Land filling

Seasonal vegetable waste Packing materials Bottle bags Land filling Fish and meat process waste Card board wood pellet polythene Straw husk fraction

Fig. 2.8 Schematic presentation of composition of organic waste from a vegetable market

Table 2.9 Physical characteristics of vegetable wastes on dry-weight basis Moisture Total solid Vegetable wastes % Ash % % References Potato (leachate/solid waste) 85–87 6–12 17–19 [1, 2, 32, 66, 96] Tomato (solid waste) 85–90 3.1–5.3 17–22 [8, 51, 71, 86] Onion (onion tops peelings and whole 82–92 4–7 91 [8, 9] bulbs) Pea (peel, shell, and solid waste) 84–88 4.8–15.5 11.11–39 [8, 17, 18, 42, 87] Sugar beet (pulp, silage, and leaves) 85 3.8–8.85 7–11 [26, 31, 50] observed that the formulation of feed with complementary waste is an effective method of overcoming the deﬁcit budget of nitrogen while generating biogas from VMOW. In order to get maximum biogas, the ratio of C/N is generally maintained between 20 and 30 [120]. However, in paper and sewage waste, maximum yield of biogas was even noticed at C/N ratio of 52. 2.3 Methane Emission and Organic Feedstocks 59

Table 2.10 Chemical characteristics of vegetable wastes on dry-weight basis Nature of waste Starch Cellulose Hemicellulose Protein References Potato (peel and mesh) 0–40 17–25 10–15 3–5 [26, 62, 64] Tomato 10–18 30–32 5–18 17–22 [3, 61, 62, 69] Carrot 1–2 13–15 12–19 5–8 [6, 19, 20, 63, 74]

Market organic waste

Total soluble organic Total biodegradable Total inorganic Total non-biodgradable component (crbohydrate, solid Compounds, protein etc.) nitrogen,carbohydrate, solid phosphorus etc

Total organic Renewable Non-Renewable Substances indirectly used Non-biodegradable Non-biodegradablesolids by methanogens Solids(plastic, (glass, china clay,metal polythene)

Total organic fraction Total volatile Used by methanogens Fatty acids,ethanol

Fig. 2.9 Composition of market organic wastes

Table 2.11 Physical characteristics of different types of vegetable wastes on a dry-weight basis Moisture Total solid Type of waste % Ash % % References Potato (leachate/solid waste) 85–87 6–12 17–19 [97–101] Tomato (solid waste) 85–90 3.1–5.7 19–22.4 [102–105] Onion (onion tops peelings and whole 82–92.6 4.7 91 [102, 106] bulbs) Pea (peel, shell, and solid waste) 84–88 4.8–15.5 11.11–39 [102, 107– 110] Sugar beet (pulp, silage, and leaves) 85 3.8–8.85 7–11 [111–113]

Table 2.12 Chemical characteristics of different types of vegetable wastes Type of waste Starch Cellulose Hemicellulose Protein References Potato (peel mesh) 30–40 17–25 10–13.5 3–5 [112, 114] Tomato 10–18 30–32 5–18 17–22 [94, 115–117] Carrot 1–12 13–52 12–19 5–8 [93, 109, 118, 119] 60 2 Biomethanization

2.3.3 Methane from Human Excreta

Globally, more than a billion people lack access to adequate sanitation. In India alone more than 160 million septic tanks and pit latrines without septic tanks contribute to surface water pollution due to lack of septage treatment facilities. In Sri Lanka, researchers recently found that only 10 % of human excreta from septic tanks were being collected and only 1 % treated. Human waste is considered a biowaste as it is a good vector for both viral and bacterial diseases. It can be a serious health hazard if it gets into sources of drinking water. The World Health Organization (WHO) reports that nearly 2.2 million people die annually from diseases caused by contaminated water. A major accomplishment of human civili- zation has been the reduction of disease transmission via human waste through the practice of hygiene and sanitation, which can employ a variety of different technologies. Human excreta (HE) is also an excellent feed material for anaerobic fermentation. Physicochemical nature of typical sample of human excreta is given in Table 2.13. The carbon nitrogen ratio (C/N) is low in comparison to the optimum value. The excess of ammonia present/produced during digestion is generally neutralized by volatile fatty acids accumulated in the digester, due to overloading. Fresh excreta is generally acidic (pH 5.24–5.73), but effluent present in the digester very often remains slightly alkaline (pH 7.02–7.78). Besides this, the night soil is rich in organic contents and nutrient and is easily susceptible for microbial digestion. For this reason, it is often responsible for environmental pollution and a host of diseases. With proper microbial amendment technique, the night soil can be subjected to resource recycling process for production of value-added gases and low-cost fertilizer. This means that biomethanation of human excreta has two important aspects: environmental sanitation and biofuel generation. Presently, in China, a mixed feed with the proper combination of pig manure, human excreta, and agricultural wastes has been used. Chinese are the first front in using human excreta at mass-scale level [122, 123]. Generally, a variety of techniques are used to handle human waste: some cities dump it in rivers, others choose to incinerate, and still others bury it in ditches. United Utilities Group Plc, Britain’s largest publicly traded water company, han- dles the sewage of 1.2 million people in Manchester and operates a sludge recycling center that runs on enough human waste to power 25,000 homes. A growing portion of China’s toilet waste is converted into fertilizer and biogas. In Beijing, 6800 tonnes of human excrement are treated each day by some estimates: enough to fill almost three Olympic-size swimming pools. A typical community biogas plant in India based on human waste is depicted in Fig. 2.10. Animal manure and kitchen waste contain a lot of organic matter, and generally, the process produces enough biogas for the family to cover at least cooking requirements. Humans produce less excreta, which contains less material that can be converted to biogas than animal dung (e.g., cows). However, toilet, if available, 2.3 Methane Emission and Organic Feedstocks 61

Table 2.13 Physicochemical Nature of constituent % nature of typical sample of Water 75/ww human excreta [121] Total solid 25/ww Bacterial debris 10–30/dw Fat 5–25/dw Inorganic solids 10–20/dw Organic compounds 13/dw Carbohydrate 10–30/dw Nitrogen materials 2–3/dw Urea 36/dw

Biogas Plant

Inlet Latrine

Outlet

Gas holder BSo slurry

Digester

Fig. 2.10 A typical fixed dome-type community biogas digester can directly be linked to the biogas plant where human feces are digested together with the other wastes. This option provides a safe treatment of human excreta and thus improves the hygienic situation of the family. The availability of a renewable green energy source reduces the use of firewood for cooking and indoor air pollution. Thus, biogas digesters have the potential to minimize health risks and environmental pollution by using human excreta as a resource for producing energy and fertilizer. Consortium of Microflora in HE Digester Irrespective of many demerits, human excreta is proven to be a good feed material for anaerobic fermentation in the biogas plant. Due to tremendous obnoxious odor plus the presence of highly pathogenic bacteria and other microorganisms, human excreta-based digester is not popular among rural area like the other types of biogas plants based on organic and agriculture wastes. However, it has been clear that during the course of operation, the pathogenic microflora is reduced to minimum. Although, there is drastic reduction in the pollution potential of human excreta, the effluent is noticed to be quite concentrated and still has chance of carrying a number of viable pathogens. Hence, it is not advised to expose the effluent indiscriminately 62 2 Biomethanization into any open drains or water bodies. Secondary treatment of the effluent and digested sludge would be required before using it as a potential biofertilizer. As the pathogenic microflora of excreta depends on the food habit and the climatic factors of a specific location, it is difficult to generalize any bioindicator (s) in order to assess pollutant load in effluent in terms of pathogenic microbes. Fresh feed material of a human excreta-based biogas digester of a warm climate country shows the following pathogens along with their representative eggs and yeasts:

Parasitic Ova Ancylostoma duodenale (hookworm) Ascaris lumbricoides (roundworm) Taenia solium (pork tapeworm) Trichuris trichiura (whipworm) Echinococcus granulosus (hydatid) Enterobius vermicularis (pinworm)

Pathogenic Bacteria Escherichia coli Staphylococcus sp. Vibrio cholerae Salmonella sp. Shigella sp. Vibrio parahaemolyticus Cysts of parasitic protozoa Entamoeba histolytica Survival rate of bacterial pathogen is noticed to be lower than the parasitic egg. The pattern of inhibition of pathogen is noticed to be independent of dilution of feed slurry and seasonal variation of temperature. But with the increase in HRT, marginal increase in killing rate of the pathogen bacteria is noticed. A digester with relatively higher HRT value shows significant reduction in the flora of pathogenic microbes and also quantity of parasitic egg. Among the parasite eggs, hookworm and roundworm ova are the most resistant to environmental stress. No significant reduction in the number of these parasitic eggs is observed with respect to either dilution or hydraulic retention time or even seasonal variation of temperature. This observation is generally noticed in an experimental digester. However, significant reduction in the quantity of microflora of hookworm and roundworm ova is noticed in a digester attached with community toilet. The population of whipworm, hydatid, pinworm, and pork tapeworm during the course of operation of digester gets reduced significantly, and the same is noticed in better way when the digester is with lower TS. The protozoa cysts are also noticed to be susceptible when the HRT is fixed for a longer duration. 2.3 Methane Emission and Organic Feedstocks 63

Microbial investigation of thoroughly sun-dried digester sludge shows no sign of viable pathogen, not even roundworm and hookworm ova. The sun-dried digester sludge is granulated. These granules resemble tea leaves. The granulated material can be size graded as per requirement. These digested granules can be fortified with chemical fertilizer. Retreatment Process of HE (a) Heat pretreatment: During this process, human excrement would be pasteurized to 70 C before entering the biodigester. This would be done best before dilution to reduce energy costs and can be done using waste steam, passive solar heating, or direct combustion of biogas or any other fuel source. The process would make more of the human excrement available for anaerobic digestion and would in fact likely increase the amount of biogas produced. Heat pretreatment can also lower the HRT. Sterilization upfront will deal with any pathogen-related effluent issues down the line and produce a biofertilizer for comestible (fit for human consumption) crops. (b) Treatment through retention time (RT): Very long retention times for sewage have the ability to virtually destroy pathogens. The amount of time human excrement should be retained varies. In a very warm climate, it is expected to retain the waste for 60–90 days; however, in cold climates (20 C and below), 150 or more days of retention are recommended. Retention time can be controlled via the biodigester HRT or by holding the effluent for an additional period of time. The option that is the most economic should be considered as well as safety factors such as the access to holding tank and any other issue that involves potential exposure to humans and animals. From safety point of view, retention methods to destroy pathogens should be confirmed by lab results before adoption. (c) Posttreatment and sterilization: Biodigester effluent may also be treated in a secondary treatment phase such as ultrafiltration, ultraviolet light treatment, a treatment wetland, composting, or aerobic treatment. Ultrafiltration consists of running the effluent through a membrane that only allows soluble to pass through. At the moment, this technology is more likely to be used in the developed world, but appropriate solutions using materials such as mangroves and other plants might be used. Ultrafiltration is practical for concentrated wastewaters that have had most solids settled out. UV treatment is a common water treatment technology but may only be practical for dilute effluents where turbidity is not an issue. A treatment of wetland provides additional treatment as well as habitat for wildlife. Essentially a movement gradient is created and planted with wetland plants that facilitate nutrient and pathogen removal. This is the way wastewaters, such as storm runoff, are naturally treated in the environment. A composting process may be used to treat the effluent; however, it must first be dried to facilitate aeration, which is land and energy intensive. Care must be made to ensure that no one breathes in the dust from the fresh effluent during this process. The effluent may also go through an aerobic treatment process to polish the effluent; however, this is expensive and 64 2 Biomethanization

intensive and removes nutrients from a productive system. Other waste treatment options may include sand filters and clarifier. (d) Pathogens controlling: As previously alluded to, some biodigester processes are able to control virtually all the pathogens found in sewage. These are thermophilic biodigesters, phase biodigester, and staged biodigester. In a thermophilic biodigester, the environment within the biodigester is so hot that many pathogens are unable to survive. The environment is also far more competitive than in a regular biodigester. Pathogens are usually acclimatized with body temperature. Fortunately, many of the organisms capable of carrying out anaerobic digestion are thermophiles or heat-loving organisms. However, caution must be made with the previously mentioned ammonia toxicity, as thermophilic biodigesters are far more sensitive to this issue than ambient and lower temperature biodigesters. A phase biodigester separates the respective phases that material must undergo during the anaerobic digestion process. Organic material undergoes hydrolysis, acidogenesis, and acetogenesis. Essentially a container can facilitate the conversion of organics to solubles (hydrolysis), the production of acids (acidogenesis and acetogenesis), or methane production (methanogenesis). In phase anaerobic digestion, two or more containers are used to separate the phases. This can be done physically (removing organics as they are hydrolyzed), chemically (inhibiting methane production or buffering acids to a pH where methanogenesis can occur), or biologically (acidifying the first reactor(s)). If a reactor is allowed to acidify to inhibit methane production, the low pH will also create an extreme environment where some pathogens are unable to live. After an acidic environment, they will be introduced to a methane-producing environment that additionally removes pathogens through microbial competition. A two-phase biodigester capable of eliminating pathogens might have an acidifying first tank, which is then fed into a thermophilic, methane-producing second tank. Stage biodigesters can work in the same way by changing the competition mechanisms in various stages (reactors) though still not quite separating the phases. (e) Applying effluent: Completely eliminating pathogens is not necessary when adequate care is given to applying the effluent. Biodigester effluent that still contains pathogens can be applied into subterranean leach field (with a clarifier), used for nonedible crops and in some cases forge crops, and applied directly to land. However, all these things require safety considerations. The amount of human exposure needs to be taken into consideration. Groundwater and water body contamination are all potential threats to releasing effluent not completely void of pathogens into the environment. Direct land application needs to take direct exposure into account such as use of land by children and adults. Nonedible crops are another option and also allow for nutrient capture. Crops could include energy crops, biomass production, and many others. Exposure to humans however is again a risk that must be accounted for. The simplest and safest way to dispose of effluent is to simply inject it in an already existing sewer system. 2.3 Methane Emission and Organic Feedstocks 65

Human Excreta Biogas Digester Monitoring In India, generally, the human excreta-based biogas plant is attached to public toilets which are commonly visited by a large number of users, varying from few hundred to more than thousand per day. The better use depends on the location and the standard of the city. For example, commonly toilets present near railway station, bus stop, hospital, and high market complex of metropolitan cities remain busy round the clock. However, there is a peak period and a lean period. So, the loading of the digester is not uniform. Besides this, dropping of contraries like cigarette, matchboxes, and small pieces of stones into the pan are subsequently flushed with water into the drain and the inlet chamber of the digester. These materials sometimes cause chocking of the inlet pipe. The lighter materials float inside the digester and help to toughen the floating scum. Keeping the aforesaid fact in view, it appears that the use of human excreta as the sole feed material of a biogas plant is much more complicated than cattle dung. Due to a widely varying food habit, human excreta are variable with respect to its physicochemical character and its pathogenic content from users to users as well as from time to time, even for the same period. At the same time, due to its obnoxious odor, there is problem of proper channelizing of this waste to the digester. Another variable which needs maximum control is the influx of water along with cleaning agents into the digester. The water from the bath and wash basins is not allowed to flow into biogas digester. Calculated volume of feed material was put into the digester once a day followed by removal of an equivalent volume of effluent. The excreta is channel- ized through perfectly covered drains into the inlet chamber and then into the digester. The digester slurry comes out of the outlet chamber and is finally discharged into the sewer. The biogas rich in methane (Table 2.14) produced into the digester is stored under the fixed dome of the digester and is piped into the engine room where it is fed into dual-fuel gensets. In order to produce 60 m3 of biogas per 24 hours, the design considerations are as follows: (a) Hydraulic retention time—30 h (b) Biogas production per user per day—0.03 m3 (c) Volume of excreta plus wash water per user—3 L (0.003 m3) (d) Expected number of user per day—200 (e) Volume of slurry in the digester for 30 days HRT—180 m3 (f) Daily gas production—60 m3 (g) Maximum volume of biogas to be stored inside the digester (50 % of the daily production)—30 m3 (h) Total volume of the digester shell—210 m3 (i) Diameter to depth ratio—1.75 to 1 (j) Maximum biogas pressure inside the digester—1.3 m (water column). 66 2 Biomethanization

Table 2.14 A typical Type of gas produced % of gas analysis data of biogas Methane 64–48 produced from a human excreta-based digester Carbon dioxide 34.52 Hydrogen sulﬁde 0.55 Others 0.65

A typical complex (2000 user) generally consists of the following facilities: Toilet cubic: 10–30 nos Bathrooms: 10 nos Wash basin: 4 nos This type of digester can produce sufficient quantity of gas and to run a low kV genset for 8 h per day. The periodic monitoring of biogas production of such digester is noticed, fairly stable throughout the year. Proper dislodging operation, i.e., removal of settle sludge from the digester, is also an important consideration while operating a biogas digester. Generally, the scum layer, present at the top of the digester, is thick and also tough. The thickness of the scum varies with the volume of the digester and working of a digester. A community digester having the capacity of 60 m3 per 24 h about 3 m high, after 3 years of use may attain scum layer of about 1.25 m thick. The compact sludge at the bottom may reach about one meter height and middle space is filled with thick slurry. Generally, a bamboo stick is used to break the scum and mix the contents of the digester. A sludge pump is used for pumping out the slurry. About 3/4th of the digester is, generally, emptied leaving behind some old mixed sludge for inoculation. The digester is then filled with water to the invert levels of inlet and outlet pipes, but top opening (if provided) is closed and the digester is ready to use again. The biogas plant helps to reduce the pollution load significantly. Comparison of physicochemical characteristics of the feed slurry and the effluent shows that there is substantial reduction in the total solid content, TS (88–96 %); volatile solid content, VS (90 %); chemical oxygen demand, COD (50,000 mg/L); and biological oxygen demand, BOD (87–97 %). Influence of Mixed Feedstock (HE Plus Vegetable Waste) In the earlier section, the pattern of biogas production from human excreta-based digester and also vegetable waste-based digester has been discussed. Reports are also available on working feasibility of a biogas digester where human excreta and vegetable waste are used either together or on alternate days. Better results are obtained when human excreta-fed digester is mixed with the vegetable waste. The effluent from the mixed feed digester (experimental) has the following characteristics: TS: 5.41 gm/liter COD: 6.52 gm/liter VS: 3.58 gm/liter BOD: 1.31 gm/liter 2.3 Methane Emission and Organic Feedstocks 67

Parasitic eggs: 33.87 gm/liter (roundworm and hookworm), Bacterial pathogen: 73.50 mg/liter (E. coli, Shigella sp., Salmonella sp., and Staphylococcus sp.). Before mixing with human excreta, the vegetable wastes are made into small size about 100 to facilitate feeding through the inlet pipe. Due to the low density of the material, direct feeding is difﬁcult. So, a manual device is used to push the fruit/ vegetable waste down the inlet pipe. Before loading, the vegetable waste is pretreated in different ways. Mostly, the vegetable waste is composed aerobically/anaerobically for few days, and the putreﬁed portion is loaded into the digester. Before doing this, care should be taken to maintain the TS to approximately 3 % level balance so as to get high yield of biogas.

2.3.4 Methane from Whey

Whey is the greenish yellow fluid left after separating curd-like material in cheese- manufacturing process. World annual production of whey is estimated to be 115 million tonnes; approximately 47 % of the produced whey is disposed into the environment [124]. Whey is a by-product of cheese and casein manufactures; it mainly consists of carbohydrates, lactose, salts, lactic acid, proteins, and fat, respectively [125, 126]. The total solid present in whey is about 6 %. One pound of manufactured cheese is accompanied by approximately 6–9 pound of whey. It has a high biological oxygen demand which is caused by its protein and carbohydrate content, and it created a problem when disposed as wastewater. It has been established that 1000 gallon of raw whey, if influx into a stream, would require the dissolved oxygen in over 4,500,000 gallon of unpolluted water for its biooxidation. The BOD of whey varies from 30,000 to 60,000 m/L [127]. The high organic constituents of whey lead to treatment and disposal problem. On the other hand, whey can potentially be converted to useful end products such as single- cell protein (SCP), food and beverages, animal feed, alcohol, and methane. Because of high BOD of whey, it will require better than 99 % treatment. Even at 99 % removal efficiency, the residual BOD in whey will vary from 300 to 600 m/L. The pH of sweet whey ranges from 5 to 7 and that of acid whey between 4 and 5. The pH of acid whey will tend to require an optimum range of pH from 6.5 to 8. On either side of this value, there are detrimental effects to the aquatic life. Methane Production Potential On stoichiometric basis, one pound of liquid whey will give 0.014 pounds of CH3 or 0.314 ft (STP) each of CH4 and CO2 in the production gas mixture with the corresponding heating value of 314 Btu/lb of whey (6280 Btu/lb of lactose, assuming 5 % lactose in whey). The product gas has low sulfur content and energy value of about 1.8 kg fuel oil/m per meter. Methane concentration in the product gases is about 63 %. 68 2 Biomethanization

CH4 CH4

Whey Feed Effluent

Return Sludge Waste Sludge

Fig. 2.11 A typical whey digester process and biogas (methane) production

A typical digester (Fig. 2.11) is composed of a container in which the mixed liquid is kept under constant agitation from where the mixed liquor goes to the settler. The settled sludge is recirculated and methane gas is collected from the gas vent. Theoretically, methane can be produced from any of the three constituents of algae—carbohydrates, proteins, and fats. Closed algal bioreactors offer a promising alternative route for biomass feedstock production for biomethane. Using these systems, microalgae can be grown in large amounts (150–300 tonnes per ha per year) using closed bioreactor systems (lower yields are obtained with open-pond systems). This quantity of biomass can theoretically yield 200,000–400,000 m2 of methane per ha per year [124].

2.3.5 Methane from Algae

A major limiting factor for the future growth of biomethane production from plant sources is the availability of biomass. Photosynthetic microalgae are a major focus of interest as the efficiency of biomass production per hectare is estimated to reach 5–30 times that of crop plants [128]. On an annual basis, large-scale cultures of microalgae and photosynthetic bacteria can convert solar energy into chemical energy of biomass more efficiently than agricultural and forest land. Besides this, large amount of biomass (more than 50 tonnes of dry weight ha year) can be obtained with a limited requirement of resources by growing marine algae [129]. Similar to biodiesel, lipid plays an important role, since their conversion capacity into biomethane is higher (1390 L biogas (72 % CH4,28%CO2) per kg organic dry substance) than that of proteins (800 L biogas (60 % CH4,40%CO2) per kg organic dry substance) and carbohydrate (746 L biogas (50 % CH4,50% CO2) per kg organic dry substance). The idea of using algae as a source of food, feed, and energy goes back more than half a century when the feasibility of using microalgae for biomethane production was published for the first time. It could convince the scientists that 2.3 Methane Emission and Organic Feedstocks 69 the process could be feasible and could be further optimized in the future. In 1960, Oswald and Golweke proposed the use of large-scale ponds for cultivating algae on wastewater nutrients and anaerobically fermenting the biomass [124], and it received a big impetus during the energy crisis of the 1970s, when projects were initiated to produce gaseous fuels (hydrogen and methane). From 1980 to 1996, the US Department of Energy supported the Aquatic Species Program (ASP), a relatively small effort (about $25 million over almost 20 years) with the specific goal of producing oil from microalgae. The ASP researchers worked primarily on growing algae in open ponds, making significant contributions to our understanding of growing algae for fuel. Thousands of different species were isolated and tested, the impacts of different nutrient and CO2 concentrations were documented, the engineering challenges of mass- producing algae were addressed, and a solid foundation of algae–fuel research was built. But in 1995, faced with financial constraints and cheap oil, the DOE made the decision to terminate the program. In recent years, things have changed. Exploding global demand for transportation fuels, concerns about “peak oil,” increasing impacts of atmospheric CO2, the USA’s increasing importation of fuel, and the energy security risks that come with that have fueled a rebirth in the interest of biofuels in general and algae-based biofuels in particular. Advances in biotechnology, such as the ability to genetically engineer algae to produce more oils and convert solar energy more efficiently, have unleashed new possibilities not feasible during the ASP years. Most of the activity in algae research and commercial production has been in the USA. With more than 100 start-ups and large corporations, along with the US government, investing billions in this new industry, the USA is the leader in advancing algae-based fuels. However, now algae biofuels are also being researched around the world in both developed and developing nations in Europe, Asia, and elsewhere. The US-based Algal Biomass Organization is a leading voice for the industry and a source for information on the companies involved in advancing the technology. Microalgae can now be grown in large amounts (150–300 tonnes per ha per year) using highly efficient closed algal bioreactors for biomass feedstock and biomethane production. This quantity of biomass can theoretically yield 200,000–400,000 m3 of methane per ha per year. However, it has to be pointed out that biomethane production from microalgae currently is not competitive with biomethane production from maize or other crops because the production of biomass is expensive (in Germany, market price for green algae Chlorella vulgaris are in the order of 100 times higher than the price for maize), and the production capacity is far too low today to feed the demand of commercial biogas plants. Methane produced in biogas facilities today is mixed with carbon dioxide gas (usually 50–75 % methane), and the high degree of impurity limits its use. As compared with other methods for producing energy from biomass, biogas digester based on algal biomass is still unexplored. In general, a process for obtaining methane from algae involves the following successive stages: 70 2 Biomethanization

First: Pretreatment of the algae, capable of producing a liquid suspension of fine solid particles, said treatment being moreover capable of partially depolymerizing the solid algae matter. Second: Running the suspension through a fluidized bed containing granules on which enzymes are immobilized which are capable of transforming the particles into sugar, said liquid-containing acidific bacteria capable of transforming said sugars into volatile fatty acids. Third: Decantation of the suspension, so as to remove any solid particles that may remain and to extract a decanted liquid. Fourth: Running the decanted liquid across a fixed bed containing methanogenic bacteria set onto a support so as to cause the liquid to release a gas mixture containing mainly methane [128, 129]. However, the energy balance of the process and its economy depend on the proper design of the whole process and of its different steps and unit operation. In spite of so much advantages of using algal biomass for generation of multiple types of biofuels (algal diesel, H2, and biogas), the algal resource as renewable still has certain disadvantages: (a) biodegradability of microalgae can be low depending on both the biochemical composition and the nature of cell wall; (b) high cellular protein results in ammonia release which can lead to potential toxicity; and (c) the presence of sodium for marine species can also affect the digester performance.

2.3.6 Methane from CO2,H2, and CO by Anaerobic Fermentation

Methane can be obtained by anaerobic fermentation of CO, CO2, and H2 in the presence of suitable methanogenic bacteria. This gaseous substrate for methane production is the major products from the gasiﬁcation of coal and biomass. The generation of methane from CO2 and H2 was ﬁrst observed by Sohngen in 1906 to proceed according to the stoichiometry [130]:

CO2 þ 4H2 ! CH4 þ 2H2O ð2:6Þ

Considerable trials performed since this early observation has not only con- ﬁrmed this stoichiometry but also reveal this biochemical pathway to be the most appropriate reaction for gaseous fuel production by methanogenic bacteria [131]. In the microbial conversion of methane, CO is oxidized to CO2

0 CO þ H2O ! H2 þ CO2, ΔG of À 4:8Kcal=mol CO2 produce ð2:7Þ

And subsequently the carbon dioxide is reduced to methane. The overall stoichiometry is as follow: 2.3 Methane Emission and Organic Feedstocks 71

0 4CO2 þ 2H2O ! CH4 þ 3CO2 þ 2O2, ΔG f À 81:44cal=mol CH4 produce ð2:8Þ

These reactions have since been experimentally confirmed in two significant experiments. In the first, CO2 and H2 were converted to methane by Methanosarcina barkeri in the presence of excess H2. In the second set of experiment, M. barkeri could convert CO to CH4 in the absence of hydrogen. These result conform reactions (2.8). Subsequently, M. thermoautotrophicum can grow on CO as its sole electron donor. Perilex et al. [132, 133] had taken trial to generate high CH4 produce with M. thermoautotrophicum. Under batch conditions, with highly efficient gas transfer reactor, the CH4 productivity was 470 L per day. In continuous culture with the same fermenters, the productivity was lower. Later, the same group could improve the production of CH4 to fermenter system equal to the Ruston-type impeller.

2.3.7 Methane from Slaughterhouse Wastes

Most of the urbanized cities all over the world have vast and extensive meat- processing industry, which generates large volumes of wastewater and solid waste. A huge quantity of animal paunch manure amounting about 15–20 million tonnes per day is generated from various slaughterhouses in the world. Thus, it requires considerable treatment before the materials can be safely discharged into the environment. It has been estimated that for every cow and pig processed, 700 and 330 L of wastewater are generated, respectively, with an increase of 25 % if further processing is carried out to produce edible products [134]. Early applications of anaerobic technology to treat slaughterhouse wastewater date back to the 1950s [135]. Since then, many physicochemical methods and anaerobic processes have been developed and implemented to effectively manage slaughterhouse wastewater [136–139]. Typically, anaerobic digestion processes are used to treat wastewaters with high or medium organic loads [108]. They are also suitable for treating efﬂuents from other types of agro-industries. Waste Fractions Solid process wastes. Manure, feces, and urine, sometimes mixed with straw and fodder (dependent on kind and duration of animal keeping before slaughtering), are the main solid wastes generated in animal-keeping areas, and unwanted carcasses, unwanted hide, skins, or pieces are noticed in slaughterhouse processing areas. Liquid process wastes. The liquid wastes produced in slaughterhouses comprise a number of different wastewater streams as well as other nonsolid wastes such as paunch manure and blood. (1) Spent water: For hygienic reasons, abattoirs use large amounts of water in animal processing, thus producing large amounts of wastewater that have to be treated. The wastewater produced in animal slaughter areas typically has a high 72 2 Biomethanization

Table 2.15 Slaughterhouse Speciﬁc amount of wastewatera Unit Average value wastewater: average amounts Wastewater/cattle 1 1000–1500 and composition Wastewater/pig 1 200–600 Wastewater parameter – – COD mg/L 1000–6000 (max. 20,000) BOD mg/L 1000–4000 (max. 10,000) Total N mg/L 200–700 (max. 950)

NH4–N mg/L 200–300 Settable solids mg/L 80–120 Temperature C 10–35 Source:[86, 139] aAmount of wastewater in small rural slaughterhouses may be signiﬁcantly smaller (approx. 300–400 L/LSU)

organic loading. Table 2.15 resents average values for wastewater amounts and composition. In general, slaughterhouse wastewater is composed of a complex mixture of fats, proteins, complex organic compounds, and water. An organic load of 5000–10,000 mg/L, known as chemical oxygen demand (COD), is typical for such wastewater. The presence of pathogenic microorganisms is also quite typical [140]. The availability and need to explore and establish the utility of cattle paunch manure have been widely reported [141, 142]. Slaugh- terhouse wastes contain mostly biodegradable matter and are malodorous; this makes it suitable for anaerobic digestion. (2) Blood: The amount of blood also depends on the species of slaughtered animals and their live weight. Blood contains a large amount of protein and is too valuable to be discharged into the sewerage system. It is utilized as a raw material for blood meal, ﬁbrin, or blood serum, with blood meal being used as fertilizer or animal feed. However, anaerobic digestion may be a good reuse alternative for the blood, not least in times of BSE (“mad cow disease”), utilizing the contained energy for electricity or heat production. (3) Paunch manure: The amount of stomach content depends on the kind of animal slaughtered and substantially on the time of last feeding and kind of last fodder. For example, in Europe, an average of 50 kg is normally calculated per cattle, while in West Africa, 75 kg was measured. The hygienic status of untreated paunch manure is critical. Rarely, species of salmonella were found in critical amounts in healthy animals besides bacteria, viruses, and parasites (worms). Especially concerning salmonella, techniques for treating paunch manure have to be considered for hygienic reasons. Typical data on a paunch manure from a cow slaughtering house is given in Table 2.16. (4) Slaughterhouse waste processing: The slaughterhouse waste is rich in proteins which is a well-known source of sulﬁde formation during anaerobic 2.3 Methane Emission and Organic Feedstocks 73

Table 2.16 Typical data on Nature of organic chemicals g/kg TS paunch manure collected Crude protein 105–173 from slaughterhouse Crude fat 15–31 Crude ﬁber 256–391 Nitrogen 20–22 C/N ratioa 17–21 Phosphorus 5.6 Potassium 4–5 Calcium 6–8 Sodium 9–15 Magnesium 0.8–1 TS% 11.3 TVS (% of TS) 87.6 pH 5.8 Total volatile fatty acids 6–8 aNondimensional

digestion. As stated earlier, sulfides are the main cause of corrosive. In addition increase in H2S in the biogas causes sulfide inhibition of the methanogens [143, 144]. With the degradation of protein, occurrence of ammonia formation is a usual process [145] and mainly responsible for increasing the pH in the digester. This increase in pH becomes a main factor for the growth limitation of some VFA-consuming methanogens [146]. The fatty acid accumulation resulted due to continuous degradation of proteins during fermentation is mainly responsible for increasing concentrations of process-inhibiting fatty acids, to the consequential pH drop, and finally to a total inhibition of methanogenesis leading to complete blockage of digestion process, further. Thus, methods to lower ammonia levels in anaerobic digesters treating high pH and temperature, the equilibrium is shifted toward the toxic ammonia, resulting in a positive correlation between toxicity effects and increasing pH and temperature [147]. Among the methanogens, the acetate- utilizing methanogens have been suggested to be responsible for 70–80 % of the methane produced [148]. However, recent results suggest that an alternative methane-producing pathway is activated at elevated levels of ammonia [149]. In this pathway, acetate is converted to hydrogen and carbon dioxide by syntrophic acetate oxidizers (SAO), followed by the subsequent reduction of carbon dioxide to methane by hydrogen-utilizing methanogens, i.e., by this pathway methane is produced by hydrogenotrophic methanogens only. Devel- opment of SAO has been shown to occur due to a selective inhibition of acetate- utilizing methanogens by ammonia, released, i.e., during the degradation of proteins [149]. In 1996, it has been noted that by adapting co-digested operation technique in slaughterhouse waste digestion process, the efficiency of biogas production 74 2 Biomethanization

increased, signiﬁcantly [150, 151]. The co-digestion plant consists of three basic parts: (1) substrate reception and storage, (2) pasteurization equipment, and (3) anaerobic digesters. Slaughterhouse waste is treated with formic acid at the slaughterhouse, and all waste is delivered to the plant by closed trucks in a grinded (12 mm) pumpable form and is either transferred into a combined homogenization and buffer tank or directly into a second, heated buffer tank. After homogenization the substrate mixture is continually pumped to the second heated buffer tank. The target temperature of the second buffer tank is above 75 C to avoid foaming, to preheat the material before pasteurization, and to provide a stable thermal disintegration of the substrate. Substrate which is delivered warm is pumped directly to the heated buffer tank to save energy on substrate heating. Before loading of the digesters, the substrate is pasteurized in a batch process for one hour at 70 C, to fully comply with the EU ABP regulation for category three materials [152]. The anaerobic digestion is carried out at mesophilic condition, at 38 C. For anaerobic treatment, the blood should be mixed with other substrates of a higher carbon content such as paunch or manure. Blood is one of the most powerful nutrient media for anaerobic bacteria known in nature; it reaches a BOD value of about 150,000 mg/L. Figure 2.12 represents the general pattern of treatment of slaughterhouse wastes and spent water and generation of biogas.

Fig. 2.12 Schematic diagram of ﬂoating drum-type biogas reactor 2.3 Methane Emission and Organic Feedstocks 75

2.3.8 Sewage Sludge

Anaerobic technology has been applied in sewage sludge treatment for over one hundred years [153–155]. In the 1980s, anaerobic system became increasingly common in the treatment of industrial wastewater containing easily degradable, nontoxic compound. The first anaerobic system in the pulp and paper industries was anaerobic lagoons, introduced toward the end of the 1970s together with the contact reactor in 1981 [156]. The first high-rate anaerobic was the VAS reactor, which was introduced in 1983 [92, 157]. In 1889, at least 33 full-scale anaerobic external treatment plants were in operation in the pulp and paper industry [158]. The methane production in anaerobic process ranges from 0.1 to 0.35 m3 methane per kg COD removed [159]. Rapid urbanization all over the world has created a serious problem of proper disposal of sewage sludge. One way of solving the problem is to make use of this waste for producing biogas. During this process CO2 also evolve. There are reports describing the proper recycling/upgradation of sewage sludge and production of nonconventional gaseous fuel. The conversion is generally carried out by two stages. The first-stage consists hydrolysis of organic wastes by fermentative bacteria. During this process, large molecules (polysaccharide, protein, etc.) to small ones mainly consist of acetate. During the second phase, the acetate is converted to methane and carbon dioxide by acetogenic bacteria. Chemical composition: Alkaline night soil sewage sludge is also highly contaminated with hazardous pathogens [160–162] belonging to plants, humans, and animals. The extent to which sludge is contaminated with dangerous biological components depends mainly on the nature of sewage-producing sludge and the type of treatment, if any, it has received. Most commonly the sludge is contaminated to a greater or less degree by metals which can have adverse effect on crops and their productivity [163]. Besides this, many toxic metals enter into the food chain through edible crop and vegetables and are responsible for health hazard. It is advised to refer Forster’s work on availability of metals in sewage sludge [164]. Type of Sewage Sludge Sewage sludge is produced from the treatment of wastewater in sewage treatment plant and consists of two basic forms—raw primary sludge and secondary sludge, also known as activated sludge. Primary sludge is also called raw sludge which comes from the bottom of the primary clarifier. Primary sludge is easily biodegradable since it consists of more easily digestible carbohydrates and fats, compared to activated sludge which consists of complex carbohydrates, proteins, and long-chain hydrocarbons [165]. So, biogas is more easily produced from primary sludge. Activated sludge is also called excess sludge or waste-activated sludge which comes from the secondary treatment. It’s a result of overproduction of microorganisms in the activated sludge process. Activated sludge is more difficult to digest than primary sludge. Sewage sludge is usually treated by one or several treatment 76 2 Biomethanization steps before it is disposed of in landfills, dumped in the ocean, or applied to land. Lime stabilization, thickening, dewatering, drying, anaerobic digestion, and composting are some treatment processes to bring sewage sludge for reclamation. As we know, large amounts of waste-activated sludge, containing organic and mineral components, are produced by municipal and industrial wastewater treatment plants. Sludge handling represents a bottleneck in wastewater treatment plants, due to environmental, economic, social, and legal factors. If handled properly, sludge can be a valuable resource for renewable energy production and a source of nutrients for agriculture. Sludge Pretreatment Process (1) Thermal pretreatment Heat applied during thermal treatment destroyed the chemical bonds of the cell wall and membrane and thus makes the proteins accessible for biological degradation. Thermal pretreatment has been studied using a wide range of temperatures ranging from 60 to 270 C. In practice, the optimum temperature is in range of 160–180 C and treatment times from 30 to 60 min; pressure associated to these temperatures may vary from 600 to 2500 kPa [166]. (2) Chemical pretreatment Chemical pretreatment is also an efficient and cost-effective method to hydrolyze the cell wall and membrane and thus increase solubility of the organic matter contained within the cells. According to different principles, chemical methods can be divided to acid and alkaline (thermal) hydrolysis and oxidation. The most frequent studies of oxidative methods are ozonation and peroxidation. Acid and alkaline hydrolysis will be introduced in the thermochemical pretreatment part. Ozone is a strong cell lytic agent, which can kill the microorganisms in activated sludge and further oxidize the organic substances released from the cells [167, 168]. Of the techniques to disintegrate sludge, ozonation of sludge is one of the effective ways and yields the highest degree of disintegration [169]. Following ozonation, the characteristics of the sludge are greatly changed. The flocs are broken down into fine, dispersed particles. Floc integration and solubilization generates a large number of microparticles dispersed in the supernatant in addition to soluble organic substances [170]. The sludge biodegradation is affected by ozone dose. Several peroxidation techniques, including the well-known Fenton peroxidation and novel reactions involving peroxymonosulfate (POMS) and dimethyldioxirane (DMDO), can also achieve a transformation of refractory COD into readily available and soluble BOD, which further enhance the biogas production. Fenton pretreatment disintegrates extracellular polymeric substances and breaks cell walls; thus, intracellular water is released. Hence, the amount of soluble COD and BOD in the sludge water increases considerably. 2+ The oxidation process utilizes activation of H2O2 by iron salts (Fe ). A major drawback of this method is the necessity of bringing the sludge to a very low PH. 2.3 Methane Emission and Organic Feedstocks 77

Recently, a new oxidant peracetic acid (PAA) has been proposed to enhance anaerobic digestion biogas production. PAA has a strong effect of oxidation on microorganism. It is reported that PAA is able to destroy the barrier of spores, dissolve the core, and make the material such as DNA protein leaking. It can be inferred that PAA can dissolve and oxidize cells to promote the release of organic matter, thereby reduce the sludge volume and improve the efficiency of subsequent anaerobic digestion [171]. (3) Thermochemical pretreatment Alkali treatment is normally combined with thermal treatment; it’s the so-called thermochemical treatment. There is no consensus on the efficiency of alkali agents. It has been noted that pretreatment with KOH was more efficient than using NaOH [172]. (4) Mechanical pretreatment Mechanical pretreatment plays an important role because it favors solubilization of particulate matters in liquid phase. In general, the most often used techniques in mechanical pretreatment are ultrasonication, grinding, and high-pressure homogenization. By these methods, the aim is to increase the degradability of organic matters by disrupting the flocs and/or lysing the bacterial cells. (5) Ultrasonic pretreatment Ultrasonication is a promising and effective mechanical pretreatment method to enhance the biodegradability of the sludge. This technology has several inherent merits like efficient sludge disintegration (>95 %), improvement in biodegradability, improved biosolids quality, increase in methane percentage in biogas, no chemical addition, less retention time, sludge reduction, and energy recovery (1 kW) of ultrasound energy which generates 7 kW of electrical energy including losses [173]. Ultrasonication enhances the sludge digestibility by disrupting the physical, chemical, and biological properties of the sludge. (6) Grinding One predominant technique is the wet milling, which is more of a grinding method. Wet milling uses small beads to rupture cell walls; the size of the beads used is thus critical for maximal sludge disintegration [174]. Of several milling devices, the ball mill using small diameter (0.2–0.25 mm) balls has the best performance [175]. The use on an agitator ball mill was studied by Kunz et al. [176]. Sludge was pressed through a cylindrical or conical space by an agitator including shear stresses high enough to break the bacterial cell walls. (7) High-pressure homogenizer One of the most frequently used methods for large-scale operation is high- pressure homogenization, compressing the sludge to 60 MPa [177]. The compressed suspension is then depressurized through a valve and projected at high speed against an impaction ring. The cells are hereby subjected to turbulence, cavitation, and shear stresses, resulting in cell disintegration [178]. Lacking of available literatures on mechanical pretreatment, the methods mentioned above are not comprehensive. However, it is seen that their efficiency of improving 78 2 Biomethanization

anaerobic digestion of waste-activated sludge is rather lower than other methods. (8) Biogas-producing potential A long aerobic digester technique has been studied for upgrading sewage sludge for gaseous fuel and fertilizer. For over three decades, this technology has been widely used in the USA and UK and many other developing countries [179, 180]. Like organic wastes, anaerobic digester of sewage sludge is also monitored based on pH, alkalinity, volatile fatty acids (VFAs), and total organic solid (TOS). The digester is generally maintained with the range 7.5–7.7 and bicarbonate alkalinity with the range of 3500–5000 mg. Concentration of VFA is measured in terms of acetic acids, and for the digestion of sludge, it is needed to be kept at the value below 200–500 mg. Generally, there is need of adjusting the pH due to interaction between ammonium and bicarbonate ions. Exogenous carbonate/bicarbonate is proved at the time of requirement, if needed. An alternative option for this is to add lime. The gas production rate depends on the nature of sludge accumulated from various resources. Irrespective of the operational methods of digester, generally, a typical production rate of gas is 1m3/kg volatile matter digested, and the methane concentration is about 65–70 % of total biogas generated. Its gross caloriﬁc value (CV) is estimated from CV ¼ 334x (% of methane) KJ M322-24MJ m3.

2.3.9 Methane from High Solid Agro-wastes

Intensive agriculture practice has caused generation of significant amount of unutilized high solids, postharvest residues, and manures. Annual biomass production from maize, wheat, rice, and sugarcane crops yields about 5358.54 million tonnes of dry biomass. Though its major part is utilized as feed for animals, a huge amount of crop residues such as grain straws, corn stalks, and rice husk are left on the field or burned. Rice husk amounts 20–25 % of the harvested rice grain on dry-weight basis, which is usually separated from rice grain at the processing centers [181]. Direct combustion of biomass yields into emission of 1599 kg carbon dioxide, 111.3 kg carbon monoxide, 9.2 kg methane, 5.6 kg hydrocarbon and 4.8 kg particulate matter per tone of dry biomass [224]. However, the direct burning of biomass in open environment is totally oxidized without 100 % utilization in energy cycle. Hence, it is not a recommendable practice for sustainable development. There- fore, this biomass must be utilized properly and effectively for production of fuels/ chemicals. This certainly helps in lowering down the overall global warming potential from other greenhouse gases, since environmental protection along with the energy generation is of a great concern in the twenty-first century. Chemical composition: Like other vegetable and organic wastes, crop residues also contain lignocellulosic materials and other substances. However, the percent of 2.4 Raw Biogas Upgradation Process 79 lignocellulosic is relatively much higher than the market vegetable wastes (Table 2.16). The optimum biodegradable C/N ratio is about 25:1 [183]. However, some variation in this ratio is noticed, but these variations remained within the range of optimal microbial activity and have no adverse effect on digester performance. Biogas Production Potential A crop residue-based digester with high TS loading rate generally produces 1.38 m3/m3 per day biogas in which the methane content is about 60 % [116]. With 25 % beef cattle manure and 75 % corn residues, gas production rates varied between 2.53 % and 0.7 m3/m3 per day during the 30-day retention time, and the overall process efficiency [184] is noticed to be about 79.2 %. High-quality compost with a C/N ratio of 16:1 is also obtainable from a crop residue-based biogas digester. Composition of Microflora The biogas production based on high solids agro- wastes from agriculture land and from agro-industry cellulosic wastes restricted mainly by the rate of hydrolysis of the polymeric compounds. These substances require microflora for digestion and biogas production, under anaerobic condition. Numerous reports are available related to the microflora composition of a digester fed with various organic wastes like piggery wastes [185], swine waste [186], sludge [187], and swine digester fluid, and even data are also available in microflora composition of volcanic environments where methanogenesis is a common feature [184]. However, very little information is available on the association of microbes during biogas production from a high solid agro-waste-based digester. Lapat and Garcia [188] reported that during the first four weeks of incubation, the total microflora increased to more than hundred times of the initial value. In the first phase of anaerobic digestion, mostly the fermentative bacteria were present in the digester. Its quantity was about 80 % of the total microflora. Non-oxygen- sensitive strictly anaerobic bacteria were about 5 % of total microflora. Among the fermentative bacteria, glucose fermentative bacteria were more numerous than lactose fermentative bacteria. The methanogenic bacteria are accounted for less than 10 % of the total microflora. Besides these microbes, very little quantity of nitrate-reducing bacteria and about 0.1 % of sulfur-reducing bacteria were also recorded.

2.4 Raw Biogas Upgradation Process

The raw biogas immediately collected from the digester is having about 60 % methane and 29 % CO2 with trace of H2S and some other impurity (Tables 2.1 and 2.2). The crude biogas contains many impurities such as water, hydrogen, sulfur, nitrogen, oxygen, ammonia, siloxanes, and particles. The degree of such impurities depends on the nature of feedstock and process followed for biogas production. To prevent corrosion and mechanical wear of the upgrading equipment itself, it can be advantageous to clean the gas before the upgrading. The following 80 2 Biomethanization are few important techniques to clean crude biogas, in medium-scale production unit to meet alternate renewable energy at community level.

2.4.1 Removal of Water

Removing moister is easier than removing CO2 and H2S from biogas (Fig. 2.13). A condensate trap at a proper location on the gas pipeline can remove water vapor as warm biogas cools by itself after leaving the digester. Water can also be removed by cooling, compression, absorption, or adsorption (SiO2, activated carbon or molecular sieves). By increasing the pressure or decreasing the temperature, water will condensate from the biogas and can thereby be removed. Other technologies for water removal are absorption in glycol solutions or the use of hygroscopic salts.

2.4.2 Removal of Nitrogen

Oxygen is not normally present in biogas since it should be consumed by the facultative aerobic microorganisms in the digester. However, if there is air present in the digester, nitrogen will still be present in the gas when leaving the digester. Oxygen and nitrogen can be present in landﬁll gas if the gas is collected using an under pressure. These gases can be removed by adsorption with activated carbon, molecular sieves, or membranes. They can also to some extent be removed in desulfurization processes or in some of the biogas-upgrading processes.

2.4.3 Removal of Ammonia

Ammonia is formed during the degradation of proteins. The amounts that are present in the gas are dependent upon the substrate composition and the pH in the digester. Ammonia is usually separated when the gas is dried or when it is upgraded. A separate cleaning step is therefore usually not necessary.

2.4.4 Removal of Siloxanes

Siloxanes are compounds containing a silicon–oxygen bond. They are used in products such as deodorants and shampoos and can therefore be found in biogas from sewage sludge treatment plants and in landﬁll gas. When siloxanes are burned, silicon oxide, a white powder, is formed which can create a problem in gas engines. Siloxanes can be removed by cooling the gas; by adsorption on activated carbon 2.4 Raw Biogas Upgradation Process 81

Water And saturated Heat exchanger Blower biogas Or And / Or And Dry Biogas Dessicant Chiller

Fig. 2.13 Slaughterhouse wastes management system (Adapted from Schuchardt et al., ref [87])

(spent after use), activated aluminum, or silica gel; or by absorption in liquid mixtures of hydrocarbons. Siloxanes can also be removed while separating hydrogen sulﬁde, as described under “removal of hydrogen sulﬁde.”

2.4.5 Removal of Particulates

Particulates can be present in biogas and landﬁll gas and can cause mechanical wear in gas engines and gas turbines. Particulates that are present in the biogas are separated by mechanical ﬁlters.

2.4.6 Removal of Hydrogen Sulﬁde

Although carbon dioxide is the major contaminant in the raw biogas during the production of biomethane, it has been shown that the removal of hydrogen sulfide can be of crucial importance for the technological and economic feasibility of the whole gas upgrading chain. Of course, this behavior strongly depends on the sulfur content of the used substrate and the continuity of the fermentation process. Hydrogen sulfide is a hazardous and corrosive gas that has to be removed from the gas prior to any further utilization, whether its grid injection or CNG-fuel production. A number of technologies are readily available to fulfill this task (Fig. 2.14a). Depending on the local circumstances of the anaerobic digestion plant and the biomethane production unit, one technology or a combination of two or even more technologies for biogas desulfurization has to be applied to realize a technically stable and economically competitive solution. Hydrogen sulfide is formed during microbiological reduction of sulfur- containing compounds (sulfates, peptides, amino acids). The concentrations of hydrogen sulfide in the biogas can be decreased by precipitation in the digester liquid or by treating the gas either in a stand-alone vessel or while removing carbon dioxide. 82 2 Biomethanization

a Purified biogas

Absorbent (iron compounds)

Water/Chemical Absorption scrubber clumn Particle Air separator Biogas

Regeneration process

Sulphur

b Desulphurised biogas

Scrubbing Column Caustic Oxidiser

Raw biogas

Fresh air

Effluent

Fig. 2.14 (a) Simpliﬁed diagrammatic presentation to remove hydrogen sulﬁde from crude biogas. (b) Flow sheet of a chemical–oxidative scrubbing plant for raw biogas desulfurization; pictures of the chemical–oxidative scrubber at the biogas plant Bruck/Leitha, Austria, with a raw biogas capacity of 300 m3/h. (Source: Vienna University of Technology, Biogas Bruck GmbH)

Natural bacteria can convert H2S into elemental sulfur in the presence of oxygen and iron. This can be done by introducing a small amount (2–5 %) of air into the head space of the digester. As a result, deposits of elemental sulfur will be formed in the digester. Even though this situation will reduce the H2S level, it will not lower it below that recommended for pipeline-quality gas. This process may be optimized by a more sophisticated design where air is bubbled through the digester feed material. It is critical that the introduction of the air be carefully controlled to avoid reducing the amount of biogas that is produced. Hydrogen sulfide reacts readily with either iron oxide or iron chloride to form insoluble iron sulfide. The reaction can be exploited by adding the iron chloride to the digester feed material or passing the biogas through a bed of iron oxide- containing material. The iron oxide comes in different forms such as rusty steel wool, iron oxide pellets, or wood pellets coated with iron oxide. The iron oxide 2.4 Raw Biogas Upgradation Process 83 media needs to be replaced periodically. The regeneration process is highly exothermic and must be controlled to avoid problems. Activated carbon impregnated with potassium iodide can catalytically react with oxygen and H2S to form water and sulfur. The reaction is best achieved at 7–8 bars (unit of pressure) and 50–70 C. Activated carbon beds also need regeneration or replacement when saturated. During the process of carbon dioxide removal by water scrubber method, both the CO2 and H2S can be scrubbed by water. The simplest method of desulfurization is the addition of oxygen or air directly into the digester or in a storage tank serving at the same time as gas holder. Thiobacilli are ubiquitous and thus systems do not require inoculation. They grow on the surface of the digestate, which offers the necessary microaerophilic surface and at the same time the necessary nutrients. They form yellow clusters of sulfur. Depending on the temperature, the reaction time, and the amount and place of the air added, the hydrogen sulfide concentration can be reduced by 95 % to less than 50 ppm. Most of the sulfide oxidizing microorganisms belong to the family of Thiobacillus. For the microbiological oxidation of sulfide, it is essential to add stoichiometric amounts of oxygen to the biogas. Depending on the concentration of hydrogen sulfide, this corresponds to 2–6 % air in biogas. Measures of safety have to be taken to avoid overdosing of air in case of pump failures. The absorption of hydrogen sulfide in caustic solutions is one of the oldest methods for gas desulfurization. Nowadays, typically sodium hydroxide is used as a caustic, and the pH is carefully controlled to adjust the separation selectivity. The task is to create and maintain a plant operation with maximized hydrogen sulfide absorption and minimized carbon dioxide absorption in order to minimize chemical consumption (carbon dioxide is to be removed with a more efficient technology). The selectivity of hydrogen sulfide versus carbon dioxide can be further increased by the application of an oxidizer to oxidize the absorbed hydrogen sulfide to elemental sulfur or sulfate, thus increasing the rate of hydrogen sulfate removal (Fig. 2.14b). Usually, hydrogen peroxide is used as an oxidizer in biogas-upgrading plants. This technique shows favorable controllability and stable operation even under strong fluctuations of raw biogas quality and quantity. Hydro- gen sulfide contents of as low as 5 ppm can be reached during stable operation. Usually, the most economic operation is to control the content of the purified gas to be around 50 ppm; the remaining hydrogen sulfide is removed by means of adsorption on metal oxides. This technology requires elaborate process control and the knowledge of dealing with the used chemical agents. It has been reported that the specific costs of this technology is highly competitive to other existing desulfurization technologies. This technology has to be considered if high or strongly fluctuating hydrogen sulfide contents have to be expected at a biomethane production site. 84 2 Biomethanization

2.4.7 In Situ Biogas Upgradation

As discussed earlier, various technologies for biogas upgrading are commercially available including water scrubbing, organic physical scrubbing, chemical adsorption, pressure-swing adsorption, and membrane separation. However, biogas- upgrading plants based on these technologies are all expensive and can only be economic favorable for large-scale biogas plant. Thus, there is a demand on developing cost-efficient upgrading technologies with focus on small or middle- sized biogas production facilities. An alternative and potentially cost-efficient technique to upgrade biogas is in situ methane enrichment (Fig. 2.15). This technology is mainly based on solubility of carbon dioxide and methane in aqueous medium. The in situ methane enrichment process increased methane concentration of the biogas from 60 % to 81 %, while removing 72 % carbon dioxide. The process also reduced hydrogen sulfide concentration by an average of 83 %. Inclusion of the wood–ash filter removed additional carbon dioxide and hydrogen sulfide from the biogas, increasing methane concentration to 97 % (the remaining balance being mainly nitrogen and water). Methane loss during this process represented <2%of total methane production. The in situ methane enrichment technique is performed by pumping the digester sludge, rich in soluble CO2, through a CO2 desorption column and then back to the digester (Fig. 2.15). The desorption of CO2 is achieved by aeration of the sludge, based on the higher solubility of CO2 compared to CH4. At a pH of 7.0 and a temperature of 35 C, e.g., CO2 is 20 times more soluble than CH4. The sludge being returned from the desorption column will not be saturated in CO2 and will have a capacity to absorb a significant portion of the CO2 produced in the anaerobic

High methane Organic feedstock content biogas Gas outlet for

Desorbed CO2 & Air Carbon dioxide Rich slurry

CO2 Anaerobic desorption Digester Column

CO2 Poor slurry Air Effluent

Fig. 2.15 Schematic picture of in situ methane enrichment based on air stripping 2.5 Full-Scale Technology for Biogas Upgrading 85 conversion of organic matter but will only absorb a small fraction of the less soluble CH4. In principal, much higher methane content can be expected as the result of in situ enrichment of digester off gas.

2.5 Full-Scale Technology for Biogas Upgrading

However, in large-scale production of biogas, mainly, four steps, i.e., (1) water washing, (2) pressure-swing adsorption, (3) selexol adsorption, and (4) amine gas treatment, are followed to upgrade crude biogas and make it effective and pollutant (CO2, water, H2S and particulate)-free. In addition to these, the use of membrane separation technology for biogas upgrading is increasing, and there are already several plants operating in Europe and the USA. Upgrading of biogas or landﬁll gas is deﬁned as removal of carbon dioxide from the gas. This will result in an increased energy density since the concentration of methane is increased. Several technologies for biogas upgrading are commercially available, and others are at the pilot or demonstration plant level. Some of these technologies are described below and reviewed in terms of recent developments.

2.5.1 Water Scrubber

Carbon dioxide has a higher solubility in water than methane. Carbon dioxide will therefore be dissolved to a higher extent than methane, particularly at lower temperatures. The most common method to remove carbon dioxide and other trace elements is passing through cascading water running counterflow to the gas (Fig. 2.16). Carbon dioxide will therefore be dissolved to a higher extent than methane, particularly at lower temperatures. In the scrubber column, carbon dioxide is dissolved in the water in the absorption column by using high pressure, normally 6–10 bars (a), while the methane concentration in the gas phase increases. The gas coming out from the water scrubber (Fig. 2.10) is rich in methane. The water leaving the absorption column is transferred to a flash tank where the dissolved gas, which contains some methane but mainly carbon dioxide, is released and transferred back to the raw gas inlet. If the water should be recycled, it is transferred to a desorption column filled with plastic packing, where it meets a counterflow of air, into which carbon dioxide will be released. The water is cooled down to achieve the large difference in solubility between methane and carbon dioxide before it is recycled back to the absorption column. 86 2 Biomethanization

Absorption column Desorption column

Air with Desorbed carbon dioxide Upgraded biomethane

Raw biogas Air

Flash column

Compressor Water bleed stream

Make-up water

Fig. 2.16 Schematic illustration of a water scrubber

Biomethane

Off-gas

Absorption column Deabsorption column

Raw biogas

Fig. 2.17 Schematic presentation of a typical amine scrubbing process to upgrade raw biogas

2.5.2 Chemical Scrubbing

The use of reactive systems for removing CO2 from biogas is not a brand new notion, but it is less common compared to other technologies such as PSA and water scrubbing. Chemical scrubbers use amine solutions (Fig. 2.17). Carbon dioxide is 2.5 Full-Scale Technology for Biogas Upgrading 87 not only absorbed in the liquid but also reacts chemically with the amine in the liquid. Since the chemical reaction is strongly selective, the methane loss might be as low as <0.1 %. Part of the liquid is lost due to evaporation and has to be replaced. The liquid in which carbon dioxide is chemically bound is regenerated by heating. The most common amines used historically for the purpose of sour gas removal (carbon dioxide and hydrogen sulﬁde) are methyldiethanolamine (MDEA), diethanolamine (DEA), and monoethanolamine (MEA) [189, 190]. If hydrogen sulﬁde is present in the raw gas, it will be absorbed in the amine scrubber solution, and higher temperatures will be needed for the regeneration. Therefore, it is advisable to remove it before absorption in the amine scrubber.

2.5.3 Membrane Technology (Gas Permeation)

Membranes for biogas upgrading are made of materials that are permeable for carbon dioxide, water, and ammonia. Hydrogen sulfide, oxygen, and nitrogen permeate through the membrane to a certain extent, and methane passes only to a very low extent. Typical membranes (Fig. 2.18) for biogas upgrading are made of polymeric materials like polysulfone, polyimide, or polydimethylsiloxane. These materials show favorable selectivity for the methane/carbon dioxide separation combined with a reasonable robustness to trace components contained in typical raw biogases. To provide sufficient membrane surface area in compact plant dimensions, these membranes are applied in form of hollow fibers combined to a number of parallel membrane modules. After the compression to the applied operating pressure, the raw biogas is cooled down for drying and removal of ammonia. After reheating with compressor waste heat, the remaining hydrogen sulfide is removed by means of adsorption on iron or zinc oxide. Finally, the gas is piped to a single- or multi-staged gas permeation unit. The numbers and interconnection of the applied membrane stages are not

CO2(+H2S)

Compressor +10- 15 %CH4

>78%CH4 Biogas H2S removal Membrane separator

CO2(+H2S)

>90%CH4

Internally staged

Fig. 2.18 Flow chart of membrane biogas puriﬁcation process 88 2 Biomethanization determined by the desired biomethane quality but by the requested methane recovery and speciﬁc compression energy demand. Modern upgrading plants with more complex design offer the possibility of very high methane recoveries and relatively low-energy demand. Even multi-compressor arrangements have been realized and proved to be economically advantageous. The operation pressure and compressor speed are both controlled to provide the desired quality and quantity of the produced biomethane stream.

2.5.4 Cryogenic Upgrading

Cryogenic separation (Fig. 2.19) of biogas is based on the fact that CO2,H2S, and all other biogas contaminants can be separated from CH4 based on the fact that each contaminant liquefies at a different temperature–pressure domain. This separation process operates at low temperatures, near À100 C, and at high pressures, almost 40 bars. These operating requirements are maintained by using a linear series of compressors and heat changers. Crude biogas streams through the first heat exchanger which cools the gas down to 70 C. This heat exchanger makes use of the product stream as cooling medium, which is energy efficient and has the advantage of preheating the upgraded biogas before leaving the plant. The first cooling step is followed by a cascade of compressors and heat exchangers which cool the inlet gas down to À100 C and compress it to 40 bars before it enters the distillation column. Finally, the

Fig. 2.19 Drying process of water-saturated crude biogas through various options 2.5 Full-Scale Technology for Biogas Upgrading 89

distillation column separates CH4 from the other contaminants, mainly H2S and CO2. The main advantage of cryogenic separation is the high purity of the upgraded biogas (99 % CH4), as well as the large quantities that can be efﬁciently processed. The main disadvantage of cryogenic separation is that cryogenic processes require the use of considerable process equipment, mainly compressors, turbines, and heat exchangers. The need for the equipment raises capital and operating costs relative to other options. The ﬁnal price of upgraded biogas using this technique is estimated to be €0.44 per Nm3 biogas [191].

2.5.5 Pressure Swing Adsorption (PSA)

Gas separation using adsorption is based on different adsorption behavior of various gas components on a solid surface under elevated pressure (Fig. 2.20). An upgrading plant, using this technique, has four, six, or nine vessels working in parallel. When the adsorbing material in one vessel becomes saturated, the raw gas ﬂow is switched to another vessel in which the adsorbing material has been regenerated. During regeneration the pressure is decreased in several steps. The gas that is desorbed during the ﬁrst and eventually the second pressure drop may be returned to the inlet of the raw gas, since it will contain some methane that was adsorbed together with carbon dioxide. The gas desorbed in the following pressure

Purge gas Compresor

PSA columns H2S Removal

Waste gas

Gasconditionig

Condensate

Fig. 2.20 Process diagram for upgrading of biogas with PSA. H2S and water vapor is separated from the raw biogas before it is fed to the adsorption column. Multiple columns work in parallel cycles for a continuous process. Figure adapted from [191] 90 2 Biomethanization reduction step is either led to the next column or, if it is almost entirely methane- free, is released to the atmosphere. Usually, different types of activated carbon or molecular sieves (zeolites) are used as the adsorbing material. These materials selectively adsorb carbon dioxide from the raw biogas, thus enriching the methane content of the gas. If hydrogen sulﬁde is present in the raw gas, it will be irreversibly adsorbed on the adsorbing material. In addition, water present in the raw gas can destroy the structure of the material. Therefore, hydrogen sulﬁde and water need to be removed before the PSA column.

2.5.6 Bioﬁltration

Biofiltration is one of the most promising clean technologies for reducing emissions of malodorous gases and other pollutants into the atmosphere (Fig. 2.21). In a biofiltration system, the gas stream is passed through a packed bed on which pollutant-degrading microbes are immobilized as biofilm. A biological filter com- bines water scrubbing and biological desulfurization. Biogas and the separated digestate meet in a countercurrent flow in a filter bed. The biogas is mixed with 4–6 % air before entry into the filter bed. The filter media offer the required surface area for scrubbing, as well as for the attachment of the desulfurizing microorganisms. Microorganisms in the biofilm convert the absorbed H2S into elemental sulfur by metabolic activity. Oxygen is the key parameter that controls the level of oxidation.

Heater Heater

Absorption Desorption column column Upgraded biomethane Cooler Compressor Off-gas

Biogs

Flash column

Fig. 2.21 A simpliﬁed schematic process ﬂow diagram of a typical organic physical scrubbing process 2.5 Full-Scale Technology for Biogas Upgrading 91

The capital costs for biological treatment of biogas are moderate and operational costs are low. This technology is widely available worldwide. However, it may be noted that the biological system is capable to remove even very high amounts of hydrogen sulfide from the biogas, but its adaptability to fluctuating hydrogen sulfide contents is not yet proven.

2.5.7 Organic Physical Scrubbing

Very similar to water scrubbing, this technology uses an organic solvent solution (e.g., polyethylene glycol) instead of water as a scrubbing liquid. The solubility of carbon dioxide is much higher in the organic solvent than in water, i.e., the value of Henry’s constant for carbon dioxide is higher. Carbon dioxide has a solubility of 0.18 M/atm in Selexol which is about five times higher than in water [192]. Carbon dioxide is about 17 times more soluble than methane in the Genosorb solvent [193] which is actually a smaller difference than for water, in which carbon dioxide is 26 times more soluble than methane. Due to the higher solubility of carbon dioxide in the solvent, the volume of solvent that must be circulated in the system decreases significantly compared to a water scrubber. The process is designed in a similar way as a water scrubber with the following two main differences: (1) the diameters of the columns are smaller since lower flow of the organic solvent is required, and (2) the organic solvent has to be heated before desorption and cooled before absorption. A schematic illustration of the process is shown in Fig. 2.22.

Clean gas Clean gas

Air B Air A C Raw gas Raw gas Raw gas

Growth Growth medium medium

Fig. 2.22 Purification of raw biogas through biofilm technology systems for removal of H2S: (A) bioscrubber, (B) biofilter, (C) biotrickling filter 92 2 Biomethanization

2.6 Factors Affecting the Biogas Generation

Anaerobic digestion depends on several different parameters for an optimum performance. Different groups of microorganisms are involved in the methane production, and suitable conditions have to be established to keep all the microorganisms in balance. Some of these parameters are pH, temperature, mixing, substrate, C/N ratio, and hydraulic retention time (HRT). Digestion is a slow process and it takes at a minimum of three weeks for the microorganisms to adapt to a new condition when there is a change in substrate or temperature.

2.6.1 Temperature and Pressure

Temperature for fermentation will greatly affect biogas production. Depending on prevailing conditions, methane can be produced within a fairly wide range of temperature. The process of anaerobic fermentation and methane-forming bacteria works best in the temperature between 29 C and 41 C or between 49 C and 60 C and pressure of about 1.1–1.2 bars absolute. This is due to fact that two different types of bacteria multiply best in these two different ranges, but the high temperature bacteria are much more sensitive to ambient inﬂuences. The rate of gas production increases with the increase in temperature but the percentage of methane reduces. It is found that temperature between 32 C and 35 C is most efﬁcient for stable and continuous production of methane. Biogas produced outside this range will have a higher percentage of carbon dioxide and other gases than within this range. The production of biogas is fastest during summer and it decreases at lower temperature during winter. If the temperature is lower than 20 C, the rate of gas production falls sharply, and it almost ceases at about 10 C. Also methanogenic microorganisms are very sensitive to temperature changes, a sudden change exceeding 30 C will affect production, and therefore one must ensure relative stability of temperature. Thus, in cold climates, it is necessary to heat the digester to about 35 C. The thermophilic mode of digester operation results in approximately 50 % higher rate of degradation and, particularly with fat-containing materials, a better availability of the substrates to the enzymes excreted by the acidogens and thus a higher biogas yield [194]. Due to the lower solubility of CO2 at higher temperatures, a 2–4 % higher CO2 concentration in biogas can be observed in thermophilic digesters. Despite some advantages of AD at higher temperatures, operating the digester at thermophilic ranges is generally considered less feasible in country context due to the additional energy inputs required as well as the lower stability of the process. 2.6 Factors Affecting the Biogas Generation 93

2.6.2 Organic Loading Rate (ORL)

Organic loading rate (OLR)/volatile solids (VS) OLR is a measure of the biological conversion capacity of the AD system. Feeding the system above its sustainable OLR results in low biogas yield due to the accumulation of inhibiting substances in the digester slurry (i.e., fatty acids). Under such circumstances, the feeding rate of the system must be reduced. OLR is a particularly important control parameter in continuous systems. Many plants have reported system failure due to overloading. OLR is expressed in kg chemical oxygen demand (COD) or volatile solids (VS) per cubic meter of reactor. It is linked with retention time for any particular feedstock and anaerobic reactor volume. Volatile solids (VS) represent the organic matter in a sample which is measured as solid content minus ash content, as obtained by complete combustion of the feed wastes. VS comprise the biodegradable VS (BVS) fraction and the refractory VS (RVS). High VS content with low RVS is more suitable for AD. Studies of anaerobic treatment of biowaste in industrialized countries describe organic loading rates in the range of 48 kg VS/m3 reactor and day, which result in VS removal in the range of 50–70 % [195]. This is ideal for continuously stirred reactors. However, for non-stirred AD systems which are predominant in developing countries, an OLR below 2 kg VS/m3 reactor and day is recommended and considered suitable.

2.6.3 Hydraulic Retention Time (HRT)

The hydraulic retention time (HRT) quantiﬁes the time the liquid fraction remains in the reactor. It is calculated by the ratio of the reactor (active slurry) volume to the input ﬂow rate of feedstock. The HRT required to allow complete AD reactions varies with different technologies, process temperature, and waste composition. Recommended HRT for wastes treated in a mesophilic digester range from 10 to 40 days. Lower retention times down to a few days only are required in digesters operated in the thermophilic range [196]. A distinction is made between hydraulic retention time (HRT) and solids retention time (SRT), but for digestion of solid waste, HRT and SRT are generally considered equal.

2.6.4 pH Value or Hydrogen Ion Concentration pH value indicates the degree of acidity or alkalinity of a solution. The pH value is represented as the logarithm of the reciprocal of the hydrogen ion concentration in gm equivalent per liter of solution. pH value in the range 0–7 represents acidic solution and in the range 7–14 indicates the alkaline solution. The microorganisms require a neutral or mildly alkaline environment—a too acidic or too alkaline 94 2 Biomethanization environment will be detrimental. Ideal pH value is between 7.0 and 8.0 but can go up or down by a further 0.5. In the initial stages of acid-forming stage of digestion, the pH value may be around 6.0 or less; however, during methane formation stage, the pH value higher than 7.0 is maintained since methane formers are sensitive to acidity. The pH value depends on the ratio of acidity and alkalinity and the carbon dioxide content in the digester, the determining factor being the density of the acids. For the normal process of fermentation, the concentration of volatile acid measured by acetic acid should be below 2000 parts per million because too high concentration will greatly inhibit the action of the methanogenic microorganisms. Lime is commonly used to raise the pH of AD systems when the process is too acidic. Alternatively, sodium bicarbonate can also be used for pH adjustment. Lime is usually much cheaper and there might be free sources of spent lime solutions from local industry. Lime however frequently leads to precipitations and clogging of pipes when used in larger quantities. Sodium bicarbonate and sodium hydroxide are fully soluble and usually do not lead to precipitations, but on the other hand they contribute to higher costs. Additionally, the availability of sodium bicarbonate or sodium hydroxide is sometimes lower than lime. For immediate action, the addition of sodium salts is recommended. For back-up pH adjustment of low pH substrates, lime might be the choice.

2.6.5 Carbon to Nitrogen Ratio

The relationship between the amount of carbon and nitrogen in organic materials is represented by the C/N ratio. The C/N ratio is an important parameter in estimating nutrient deﬁciency and ammonia inhibition [40]. Optimal C/N ratios in anaerobic digesters are between 16 and 25 [194]. A high C/N ratio is an indication of rapid consumption of nitrogen by methanogens, which then results in lower gas production. On the other hand, a low C/N ratio causes ammonia accumulation and pH values then may exceed 8.5. Such conditions can be toxic to methanogenic bacteria [196]. Although methanogenic bacteria can adapt to very high ammonia concentrations, this only happens if concentrations are increased gradually allowing time for adaptation. Optimum C/N ratios can be ensured by mixing different feedstock materials, with high (e.g., organic solid waste) and low (e.g., sewage or animal manure) C/N ratios to achieve an ideal C/N ratio level.

2.6.6 Nutrients Concentration

The major nutrients required by the bacteria in the digester are N2,P,S,C,H2, and O2 to accelerate the anaerobic digestion rate. Thus, it is necessary that the major nutrients are supplied in correct chemical form and concentrations. The carbon in 2.6 Factors Affecting the Biogas Generation 95 carbohydrates supplies the energy, and the nitrogen in proteins is favorable for methane formation. The proteinaceous materials produce little quantity of gas.

2.6.7 Reaction Period

Under optimum condition, 80–90 % of total gas production is obtained within a period of 3–4 weeks. The size of the fermentation tank also decides the reaction period. It is found that the biogas production per unit volume of digester is high when its diameter to depth ratio ranges between 0.66 and 1.

2.6.8 Stirring or Agitation of the Content of Digester

Some method of stirring the slurry in a digester is always advantageous, not essential. If not stirred, the slurry will tend to settle out and form a hard scum on the surface, which will prevent release of biogas. This problem is much greater with vegetable waste than with manure, which will tend to remain in suspension and have better contact with the bacteria as a result. Continuous feeding causes fewer problems in this direction, since the new charge will break up the surface and provide a rudimentary stirring action. Since bacteria in digester have very limited reach to their food, it is necessary that slurry is properly mixed and bacteria get their food supply. It is found that occasional mixing allows the masses that ﬂoat at the top in the form of scum mixing with the deposits at the bottom. It helps in improving fermentation.

2.6.9 Inoculation and Start-Up

When starting the digester for the first time, the digester needs to be inoculated with bacteria necessary for the anaerobic process. Diluted cow dung (optimally 1:1 ratio with water) is an ideal inoculate. Typically, the minimum cow dung required for good inoculation amounts to 10 % of the total active reactor volume. In general, the more cow dung used for inoculation, the better. During the start-up phase, the bacteria population needs to be gradually acclimatized to the feedstock. This can be achieved by progressively increasing the daily feeding load which allows time to achieve a balanced microorganism population. Initial overloading presents a risk to the overall anaerobic process. Overloading results from either feeding too much biodegradable organic matter compared to the active population capable of digesting it or rapidly changing digesters conditions (e.g., abrupt change of temperature, accumulation of toxic substances, flow rate increase). Such disturbances specifically affect methanogenic bacteria, whereas the acidogenic bacteria, which 96 2 Biomethanization are more tolerant, continue to work and produce acids. This eventually leads to an acidification of the digester which inhibits the activity of methanogens. Such an imbalance of acidogenic versus methanogenic bacteria can result in digester failure. Addition of manure can avoid this as it increases the buffer capacity, thereby reducing the risk of acidification. The gas that is produced in the first weeks after start-up is mainly carbon dioxide (CO2). This gas is not flammable and can be released. After a few days, the methane content of the gas will have sufficiently increased to a level that can sustain a flame (CH4 > 45 Vol.%) and lead to high-quality biogas (55–70 Vol.%).

2.6.10 Inhibition

When planning and operating a biogas plant, aspects which inhibit the anaerobic process need to be considered. Some compounds at high concentrations can be toxic to the anaerobic process. Generally speaking, inhibition depends on the concentration of the inhibitors, the composition of the substrate, and the adaptation of the bacteria to the inhibitor. Deublein and Steinhauser [194] list the following typical inhibitors: oxygen, hydrogen sulﬁde (H2S), organic acids, free ammonia, heavy metals, tannins/saponins/mimosine, and other hazardous substances such as disinfectants (from hospitals or industry), herbicides, insecticides (from agriculture, market, gardens, households), and antibiotics. Ammonia nitrogen is often referred to as one of the common inhibiting substances of AD. Ammonia inhibition can take place at a broad range of concentrations. Different studies report ammonia inhibition between 1400 and 17,000 mg N/L of total inorganic nitrogen [144]. In anaerobic reactors, the total inorganic nitrogen consists mainly of ammonia (NH3) and the protonized form of ammonium (NH4+). At normal pH ranges, the biggest share of the total inorganic nitrogen is in the form of ammonium. With increasing pH value and temperature, the share of ammonia increases. The undissociated ammonia form diffuses through the cell membranes and inhibits cell functioning by disrupting the proton and potassium balance inside the cell [197]. This inhibition will cause an imbalance and accumulation of intermediate digestion products such as volatile fatty acids (VFA) which can result in acidiﬁcation of the digester. Generally, it is agreed that with long enough adaptation periods, anaerobic microorganisms can tolerate higher ammonium concentrations than those typically measured. However, this may result in a reduction in methane production. 2.7 Pretreatment for Enhanced Biogas Production 97

2.7 Pretreatment for Enhanced Biogas Production

2.7.1 Mechanical Pretreatment

In mechanical pretreatment process, the desired biomass is subjected to milling process to bring smaller pieces in order to increase and expose maximum area of biomass for enzymatic and other biochemical interaction. The milling process can be carried out on wet- or dried-based milling type. Generally, extrude and hammer mill are used for dry materials, and colloid mill and ﬁbrillator are used for wet wastes biomass like paper pulp, wet paper, etc.

2.7.2 Thermal Pretreatment

High Temperature Treatment In this process, the target waste biomass is exposed to elevated temperature at about 150–180 C for a short time. By doing so, the lignin component breaks down to hemicelluloses with the formation of acids. The acidic environment inside the digester acts as catalysts in the further break down and hydrolysis of hemicelluloses and accelerates the solubilization of hemicelluloses’s oligomers [198, 199]. The only risk in this pyrocellulose cess of treatment is the formation of inhibitory products such as such as phenolic and heterocyclic compounds, furfural, and HMF [200, 201]. These products inhibit the growth and development of the consortium of microbes involved in anaerobic digestion. So, it is better to avoid pretreatment at temperatures of 250 C and above [202, 203]. Steam Pretreatment Steam treatment process is performed for a short period of time, in minutes, under a pressure about 33.4 bars, and cool down to ambient temperature, instantaneously. The main purpose of this treatment is to get 80–100 % of the hemicellulose fraction solubilized making the cellulose fraction accessible to enzymatic hydrolysis [204]. The critical factors governing this process are retention time (RT) and temperature [203]. Mainly, RT depends on the moisture contents. The pressure difference results in the evaporator of the moisture content in the biomass and causes the explosion [205]. The treatment conditions used are often described by using the severity factor (log R0) [206]: log R0 ¼ log t: eðÞTÀ100 =14:75 where t ¼ resident time (min) T ¼ reaction temperature in degrees Celsius 98 2 Biomethanization

In order to optimize treatment conditions for lignocellulosic materials, it is important to be able to relate the net product yield to the pretreatment severity [207].

2.7.3 Chemical Pretreatment

Acid Hydrolysis This process is carried out either by dilute acid or strong acid treatment. In practice, dilute acid pretreatment is more affective compared to strong acid treatment [208, 209]. Dilute acid hydrolysis process followed for a longer period at elevated temperature (T > 160 C) with continuous ﬂow with low solid loading and short retention times (e.g., 5 min) or low temperatures (T 160 C) and batch process with high solid loading at longer retention times (e.g., 30–90 min) [210, 211]. A variety of acids (i.e., dilute sulfuric acid, dilute nitric acid, dilute hydrochloric acid, dilute phosphoric acid, and peracetic acid) are used for such process, depending on the type of feedstock (softwood, hardwood, herbaceous crops, agricultural residues, wastepaper, and municipal solid waste) to be used. The acid hydrolysis process is noticed to be more effective when the biomass is rich in hemicellulose content, as the removal of the hemicellulose fraction increases the porosity of the material enhancing the digestibility [212, 213]. Strong acid hydrolysis process is performed under low temperature by using SO4 and HCl (30–72 %) [214]. The strong acids are powerful agent for cellulose hydrolysis and give high sugar yields (almost 100 % of the theoretical hexose yield). The main drawback of using strong acid is its corrosive nature, and at the end of process, the residual acid is to be recovered and steam produced must be neutralized. Due to its high cost, this pretreatment method is not economically feasible for commercial use.

2.7.4 Alkali Pretreatment

The rigid structure of lignocellulosic biomass is resistance to hydrolysis. Alkali treatment (lime or sodium hydroxide (NaOH)) causes swelling of lignocelluloses [215] and partial lignin solubilization and enhances biogas production [144]. In general, this pretreatment technology is economically unattractive due to the high costs of alkalis [144], but it may be useful for acidic and lignin-rich substrates that could otherwise not be anaerobically digested. 2.7 Pretreatment for Enhanced Biogas Production 99

2.7.5 Oxidative Pretreatment

This process is similar to alkaline pretreatment. Hydrogen peroxide, ozone, and ammonium are being used, individually, for such treatment [216]. This pretreatment is also not carried out at large scale, probably due to high costs [217]. But it may be useful for acidic and lignin-rich substrates that could otherwise not be anaerobically digested.

2.7.6 Ionic Liquid Pretreatment

In this process, the cellulose component of biomass is disintegrated and dissolute and increases the efficiency of enhanced biogas production [218]. Almost 100 % dissolution of cellulose occurs, under mild conditions of the used ionic liquids, During this process, the hydrogen and oxygen atoms of cellulose hydroxyl groups interact with ionic liquids, leading to the formation of electron donor– electron acceptor (EDA) complex [219]. The interaction between -OH and ionic liquids finally results in the dissolution of cellulose. The solubilized cellulose component is then recovered by precipitation, using anti-solvents such as ethanol, methanol, acetone, or water. This pretreatment process decreases the crystallinity of cellulose, remarkably [220]. The main advantages of ionic liquids are such as low toxicity, thermal stability, broad selection of anion and cation combinations, low viscosity, high reaction rate, and nonflammable properties [221]. The most commonly used liquid agents include N-methylmorpholine-N-oxide monohydrate (NMMO), 1-allyl-3-methylimidazolium chloride (AMIMCI), and l-N-bytyl-3- methyl (tetradecyl) ammonium chloride (BDTACI) [222–224].

2.7.7 Biological Pretreatment

Generally, mechanical, chemical, and physical pretreatment processes are tedious and required high-energy input. Besides this, expensive equipment are required to perform these processes. In contrast, biological pretreatment process to enhance the biodegradability of organic matters and consequence methane production offers advantages such as low capital cost and low-energy demand. Lignin component of biomass is a major constrain in biomass degradation and subsequently methane production. In this context, microbes, especially brown, white, and soft-rot fungi, are most suitable for degradation of hemicelluloses and lignin, but due to its high resistance, only a small amount of cellulose will be degraded. Ceriporiopsis subvermispora is the best biological agent to degrade lignin without much effect to cellulose component. Other examples are 100 2 Biomethanization

Phanerochaete chrysosporium, Trametes versicolor, Trametes hirsuta, and Bjerkandera adusta.

2.8 Type of Biogas Digester

Household Digesters Architecture of a biogas digester range across a wide spectrum from simple to extremely complex. Long back, Struckey [225] in his excellent review classified the biogas digester on the basis of its application like domestic, commercial, intensive animal feed lots and industrial. The use of biogas in the first two areas of application is common in developing countries. The latter two areas have rarely been exploited, and there are very limited units presently being under operation. Irrespective of history of application of anaerobic techniques, the Chinese people have taken the first credit of having world’s largest biogas program, and the same is followed by India. Probably, the history of using biogas in China is the longest one, although the first biogas plants were designed in 1939 in India by Khadi and Village Industry Commission (KVIC). As can be seen from (Fig. 2.23), a typical reactor consists of fermentation tank, gas storage partition, balancing chimney, mixing pit, and mechanical mixing device. There is an additional tube in the tank to locate the thermometer and to gather the waste samples. Inlet and outlet tubes have lids. On requirement basis, the volume of digester may vary. In India, the top of the digester (mostly used for community purpose) is dome shaped and made of concrete materials. The bottom is arch type with vertical side walls either made with bricks or concrete. During the design of the digester, care is taken to accom- modate maximum quantity of scum, generated during anaerobic fermentation. The inner and outer walls of the digester are insulated with locally available materials.

Fig. 2.23 Schematic diagram of ﬁxed dome-type biogas reactor 2.8 Type of Biogas Digester 101

The space left between the top fixed dome-shaped cover and ground surface is packed with locally available cheap insulator. However, the insulation provision is not compulsory during construction of any digester. When the digester volume is kept 35 m, the gas storage should be of 18 m in volume. If the biogas is not used over long period, organic wastes can overflow into the balancing chimney. To avoid such problem, a metal gas holder is connected to the tank which is installed close to the tank. One end of the inlet tube is opened at the bottom part of the digester and the other end is connected to a mixing pit (inlet chamber). The mixing pit is made with concrete or brick. The shape of the pit is either rectangular type or prism type. The inlet tube is either concrete or PVC type or steel tube. The storage capacity of mixing pit for a 35 m digester is about 25 m. On the basis of requirement and geographical location, the design and construction of biogas plant is made. As an example, a digester used in mountainous regions is designed to have less gas volume in order to avoid gas loss. The people of tropical countries prefer to have biogas plants underground due to the geothermal energy [226]. The most common biogas plant seen in China is fixed dome type, whereas the floating drum type is most common, even today [227]. Meanwhile, plug flow digesters has drawn attention due to its portability and easy operation.

2.8.1 Fixed Dome Digesters

The fixed dome type is also known as “Chinese” or “hydraulic” digester and most commonly used in China (Fig. 2.23). Organic wastes (agricultural wastes, cattle wastes, vegetable wastes, night soil) are generally loaded every 6 months after the digester has been partially emptied to provide fertilizer for crop planting. The gas produced is usually stored in the fixed dome, and this displaces the liquid contents into the inlet and outlet chamber, often leading to gas pressure as high as in of water. This high gas pressure sometimes creates problem like gas leaks. Besides this, the gas produced is stored in RMP gas bags (1–1.5 m3). While the bag is expensive, this method reduces the gas leaks. Fixed dome digesters are usually built underground. The size of the digester depends on the location, number of households, and the amount of substrate available every day. For instance, the size of these digesters can typically vary between 4 and 20 m3 in Nepal [228], between 6 and 10 m3 in China [229], and between 1 and 150 m3 in India [230], and in Nigeria it is around 6 m3 for a family of nine [231]. Instead of having a digester for each individual home, a large volume digester is used to produce biogas for 10–20 homes and is called community-type biogas digesters. In countries where houses are clustered as in Nigeria, these types of biogas digesters are more feasible [232]. There are mainly three types of fixed dome digester common in developing countries. Figure 2.24 depicts schematic sketch of (a) a janta model fixed dome digester and its modifications, (b) a deenbandhu model fixed dome digester and its 102 2 Biomethanization

Fig. 2.24 Schematic presentation of plug flow biogas digester modifications, and (c) a modified fixed dome digester with straight and curved inlet and outlet tube. Fixed dome models developed in India include the janta and deenbandhu models. The janta model was introduced in 1978. It consists of a shallow well with a dome roof on top. The inlet and outlet were kept above the dome with the gas pipe fitted on top of the dome. The disadvantages of the janta model includes short circulating path of the slurry, escape of undigested slurry at the top, and less volume of gas produced due to the increased gas pressure [233]. Action for Food Production (AFPRO) launched a modified janta model called the deenbandhu model in 1984. It consists of two spheres of different diameters. The lower sphere acts as a fermentation unit, while the upper one is the storage unit. This model was developed to reduce the price without decreasing the efficiency of the process [234].

2.8.2 Floating Drum Digester

This type of biogas digester is very common in India. This biogas digester consists of a cylindrical reactor with an H/D ratio between 2.5 and 4.1, and the gas production is trapped under a ﬂoating cover with a volume of approximately 50 % of total gas production.

2.8.3 Plug Flow Digesters

A plug flow digester vessel is a long narrow (typically a 5:1 ratio; five times as long as the width) insulated and heated tank made of reinforced concrete, steel, or fiberglass with a gas-tight cover to capture the biogas (Fig. 2.24). These digesters can operate at a mesophilic or thermophilic temperature. The plug flow digester has no internal agitation and is loaded with thick manure of 11–14 % total solids. This type of digester works well with a scrape manure management system with little bedding and no sand. Retention time is usually 15–20 days. In theory, manure in a plug flow digester does not mix longitudinally on its trip through the digester but can be imagined to flow as a plug, advancing toward the outlet whenever new manure is added. When the manure reaches the outlet, it 2.8 Type of Biogas Digester 103 discharges over an outlet weir arranged to maintain a gas-tight atmosphere but still allow the effluent to flow out. In actuality, the manure does not remain as a plug, and portions of the manure flow through the digester faster than others, and some settles or floats and remains in the digester. Biogas produced by the digester is used to heat the digester to the desired temperature. Excess biogas can be used to run an engine generator. Heat can also be recovered from the engine generator and used for space or floor heating, water heating, or steam production to offset the cost of purchased electricity, propane, natural gas, or fuel oil used on the farm for daily operations. The gas production rate is noticed to be much higher as compared to fixed dome or floating-type digester. This type of reactor is very uncommon in developing countries although Mexico has developed a similar type of digester known as “Xochicalli” [235].

2.8.4 Biogas Digester

It is essentially a long cylinder (L/D ¼ 3–14) made of PVC or nylon (neoprene coated) or RMP. The bag sits in a trench about half the diameter of the bag and hence is exposed to changes in ambient temperature. The gas is stored either in gas bag or in a reactor under the ﬂexible membrane. Due to their plug ﬂow characteristics and the fact that they can easily trap solar radiation, their volumetric gas production rates can be quite high. It has been noticed that [80] the average temperature inside the digester is generally 2–7 C higher than dome-type digester. Due to this reason, the volumetric gas routes can be from 50 to 300 % higher in the bag (0.24–0.61 V/L day) [236].

2.8.5 The Batch Reactor

Batch plants are filled and then emptied completely after a fixed retention time. Each design and each fermentation material is suitable for batch filling, but batch plants require high labor input. This type of reactor is simple in design. The biogas production varies considerably with the digestion time. For continuous supply of gas, a number of these units have to be operated simultaneously. A wide range of organic solids (0–10 or even 20 %) can be loaded in the type of digester. A digester with high concentration of solid load is known as “dry fermentation.” It has been reported that the dry fermentation can proceed at Ts concentration up to 32 %. This type of biogas reactor is suitable in the desert area where the problem of scarcity of water is noticed very often. This technique is very low capital investment oriented and also very simple in design and operation. As a major disadvantage, their gas output is not steady. 104 2 Biomethanization

2.8.6 Continuous Biogas Digester

Continuous plants are fed and emptied continuously. They empty automatically through the overflow whenever new material is filled in. Therefore, the substrate must be fluid and homogeneous. Continuous plants are suitable for rural households as the necessary work fits well into the daily routine. Gas production is constant and higher than in batch plants. Today, nearly all biogas plants are operating on a continuous mode.

2.8.7 Anaerobic Bafﬂe Reactor

An anaerobic baffled reactor (ABR) is an improved septic tank with a series of baffles under which the wastewater is forced to flow [237]. It is a simple rectangular tank divided into five to six chambers by means of wall from the roof and bottom of the tank. The liquid flow direction is maintained upward and downward, alternatively. During upward movement, the waste passes through an anaerobic sludge blanket. As a result, the waste is in sufficient contact with active biowastes, but due to the baffled nature of the design, most of the biowaste is retained within the reactor even under quite large hydraulic shocks. The upflow chambers provide enhanced removal and digestion of organic matter. BOD may be reduced by up to 90 %, which is far superior to its removal in a conventional septic tank. Due to its physical configuration, this type of reactor appears to be able to treat wastes with quite high solid contents (sewage), and hence it may be a better option under certain circumstances in developing countries [238, 239].

2.8.8 Anaerobic Filter-Type Reactor

An anaerobic filter is a fixed-bed biological reactor with one or more filtration chambers in series. As wastewater flows through the filter, particles are trapped and organic matter degraded by the active biomass that is attached to the surface of the filter material. Anaerobic filters are widely used as secondary treatment in household black or gray water systems and improve the solid removal compared to septic tank or anaerobic baffled reactors. Since anaerobic filters work by anaerobic digestion, they can be designed as anaerobic digesters to recover the produced biogas. As septic tanks or anaerobic baffled reactors, anaerobic filters are based on the combination of a physical treatment (settling) and a biological treatment. It com- prises a watertight tank containing several layers of submerged media, which provide surface area for bacteria to settle. As the wastewater flows through the 2.9 Biogas Utilization and Problems 105

ﬁlter usually from bottom to top (up-low), it comes into contact with the biomass on the ﬁlter and is subjected to anaerobic degradation [240].

2.9 Biogas Utilization and Problems

2.9.1 Biogas for Cooking and Lighting

In rural area, biogas is used for cooking, heating, or lighting by mantle-type lamp or for generating electricity. Practice of cooking with biogas particularly in village area is the best method of conserving energy and exploitation of rural resources based on agricultural, cattle, and vegetable wastes. The designing of biogas burners are slightly different from the regular type of LPG burners. In India, at present these burners are available in a wide range of capacity varying from 227 L (8 cft) to 2840 L (100 cft) biogas consumption per hour. Due to the presence of H2S in the raw biogas, mild smell of hydrogen sulﬁde comes out while burning biogas.

2.9.2 Biogas-Based Diesel Engine

In India, many voluntary organizations are presently giving consultancy for generation of Motive Power using the biogas in dual-fuel internal combustion (IC) engine. Such engines are basically diesel-fuelled compressing ignition engines in which the charge is ignited spontaneously because of rise in temperature due to compression. Air mixed with biogas is aspirated into the engine, and the mixture is then compressed, elevating its temperature to about 350 C. This temperature, which is the self-ignition temperature of diesel oil, is much below that of methane, which is over 600 C. Therefore, in order to initiate combustion of the charge, a small quantity of diesel is injected into the cylinder just before the end of compression. The charge is thus ignited and the process is continued smoothly. Diesel consumption of the engine drops to about 20 % of its rate of consumption when sufﬁcient biogas is available. These engines consume approximately 426 L (15 cft) of biogas per BHP per hour. Gas turbines can also be used, but small turbines are difﬁcult to run economically and are, in general, suitable for use with public toilet- based biogas plants. Dual-fuel engines can be coupled to air compressors or used to drive generators. 106 2 Biomethanization

2.9.3 Electricity Generation from Biogas

The average caloriﬁc value of biogas is about 21–23.5 MJ/m3, so that 1 m3 of biogas corresponds to 0.5–0.6 L diesel fuel or about 6 kWh (FNR 2009). In Germany, cow manure and energy crops are the main forms of feedstock. About two livestock units (corresponding to about 2 cows or 12 rearing pigs) plus 1 ha of maize and grass are expected to yield a constant output of about 2 kWhel (48 kWhel per day). An IC engine coupled to an alternator is called a dual-fuel genset. A public convenience visited by about 2000 persons per day on an average would produce approximately 60 m3 of biogas which can run a 10 kV genset for eight hours a day, producing about 65 units of power. With this electricity street lighting can be provided for 3 km using tube light or for 2 km using high-pressure mercury vapor lamps. So, conversion into electrical energy is advisable when 60 m3 or more of biogas is produced per day. In the USA, about 80 % projects are currently involved in electricity production from biogas plants linked to dairy farms. A large number of biogas energy projects are noticed in New York, Pennsylvania, Vermont, and Wisconsin, out of about 8000 biogas energy project being under operation condition. Since 2011, the University of Wisconsin at Oshkosh has been operating a $3.5 million digester that uses a combination of agricultural waste, yard waste, and supermarket waste. When the biodigester goes into full production in April, it should process some 8000 tonnes of organic which annually provide as much as 10 % of the University’s electric need.

2.9.4 Biogas for Automobile

Biogas is an excellent fuel for the spark ignition engine having high antiknocking performance, without any combustion complicacy. It is also relatively easy to convert existing petrol-driven engine to run on methane. Therefore, the feasibility of utilizing digester gas to power automobiles arises. Utilization of this gas after liquefaction is not advisable due to its uneconomic conversion technology, presently available. The gas can be compressed and used, but scrubbing is needed before compression (to 150 bars). At ordinary pressure, it is least convenient for utilization as the volume of gas required to travel even short distances becomes substantial, 0.75 m ¼ 1 L of petrol. The CO2 and H2S contents are usually removed by washing, and gas in this form can be used to power motor gases, car, and small agricultural vehicles. 2.10 Biogas Utilization Problems 107

2.9.5 Optimization of Process for Biogas Production

There have been many attempts to rationally optimize various process factors affecting biogas production, since long. In this regard, about two decades back, Valinin in 1994 [241] suggested a universal model for optimum biogas production taking into account all important factors of the anaerobic process. At industrial level, even a small improvement in the process gives a better yield which may be beneficial commercially, making process optimization a major area of research in the field of industrial biotechnology [242]. Therefore, there is a need for optimization of accurate process parameters, which improves the production of the biogas significantly [242, 243].

2.10 Biogas Utilization Problems

2.10.1 Choice of Digester

Various types of wastes with high BOD load are being used for biogas generation. On the basis of volume of digester and quality of organic load, retention time, during operation of plant, is of the order of the day. Performance of these reactors on sewage sludge is relatively well documented, while information on other waste is much more sparse. Meanwhile, China, India, and Korea have developed high quality of digester to be suitable for both concentrated and dilute wastes. Besides, hybrid types of plants for liquefaction and gas production are also under intensive trials.

2.10.2 Supply of Feed

There is likelihood of breakdown and blockage of digester particularly because of the nature of the waste being digested. Vegetable and animal wastes contain lignocellulosic ﬁbers, undigested cereal grains, animal hair, etc., which can easily close pipes and pumps. Although this sort of problem is well experienced earlier and ought to be considered at the designing stage, in practice it can still cause problems if stringent management of the digester is not maintained. It has been noticed that a small-type digester (15–150 L) with agriculture wastes used to be stable in spite of wide variation in loading and short period of no loading, in one case up to about 6 weeks. A piggery waste-based digester having the capacity of 13 m, when subjected to intermittent running, showed stable biogas production for long period [244, 245]. A potato waste-based digester is said to be capable of “campaign operation” in that once the pretreated sludge is to obtain, the reactor could be shut down for a period of 8 months. During this period, no loss in 108 2 Biomethanization

Table 2.17 A comparison of energy contents between organic wastes and medium fuel oil Organic waste Calorific value (TJ) Medium fuel oil equivalent (m) Municipal refuse 1.0 Â 10 2.45 Â 10 Cattle manure 3.0 Â 10 7.35 Â 10 Poultry manure 1.8 Â 10 4.41 Â 10 Pig slurry 1.2 Â 10 2.94 Â 10 Sewage sludge 1.0 Â 10 3.43 Â 10 Total 8.4 Â 10 2.085 Â 10 biological activity is experienced, and start-up is said to be fast when the system is next required. Wide variability in both the quality and quantity of the feedstock can also enable the microbial flora to produce a significant reduction in the operational performance in relation to a steady state of operation. This situation could well occur in the newer applications of anaerobic digestion such as food industry wastes which are both seasonal and highly variable. Fortunately, many NGOs in India have already developed techniques to commercialize the vegetable waste-based biogas digester for generation of electricity.

2.10.3 Waste Collection Transport and Storage

Plenty of data is available on waste recycling and biogas generation. However, such important factors as collection, transport, and proper storage of the waste are often ignored. Waste like sewage sludge and animal wastes are having around 90 % water. Transportation of the semiliquid wastes under perfect hygienic conditions is uneconomical due to the low-energy per unit volume of the waste and the operational cost of tankers. Plenty of literature is available on energy content of different types of organic wastes in relation to fuel oil (Table 2.17). Due to such practice, it has been difﬁcult to assess the viability of anaerobic digestion at large- scale level.

2.10.4 Nature of Biogas

Due to uneven composition of organic wastes, the caloriﬁc values of biogas vary, accordingly. In the earlier section of this chapter, the physicochemical characteristic of biogas is given in detail. In addition, scrubbing of biogas to upgrade its purity is also discussed. 2.10 Biogas Utilization Problems 109

2.10.5 Gas Flammability

Generally, the problem of flammability is ignored during operation of a plant. Influx of air into the digester causes explosion risks and also lowers the calorific value of the gas. The explosive range is typically 6.12 % v/v with air. Therefore, the leakage of the gas into air is more dangerous than leakage of air into gas. The lower limit of explosion is stated to be around 6 % v/v with air, whereas gas concentration as low as 1.2 % v/v should be considered dangerous. During desludging of the digester or any ranging, maximum care should be taken to replace the digester or any repairing, and maximum care should be taken to replace the residual gas in the holder by air. International standard fitting should be regularized while designing the digester. Apart from this joining of gas inlet and outlet pipe, condensate trap systems should be of international standard. So it will be easy while transferring the technology from one state to other. If the use of biogas becomes widespread, safety regulations will have to be laid down as suggested by British Anaerobic and Biomass Association Ltd.

2.10.6 Gas Compressibility

Methane has low-energy content (37.3 MJ/m3) as compared to butane (110.00 MJ/m3) or propane (87.8 MJ/m3). So, a large volume of gas is needed to produce the same amount of heat or motive power. The only option left over is compression and for the same purpose, the gas should be made free of volatile organic, CO2, and H2S. It has been reported [246] that bottles for motor vehicles, typically of capacity 0.05 m3 and 63 kg weight, would carry only 12 m3 methane (1 atm 20 C) at 200 atm pressure equivalent to 3.5 gallons of petrol.

2.10.7 Biogas Storage

Biogas that has been upgraded to biomethane by removing H2S, moisture, and CO2 can be used as a vehicular fuel. Since production of such fuel typically exceeds immediate on-site demand, the biomethane must be stored for future use, usually either as compressed biomethane (CBM) or liqueﬁed biomethane (LBM). Because most farms will produce more biomethane than they can use on-site, the excess biomethane must be transported to a location where it can be used or further distributed. Biogas, like natural gas can be stored both at site of production and use for automobile and other purpose. Table 2.18 summarizes the biogas-storing process and systems. 110 2 Biomethanization

Table 2.18 On-farm storage options for biogas and biomethane Pressure Storage Purpose of storage (psi) device Material used Size Short and intermediate storage <0.1 Floating Reinforced and Variable vol- on farm house (currently used cover non-reinforced ume usually on farm for biogas storage) plastic rubber for less than one day <2 Gas bag 150–11,000 Water- Steel 35,000 sealed gasholder Weighted Reinforced and 800–28,000 gas bag non-reinforced Floating roofplastic rubber Variable volume usually for less than one day Possible means of storage 10–29,000 Propane or Steel Variable vol- For late or off-farm use butanol ume usually (could be used for for less than biomethane) one day 72,900 Commercial Alloy steel 350 gas cylinder Source: Ross et al. [247]. 3 psi ¼ Pounds per square inch, ambient conditions ft ¼ Cubic feet

(1) Low-Pressure Storage of Biogas: Floating gas holders on the digester form a low-pressure storage option for biogas systems. Flexible membrane materials commonly used for these gas holders include high-density polyethylene (HDPE), low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), and chlorosulfonated polyethylene-covered polyester. Thicknesses for cover materials typically vary from 18 to 100 mils (0.5–2.5 mm). In addition, gas bags of varying sizes are available and can be added to the system. These systems typically operate at pressures up to 10 in. water column (less than 2 psi). Floating gas holders can be made of steel, fiberglass, or a flexible fabric. A separate tank may be used with a floating gas holder for the storage of the digestate and also storage of the raw biogas. Flexible membrane materials commonly used for these gas holders include high-density polyethylene (HDPE), low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), and chlorosulfonated polyethylene- covered polyester (such as Hypalon®, a registered product of DuPont Dow Elastomers L.L.C.). Thicknesses for cover materials typically vary from 18 to 100 mils (0.5–2.5 mm). In addition, gas bags of varying sizes are available and can be added to the system. These bags are manufactured from the same materials mentioned above and may be protected from puncture damage by installing them as liners for steel or concrete tanks. (2) Medium-Pressure Storage of Cleaned Biogas: In medium-pressure storing system, a pressure about 2–200 psi is maintained. Tank vessel made of propane 2.10 Biogas Utilization Problems 111

with 250 psi pressure-bearing capacity is, generally, used for gas storage. Typical propane gas tanks are rated to 250 psi. Compressing biogas to this pressure range uses about 5 kWh per 1000 ft. Assuming the biogas is 60 % methane and a heat rate of 13,600 Btu/kWh, the energy needed for compression is approximately 10 % of the energy content of the stored biogas. (3) High-Pressure Storage of Compressed Biomethane: After scrubbing. Biomethane can be stored under high pressure between 2000 and 5000 psi. For this purpose commercially available certified steel cylinder can be used, as practiced in natural compressed gas storing. Compression to 2000 psi requires nearly 14 kWh per 1000 ft of biomethane. If the biogas is upgraded to 97 % methane and the assumed heat rate is 12,000 Btu/kWh, the energy needed for compression amounts to 17 % of the energy content of the gas. Due to low-energy density at atmospheric pressure, compressed biomethane (CBM) can be easily transported on road as in case of CNG, at high pressure ranging from 3000 to 3600 psi. The compressor receives the low-pressure biomethane from the storage tank and compresses it to 3600 to 5000 psi. The compressor should be specified to handle the output flow rate from the biogas- upgrading equipment. For example, a dairy with 1000 cows would yield a flow rate of approximately 2000 ft3 raw biogas/hour. There are several manufacturers of commercially available compressors in this range (e.g., Bauer Com- pressors and GreenField Compression). (4) Storage of Liquefied Biomethane: As compared to compressed biomethane (CBM), the liquefied biomethane (LBM) is a better option for transfer as in the case of CNG. Generally, CNG tanker trucks which normally have a 10,000- gallon capacity are, also, used for LBM transport. One of the main disadvantages of LNG and thus LBM is that the cryogenic liquid will heat up during storage, which will result in loss of LBM to evaporation through a release valve on the tank. To minimize these losses, LBM should be used fairly quickly after production. It is generally recommended that LBM be stored for no more than a week before it is either used or transported to a fueling station. Storage for a longer period will result in an economically unacceptable level of evaporative loss. Since standard LNG tankers carry about 10,000 gallons, a small-scale liquefaction facility should produce at least 3000 gallons of LBM per day. However, the production of this much LBM requires approximately 8000 cows—which could only be found at an extremely large dairy or a central digester facility.

2.10.8 Social Problem

One of the most important factors that have limited the diffusion of the biogas technology is poverty. The needs of rural people are often poorly understood. So, introduction of biogas technology is likely to be initiated. Besides this, one more striking feature in the information on why some innovations are successful and 112 2 Biomethanization others fail? Rural societies are being increasingly recognized as very complex systems, and the failure of much technical intervention appears to arise from a misunderstanding by foreigners of what rural people see as their problem. There are many social factors which tend to inhibit the further popularization of biogas use. In some societies there exist social restriction about the handling of excreta particularly night soil. As state level it is essential that strong government interest and backing is necessary if biogas is to be popularized quickly. This should be incorporated while taking national policy decision during planning of future of a state. Unfortu- nately, with the exception of China, this infrastructure is often weak and leads to uneven performance.

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3.1 Biodiesel from Microalgae

Ensuring secure, affordable supplies of energy and tackling climate change are central, interlinked global challenges facing the international community. Social crisis and political instability in oil-producing countries have led to frequent price spikes, both in developed and developing countries. Surprisingly, it has been noticed that the transportation sector consumes about 30 % of the world’s energy utilization, most of which is in the form of liquid fuels [1]. Major part of the global energy consumption is derived from petroleum-based liquid fuels. Hence, more attention should be given on the renewable energy sources of liquid fuels derived from biomass [2, 3]. Biofuels can play a critical role in solving energy crisis and environmental pollution [4, 5]. The most common biofuels are ethanol produced from sugar and starch crops and biodiesel produced from vegetable oils and animal fats [6, 7]. As arable lands are used for biodiesel production, it may lead to food scarcity. It also may cause deforestation because of the excessive demand of land for biodiesel production. The water demand for some biodiesel crops could put unsustainable pressure on local water resources. In addition, petro crop cultivation and management are not only labor intensive but also expensive. On the basis of bioresources availability and geographical conditions, three generations of biodiesel feedstock are available. The first generation of biodiesel is derived from soybeans, coconut, sunflower, rapeseed, palm oil, etc. The second- generation biodiesel feedstock includes Jatropha,mahua,cassava,Miscanthus, jojoba oil, salmon oil, tobacco seed, straw, etc. Microalgae are categorized as third- generation biodiesel, widely available in freshwater and saline water ecosystems. For the first generation of biodiesel, feedstock needs huge area of arable agricultural lands, which may, consequently, have impact on global food security and a threat to forest ecosystem. The second-generation biofuels are produced from nonedible components. It is intended to produce from the woody part of nonedible plants that do not compete with food production. However, converting the woody

© Springer International Publishing Switzerland 2016 123 B. Kumara Behera, A. Varma, Microbial Resources for Sustainable Energy, DOI 10.1007/978-3-319-33778-4_3 124 3 From Algae to Liquid Fuels biomass into fermentable sugars requires sophisticated and expensive equipment for commercialization. Therefore, second-generation biofuels cannot be produced economically in large scale [8]. They also need arable lands which may cause food scarcity and deforestation. The practice of implementing renewable fuel especially algal biodiesel has two major beneﬁts of improving energy security and mitigating the carbon dioxide emission from industrial activity [9]. In this connection, microalgae provide an exceptional opportunity over terrestrial plant-based biofuel, such as high biomass productivity and the ability to grow in cultivation ponds or photobioreactors on nonarable lands using saline water or wastewater sources.

3.1.1 What Is Microalgae?

Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms. They range from microscopic to large seaweeds (Fig. 3.1), such as giant kelp more than hundred feet in length. Microalgae include both cyanobacteria (similar to bacteria and formerly called “blue-green algae”) as well as green, brown, and red algae. Naturally, they can grow rapidly in fresh- or salt water due to their unicellular or simple multicellular structure. Because of their simple cellular structure, they are very efﬁcient converters of solar energy. Most microalgae grow through photosynthesis—by converting sunlight, CO2, and a few nutrients, including nitrogen and phosphorus into a material known as biomass. This is called “autotrophic” growth. Other algae can grow in the dark using sugar or starch (called “heterotrophic” growth) or even combine both modes (called “mixotrophic” growth). Interestingly, the entire growth cycle of algae is complete within few days [9]. As the cells of microalgae grow in aqueous suspension, they have efﬁcient access of water, CO2, and other nutrients [10]. Microalgae are one of the oldest living organisms in our planet. Several species of them have oil content up to 80 % of their dry body weight. Table 3.1 shows lipid contents of different microalgal species [11, 12].

3.1.2 Beneﬁt of Using Microalgae for Biodiesel Production

Microalgae are, only, sustainable resources for biodiesel, as compared to other feedstock used for green energy program. Both the prokaryote and eukaryote microalgae are having sexual and asexual reproduction. Compared to petro crop, microalgae mass culture management is tussle-free due to their variable habit and habitat [13]. Solar energy trapping and its conversion to biochemical energy in algae are more effective, compared to other plants. It is about 3–8 % against 0.5 % for terrestrial plants. Microalgae are seasonal independent in growth and development process and capable of producing oil throughout the year. The oil production capacity of microalgae per unit biomass is signiﬁcantly higher than petro crops [14]. On biomass basis, microalgae possess 20–50 % higher oil content than any petro crops. On cultivated land requirement basis, microalgae produce 15–300 times more oil than regular petro crops on area basis. In addition, the growth rate in algae is quicker 3.1 Biodiesel from Microalgae 125

Fig. 3.1 Morphology of different types of algae (Source: www.slideshare.net)

Table 3.1 Lipid contents of Microalgal species Lipid content (% dry weight) different microalgal species Botryococcus braunii 25–75 Chlorella 18–57 Chlorella emersonii 25–63 Chlorella sp. 10–48 Dunaliella sp. 18–67 Dunaliella tertiolecta 18–71 Nannochloris sp. 20–56 Nannochloropsis sp. 12–53 Neochloris oleoabundans 29–65 Phaeodactylum tricornutum 18–57 Scenedesmus obliquus 11–55 Schizochytrium sp. 50–77 126 3 From Algae to Liquid Fuels than switch grass, which is the fastest growing terrestrial crop [15]. Microalgae are seen with luxury growth habit in freshwater, seawater, wastewater, and nonarable land and do not compete with food crops for land and water management [16–20]. Table 3.2 shows comparison of different sources of biodiesel [21–32]. To produce 1 kg of algae biodiesel, 1.8 kg of CO2 is needed. Besides getting biodiesel, many other value-added by-products like edible protein, antioxidants, pigments, natural dye, high-value bioactive compounds, and other fine biochemicals can be derived from residual algal biomass, after biodiesel extraction [33–35]. The CO2 mitigation rate in microalgae on biomass basis is between 50 and 65 % on cloudy days and about 80–95 % on sunny days. The efficiency in CO2 mitigation varies with algae species and geographical factor and significantly higher than terrestrial plants [36–39]. Some of the microalgae have high potential to absorb CO2 from flue gas, even in the range of 6–8 % CO2 concentration. The industrial flue gases are, generally, rich in carbon dioxide and can be used for growth and development of algae in mass cultivation process [4], thus reducing the GHG emissions of a company or process while producing biodiesel [40]. Spent wash water containing NH, NO3, and PO4 can also be used in mass cultivation of algae. After oil extraction the resulting algae biomass can be processed into ethanol, methane, livestock feed, used as organic fertilizer due to its high N:P ratio, or simply burned for energy cogeneration (electricity and heat) [4]. Biodiesel feedstock plants absorb CO2 through photosynthesis, which is more than it discharges by the combustion process. Therefore, it can maintain ecological balance more effectively compared to petroleum diesel. Table 3.2 shows average emission impacts of biodiesel blends compared to petroleum diesel [41, 42]. Biodiesel gives more clean combustion than petroleum diesel as it possesses high cetane number. The cetane number is a measurement of the combustion quality of diesel fuel during compression ignition. So the flammability of biodiesel is better than that of diesel oil. Biodiesel can be transported safely due to its high flash point. Biodiesel acts as a better lubricant and detergent than petroleum diesel. Table 3.3 shows comparison of different properties of diesel and biodiesel [44, 45]. Many microalgal species can be induced to accumulate substantial quantities of lipids [5], thus contributing to a high oil yield. The average lipid content varies between 1 and 70 %, but under certain conditions some species can reach 90 % of dry weight [46–48].

3.1.3 Algae for Value-Added Products

Algae are emerging to be one of the most sustainable resources for a variety of products, and new ones are constantly being discovered. With the recent energy security problem all over the world, there is growing interest in utilizing the algal biomass in an optimal manner, not just to produce biofuels (biodiesel, ethanol, hydrogen, biogas) but also to manufacture coproducts of the process. Such an optimal utilization could make algae-based biofuels more economically viable. So the topmost of algal oil-producing companies, as mentioned elsewhere in this book, are also involved in the production of value-added biochemicals (Table 3.4) . idee rmMcola 127 Microalgae from Biodiesel 3.1

Table 3.2 Comparison of different properties of diesel and biodiesel Density at 15 C Viscosity at 40 C Sulfur Carbon Hydrogen Oxygen Flash Cetane Lower caloriﬁc value Fuel (g/cm3) (mm2/s) (%) (%) (%) (%) point number (MJ/kg) Diesel 0.834 2.83 0.034 86.2 13.8 – 62 47 42.59 Biodiesel 0.8834 4.47 <0.005 76.1 11.8 12.1 178 56 37.243 128 3 From Algae to Liquid Fuels

Table 3.3 Average emission impacts of biodiesel blends compared to petroleum diesel [43] Biodiesel blends B20 (%) B40 (%) B60 (%) B80 (%) B100 (%) Unburned hydrocarbons À20 À35 À49 À59 À67 Carbon monoxide À12 À22 À32 À40 À48 Particulate matter À12 À22 À32 À40 À47 NOX +2 +4 +6 +8 +10

Table 3.4 Value-added products and applications from microalgae Species Product Application areas Chlorella vulgaris Biomass, pigments Health food, food supplement Chlorella spp. Chlorella ellipsoidea Coccomyxa acidophila Lutein, β-carotene Pharmaceuticals, nutrition Coelastrella striolata Canthaxanthin, astaxanthin, Pharmaceuticals, nutrition, var. multistriata β-carotene cosmetics Crypthecodinium cohnii Docosahexaenoic acid Pharmaceuticals, nutrition Diacronema vlkianum Fatty acids Pharmaceuticals, nutrition Dunaliella salina Carotenoids, β-carotene Health food, food supplement, feed Galdieria sulphuraria Phycocyanin Pharmaceuticals, nutrition Haematococcus Carotenoids, astaxanthin, can- Health food, pharmaceuticals, feed pluvialis thaxanthin, lutein additives Isochrysis galbana Fatty acids, carotenoids, Pharmaceuticals, nutrition, cos- fucoxanthin metics, animal nutrition Lyngbya majuscula Immune modulators Pharmaceuticals, nutrition Muriellopsis sp. Lutein Pharmaceuticals, nutrition Nannochloropsis Eicosapentaenoic acid Pharmaceuticals, nutrition gaditana Nannochloropsis sp. Odontella aurita Fatty acids Pharmaceuticals, cosmetics, baby food Parietochloris incise Arachidonic acid Nutritional supplement Phaeodactylum Lipids, eicosapentaenoic acid, Nutrition, fuel production tricornutum fatty acids Porphyridium cruentum Arachidonic acid, Pharmaceuticals, cosmetics, polysaccharides nutrition Scenedesmus Lutein, β-carotene Pharmaceuticals, nutrition, almeriensis cosmetics Schizochytrium sp. Docosahexaenoic acid Pharmaceuticals, nutrition Spirulina platensis Phycocyanin, γ-linolenic acid, Health food, cosmetics biomass protein Ulkenia spp. Docosahexaenoic acid Pharmaceuticals, nutrition Microalgal species of high-value compounds extraction and applications [49, 50] 3.1 Biodiesel from Microalgae 129

Table 3.5 Various process technology to derive algal fuels Final product Processes Biodiesel Oil extraction and transesterification Ethanol Fermentation Methane Anaerobic digestion of biomass; methanation of syngas produced from biomass Hydrogen Triggering biochemical processes in algae; gasification/pyrolysis of biomass and processing of resulting syngas Heat and Direct combustion of algal biomass; gasification of biomass electricity Other hydrocarbon Gasification/pyrolysis of biomass and processing of resulting syngas fuels rather than remaining strict in the algae-to-fuel domain. Hence, as a starting point, many of these companies are exploring the possibility of getting high-value, nonfuel products from algae. Besides oil the starch constituent of the biomass makes an appropriate feedstock for the production of ethanol. The leftover biomass could then be used for conventional animal, fish or poultry feed, or for other nutraceuticals. There are emerging technologies using which the residue could be used to make products such as bioplastics. This allows them to generate profits fairly early into their venture while at the same time ensuring that they are able to continue with their efforts in sustainable microalgae fuel production (Table 3.5) through different techniques.

3.1.4 Viability of Microalgae for Biodiesel

Microalgae possess great versatility as an energy source. The algal biomass can directly be used for energy through gasification, pyrolysis, and liquefaction process or be best used as sustainable and reliable biomaterial for its high oil content. Although lots of industries are in progressive direction to commercialize algal diesel, still research is going on cost of cultivation conversion process to use algal diesel as a popular substitute of petrol and diesel which are under extinction [51, 52]. One of the key reasons why algae are considered as feedstock for oil is their yields. DOE (Department of Energy, Government of USA) has reported that algae yield 30 times more energy per acre than land crops such as soybeans, and some estimate even higher yields up to 15,000 gal per acre. Aside from keeping the earth clean and free from pollution, these algal biodiesel helps to utilize a resource that is available in abundance just waiting to be harnessed and exploited. In this connection it has been assumed that 10 million acres of land would need to be used for biodiesel cultivation in the USA in order to produce biodiesel to replace all the petrodiesel used currently in that country. This is just 1 % of the total land used 130 3 From Algae to Liquid Fuels today for farming and grazing together the land in the USA (about 1 billion acres). This clearly shows that algae are a superior alternative as a feedstock for large-scale biodiesel production. Fortunately, the recent algae biodiesel scenario in developed countries has brought new revolution in algae biodiesel technology and has sprouted new hope for algae as, the only, sustainable resource for third-generation green energy. Keeping this assumption in view, the USA is in progress to have energy security through algal biofuels. The renewable energy department’s Bioenergy Technolo- gies Office (BETO) is desperately busy in exploring the immediate opportunity to use algae into advanced biofuel such as renewable diesel and jet fuel. BETO has a target to produce 2,5000 gallon of algae biofuel (per acre equivalent) by 2018. The longer-term target is to have yields that enable 5000 gal by 2022. Under algae biodiesel development project, BETO provides cost-shared funding to the following biorefinery projects for algal biofuels: • Sapphire Energy Inc. (at demonstration level) • Algenol Biofuels Inc. (to operate at pilot scale) • Solazyme Inc. (to operate at pilot level) Meanwhile, Solazyme Integrated Biorefinery (SzIBR) developed innovative process that harnesses the capacity of some algae to produce above 75 % of the dry weight of the cells as oil. In the SzIBR process, the microalgae grow luxuriously in fermentation vessel in dark condition having very high cell density. The heterotrophic algae metabolize carbon substrates provided in the growth media and convert them to triglyceride nearly similar in composition to vegetable oil. The oil production capacity of algae is very high as compared to autotrophic algae. Solazyme has already manufactured thousands of gallons of oil into algae biofuel that comply with ASTM standard without the use of any blending oil. This project has potential to adjust 88 high-skilled scientists per year, directly. In addition, it also provides job opportunity to 256 people, indirectly [53]. Sapphire Energy Integrated Algal Biorefinery (SEIABR) project is in operation condition at Columbus, New Mexico. Under full capacity working condition, the algae will fix about 56 metric tonnes of carbon dioxide per day which is equivalent to 100 barrels of algae biodiesel per day or about 1 million gallons per year of finished biofuel product [54]. The first commercial biorefinery will commence in 2018, having production capacity of about 5000 barrels of oil per day. This biodiesel is targeted to be used as jet fuel and diesel fuel. In 2010, in order to test the technical feasibility and efficacy of using 50–50 blend of algae biofuel and standard marine petroleum fuel, without any alternation in engine configuration, the US defense organization under “The Navy’s Green Fleet Initiative” program tested 20,000 gal of algae biofuel on defense marine ship, successfully. Since 2010, the Defense Advanced Research Projects Agency (DARPA) has been in working process to exploring the possibility of getting algae biodiesel from Yangtze River, located in Beijing, China. Barbara McQuiston [55], special assistant for energy at DARPA, revealed the fact that DARPA’s research projects have already extracted oil from algal river at a cost of $2 per 3.1 Biodiesel from Microalgae 131 gallon. It is now on track to begin large-scale refining of that oil into jet fuel, at a cost of less than $3 a gallon. The work is part of a broader Pentagon effort to reduce the military’s thirst for oil, which runs between 60 and 75 million barrels of oil a year. Much of that is used to keep the US Air Force in flight. Commercial airlines— such as Continental and Virgin Atlantic—have also been looking at the viability of an algae-based jet fuel. In India, Reliance Industries and Holdings Ltd. in a joint venture program with Algae.Tec Ltd., an Australian producer of algal oils, will develop a pilot project in India that will produce two barrels of biofuel a day with the investment of A$1.5 million ($1.3 million) in Algae.Tec and A$1.2 million more over the next 2 years. Algae.Tec’s algal oils can be used to make biofuel for vehicles and jets. India is aiming for 20 % of its fuel to come from clean sources by 2017. However, the final cost of algae biodiesel produced is not yet declared [56]. Recently, a South Australian-based company Muradel demonstrated a commercial plant with the potential to produce 80 million liters of an environmentally sustainable fossil crude equivalent. The $10.7 million demonstration plant, located in coastal town of Whyalla, will produce 30,000 L of oil a year using Muradel’s Green Black technology that produces biofuel from naturally occurring marine microalgae. At the commercial scale, Muradel expects production costs would be on par with the cost of producing fossil fuels for transport. The planned 1000 ha commercial plant would also create at least 100 new skilled and operational jobs in the Whyalla region [57]. Murdoch University in collaboration with the University of Adelaide [58], recently, completed a pilot-scale project on algae biodiesel having capacity of 50 tonnes per hectare per year. This joint project worth about $3.3 million was partly supported ($1.83 million) by the Australian government as part of the Asia- Pacific Partnership on Clean Development and Climate. The unique salient feature of the project is that all aspects of algae biodiesel production, i.e., the process of microalgae biofuel production, from microalgae culture, harvesting of the algae, and extraction of oil suitable for biofuels production, are intensively studied from techno-economical feasibility point of view. Due to the project’s success, construction of a multimillion dollar pilot plant to test the whole process on a larger scale will now begin in Karratha in the northwest of Western Australia in January 2016 and is expected to be operational by July. The main target of the project is to bring down the cost of algal diesel to less than $1 a kilo.

3.1.5 Overview of Algal Biodiesel Supply Chain

Supply chain management is a business strategy to improve operational and customer value by optimizing the flow of products, services, and related information from source to customer. Processes of creating and fulfilling the market’s demand for goods and services in a supply chain as well as a trading partner community engaged in a common goal of satisfying the end customer. Therefore, 132 3 From Algae to Liquid Fuels it is an organization of the overall business processes to enable the profitable transformation of raw materials or products into finished products and their timely distribution to meet customer demand. Basically, it is biomass process management starting from cultivation to biodiesel production and sell. The first step in supply chain is mass cultivation of micro- and macroalgae by closed or open system, depending on the nature of algae and place of cultivation. Via natural system the algae can also be collected. The second step is harvesting, from manual harvesting, to methods using continuous automated flow for some microalgae. The third step is biomass pretreatment, including cleaning, desalination, dewatering, and drying (when needed). A further step is grouped under downstream processing that can vary depending on the energy generation route selected. Finally, the market aspects from a biofuel end-user perspective are considered. Algal biodiesel production is mainly governed by small-scale level, restricted to particular locality, and commercial-scale production activity being organized both at national and international stage. Recently, it has been observed that big algal diesel projects are in an initial stage of development with the joint collaboration of different countries. This type of joint venture in algae biodiesel supply chain may bring new strategy in bringing better energy security in developing countries that import petrol and diesel from Arabian countries. In this connection, it is immediately required to pay much attention in large-scale operation of “algae to biodiesel” project and its efficiency supply chain operation. The main operations along the supply chain need to ensure constant, stable, and competitively priced feedstock supply for energy conversion plants. These operations are coordinated and optimized by the design of the overall supply system as illustrated in Fig. 3.2.This operation needs the following process to be monitored under international standard conditions, and the same has been explained in the next section.

3.2 Large-Scale Microalgae Production

3.2.1 Microalgae Cultivation

Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms. Natu- rally they can grow rapidly in fresh- or salt water due to their unicellular or simple multicellular structure. Microalgae are one of the oldest living organisms in our planet. Microalgae have more than 300,000 species. Algae can also be cultivated using photoautotrophic (photosynthetic), heterotrophic, or mixotrophic growth techniques. Heterotrophic growth is based on the cellular consumption of organic carbon instead of light, and mixotrophic growth uses the combination of these energy sources. Autotrophic microalgae are cultivated on land in large ponds or in enclosed so-called photobioreactors, using enriched CO2. The CO2 can come in the form of ﬂue gases from power plants or be obtained from other fossil-fuel 3.2 Large-Scale Microalgae Production 133

Algal lipid extraction

Left over Algalbiomass Dewatering & Drying After solvent extraction

Process

ALGAL BIODIESEL

Value-added chemicals Biofertilizer CO 2 Methane

Electricity

Fig. 3.2 Overview of algal biodiesel supply chain combustion and biological processes. They thus can help recycle this speciﬁc greenhouse gas and can help reduce greenhouse gas emissions overall when the algal biomass is converted into biofuels (Fig. 3.3a–d). Heterotrophic microalgae are grown in large fermenters (Fig. 3.3e) using sugar or starch, similar to the corn ethanol fermentation already providing almost 10 % of our liquid transportation fuels. The following are the important factors that determine the growth rate of algae, irrespective of their growth conditions. A typical model on microalgae cultivation and production of biofuels and other value-added chemicals is depicted in Fig. 3.4: • Light—Light is needed for the photosynthesis process (for autotrophic grown algae). • Temperature—There is an ideal temperature range that is required for algae to grow. • Medium/nutrients—Composition of the water is an important consideration (including salinity). 134 3 From Algae to Liquid Fuels

Fig. 3.3 Algae cultivation methods. (a) Algal ponds of 0.5 ha and 1 ha are part of the first commercial-scale algal biofuel facility in the USA at Sapphire Energy’s Integrated Algal Biorefinery. They cover an area of 400 m wide by 1600 m long at a location near Columbus, New Mexico. (b) A single 1-million-liter paddle-wheel-driven pond from the Columbus facility. (c) A pilot-scale flat-panel photobioreactor developed at the Laboratory for Algae Research and Biotechnology at Arizona State University in Mesa (image courtesy of Q. Hu). (d) A commercial- scale tubular photobioreactor designed and constructed by IGV and operated by Salata in Germany (image courtesy of C. Grewe). (e) An industrial-scale fermentation tank for heterotrophic cultivation of microalgae at Martek Biosciences, part of DSM in Heerlen, the Netherlands (image courtesy of D. Dong)

Fig. 3.4 A general model on microalgae cultivation for algal biodiesel production (Image courtesy of source: vult.sierracclub.org) 3.2 Large-Scale Microalgae Production 135

• pH—Algae typically need a pH between 7 and 9 to have an optimum growth rate. • Algae type—Different types of algae have different growth rates. • Aeration—The algae need to have contact with air, for its CO2 requirements. • Mixing—Mixing prevents sedimentation of algae and makes sure all cells are equally exposed to light. • Photoperiod—Light and dark cycles.

3.2.2 Algae Cultivation Methods

Over the last decade, constant and innovative research and development is taking place in the area of algae cultivation technology. The presently used cultivation system can be subdivided into two major types: (i) Open/half-open production system (Open pond, Raceway ponds, Long lines) (ii) Close-type photobioreactor a) Tubular photobioreactor b) Flat-plate photobioreactor c) Vertical column photobioreactor d) Algal fermenter (i) Open Ponds and Raceway Ponds Open ponds (Fig. 3.5) otherwise known as raceway ponds are shallow artiﬁcial pools, used in algae cultivation exposed to natural conditions. Algae cultivation in open-pond production systems has been used since the 1950s [59]. The pond is divided into a rectangular grid, with each rectangle containing one channel in the shape of an oval, like an automotive raceway circuit. Each rectangle contains a

Fig. 3.5 Aerial view of (a) open ponds and (b) raceways (http://allaboutalgae.com/open-pond) 136 3 From Algae to Liquid Fuels paddle wheel in order to keep the continuity of water flow around the circuit [60]. Even natural lake, lagoon, and pond (Fig. 3.5) can be used for algae cultivation under certain monitoring conditions. Compared to closed system, open ponds are easy to construct and manage, from economical point of view. Raceway ponds are the most commonly used artificial system [61]. They are typically made of a closed loop, oval-shaped recirculation channels, generally between 0.2 and 0.5 m deep, with mixing and circulation required to stabilize algae growth and productivity. Raceway ponds are usually built in concrete, but compacted earth-lined ponds with white plastic have also been used. As open ponds are exposed to natural climate, the maintenance of temperature, poor diffusion rate of carbon dioxide to the atmosphere, and requirement of extensive areas are some chronic problems associated with such practice. In addition, contamination of bacteria and other fast-growing heterotrophs restricts the growth and development of algae and hampers its commercial production. Also, due to inefficient stirring mechanisms in open cultivation systems, their mass transfer rates are very poor resulting to low biomass productivity. The artificial raceway ponds are shallow water bodies where supply of carbon dioxide, feeding of nutrients, and diffusion of oxygen are being monitored by means of suitable mechanical devices. The ponds are usually kept shallow because the algae need to be exposed to sunlight, and sunlight can only penetrate the pond water to a limited depth. Compared to closed photobioreactors, open pond is the cheaper method of large-scale algal biomass production. Open-pond production does not necessarily compete for land with existing agricultural crops, since they can be implemented in areas with marginal crop production potential [61]. They also have lower energy input requirement, and regular maintenance and cleaning are easier [62] and therefore may have the potential to return large net energy production. The open systems, in order to increase their efficiency, are generally designed as a continuous culture in which a fixed supply of culture medium or influent ensures constant dilution of the system. The organisms adapt their growth rate to this dilution regime, with the organism best adapted to the environment prevailing in the system winning the competition with the other organisms. Inconsistent data are available and achievable with open-pond systems. A trial on raceway pond having 450 m2 and 0.30 m deep produced biomass dry weight of 8.2 g m2 per day in Malaga, Spain [57]. With this system Jimeńez et al. [61] extrapolated an annual dry weight biomass production rate of 30 tonnes per hectare. By using similar depth of culture, Becker [63] achieved biomass productivity in the range of 10–25 g m2 per day, and such high productivity was also noticed by Setlik et al. for Chlorella in an inclined system. Weissman and Tillett [64] operated an outdoor open pond (0.1 ha) in New Mexico, USA, and attained an average annual dry weight biomass production rate of 37 tonnes per hectare with a mixed species culture (four species), highest yields were confined to the warmest months of the year. Japan started the first large-scale cultivation of Chlorella vulgaris (Chlorella) [65]. Then extensive production of Arthrospira platensis (Spirulina) was first set in Mexico, and today, the major share of industrial microalgae production is located in 3.2 Large-Scale Microalgae Production 137 the Asia Pacific Rim, including China and India [66]. The industrial scale world production of the three major strains, including Spirulina spp., Chlorella spp., and Dunaliella spp., accounted for 8000 t of dry weight/year, and among them Spirulina was estimated to be over 3000 t of dry weight/year [67]. These biomasses are marketed as health food, nutraceutical products, and for cosmetics [68] without paying much attention on algal biodiesel. Presently, in the desert scrub outside of Columbus, New Mexico, Sapphire Energy is commissioning the most advanced, open-pond algae production facility in the world. Sapphire Energy’s algae farm is the world’s first commercial demonstration-scale algae conversion farm, integrating the entire value chain of algae-based production, from cultivation, to production, to extraction. Presently, San Diego-based Sapphire Energy is pioneering an entirely new industry—green crude production—with the potential to profoundly change America’s energy and petrochemical landscape for the better. Sapphire’s products and processes in this category differ significantly from other forms of biofuel because they are made solely from photosynthetic microorganisms (algae), using sunlight and CO2 as their feedstock; are not dependent on food crops or valuable farmland; do not use potable water; do not result in biodiesel or ethanol; enhance and replace petroleum-based products; and are low carbon. Green crude can be refined into the three most important liquid fuels used by our society: gasoline, diesel, and jet fuel. The fuels meet ASTM standards and are compatible with the existing petroleum infrastructure, from refinement through distribution, and the retail supply chain. Green crude is an algae-based renewable oil developed as drop-in replacement for petroleum. It can be refined into gasoline, diesel fuel, and aviation fuel in existing refineries for use in existing engines. In addition, Sapphire and Linde will commercialize a new industrial scale conversion technology needed to upgrade algae biomass into crude oil. Together, the companies will refine the hydrothermal treatment process developed and operated today by Sapphire Energy at pilot scale. When Sapphire reaches commercial readiness, its plan is to be price competitive with traditional crude. Current industry estimates are for algae-based green crude production to result in a $75 and $85/barrel cost at commercial scale. In the initial testing by Sapphire Energy, green crude oil was upgraded into on-spec ASTM 975 diesel fuel, proving its compatibility with the existing network of pipelines, refineries, and transport systems. The company expects to be at commercial demonstration scale in 2015, commercial scale in 2018, and is eventually projected to produce 1 billion gallons per year by 2025. (ii) Closed-Type Photobioreactor Since 1950, after the Second World War, continuous attempts have been going on to design the most efficient photobioreactors to produce maximum algal biomass. A photobioreactor receives natural light and exogenously supplied oxygen, carbon dioxide, and nutrients, on the basis of the types of algae to be grown. Thus, a photobioreactor allows much higher growth rates and purity levels than anywhere in natural or habitats similar to nature. In order to reduce the process cost, people 138 3 From Algae to Liquid Fuels have started using nutrient-rich wastewater and flue gas carbon dioxide in a photobioreactor, after proper filtration process. In a closed photobioreactor, the evaporation loss of water is perfectly regulated. Besides this, the risk of contamination through landing water birds or dust is minimized. All modern photobioreactors have tried to balance between a thin layer of culture suspension, optimized light application, low-pumping energy consumption, capital expenditure, and microbial purity. Many different systems have been tested, but only a few approaches were able to perform at an industrial scale. PBRs facilitate better control of culture environment such as carbon dioxide supply, water supply, optimal temperature, efficient exposure to light, culture density, pH levels, gas supply rate, mixing regime, etc. Due to large surface-to- volume ratio, PBRs provide high efficiency of light capturing chance to algae. Compared to bags of algae PBR culture, the culture density of algae produced is 10–20 times greater by reducing water evaporation rate, even to zero. PBRs are space saving and can be mounted vertically, horizontally, or at an angle, indoors, or outdoors. Nowadays, due to the provision of self-cleaning, mechanisms can dramatically reduce fouling. However, instead of having all the good quality, the practice of algae through PBR poses certain disadvantages. Initial capital cost in PBR project is very high. This is one of the most important bottleneck that is hindering the progress of algae– fuel industry. Despite higher biomass concentration and better control of culture parameters, data accumulated in the last two decades have shown that the productivity and production cost in some enclosed photobioreactor systems are not much better than those achievable in open-pond cultures. The technical difficulty in sterilizing these photobioreactors has hindered their application for algae culture for specific end products such as high-value pharmaceutical products. PBRs are complex systems composed of several subsystems. The key systems are light system, optical transmission system, air handling and gas exchange systems, mixing system, nutrient system, instrumentation system, and electrical system. Besides these main components, there are few more subcomponents like oxygen and CO2 sensors, temperature sensor, pH sensor, light sensor, conductivity sensor, recirculation pump, harvest pump, CO2 injection valve, substrate pump, filtrate recirculation valve, water inlet valve, purge valve, connectors and hoses, oxygen release system, PLC control panel, and feeding tank that help in regulating the working conditions of PBR effectively. Algae can be grown cheaply in saltwa- ter ponds of the desert or even more efficiently in proprietary photobioreactors (which solve a lot of the problems encountered in open ponds for a few more dollars on the initial investment). It’s conceivable that the photobioreactors could be placed in a desert environment, although one of the challenges for growing algae is to keep the water at a very consistent temperature of around 70 Fahrenheit so that will likely also influence optimal placement of the photobioreactors. As already stated several times, the primary inputs for growing algae are water, CO2, and sunlight. This activity would be best accomplished closer to the desert, where seasonal sunlight levels and temperatures do not vary as much as they do 3.2 Large-Scale Microalgae Production 139 further away from the equator. Another possible method to increase production would be to put the photobioreactors near a conventional coal-burning electric plant and harvest the significant amounts of CO2 generated by the plant. As attractive as it sounds, the production of biodiesel should not depend on the coal plant operating indefinitely since that wouldn’t be a sustainable long-term strategy. Algae strains suitable for the desert cultivation include: • Haematococcus pluvialis • Microcoleus vaginatus • Chlamydomonas perigranulata • Synechocystis sp. Valcent’s HDVB bioreactor system which can be deployed on nonarable land requires very little water due to its closed circuit process, does not incur significant labor costs, and does not employ fossil fuel-burning equipment. A number of companies such as Petrosun and BioFields are considering large-scale algae farms in desert regions. The conventional type of glass reactor can be used for algal culture, after little alteration. However, such type of reactor can only be used in the laboratory, due to its heavy cost and expensive operation condition. This type is quite common in laboratory scale, but it has never been established in bigger scale, due to its limited vessel size. So, on the basis of design, operational conditions, and types of algae to be used, four main categories most generally suitable for large-scale cultivation are (a) tubular/horizontal, (b) flat plate or flat panel, (c) vertical column reactors, and (d) algal fermenter. (a) Tubular Photobioreactor Tubular photobioreactor is one of the most suitable types for outdoor mass cultures. Most outdoor tubular photobioreactors are usually constructed with either glass or plastic tube, and their cultures are recirculated either with pump or preferably with airlift system. They can be in the form of horizontal/serpentine, vertical, near- horizontal, conical, and inclined photobioreactor (Fig. 3.6). Aeration and mixing of the cultures in tubular photobioreactors are usually done by air-pump or airlift systems. Tubular photobioreactors consist of straight, coiled, or looped transparent tubing arranged in various ways for maximizing sunlight capture [69]. Properly designed tubular photobioreactors completely isolate the culture from potentially contaminating external environments, hence, allowing extended duration monoalgal culture. General operation process of tubular photobioreactor is depicted in Fig. 3.6. (1) The feeding of algae to tubular system of a photobioreactor is linked with a feeding vessel through peristaltic pump which moderates the flow of the algae into the tubes. Generally, sparging of carbon dioxide and influx of nutrients to tubular system is carried out through feeding vessel or at the beginning and end junction of phototubes. (2) The tubular photobioreactor itself is used to promote biological growth by controlling environmental parameters including light. The tubes are made of 140 3 From Algae to Liquid Fuels

Fig. 3.6 (a) Photograph of vertical inclined photobioreactors used for microalgae production at pilot level. (b) Photograph of a horizontal type of tubular photobioreactor commercially used for microalgae production to manufacture algal diesel. (c) Diagrammatic view of tubular photobioreactors with monitoring devices to regulate microalgae growth and development process (www.ecopedia.com). (d) Photograph of vertical photobioreactors used for microalgae production at pilot level (www.cambia.org)

acrylic or borosilicate glass with the provision of supply of light and dark photo period. Algae cultures are recirculated either with a mechanical pump or airlift system, the latter allowing CO2 and O2 to be exchanged between the liquid medium and aeration gas as well as providing a mechanism for mixing. Mixing is very important to encourage gas exchange in the tubes. The tubular photobioreactor has a built-in cleaning system that internally cleans the tubes without stopping the production. (3) After the algae have completed the flow through the tubular system of photobioreactor, it passes back to the feeding vessel. As it progresses through the hoses, the oxygen sensors monitor how much oxygen has built up in the plant, and this oxygen is released in the feeding vessel itself. It is also at this stage that the optical cell density sensor determines the harvesting rate. (4) When the algae are ready for harvesting, they pass through the connected filtering system. This filter collects the algae that are ready for processing, 3.2 Large-Scale Microalgae Production 141

while the remaining algae pass back to the feeding vessel, and continuity in flow is maintained, consistently. (5) Tubular photobioreactor are very suitable for outdoor mass cultures of algae since they have large illumination surface area. On the other hand, one of the major limitations of tubular is poor mass transfer. It should be noted that mass transfer (oxygen buildup) becomes a problem when tubular photobioreactors are scaled up. For instance, some studies have shown that very high dissolved oxygen (DO) levels are easily reached in tubular photobioreactors [70, 71]. Tubular photobioreactors were designed limitations on length of the tubes, which is dependent on potential O2 accumulation, CO2 depletion, and pH variation in the systems [72]. In order to have noncontaminated pure culture, more attention was paid on closed-type photobioreactors, where the regulatory aspects of microalgae growth and development could be well managed. In this connection, several types of photobioreactors were designed [70, 71, 73–76] and tested for better growth and development of algae, since the pioneering work of Tamiya et al. (1953) [77]. Japan has taken credit of demonstrating first large-scale cultivation of Chlorella vulgaris (Chlorella)[65]. Then extensive production of Arthrospira platensis (Spirulina) was first set in Mexico, and today, the major share of industrial microalgae production is located in the Asia-Pacific Rim, including China and India [66]. Spirulina spp., Chlorella spp., and Dunaliella spp. were used for the first time to produce health food, nutraceutical products, and for cosmetics at commercial level [67, 68] without paying much attention on algal biodiesel. In developed countries, large-scale microalgal biomass productions use open-type production systems, such as artificial ponds, pools, or raceways, often placed under greenhouses, which involve extra building costs [78]. Meanwhile, various PBR designs have been developed: plastic bags [79], stirred tanks, bubble columns [80], airlifts, large surface area flat glass panels or thin layer reactors [81], horizontal glass or light transparent plastic tubes connected together, vertically or horizontally, in a closed serpentine system. Sophisticated internally illuminated photobioreactors (IIPBRs), equipped with optical fibers to increase permanent light availability, were also developed [82, 83]. However, these PBRs also entail high construction costs [84–86]. The world’s first and largest tubular production facility for microalgae in Klotze (Saxony-Anhalt, Germany) with 20 photobioreactors and around 700,000 liter volume on a 1.2 ha (2.9 acre) site was developed by GICON with the technical collaboration of Anhalt University of Applied Sciences. In 2013, the Central Germany Biosolar Center was awarded the Hugo Junkers prize, an award from the State of Saxony-Anhalt for the “Most Innovative Alliance” between academia and industry. Compared to conventional photobioreactor systems, the GICON system operates with a high level of reliability and low maintenance requirements. Biofouling is minimized by utilizing a new tube material which was specifically developed for algae cultivation applications. As a result, the system is more reliable and efficient because the important light entrance areas are not blocked by surfaces 142 3 From Algae to Liquid Fuels with biofouling. The tube material also provides effective protection against harmful UV radiation. The flexibility of the tubing offers high mechanical stress resistance and is resistant to organic solvents, salt water, acids, and alkalis. Last but not the least, the reactors are characterized by low specific energy consumption and low maintenance costs. The GICON® photobioreactor TBR 250—also referred to as “Christmas tree” reactor (Fig. 3.7) due to its biomimetic shape—ensures optimal light supply, thanks to the truncated conical geometry. Self-shading of the algae is prevented, and the structure’s angle can be adjusted to achieve optimal light impact. The system design features a tubular reactor, which is preferential for pharmaceutical and food applications. Carbon dioxide and nutrients can be predicatively added in relation to growth rates. The innovative, geometrically variable structure with a flexible, double-wall tube system protects the algae from extreme temperature fluctuations. The controllable temperature management allows for adapted use at a variety of sites in different climate zones while achieving clearly defined product quality. In addition to rapid growth, another positive point in algae is the ability to effectively bind carbon dioxide and use it as a carbon source. Carbon dioxide can thus be bound at the very site it is produced, which reduces the likelihood of damaging environmental effects. Algal biomass accrued in a photobioreactor can be used in a variety of ways, including recycling as a raw material in the pharmaceutical and cosmetic industries, as a nutritional supplement, and for the production of fuels and animal feed. One of the most salient features of a GICON photobioreactor is its optical fiber light supply technology (Fig. 3.8) by which optimum light input for the highest growth rates of algae is managed. Required photon of light is correctly directed directly to where it’s needed: from optimal external illumination to systems within the algae cultivation system. In this technique, light guides transmit only light, not heat, thus avoiding temperature levels that are detrimental to algae development.

Fig. 3.7 A “Christmas tree”-shaped photobioreactor test station at Anhalt University in Kothen is the most recent implementation of a novel and innovative photobioreactor 3.2 Large-Scale Microalgae Production 143

Fig. 3.8 Supply of indirect light to microalgae grown in tubular-type photobioreactor

Fiber optic light guides transmit only light, not electricity; therefore, they can be easily used in wet environments. With this technology, easy adjustment of light intensity and color temperature to the special requirements of varying algae strains is managed effectively. It is also possible to bundle natural sunlight and guide it directly to the algae. (b) Flat-Plate Photobioreactors In India, Algenol and Reliance Industries have successfully deployed India’s first Algenol algae production platform. The demonstration module, i.e., closed-type photobioreactors (vertical flat plates and vertical hanged bags plastic) (Fig. 3.9), is located near the Reliance Jamnagar Refinery, the world’s largest oil refinery with a nameplate capacity of 668,000 barrels per day at a 7500 acre complex in Gujarat state at the western extreme of India. Generally, flat-plate photobioreactors are made of transparent materials for maximum utilization of solar light energy. Accumulation of dissolved oxygen concentrations in flat-plate photobioreactors is relatively low compared to horizontal tubular photobioreactors. It has been reported that with flat-plate photobioreactors, high photosynthetic efficiencies can be achieved. Flat-plate 144 3 From Algae to Liquid Fuels

Fig. 3.9 (a) Closed type of vertical plate photobioreactors and (b) vertical plastic bags hang-type photobioreactors developed and commissioned by Reliance Industry, India, with the technical collaboration of Algenol, Germany. It is located near the Reliance Jamnagar Refinery, the world’s largest oil refinery with a nameplate capacity of 668,000 barrels per day at a 7500 acre complex in Gujarat state at the western extreme of India (Source: www.biofuelsdigest.com) photobioreactors are very suitable for mass cultures of algae. The following are few salient features of flat-plate photobioreactor: • Scale-up requires many compartments and support materials. • Difficulty in controlling culture temperature. • Some degree of wall growth. • Possibility of hydrodynamic stress to some algal strains. The demonstration has completed several production cycles of Algenol’s wild- type host algae but ultimately could demonstrate the fuel production capabilities of Algenol’s advanced fuel-producing algae and systems. The Algenol fuel production process is designed to convert 1 tonne of CO2 into 144 gal of fuel while recycling CO2 from industrial processes and converting 85 % of the CO2 used into ethanol, gasoline, and diesel and jet fuels. The advanced fuel-producing algae technology is successfully operating at Algenol’s Fort Myers, Florida headquarters, also. (c) Vertical Column Photobioreactor Column PBRs are occasionally stirred tank reactors but more often bubble columns or airlifts. The columns are placed vertically, aerated from the bottom, and illuminated through transparent walls or internally. Column bioreactors offer the most efficient mixing, the highest volumetric gas transfer rates, and the best controllable 3.2 Large-Scale Microalgae Production 145 growth conditions. They are low cost, compact, and easy to operate. Their performance (i.e., final biomass concentration and specific growth rate) compares favor- ably with the values typically reported for tubular PBRs. Vertical bubble columns and airlift cylinders can attain substantially increased radial movement of fluid that is necessary for improved light–dark cycling. Light availability in bubble columns and airlift (devices is influenced by aeration rate, gas holdup, and the liquid velocity mixing) turbulence. Bubble columns and airlift photobioreactors can be useful for culturing phototrophic organisms requiring light as a nutrient. These reactor designs have a low surface/volume, but substantially have greater gas holdups than horizontal reactors and a much more chaotic gas–liquid flow. Consequently, cultures suffer less from photo-inhibition and photo-oxidation and experience a more adequate light–dark cycle. The photosynthetically generated oxygen also needs to be removed, as excessive dissolved oxygen suppresses photosynthesis. Oxygen removal capacity is governed by the magnitude of the overall gas–liquid mass transfer coefficient. At present, bubble columns and airlift reactors are not much used as photobioreactors, except for laboratory investigational purposes. (d) Algal Fermenter Algal fermenters are like conventional type of fermenters (Fig. 3.3e) with minor modification of stirrer. For the first time in the world’s history, Solazyme could be able to produce variety of oils from microalgae through heterotrophic condition without the input of solar energy and carbon dioxide (Fig. 3.10). Solazyme uses a variety of sugar feedstocks at a single manufacturing facility using conventional type of fermenter with other standard industrial equipments, thus providing exceptional input, output, and capital flexibility (Fig. 3.11). In order to economize the algal oil production, Solazyme has developed integrated microalgae processing technology to produce algal diesel along with whole algal product, such as AlgaVia™ whole algal protein. Then, through industrial fermentation, the microalgae convert sugars into the desired oil or whole algal product. Fermentation helps accelerate microalgae’s natural biological processes, allowing Solazyme to produce large amounts of a desired product in a matter of days. With this contained process, one can decouple the production of oil from geography and reduce the ecosystem damage that unsustainable oils have caused around the world. Thus, our algal oils can replace fossil fuels at one end of the spectrum and unsustainable plant-based oils at the other. Following are variety of oil being produced through heterotrophic cultivation of microalgae through conventional fermentation technology: Oleic acid—This class levels of oleic acid (C18:1) and low levels of polyunsaturates (C18:2, C18:3) provide benefits such as high lubricity, stability, renewability, low VOC, and biodegradability. Lauric acid—The fatty acid chain lengths that are differentiated from palm kernel and coconut oils by accentuating specific targeted fatty acids between C10 and C14, all while retaining the structuring characteristics of those oils. 146 3 From Algae to Liquid Fuels

Fig. 3.10 (a) Solazyme technology for algal biodiesel production through fermentation by growing microalgae under heterotrophic conditions using variety of sugars as carbon feedstock (www.grandemotte.ﬁles.wordpress.com) and (b) conventional type of fermenter for growing microalgae (Source: g.footcdn.cm). (c) The US Navy tested Solazyme’s algae diesel (Source: a57.foxnews.com). (d) Soladiesel from microalgae and cellulosic part of algal biomass (www. gigaomz.ﬁles.worpress)

Fig. 3.11 Solazyme’s manufacturing process to produce a wide range of products from a variety of sugar feedstocks at a single manufacturing facility using standard industrial equipment, thus providing exceptional input, output, and capital ﬂexibility 3.3 Harvesting and Biomass Concentration 147

Mid-chain oils—This type of oils targets optimized levels of C8 (caprylic acid) and C10 (capric acid). As an example, one unique and valuable oil in this family already has over 50 % C8–C10. Before this oil was created, the only way to arrive at a comparable oil was extensive and expensive chemical processing. Structured fat family—Solazyme’s ability to control the position of fatty acids has enabled the production of a first-in-kind algal “butter” with a structure similar to cocoa butter, resulting in a true “chocolate”-melting profile and mouth feel. Long-chain hydrocarbons—It includes the current development of highly unique oils with high levels of erucic acid (C22:1), with the benefit of not having to segregate high erucic rapeseed oil from the food supply. Peoria was Solazyme’s first wholly owned renewable oil production facility. The facility’s integrated biorefinery was partially funded by a US Department of Energy (DOE) grant to demonstrate commercial-scale production of algal-based fuels. Today, this facility is commercially producing cosmetic and food ingredients, as well as providing routine production of multiton sampling and commercial quantities. Peoria has a nameplate capacity of nearly 2000 metric tonnes per year. Solazyme began commercial-scale production in Clinton, IA, in early 2014, after reinstating a formerly idled fermentation facility. The Clinton location serves as a commercial-scale operation for the company, producing oils for a wide range of industries and partners. Clinton has a nameplate capacity of 20,000 metric tonnes of oil per year, with the potential to expand to 100,000 metric tonnes of oil per year. Solazyme Bunge Renewable Oils is joint venture with longtime partner, Bunge. Located in Orindiu´va, Brazil, this commercial manufacturing facility is colocated with Bunge’s Bonsucro® sugarcane mill. The facility is also powered by bagasse and built to produce surplus energy that may be fed back to the grid, making it a closed-loop system. At full nameplate capacity, the facility is expected to produce 100,000 metric tonnes of oil per year.

3.3 Harvesting and Biomass Concentration

3.3.1 Concept

The term algae harvesting refers to concentration of diluted algae suspension until a thick algal paste is obtained. Harvesting of microalgae from algae cultivation pond or photobioreactors employs several techniques to concentrate the algae followed by harvesting. Normally harvesting of microalgae can be a single step process or two steps process which involves harvesting and dewatering. Harvesting microalgae is difficult because of the small size of the algae. Choosing the effective harvesting process for a particular strain depends on size and properties of algae strain. Most open ponds use a semicontinuous harvest strategy that removes roughly half of the algae, alleviating the need for reinoculation. The algae are harvested after each doubling, so growth of a single generation per day would 148 3 From Algae to Liquid Fuels equate to a single harvest per day [94]. Alternatively, continuous systems to keep algae at a constant density and batch or complete systems are reminiscent of agricultural crops, removing nearly all algae during a single harvest. The most rapidly growing algal species are frequently very small and often motile unicells. These are the most difficult to harvest. Thus, it is necessary to maintain an effective interaction between the development of harvesting technologies and the selection of algal species for mass culture. So, based on microalgae culture methods and biomass density, mainly, there are two ways, i.e., (1) bulk harvesting and (2) thickening, to separate algal biomass from cultivation medium [88]. Extensive reports are available on techniques and processes of microalgae cultivation, harvesting, and dewatering [37, 50, 89–91]. Microalgae cells harvest from cultivation broth has always been one of the major obstacles for the algae-to- fuel approach. Therefore, harvesting microalgal biomass is considered a challenging issue for commercial-scale production of algal biofuels. Techniques for harvesting microalgae include settling or flotation, centrifugation, and filtration. These processes are aided by cell flocculation, either through the addition of chemical flocculants or through culture autoflocculation. Flocculation causes the cells to become aggregated into larger clumps which are more easily filtered and/or settle more rapidly. The ease in harvesting the algae depends primarily on the organism’s size, which determines how easily the species can be settled and filtered.

3.3.2 Harvesting Methods

Based on microalgae cultivation methods and density of algal biomass, the process of algae separation can be categorized in two steps, i.e., (1) bulk harvesting and (2) thickening.

3.3.2.1 Bulk Harvesting

This process is involved to separate algal biomass from cultivation medium, i.e., bulk suspension. The concentration factors for this operation are generally 100–800 times to reach 2–7 % total solid matter. This is mainly carried by any one of the following three processes: (i) flocculation, (ii) flotation, and (iii) gravity sedimentation. (i) Flocculation Flocculation is a preparatory step prior to other harvesting methods such as filtration, flotation, or gravity sedimentation [92]. Since microalgae cells carry a negative charge that prevents natural aggregation of cells in suspension, addition of flocculants such as multivalent cations and cationic polymers neutralizes or reduces 3.3 Harvesting and Biomass Concentration 149 the negative charge. It may also physically link one or more particles through a process called bridging, to facilitate the aggregation [92]. Multivalent metal salts like ferric chloride (FeCl3), aluminum sulfate (Al2(SO4)3), and ferric sulfate (Fe2(SO4)3) are suitable flocculants. Knuckey et al. [93] developed a process that entailed adjustment of the algae culture pH between 10 and 10.6 using NaOH, followed by addition of a nonionic polymer Magnafloc LT-25. The flocculate was harvested by siphoning off surface water after a settling period and subsequently neutralized to give a final biomass concentration of 6–7 g l-1. The process was successfully applied to a range of species with flocculation efficiencies of >80 %. Divakaran and Pillai [94] successfully used chitosan as a bioflocculant. The efficacy of the method was very sensitive to pH, registering maximum flocculation at pH 7.0 for the freshwater species and lower for the marine species. The residual water could be reused to produce fresh algae cultures. Gentle, acoustically induced aggregation followed by enhanced sedimentation can also be used to harvest microalgae biomass. Bosma et al. [95] successfully used ultrasound to optimize the aggregation efficiency and concentration factor. They achieved 92 % separation efficiency and a concentration factor of 20 times (the factor by which the original liquid mixture has been concentrated). The main advantages of ultrasonic harvesting are that it can be operated continuously without inducing shear stress on the biomass, which could destroy potentially valuable metabolites, and it is a non-fouling technique [95]. Successful applications in the medical sector [96] provide basis for further investigations on potential applications in algal biomass harvesting. The possibility to induce autoflocculation by pH adjustment was investigated on several freshwater (Chlorella and Scenedesmus) and marine (Phaeodactylum and Nannochloropsis) species. Results show that there is a significant flocculation effect when the pH > 10.5, independent of biomass concentration. The use of biodegradable organic flocculants, such as cationic starch, can be interesting. The types of flocculants do not contaminate the algal biomass and often require low doses. In our experiments, cationic starch was an efficient flocculant for freshwater species (Parachlorella and Scenedesmus). The results depend on the type of cationic starch but are independent of pH in the range of 5–10. Electroflocculation using different types of electrodes has been tested to harvest freshwater and marine algae resulting in an overall flocculation efficiency higher than 90 %. In future experiments, important parameters such as algal species, operation time, electrode type, and current intensity will be optimized in collaboration with KaHo St. Lieven. However, besides the high cost of chemical flocculants and possible pollution effects that may generate, the chemical reactions are highly sensitive to pH, and the high doses of flocculants required produce large amounts of sludge and may leave a residue in the treated effluent. (ii) Flotation Flotation technique is applied to induce air bubbles to reach the proximity of algal cells and make them float and aggregate [97, 98]. For some microalgal strains, 150 3 From Algae to Liquid Fuels natural float occurs at the surface of the water when their lipid content increases. Various methods applied for floating of microalgae are based on the method of bubble production: dissolved air flotation (DAF), electrolytic flotation, and dispersed air flotation [99]. Generally, DAF is applied for wastewater treatment rather than for microalgal biomass production [100, 101]. By electrolytic flotation, gas bubbles are generated. Because of the use of electricity, this method is very energy intensive. For dispersed air flotation, foam flotation, large bubbles (1 mm) moving through porous media, or froth flotation, a combination of agitation and air injection, are applied [99]. If small bubbles are required, the energy usage for the flotation processes will be very high, which inevitably results in high operational costs and therefore a required high investment. The costs can be even greater when the costs of flocculants are included. Among the various method of harvesting, flotation is the most promising method for harvesting microalgae as higher solid concentrations can be obtained. However, not much technical reports are available on techno-economical aspect of floatation methodology on microalgae harvesting. (iii) Coagulation Harvesting microalgae from water presents a major hurdle in the realization of the “algae to bioproducts” goal. The major techniques, as discussed, currently employed in microalgae harvesting and recovery include centrifugation, flocculation, filtration and screening, gravity sedimentation, flotation, and electrophoresis techniques. Of all the harvesting methods, precipitation of microalgae is shown to be the most efficient method for harvesting biomass on a large scale. Research focuses on synthesizing cationic starch which serves as an organic coagulant/flocculant for harvesting algal biomass. Starch is a natural polymer manufactured in plants as an energy reservoir. Besides being abundant, starch is inexpensive and biodegradable. Starch can be modified by cationic groups such as quaternary ammonium, amines, imines, etc. to exhibit positive charges resulting in cationic starch. The cationic starch thus formed acts as a polyelectrolyte in which it helps in precipitating algae by subsequent coagulation (charge neutralization) and flocculation (bridging) owing to the inherent polymer structure of starch. Microalgae cells can also be coagulated using chitosan and the optimization of this process. Chitosan is a natural and environmentally friendly biopolymer created by the extensive deacetylation of chitin from shrimp, crab, and crawfish. Although conventional chemical coagulants such as alum may have negative impacts on human health, the use of chitosan as coagulant is suitable to harvest live microalgae resulted in restabilization of the microalgae cultures, reducing the efficiency of the process. But no significant report is available on commercial application of chitosan for coagulation of microalgae. Reports are also available on the use of chemical agents, i.e., metal coagulants such as flocculants for microalgae harvesting [102–105]. In ECF, iron or aluminum ions are released from a sacrificial anode through electrolytic oxidation. Compared to coagulation–flocculation with Fe3þ or Al3þ salts, ECF has the advantage that no anions such as chlorine and sulfate are introduced in the process water. The electrolytic oxidation of the sacrificial anode, however, requires electricity. So 3.3 Harvesting and Biomass Concentration 151 far, the use of ECF for harvesting microalgal biomass has not been thoroughly evaluated.

3.3.2.2 Thickening

The aim is to concentrate the slurry through techniques such as centrifugation, filtration, and ultrasonic aggregation and, hence, is generally a more energy intensive step than bulk harvesting. (i) Centrifuge Centrifuge is a useful device for both biolipid extraction from algae and chemical separation in biodiesel. Coupled with a homogenizer, one may be able to separate biolipids and other useful materials from algae. The force applied in this process may be from 4000 to 14,000 times gravitational force. Various centrifugal methods have been applied for microalgae separation, such as tubular centrifuge, multichamber centrifuges, imperforate basket centrifuge, decanter, solid-retaining disk centrifuge, nozzle-type centrifuge, solid-ejecting-type disk centrifuge, and hydro-cyclone. However, continuous-flow centrifugation with the classical forest rotor is widely used for commercial purpose. This method is reasonably efficient, but sensitive algal cells may be damaged by pelleting against the rotor wall, and the method is essentially unselective; all particles with a sedimentation rate above some limiting value will be collected [105–107]. The recovery of the biomass in a sedimenting centrifuge depends on the settling characteristics of the cells, the residence time of the cell slurry in the centrifuge, and the settling depth. Settling depth can be kept small through the design of the centrifuge. The residence time of the slurry in the centrifuge can be controlled by controlling the flow rate. Heasman et al. 2000 [108] evaluated the extent of cell recovery and the effects of cell viability at different conditions of centrifugation. Nine different strains of microalgae were assessed [108]. A cell harvest efficiency of >95 % was obtained only at 13,000 g. The harvest efficiency declined to 60 % at 6000 g and 40 % at 1300 g. Cell viability depended significantly on the algal species and the method of centrifugation. (ii) Sedimentation Gravity sedimentation is a common harvesting method for algae biomass in wastewater treatment. This method is based on Stoke’s law which determines the characteristics of suspended solid by density, radius, and sedimentation velocity of algae cells [31]. Stoke’s law can only be used for non-flocculated particles and is not applicable for flocculated particles because of the complicated structure of the flocs [99]. In an algal cell suspension, the rate of sedimentation of cell mainly depends on cell diameter and degree of aggregation of cells [31]. The aggregation of cells depends on the nature of physicochemical treatment to enhance flocculation. Gravity sedimentation followed by artificial-induced flocculation is one of the most common practices for first-stage (1 to 5 % solids) algae biomass harvesting [92, 109]. This practice holds good for macroscopic algae. In the case of 152 3 From Algae to Liquid Fuels microalgae, the gravity settling rate is slow due to tiny structure of cells and attachment of small air bubbles that make the algae flocculate. So application of this method may not be much useful. However, by using suitable flocculating agents, the sedimentation rate can be increased by increasing the gravitational force via centrifugation. By this means one can achieve high-biomass recovery (>95 %) and can be suitable for a wide range of microalgae. But no report is presently available on its techno-economical viability in commercial purpose. (iii) Biomass Filtration Filtration is carried out commonly on membranes of modified cellulose, with the aid of a suction pump. The greatest advantage of this method as a concentrating device is that it is able to collect microalgae or cells of very low density. However, concentration by filtration is limited to small volumes and leads to the eventual clogging of the filter by the packed cells when vacuum is applied. Several methods have been devised which avoid these problems. One involves the use of a reverse- flow vacuum in which the pressure operates from above, making the process gentler and avoiding the packing of cells. This method itself has been modified to allow a relatively large volume of water to be concentrated in a short period of time (20 L to 300 mL in 3 h). A second process uses a direct vacuum but involves a stirring blade in the flask above the filter which prevents the particles from settling at all during the concentration process. Due to variability in the size of algae, ranging from <30 mm to <70 mm, the porosity of membrane and pressure applied vary. A conventional filtration process is most appropriate for harvesting of relatively large (>70 mm) microalgae such as Coelastrum and Spirulina. It cannot be used to harvest algal species approaching bacterial dimensions (<30 mm) like Scenedesmus, Dunaliella, and Chlorella [110]. Conventional filtration operates under pressure or suction; filtration aids such as diatomaceous earth or cellulose can be used to improve efficiency [111]. It has been reported that by conventional technique, 245-fold algal biomass of Coelastrum proboscideum could have achieved to produce a sludge with 27 % solid [92]. For recovery of smaller algae cells (<30 mm), membrane microfiltration and ultrafiltration (a form of membrane filtration using hydrostatic pressure) are technically viable alternatives to conventional filtration [112, 113]. It is suitable for fragile cells that require low transmembrane pressure and low cross-flow velocity conditions [114]. For processing of low broth volumes (<2m3 per day), membrane filtration can be more cost-effective compared to centrifugation. Owing to the cost for membrane replacement and pumping in larger scales of production (>20 m3 per day), centrifugation may be a more economic method of harvesting the biomass [115].

3.3.2.3 Gravity Filtration

A simple and cheap method for separating suspended solids from liquid is by gravity filtration. This process has been applied for the harvesting of larger algal 3.3 Harvesting and Biomass Concentration 153 species such as the cultivation of Spirulina in rural communities in India and Taiwan. Spirulina are blue-green algae in which cells can grow as large as 500 μm, clearly visible to the naked eye. Difficulties arise when the process is applied to fine and high-plasticity species such as Scenedesmus; yet some success has been attributed to colony-forming algae in conjunction with the addition of chemical flocculants [63]. Various mechanically agitated screens have been designed, such as vibrating screens and rotating screens. Vibrating screens with pore sizes down to 25 μm (500-mesh fabrics) are used in Israel for harvesting of Spirulina with an efficiency of 95 % [63]. A disadvantage of this system is the potential for rubbing to cause rupture of algal cells. This is a particular problem for fragile algal species such as Dunaliella. (iv) Ultrasonic Aggregation Ultrasound can be used to harvest microalgae. The separation process is based on gentle acoustically induced aggregation followed by enhanced sedimentation. It has been unexpectedly discovered that when nanosecond electric pulses are applied to a solution containing algae, the algae aggregate without the need for the addition of an aggregation agent. While not wishing to be bound by theory and while not necessary for practicing the invention, it is believed that the nanosecond pulsed electric fields produce nonporous which allow transport of ions, such as sodium, potassium, and calcium, across the algal cell membrane, thereby neutral- izing the repulsion between individual algae. However, the nanopores produced by the nanosecond electric pulses are not large enough to allow lipids found in the algae to be released. Nanosecond pulsed electric fields of high field strength may also change membrane morphology and in particular dipole alignments in the membrane. In addition, nanosecond pulsed electric fields may change surface charge layers and the distribution of free and bound charges in the matrix in general.

3.3.3 Dehydration Processes

The harvested biomass slurry (typical 5–15 % dry solid content) is perishable and must be processed rapidly after harvest; dehydration or drying is commonly used to extend the viability depending on the final product required. Methods that have been used include sun drying, low-pressure shelf drying, spray drying, drum drying fluidized bed drying, freeze drying, and Refractance Window Drying Technology drying. Sun drying is the cheapest dehydration method, but main disadvantages include long drying times, the requirement for large drying surfaces, and the risk of material loss. Spray drying is commonly used for extraction of high-value products, but it is relatively expensive and can cause significant deterioration of some algal pigments. Spray drying has the advantage of producing finely powdered microalgae. As with drum drying, open-cycle spray drying with direct-fired air heaters exposes microalgae to elevated temperatures (140–200 F) and high oxygen concentrations (19–20 %) and offers no means for recovery of carbon 154 3 From Algae to Liquid Fuels dioxide produced in the dryer’s burner. Freeze drying is equally expensive, especially for large-scale operations, but it eases extraction of oils. Intracellular elements such as oils are difficult to extract from wet biomass with solvents without cell disruption, but are extracted more easily from freeze-dried biomass [116– 118]. Drum drying exposes the algal slurry to long periods of elevated temperatures and produces a flake that must be milled to obtain a fine powder.

3.4 Extraction and Puriﬁcation of Biofuels

3.4.1 Lipid Contents of Algal Biomass

Microalgae produce a variety of lipids such as neutral lipids, polar lipids, wax esters, sterols, and hydrocarbons, as well as prenyl derivatives such as tocopherols, carotenoids, terpenes, and quinone and pyrrole derivatives such as the chlorophylls. Microalgae lipids can be categorized into two classes, i.e., storage lipids (nonpolar lipids) and structural lipids (polar lipids). Storage lipids are mainly in the form of TAG made of predominately saturated FAs and some unsaturated FAs which can be transesterified to produce biodiesel. Structural lipids typically have a high content of polyunsaturated fatty acids (PUFAs), which are also essential nutrients for aquatic animals and humans. Polar lipids (phospholipids) and sterols are important structural components of cell membranes which act as a selective permeable barrier for cells and organelles. Triacylglycerides (TAGs) generally serve as energy storage in microalgae that, once extracted, can be easily converted into biodiesel through transesterification reactions [119, 120]. These neutral lipids bear a common structure of triple esters where usually three long-chain fatty acids (FAs) are coupled to a glycerol molecule. Transesterification displaces glycerol with small alcohols (e.g., methanol). Recently, the rise in petroleum prices and the need to reduce greenhouse gas emission have seen a renewed interest in large-scale biodiesel production [121]. These lipids maintain specific membrane functions, provide the matrix for a wide variety of metabolic processes, and participate directly in membrane fusion events. In addition to a structural function, some polar lipids may act as key intermediates (or precursors of intermediates) in cell signaling pathways (e.g., inositol lipids, sphingolipids, oxidative products) and play a role in responding to changes in the environment. Of the nonpolar lipids, TAGs are abundant storage products, which can be easily catabolized to provide metabolic energy [122]. In general, TAGs are mostly synthesized in the light, stored in cytosolic lipid bodies, and then reutilized for polar lipid synthesis in the dark [123]. Microalgal TAGs are generally characterized by both saturated and monounsaturated FAs. However, some oil-rich species have demonstrated a capacity to accumulate high levels of long-chain polyunsaturated fatty acids (PUFAs) as TAG [124, 125]. A detailed study on both accumulation of TAG in the green microalga Parietochloris incisa 3.4 Extraction and Purification of Biofuels 155 and storage into chloroplastic lipids (following recovery from nitrogen starvation) led to the conclusion that TAGs may play an additional role beyond being an energy storage product in this alga [125, 126]. Hence, PUFA-rich TAGs are metabolically active and are suggested to act as a reservoir for specific fatty acids. In response to a sudden change in the environmental condition, when the de novo synthesis of PUFA may be slower, PUFA-rich TAG may donate specific acyl groups to monogalactosyldiacylglycerol (MGDG) and other polar lipids to enable rapid adaptive membrane reorganization [126, 127]. The lipid content of microalgae can be increased by stress induction technique to a greater extend [128], thus contributing to a high oil yield. The average lipid content varies between 1 and 70 %, but under certain conditions some species can reach 90 % of dry weight [46, 47, 50, 87]. Table 3.6 shows both lipid content and biomass productivities of different marine and freshwater microalgal species [50, 62, 87, 113, 129–132, 137–161]. Some alga like Botryococcus braunii produces about 75 % lipids by weight of dry biomass but associated with low growth rate, whereas in Chlorella, Crypthecodinium, Cylindrotheca, Dunaliella, Isochrysis, Nannochloris, Nannochloropsis, Neochloris, Nitzschia, Phaeodactylum, Porphyridium, Schizochytrium, and Tetraselmis, the lipid content varies from 20 to 50 % with higher growth rate. Thomas et al. [160] report the fatty acid contents of few freshwater microalgal species showing that all of them synthesized C14:0, C16:0, C18:1, C18:2, and C18:3 fatty acids. His group has observed that the relative intensity of other individual fatty acid chains is species specific, e.g., C16:4 and C18:4 in Ankistrodesmus sp., C18:4 and C22:6 in Isochrysis sp., C16:2, C16:3, and C20:5 in Nannochloris sp., C16:2, C16:3, and C20:5 in Nitzschia sp. This variation in lipid composition could have been due to variation in nutritional and environmental factors and cultivation conditions. For example, nitrogen deficiency and salt stress induced the accumulation of C18:1 in all treated species and to some extent C20:5 in B. braunii [160]. Other authors also reported a differentiation between fatty acid composition of various algae species [162–164]. Although the microalgae oil yield is strain dependent, it is generally much greater than other vegetable oil crops, as shown in Table 3.7 that compares the biodiesel production efficiencies and land use of microalgae and other vegetable oil crops, including the amount of oil content in a dry weight basis and the oil yield per hectare, per year [47, 165–174].

Table 3.6 Various process technologies to derive algae–fuels Type of biofuel Processing technology Biodiesel Solvent extraction followed by transesteriﬁcation Ethanol Anaerobic fermentation of residual biomass or by using genetically engineered algae, directly Hydrogen Regulating biochemical processes in algae; gasiﬁcation/pyrolysis Electricity/heat Direct combustion Algal methane to electricity 156 3 From Algae to Liquid Fuels

Table 3.7 Lipid content and productivities of different microalgal species [43, 129–136] Marine and freshwater microalgal species Lipid content (% dry weight biomass) Ankistrodesmus sp. 24.0–31.0 Botryococcus braunii 25.0–75.0 Chaetoceros muelleri 33.6 Chaetoceros calcitrans 14.6–16.4 Chlorella emersonii 25.0–63.0 Chlorella protothecoides 14.6–57 Chlorella sorokiniana 19.0–22 Chlorella vulgaris 5.0–58.0 Chlorella sp. 10.0–48 Chlorella pyrenoidosa 2.0–2 Chlorella 18.0–57 Chlorococcum sp. 19.3 Crypthecodinium cohnii 20 Dunaliella salina 6.0–25 Dunaliella primolecta 23 Dunaliella tertiolecta 16.7 Dunaliella sp. 17.5–67 Ellipsidion sp. 27.4 Euglena gracilis 14.0–20.0 Haematococcus pluvialis 25.0–0.05 Isochrysis galbana 7.0–40.0 Isochrysis sp. 7.1–33 Monodus subterraneus 16.0 Monallanthus salina 20.0–22.0 Nannochloris sp. 20.0–56.0 Nannochloropsis oculata 22.7–29.7 Nannochloropsis sp. 12.0–53.0 Neochloris oleoabundans 29.0–65 Nitzschia sp. 16.0–47 Oocystis pusilla 10.5 Pavlova salina 30.9 Pavlova lutheri 35.5 Phaeodactylum tricornutum 18.0–57 Porphyridium cruentum 9.0–18.8 Scenedesmus obliquus 11.0–55.0 Scenedesmus quadricauda 1.9–18 Scenedesmus sp. 19.6–21 Skeletonema sp. 13.3–31 Skeletonema costatum 13 Spirulina platensis 4.0–16 Spirulina maxima 4.0–9.0 Thalassiosira pseudonana 20.6 Tetraselmis suecica 8.5–23 Tetraselmis sp. 12.6–14 3.4 Extraction and Puriﬁcation of Biofuels 157

3.4.2 Algae Lipid Extraction

Following cell concentration to 200 g/L, the algal material is subjected to wet extraction process. The first step in this process is to complete or nearly complete destruction or removal of the cell wall. Microalgal cell walls contain cellulose, which make their complete lysis by organic solvents alone difficult or impossible. In addition, vesicle walls are made of lipid mono-bilayers, which must also be disrupted or lysed, but are susceptible to chemical lysis by organic solvents and aqueous detergents. Because chemical lysis requires toxic chemicals that must be separated from the desired products, cell concentrates are usually lysed nonchemically using one or more of high-pressure homogenization, supercritical fluid homogenization, electroporation, and radiation, all of which are energy intensive because of the dissipative effects of the intervening aqueous media. Lipids are then usually removed from the cell lysate via solvent extraction process. Algal lipids extraction process is completely different from the conventional methods, generally used for terrestrial oilseed crops [175, 176]. Therefore, oil extraction from algae is performed by using organic solvents, electroporation, ultrasonic, and supercritical CO2 methods.

3.4.2.1 Solvent Extraction

Since 1959, after the work of Folch et al. [110], extensive reports are available on total lipid extraction process from plants and microbes. But the main drawback of solvent extraction was the use of chloroform in total lipid extraction from endogenous cells. In solvent extraction process, some protein molecules are left with the lipid extract. These residual proteins must be separated before further processing of crude lipids for biodiesel. In 1959, Blish and Dyer [177] could find out the way for protein separation from the crude lipid extract. Nowadays, this method with some modification is widely used for estimation of algal lipids spectrophotometrically. In order to make this method more efficient, instead of water, 1 M NaCl is used to prevent binding of acidic lipids to denaturize lipids. It has been observed that addition of 0.2 M phosphoric acid and HCl improves lipid recovery with a shorter separation time in comparison with the earlier extraction methods. Hajra’s method [178] of lipid extraction was demonstrated to be the most efficient procedure for the extraction of plant sphingolipids [179]. The last decade has witnessed lot of improve methods [180–182] to increase the efficiency of total lipid extraction from various biological samples. 158 3 From Algae to Liquid Fuels

3.4.3 Transesteriﬁcation for Biodiesel Production

Transesterification is used to chemically convert algal oil or lipids to fatty acid methyl esters (FAME), which lowers the viscosity of the oil while producing a fuel which more closely resembles petroleum-derived diesel. Algal oil is converted into biodiesel through a transesterification process. Oil extracted from the algae is mixed with alcohol and an acid or a base to produce the fatty acid methyl esters that make up the biodiesel. An excess of methanol is used to force the reaction to favor the right side of the equation. The excess methanol is later recovered and reused [183]. If biomass is grown in a sustained way, its combustion has no impact on the CO2 balance in the atmosphere, because the CO2 emitted by the burning of biomass is offset by the CO2 fixed by photosynthesis.

The process of producing microalgal oil consists of producing microalgal biomass that requires light, carbon dioxide, water, and inorganic nutrients (nitrates, phosphates, and iron). About half of the dry weight of microalgal biomass is carbon, which is usually derived from carbon dioxide. Therefore, producing 100 tonnes of algal biomass fixes roughly 183 tonnes of carbon dioxide. Optimal temperature for growing many microalgae is between 293 and 303 K. A temperature outside this range could kill or damage the cells. Algae lipids react by transesterification to produce biodiesel. Transesterification reaction of triglycerides with an alcohol in the presence of a catalyst produces fatty acid chains (biodiesel) and glycerol [184–186]. Methanol, ethanol, propanol, and butanol are common alcohols used for transesterification reaction [187]. Limitations in the transesterification reaction are mainly due to oil impurities, reaction conditions (time and temperature), and catalyst nature (acid, basic, enzymes) [188, 189]. Transesterification using methanol and ethanol produces fatty acid methyl esters (FAME) and fatty acid ethyl esters (FAEE), respectively. Methanol is preferred for being more economic and has lower reaction times compared with higher alcohol molecules. The stoichiometry of reaction requires 3 mol of methanol and 1 mol of triglyceride to give 3 mol of FAME and 1 mol of glycerol [190]. For a maximum performance, this ratio must be greater than the stoichiometric ratio since the reaction is reversible. Therefore, an excess of alcohol shifts the equilibrium to the product said. 3.4 Extraction and Purification of Biofuels 159

3.4.3.1 In Situ Transesteriﬁcation

In situ transesterification is a single process where lipid extraction from dewatered algae and transesterification of lipids to biodiesel are carried out simultaneously in sub- or supercritical methanol, as shown in Fig. 3.12. The algal oil extracted contains free fatty acids and triglycerides. The fatty acid profiles of algal oils vary widely, depending on the strains of microalgae, the level of maturity of the microalgal biomass harvested, and the conditions of oil extraction. In this process methanol is used as a reactant and also a solvent. The reaction is carried out at 239.5 C and 8.14 MPa (1180 psi). Under this condition, the triglycerides and free fatty acids can be converted to methyl esters with the potential of using no catalysts, homogenous, or heterogeneous. After reaction, the methanol can be easily separated from the biodiesel because at room temperature, methanol has a very limited solubility in biodiesel and is thus immiscible. The methanol will be cycled back and reused. Figure 3.13 represents a schematic diagram of in situ transesterification and other integrated processes for methanol reused and removal of algal-spent biomass to be used as biofertilizer or used for obtaining value-added

Recovered Methanol methanol

Transes- Algal spent Methanol Microalgae terification Residue Dewatering reacor Recovery

Crude Glycerol Algaldiesel

Fig. 3.12 Proposed biodiesel production from microalgal oil

Fig. 3.13 Schematic diagram of the reactor system for this project 160 3 From Algae to Liquid Fuels chemicals. The small quantity of methanol in biodiesel can be removed using established technologies such as ﬂash evaporation; therefore, that will not be researched in this project. The leftover microalgae debris will be separated from the liquids via simple ﬁltration.

3.5 Bioethanol from Algae

3.5.1 Introduction

Algae are among the most potentially signiﬁcant sources of sustainable biofuels in the future as renewable energy. Besides high lipid content, some algae are rich in starch/cellulose (Table 3.8). So variety of fuels, i.e., ethanol, methanol, algal diesel, biogas, and hydrogen can be obtained directly or indirectly through

Table 3.8 Comparison of microalgae with other biodiesel feedstocks [129, 133, 145] Biodiesel Plan corn/maize (Zea Seed oil content Oil yield Land use (m2 productivity mays L.)[44, 66, 139, (% oil by wt in (L oil/ year/kg (kg biodiesel/ 155] t source biomass) ha year) biodiesel) ha year) Corn/maize (Zea mays 44 172 66 152 L.) Hemp (Cannabis sativa 33 363 31 321 L.) Soybean (Glycine max 18 636 18 562 L.) Jatropha (Jatropha 28 741 15 656 curcas L.) Camelina (Camelina 42 915 12 809 sativa L.) Canola/rapeseed (Bras- 41 974 12 862 sica napus L.) Sunﬂower (Helianthus 40 1070 11 946 annuus L.) Sunﬂower (Helianthus 40 1070 11 948 annuus L.) Castor (Ricinus 48 1307 9 1156 communis) Palm oil (Elaeis 35 5266 2 4747 guineensis) Microalgae (low oil 30 55,700 0.2 51,921 content) Microalgae (medium oil 50 97,800 0.2 86,515 content) Microalgae (high oil 70 136,400 0.1 121,104 content) 3.5 Bioethanol from Algae 161

Mass scale algae cultivation Cellulk harvesting Algal biomass dehydration Algal lipid extraction • Open ponds/Race ways • Bulk harvesting Algal lipids to algal diesel • Close type photobioreactors • Thickening (Triglyceride refining) • Algal fermenter

Algal biodiesel for : Automobile/jet Energy fuel/electricity

• Elecricity • Heat Algal spent residue/ Anaerobic digestion Biogas Trace amount oil

Green fertilizer

Fig. 3.14 Process block diagram for algal lipids to biodiesel through an eco-friendly integrated process physicochemical processes (Fig. 3.14). However, the composition of carbohydrates mainly varies on the basis of types of algae and their respective habits and habitats. These algal carbohydrates can be converted to bioethanol and be used as an ingredient in petrol. Ethanol is better known as alcohol. The word “bioethanol” means ethanol that can be made from biomass mainly for transport use. Macroalgae can be used to make bioethanol. Macroalgae that contain a high amount of sugar are used in the production of bioethanol. The macroalgae are cut and treated to free up the sugars within the algae. These sugars decompose to a simple sugar called “glucose.” The macroalgae is now a “feedstock” for the next step. Yeast is then added to the process. During this time there is a chemical reaction produced by the yeast called “fermentation.” Fermentation is when the yeast uses glucose and produces ethanol and other components. The ethanol is then separated from the other components by heating it up until it boils and then cooling the vapors. This is called distillation. The bioethanol undergoes a further ﬁltering process to remove water so it can then be used as an ingredient with petrol. Thus, both macroalgae and microalgae are optimal source for third-generation biofuel (Fig. 3.15) due to the fact that they are high in carbohydrates/polysaccharides and thin cellulose walls and are viable means for environmental and economic sustainability due to their luxury growth in rapid manner (Table 3.9). The ethanol yield of different types of algae differs from species to species and their respective habits and habitats. Some prominent strains of macroalgae like Sargassum, Gracilaria, Prymnesium parvum, and Euglena gracilis have high carbohydrate content and hence are promising candidates for ethanol production. However, microalgae are currently being used as an ideal biofuel feedstock due to their rapid growth rate, CO2 ﬁxation capacity, and high production capacity of lipid. Moreover, they do not compete with food or feed crops and can be cultivated on nonarable land [47, 87, 193]. 162 3 From Algae to Liquid Fuels

Biofuels

Primary Secondary

Firewood, wood 1st generation 2nd generation 3rd chips, pellets, Bioethanol or butanol by Bioethanol and biodiesel generation animal waste, fermentation of starch produced from Biodiesel from forest and crop (from wheat, barley, conventional microalgae residues, landfill corn, technologies Bioethanol gas potato) or sugars but based on novel from (from sugarcane, sugar starch, microalgae and beet, etc.) oil and sugar crops such seaweeds Biodiesel by as Hydrogen transesterification of oil Jatropha, cassava or from green crops (rapeseed, Miscanthus; microalgae and soybeans, Bioethanol, biobutanol, microbes sunflower, palm, syndiesel produced from coconut, lignocellulosic materials used cooking oil, animal (e.g. straw, wood, and fats, etc.) grass)

Fig. 3.15 Classiﬁcation of biofuels

Table 3.9 Biomass composition of microalgae expressed on a dry matter basis ([13, 14, 191]) Strain Protein Carbohydrates Lipid Anabaena cylindrica 43–56 25–30 4–7 Botryococcus braunii 40 2 33 Chlamydomonas reinhardtii 48 17 21 Chlorella pyrenoidosa 57 26 2 Chlorella vulgaris 41–58 12–17 10–22 Dunaliella bioculata 49 4 8 Dunaliella salina 57 32 6 Dunaliella tertiolecta 29 14 11 Euglena gracilis 39–61 14–18 14–20 Porphyridium cruentum 28–39 40–57 9–14 Prymnesium parvum 28–45 25–33 22–39 Scenedesmus dimorphus 8–18 21–52 16–40 Scenedesmus obliquus 50–56 10–17 12–14 Scenedesmus quadricauda 47 – 1.9 Spirogyra sp. 6–20 33–64 11–21 Spirulina maxima 60–71 13–16 6–7 Spirulina platensis 42–63 8–14 4–11 Synechococcus sp. 63 15 11 Tetraselmis maculata 52 15 3 3.5 Bioethanol from Algae 163

3.5.2 Microalgae as a Potential Source of Bioethanol

The unicellular marine microalgae are an abounding resource for carotenoids, lipids, and polysaccharides and were widely investigated in the fields of food supplements and biofuel production [194]. Terrestrial plants in temperate climates are having solar energy capturing efficiency about 1 %, whereas microalgae can convert up to 5 % of the solar energy into chemical energy [195]. Certain species of microalgae have the ability of producing high levels of carbohydrates instead of lipids as reserve polymers. These species are ideal candidates for the production of bioethanol as carbohydrates from microalgae can be extracted to produce fermentable sugars. It has been reported that approximately 5000–15,000 gal of ethanol/ acre/year (46,760–140,290 L/ha) can be produced from microalgae. This yield is several orders of magnitude larger than yields obtained for other feedstocks (Table 3.10). Microalgae provide feedstock for renewable liquid fuels such as biodiesel and bioethanol [133]. Microalgal biomass contains three main components: proteins, carbohydrates, and lipids (oil) [196]. The biomass composition of various microalgae in terms of those main components is shown in Tables (3.11 and 3.12). An integrated production of biofuels from microalgae (Fig. 3.16) may be helpful in finding out a better option for renewable energy, in sustain pattern. The integrated project based on algal biomass includes a microalgal cultivation step,

Table 3.10 Baseline algal growth assumptions Open pond PBR Scale (MM gal/year algal oil) 10 10 Algae productivity 25 (g/m2/day) 1.25 (kg/m3/day) Algal cell density (g/L) 0.5 4 Lipid yield (dry wt.%) 25 % 25 % Operating days/year [330] Productivity is on an areal basis (g/m2/day) for open ponds and a volumetric basis (kg/m3/day) for PBRs [192]

Table 3.11 Ethanol yield from different sources Source Ethanol yield (gal/acre) Ethanol yield (L/ha) Corn stover 112–150 1 050–1400 Wheat 277.2 590 Cassava 354 3310 Sweet sorghum 326–435.3 050–4070 Corn 370–430.3 460–4020 Sugar beet 536–714.5 010–6680 Switchgrass 1150 10,760 Microalgae 5000–15,000 46,760–140,290 Note: The above table was reviewed by Mussatto et al. [240] 164 3 From Algae to Liquid Fuels

Table 3.12 Potential algae for bio-ethanol production Microalgae Cyanobacteria Macroalgae Chlamydomonas reinhardtii Nostoc muscorum Ulva rigida Chlorella sp. N. maculiform Caulerpa geminat Chlorococcum sp. N. paludosum Enteromorpha sp. Scenedesmus obliquus Phormidium angustissimum Gelidium sp. S. acuminatus Spirulina fusiformis Kappaphycus sp. S. acutiformis S. maxima Gigartina livida S. acutus Synechococcus sp. Saccharina latissima S. arcuatus Sacchorhiza sp. S. armatus Laminaria hyperbore Spirogyra sp. Sargassum sp. Oedogonium sp. Alaria sp.

Fig. 3.16 An integrated schematic proposal for getting various types of biofuels from microalgae biomass followed by the separation of the cells from the growth medium and subsequent lipid extraction for biodiesel production through transesteriﬁcation, and the leftover algal residual cake can be further process for bioethanol. At present, there is intensive research on the use of microalgae to produce biofuels. The main attention is given on species diversity and improvements of cultivation process to produce microalgae rich in carbohydrates for ethanol production. However, there is also an interest on use of microalgae to produce biofuels through other biomass energy conversion technologies such as anaerobic digestion 3.5 Bioethanol from Algae 165

[197, 198], thermochemical technologies [199, 200], and anaerobic fermentation [201]. Especially the latter is a biological technology in which the only substrate for fermentation is carbohydrates. Due to a wide variation in carbohydrate contents in microalgae, not much encouraging research is noticed [201–211]. However, Chlo- rella vulgaris has been considered as a potential raw material for bioethanol production because it can accumulate high levels of starch [53, 210] and it can accumulate up to 37 % (dry weight) of starch. However, higher starch contents can also be obtained for optimized culture conditions [212]. Microalgal biomass composition can be manipulated by applying various stress cultivation conditions. Under stress conditions, such as nutrient starvation or high light intensity, some microalgal species accumulate carbohydrates in their biomass, which can amount to a significantly high level (a content of up to 65 %) [213, 214]. When Chlamydomonas reinhardtii was grown under high illumination of 450 μE/m2 s and with the addition of acetic acid for pH regulation, the carbohydrate deposit in this alga was noticed to increase about 60 % of dry biomass [45, 215]. In the same way, the carbohydrate accumulation in Scenedesmus obliquus and Chlorella vulgaris could increase significantly high under nitrogen starvation condition [216, 217]. Arthrospira platensis is a filamentous cyanobacterium having high growth rates, as compared with other algae. Generally, the carbohydrate content of A. platensis is 10 %–20 %, but under phosphorus limitation, carbohydrate accumulation increases to about 60 %–65 % of dry biomass. Besides this, the bioflocculation capability of this alga can be increased by monitoring the nutrient supply [218]. The limitation for microalgal carbohydrates applied to bioethanol production is that ethanologenic yeasts can ferment only simple sugars, such as glucose, fructose, etc. Consequently, microalgae carbohydrates (sugar polymers) have first to be hydrolyzed (saccharified) to simple sugars, so that they can be fermented by the ethanologenic yeasts [219]. Several hydrolysis methods have been developed and used for biomass carbohydrate saccharification. The most common method for hydrolysis of carbohydrates from, e.g., cellulosic biomass, for bioethanol production is the use of enzymes. However, acid hydrolysis is also frequently studied [220–222]. Due to lack of lignin, hemicelluloses, and even cellulose [223], A. platensis is easily compatible with the hydrolysis process. A. platensis contains carbohydrates mainly in the form of structure components of cell walls (peptido- glycans) and in the form of intracellular storage components (glycogen granules) [224]. Chlorella vulgaris has been considered as a potential raw material for bioethanol production because it can accumulate high levels of starch [212]. Spirogyra species and Chlorococcum sp. have been shown to accumulate high levels of polysaccharides both in their complex cell walls and as starch. This starch accumulation can be used in the production of bioethanol [37, 225]. Harun et al. [37] have shown that the green alga Chlorococcum sp. produces 60 % higher ethanol concentrations for samples that are pre-extracted for lipids versus those that remain as dried intact cells. Results showed a maximum bioethanol concentration of 3.83 g l-1 obtained from 10 g l-1 of lipid-extracted microalgae debris [48, 54, 226, 227]. It has also been reported that dark fermentation in the marine green alga Chlorococcum littorale was able to produce 450 μmol ethanol g-1 at 30 C 166 3 From Algae to Liquid Fuels

[55, 228]. This indicates that microalgae can be used for the production of both lipid-based biofuels and for ethanol biofuels from the same biomass as a means to increase their overall economic value [213]. However, the production of bioethanol from microalgae is still under investigation, and this technology has not yet been commercialized [226].

3.5.3 Ethanol Production from Macroalgae

On the basis of pigment content, the seaweeds can be classified into three groups, i.e., green, brown, and red. All types of seaweeds contain low quantity of various types of glucans which are polysaccharides composed of glucose. A unit of seaweed contains more potential ethanol than corn or switchgrass. Generally, seaweeds are having low lignin or completely lack of lignin [229]. Yanagisawa et al. [230] investigated new method to produce high concentration of ethanol from seaweeds. An ethanol yield of more than 3 % was obtained from these seaweeds. Today, the most important microorganisms applied for ethanol production are the yeast Sac- charomyces cerevisiae and the bacterium Zymomonas mobilis. However, both these ethanol producers have a very narrow substrate range. Horn et al. [176] have reported that Zymobacter palmae could produce ethanol from mannitol in seaweed extract, if some supply of oxygen was provided. In addition, the possibility of ethanol production from Laminaria hyperborea extracts was evaluated, focusing on the yeast Pichia angophorae and its potential of utilizing both mannitol and laminaran as substrates. It has been noticed that Pichia angophorae was able to utilize both mannitol and laminaran for ethanol production [231]. While having low lignin content, macroalgae contain significant amount of sugars (at least 50 %) that could be used in fermentation for bioethanol production [232]. However, in certain marine red algae, the carbohydrate content is influenced by the presence of agar, a polymer of galactose and galactopyranose. Current research seeks to develop methods of saccharification to unlock galactose from the agar and further release glucose from cellulose leading to higher ethanol yields during fermentation [232, 233].

3.5.4 Algae Ethanol Commercialization

Currently, ethanol is produced in the USA from corn, with over 13 billion gallons produced in 2010. Most ethanol is blended into gasoline and then sold at a 10 % level referred to as E10. E85—a blend of 85 % ethanol and 15 % traditional gasoline—is an alternative fuel designed for use in ﬂexible-fuel vehicles (FFVs). There are more than 8.5 million FFVs on America’s roads today. Interestingly, it has been noticed that blue-green algae do produce small amounts of ethanol naturally (Fig. 3.17), but only under anaerobic conditions when the cyanobacteria are starved or in the dark. Researchers of Algenol Biofuel, Florida, have taken beneﬁt of this phenomenon and tried to develop new mutants that can produce 3.5 Bioethanol from Algae 167

Fig. 3.17 Schematic diagram on ethanol biosynthesis in blue-green algae (Direct to ethanol technology is based on an intracellular photosynthetic process in cyanobacteria). Source: www. greenpacks.org 500 Â 460 Search by image higher quantity of ethanol under sunlight through photosynthesis, first by turning carbon dioxide and water into sugars and then by boosting and controlling the enzymes that synthesize those sugars into ethanol. Recently, Algenol and Dow Chemical jointly have undertaken a demonstration project that could end up producing 100,000 gal of ethanol annually. The photosynthetic algae turn sunlight, carbon dioxide, and water into sugars that are synthesized into ethanol within the algae cells (Fig. 3.18). The researchers of Algenol company have strong confidence that it can efficiently produce commercial quantities of ethanol directly from algae without the need for freshwater or agricultural lands—a novel approach that has captured the interest and backing of Dow Chem- ical, the chemical giant-based company in Midland, MI. There are dozens of companies in the market trying to produce biofuels from algae, but most to date have focused on growing and harvesting the microorganisms to extract their oil and then refining that oil into biodiesel or jet fuel. Instead, Algenol has chosen to genetically enhance certain strains of blue-green algae, also known as cyanobacteria, to convert as much carbon dioxide as possible into ethanol using a 168 3 From Algae to Liquid Fuels

CO2 Glucose G6P Ru5P Light CO2

O2 RuBP H2 sucrose F6P GLX PSI&PSII Calvin Cycle Isoprene GAP GLY NADPH & Lipids ATP

SER 3PG Fatty Acyl-ACP

Isobutanol Ethanol

D,L-Lactate PEP Mal-CoA PYR CO 3HB PHB 2 CO2 Fatty Acetyl-CoA Acetone Aldehyde Butanol CO2 CIT CO OAA Alkane/Alkene ICT GLX MAL Free Fatty Acids CO2 AKG Ethylene SUC

SucCoA CO2 Cyanophycin SSA Fatty alcohol

Fig. 3.18 Central metabolic pathways and products from Synechocystis 6803. The functions of several pathways (marked as dot lines) are still not veriﬁed. Abbreviation: 3PG 3-phosphoglycerate; 3HB 3-hydroxybutyrate; AKG α-ketoglutarate; CIT citrate; F6P fructose 6-phosphate; G6P glucose 6-phosphate; GAP glyceraldehyde 3-phosphate; GLX glyoxylate; GLY glycine; ICT isocitrate; MAL malate; Mal-CoA malonyl-CoA; OAA oxoacetate; PEP phos- phoenolpyruvate; PHB polyhydroxybutyrate; PSI and PSII photosystem I and photosystem II; PYR pyruvate; Ru5P ribulose-5-phosphate; RuBP ribulose-1,5-diphosphate; SER serine; SUC succinate; SucCoA succinyl-CoA; SSA succinic semialdehyde 3.5 Bioethanol from Algae 169

Fig. 3.19 Schematic diagram of obtaining ethanol, directly from algae using ordinary evaporation and condensation within the bioreactor; the ethanol can be collected without emptying the containers and/or killing the algae and then be processed to jet fuel, biodiesel, or plastic monomers in existing processes. Source: Algenol process that doesn’t require harvesting to collect the fuel. The ethanol produced inside the cells will seep out of each cell and evaporate into the headspace of the bioreactor (Fig. 3.19). The cost interesting aspect of this project is that it does not have to harvest its algae to extract the ethanol, eliminating a step that has proved costly and complex for other algae-to-biofuel start-ups [234–237]. The companies recently announced plans to build and operate a demonstration plant on 24 acres of property at Dow’s sprawling Freeport, TX, manufacturing site. The plant will consist of 3100 horizontal bioreactors, each about 5 ft wide and 50 ft long and capable of holding 4000 L. The EU-funded project DEMA (Direct Ethanol from MicroAlgae) is in progress to produce bioethanol directly from metabolically engineered strain of the cyanobacterium, Synechocystis sp. PCC 6803 commonly found in almost every terrestrial and aquatic habitat, including in oceans, lakes and damp soil, and on rocks—for less than €0.40/L (US$2.00/gal). Mainly, the project is being operated in two physical manners to achieve the goal for economizing the ethanol production process. In ﬁrst phase, the bioethanol production could reach > 1–2 % (v/v) by direct transform CO2,H2O, and sunlight into bioethanol through metabolically engineered algal strains. In the second phase, the bioethanol is continuously extracted from the 170 3 From Algae to Liquid Fuels culture media via a membrane technology process exploiting existing EU expertise and technology. The project is being organized by the University of Limerick in Ireland with a budget of €5 million (US$6.6 million) from the EU under the energy strand of the Seventh Framework Programme (FP7). Nine partners from both academia and industry from six EU countries are intensely involved in this project. Seaweed Bioethanol Production in Japan, titled the “Ocean Sunrise Project,” aims to produce seaweed bioethanol by farming and harvesting Sargassum horneri, utilizing 4.47 million km2 (sixth largest in the world) of unused areas of the exclusive economic zone (EEZ) and maritime belts of Japan. This is considered to be vital for Japan to produce biofuels from these oceanic resources. Through seaweed bioethanol production, the project aims to combat global warming by contributing an alternative energy to fossil fuel. The primary form of sugar in these seaweeds is called alginate. Unfortunately, no bacteria species was known that could convert alginate to ethanol. Adam Wargacki and colleagues at the Bio Architecture Lab (BAL) genetically engineered a new strain of E. coli bacteria, which can feed on the sugars found in brown seaweed and transform the sugars into ethanol. The ethanol is created in a similar process to making beer. The alginate sugars are fed to the bacteria in an environment without oxygen. If oxygen were present, bacteria would transform the sugar into carbon dioxide, the same things humans do when we eat food. However, in the absence of oxygen, bacteria ferment the sugar and produce ethanol. According to the report, about 200 ml of bioethanol was obtained from 1 kg of seaweeds by this process [238]. The Sendai Power Station of Tohoku Electric Power Co., Inc., reported that some 300 tonnes of seaweeds are sucked into the cooling water intake and are discharged as waste every year. Applying the abovementioned new technology, the waste seaweeds could be utilized as a marine biomass resource, and the bioethanol thus obtained could be burnt as fuel at the power station. The most amazing thing about this project is that sea algae and seaweeds can yield biofuels at quite higher rate than any plants on terrestrial or aquatic ecosystem. Soybean oil and palm oil can be produced in 1900 ‘ (liter) and 5950 ‘ of biofuel per year on one hectare of land. Algae, however, can yield 98,500 ‘ that is many times as much! In addition some algae can absorb nearly ten times as much CO2 as any land-born plants can.

3.5.5 Processing Ethanol from Algae

The microalgae which are minute photosynthetic organisms typiﬁed by Chlorella, Dunaliella, Chlamydomonas, Scenedesmus, Spirulina, and the like are known to contain a large amount (more than 50 % of the dry weight) of starch and glycogen useful as raw materials for the production of ethanol. These microalgae can be used as starting materials for the production of ethanol [226, 234]. In the ﬁrst step, a microalga is grown by culturing it photoautotrophically in the presence of light through carbon dioxide assimilation by photosynthesis or by culturing it heterotrophically in the dark and in the presence of organic materials References 171 such as sugars and organic acids. In the second step, since the grown microalga stores starch mainly in the cells thereof, the starch is released from the cells with the aid of a mechanical means (e.g., ultrasonic or explosive disintegration) or an enzyme for the dissolution of cell walls [239]. Then, the starch is separated by extraction with water or an organic solvent. In the third stage, Saccharomyces cerevisiae is added to the biomass to begin fermentation. The product of fermentation is ethanol. The ethanol is drained from the tank and pumped to a holding tank to be fed to a distillation unit.

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4.1 Introduction

Microorganisms may be harnessed through MFC to convert organic materials into electricity and hydrogen. The MFC is considered to be a promising sustainable technology to meet increasing energy needs, especially by using wastewaters as substrates, resulting in electricity and clean water as final products. So, MFCs may well have a place in the future energy paradigm, as well as in bioremediation and industrial–chemical and hydrogen production (Fig. 4.1). The MFC can convert biomass spontaneously into electricity through the metabolic activity of the microorganisms. In a typical MFC (MFC), microbes transfer electrons through a traditional electron transport chain onto an electrode surface-generating electricity while producing a proton motive force for ATP generation. MFC-based research continues to expand knowledge about the diversity of extracellular electron transfer processes, mechanisms used in such processes, and biofilm ecology. Although gram-positive bacteria can generate electrical energy in MFCs, how they transfer electrons without outer membranes is a mystery. The MFC get right to the heart of many of the principles we run into in the discussion of bioenergy. So, a typical MFC is an excellent way to illustrate electron transfer principles and to discuss subjects such as reduction and oxidation (Fig. 4.2). In brief, microbial fuel cells work by creating situations in which bacteria can feed off a substrate or food (such as an agricultural or industrial waste or human sewage waste). The bacteria are kept in an environment lacking oxygen which would normally slow down their growth. This is because oxygen normally acts as an electron acceptor. What happens in the metabolism of the substrates from the waste stream is that electrons in sugars, fats, proteins, or other bioavailable molecules are taken by the bacteria and transferred through a bacterial metabolic pathway in such a way that the electrons can be used by the cells for energy (Table 4.1). Oxygen acts as the driving force in this process as it has a high affinity for the electrons, and it is the ultimate acceptor of the electrons. So without oxygen

Fig. 4.1 A conceptual schematic diagram of direct electricity generation from microbial fuel cell and also showing its use in environment cleaning and water reclamation process [1] (Wen-Wei Li, Han-Qing Yu, and Zhen He (2014)) toward sustainable wastewater treatment by using microbial fuel cell-centered technologies (Energy Environ. Sci., 2014, 7, 911–924)

e- e-

Materia orgánica

- - - H MED e e

NAD+ H O MED 2 NADH

CO2 O NAD+ 2 - H- H

Ánodo Cátodo Bacteria Membrana

Fig. 4.2 Schematic diagram of microbial fuel cell transfer of electrons from bacteria to anode, which is subsequently transferred to cathode linked through electric wire. The two terminals of wire are connected to a bulb. The proton passes through the cathode and helps in the reduction of oxygen to water molecule. (Oxygen is the most suitable electron accepter for a microbial fuel cell due to its high oxidation potential, availability, sustainability, and lack of chemicals.) (Source: www.seminarsonly.com. Ref. [2]) 4.1 Introduction 183

Table 4.1 Generation power and current in MFC with different types of mediators and microorganisms (References [3, 4, 5, 6]) Current References density Power density Microbes used Substrate Mediator Najafpour 1 600 mA m–2 190mWm–2 Saccharomyces Glucose Neutral red et al. [5] cerevisiae Ringeisen 4 4.4 mA m–2 2 2.2 mW m–2 Shewanella Lactate Anthraquinone- et al. [3] oneidensis 2,6-disulfonate (AQDS) Thygesen 85 mA m–2 2 8 mW m–2 Domestic Glucose Humic acid et al. [7] wastewater Veg and 589mAm–2 – Streptococcus Glucose Ferric chelate Fernańdez lactis complex [6] Thygesen 589mAm–2 123mWm–2 Domestic Acetate Humic acid et al. [7] wastewater Rabaey – 479 m W m–2 Mixed Sucrose Mediator-less et al. [8] consortium Glucose Thygesen 145mAm–2 3 2 mW m–2 Domestic Xylose Humic acid et al. [7] wastewater present, none of this electron transfer (known as aerobic respiration) can occur. The cells have ways of using the substrate molecules in the waste stream in the absence of oxygen, but the amount of energy they receive is greatly reduced, and growth under these conditions is limited. An electrode provided by a microbial fuel cell solves this lack of oxygen problem for the bacteria. Bacteria transfer their electrons to the electrode that is linked by a wire to a second electrode in an oxygen-containing environment. The electrons ultimately get transferred to oxygen, which is what the bacteria need for optimal growth, but not before utilizing those electrons in the form of an electric current to power an electronic device. Of course we could always burn the substrate molecules and use the energy to heat water in a steam-powered generator to create electricity, but this would not be as efficient and the energy return is much less. In the past two decades, high-rate anaerobic processes are finding increasing application for the treatment of domestic as well as industrial wastewaters. The major advantages these systems offer over conventional aerobic treatment are no energy requirement for oxygen supply, less sludge production, and recovery of methane gas. In MFCs (Table 4.2), microorganisms interact with electrodes using electrons, which are either removed or supplied through an electrical circuit to generate electricity. For MFCs to be considered more than a laboratory novelty, standardization of data expression is necessary to allow reliable and accurate comparison of results. Advances in the hardware, operation, and microbial components are also needed. However, MFCs are valuable research tools for characterizing the physiology and ecology of extracellular electron transfer, modeling electron flow in complex microbial ecosystems, and framing and testing ecology theory. 184 4 Microbial Fuel Cell (MFC)

Table 4.2 Coulombic efficiencies and maximum power densities or output achieved in MFCs with undefined mixed cultures in the anode chamber using various anode volumes, substrates, and external resistances Synthetic or real Anode External Coulombic wastewater volume Concentration Maximum resistor efficiency substrate type (mg/L) (mg/L) power output (W) (%) Acetate 560 450 48 W/m2 20 98 Glucose 40 2000 3600 mW/m2 <100 89 Glucose 240 NP (g) 4310 mW/m2 <100 81 Glucose 390 467 35 mW/m2 20 74 Acetate 22 1000 286 mW/m2 33 65 Butyrate 22 1000 220 mW/m2 33 50 Hospital 390 332 25 W/m2 NP(g) 36 wastewater Acetate 28 800 506 mW/m2 218 29 Starch 22 1000 242 mW/m2 33 21 BSA 28 1100 354 mW/m2 >50 20 Municipal 390 429 10 W/m2 75 20 wastewater Dextran 22 1000 150 mW/m2 33 17 Sucrose 440 800 29 W/m2 20 14.2 Glucose Glucose 1000 212 mW/m2 33 14 Sucrose Sucrose 1000 170 mW/m2 66 8.1 Swine waste (solu- 28 8320 261 mW/m2 200 8 ble) portion Butyrate 28 1000 305 mW/m2 1000 7.8 Municipal 22 379 70 mW/m2 420 6 wastewater Peptone 28 500 269 mW/m2 750 6.0 Slaughter 28 1420 80 mW/m2 750 5.2 wastewater Brewery 54,000 1168 5 W/m2 10 3.6 wastewater The data are ordered based on coulombic efficiency with the highest percentages on the top Real wastewater substrate solutions (without pre-acidification) are given in bold Adapted from Jeffrey et al. (2008). References: [9–17]

4.2 Fuel Cell Versus MFC

A fuel cell is an electrochemical device capable of the direct conversion of chemical energy into electrical energy [18]. Fuel cells can operate virtually continuously as long as the necessary flows are maintained [19]. The term fuel cell is often specifically applied to hydrogen–oxygen fuel cells. In order to generate electricity, + electrons are stripped from hydrogen gas (H2) molecules to form protons (H ). The protons then traverse a proton exchange membrane (PEM), combining with oxygen (O2) to form water. Many combinations of fuel and oxidant are possible in a fuel 4.2 Fuel Cell Versus MFC 185 cell. It produces electricity from an external fuel (on the anode side) from which electrons are withdrawn and then transferred to an oxidant (on the cathode side). The circuit is closed by an ion or proton exchange connection between the fuel and oxidant chamber. Fuel cells are different from batteries in that they consume reactant, which must be constantly fed into the device, whereas, batteries store electrical energy chemically in a closed system. A battery eventually runs out because there is nothing more to oxidize. Inside a battery, there are two metal plates or posts called electrodes (anode and cathode) where chemical reactions take place and produce electrons. Also inside is a solution called an electrolyte (the lemon juice), which allows positive charges to move in the solution and balances the movement of electrons. In the anode there is a chemical reaction that oxidizes the metal and releases cations and electrons. The cations flow into the electrolyte toward the cathode, and the electrons flow through the wire producing electricity. The anode is commonly referred as the negative electrode and the cathode as the positive electrode. There is also a porous separator in between both electrodes to prevent a short circuit. The fuel cells also differ from internal combustion devices, where a fuel is burned and gas is expanded to do work, in that they convert chemical energy directly into electrical energy. The development of fuel cells offer many advantages over other types of power sources in that they demonstrate a high efficiency, do not emit pollutants, and provide a truly sustainable power output if hydrogen is produced from a renewable energy source [19]. An MFC, also known as a biological fuel cell, is an electrochemical device that generates electricity by utilizing electrons generated from oxidation and reduction process of bacterial respiration activity [18–21]. In this process, microbial fuel cells rely on living biocatalysts to facilitate the movement of electrons throughout their systems instead of the traditional chemically catalyzed oxidation of a fuel at the anode and reduction at the cathode. The performance of an MFC is accompanied by three steps while generating direct electricity. In the first stage of metabolism, glucose is converted by glycolysis to two molecules of pyruvate, during which ATP and NADH are produced. This occurs in the cytosol of the cell. In the second phase, especially in animal cells, pyruvate is catabolized in the mitochondrial cells to CO2 and acetyl-CoA. Subsequently, acetyl-CoA is transferred to oxaloacetate, after which it participates in the citric acid cycle, also known as the tricarboxylic acid cycle or the Krebs cycle. CO2 and high-energy electrons stored as NADH are produced as end products. The molecular oxygen is an essential part to regenerate NAD+ and keeps on continuing the Kreb cycle. In the membrane-bound electron transport chain, NADH passes its high-energy electrons to O2, which would later produce water, H2O. Lastly, in the third phase, membrane-bound oxidative phosphorylation occurs when glucose is catabolized to generate energy. In eukaryotic cells, the process occurs within the mitochondrial membrane, while in prokaryotes, it occurs in the cellular membrane. It is also referred to as the electron transport chain. The process involves the transfer of electrons between electron carriers in order of reduction potential. As the electrons move toward the terminal-oxidizing agent, protons are pumped across the membrane (from inside to outside), creating a transmembrane 186 4 Microbial Fuel Cell (MFC)

Fig. 4.3 Bacterial interactions with electrodes. Mediator shuttling for bacteria to produce electricity in MFCs; the cells need to transfer electrons generated along their membranes to their surfaces (Ref. [22])

Enlarge view of anode surface 4.2 Fuel Cell Versus MFC 187 proton concentration gradient. As protons move back across the membrane, ADP is phosphorylated to form ATP. Electron carriers include NADH, FADH2, and QH2, which are coenzymes that reduce O2. Electrons are transferred to the anode by electron mediators or shuttles (Fig. 4.3)[23, 24], by direct membrane-associated electron transfer [25] or by so-called nanowires [20, 26, 27] produced by the bacteria, or perhaps by other as yet undiscovered means. Chemical mediators, such as neutral red or anthraquinone-2,6-disulfonate (AQDS), are used to help transfer of electrons to anode [28, 29]. Without the use of any exogenous mediators, the MFC is classiﬁed as a “mediator-less” MFC even though the mechanism of electron transfer may not be known [28]. The direct transfer of electrons to an anode by microorganisms was until recently thought impossible, and the production of useful current was only possible through the use of exogenous mediators, which can be expensive [30], toxic, and unstable [31]. These mediators function by acting as an oxidizing agent for respiratory proteins in the outer membrane of the electricigenic bacteria and subsequently transferring the electrons obtained to an anode [32]. However, some organisms have been shown to have developed physiological processes allowing them to utilize an anode as a terminal electron acceptor without the addition of an exogenous mediator [31]. If certain bacteria are grown under anaerobic conditions (without the presence of oxygen), they can transfer electrons to a carbon electrode (anode). The electrons then move across a wire under a load (resistor) to the cathode where they combine with protons and oxygen to form water [29]. When these electrons ﬂow from the anode to the cathode, they generate the current and voltage to make electricity (Fig. 4.4).

Fig. 4.4 Schematic representation of the electron transport process from the inner membrane to the electrode in G. sulfurreducens bioﬁlms. Beyond the respiratory chain, electrons are transferred to PpcA in the periplasm, which is proposed to interact with the OmcB in the outer membrane. For extracellular electron transport, two mechanisms, explained in detail in the text, are proposed: the metallic-like conduction model (a) and the electron superexchange model (b). The electrochemical potential windows of the involved proteins, the measured open circuit potential, and the redox potential of the NAD(P)/NAD(P)H couple are shown on the right as a reference (not to scale) IM, inner membrane; OM, outer membrane; SHE, standard hydrogen electrode (Ref. [33]) 188 4 Microbial Fuel Cell (MFC)

Instead of utilizing an anode as an electron acceptor, the microbes can use a cathode, to which a low level of current is applied, as an electron donor. These electrons can then be used by the organisms to reduce and synthesize useful organic molecules, such as butanol, allowing electrical energy to be changed into chemical energy [34]. In order for MFCs to reach the point where they can produce electricity at a price competitive with other sources of energy, a greater understanding of MFC design and the interactions that occur between electrigens and the fuel cell anode needs to be obtained. One key area of research is in anode engineering and design. The number of organisms that have access to the anode, and therefore the number of electrons that can be transferred, is limited by the surface area of the anode. In addition, the internal resistance of the MFC can have a significant impact on power output. To overcome these issues, advanced materials, such as intertwined carbon nanotube textile fibers, have been developed. Carbon nanotube textile anodes not only provide a dramatic increase in the area available to receive electrons but also greatly increase the efficiency of the system due to the decrease in resistance resulting from the increased surface area [28]. Microbial fuel cell (MFC) utilizes microbial respiration to convert chemical energy into electrical energy. Like any electrochemical cell, an MFC requires an anode wired to a cathode to facilitate the flow of electrons to the cathode and an electrolytic medium to allow positive ions to diffuse to the cathode [20]. Unlike a purely chemical electrical cell, however, there is no metal catalyst required at the anode. Instead, in an MFC, the anode is exposed to a culture of electricigenic bacteria, which obtain electrons from organic molecules. These bacteria use the anode as an external terminal electron acceptor, as the electrons transferred ultimately travel to the cathode where they reduce oxygen [29]. Since the most inexpensive and abundant sources of carbon available are often industrial wastewaters, MFCs have great potential for improving the efficiency of bioremediation for industrial wastes, by allowing energy to be recovered during effluent processing [35]. To increase the efficiency of electron transfer from the plasma membrane to the electrodes, electron mediators (electronophores) have been employed. These molecules have special properties, which permit their insertion across the plasma membrane of microbes. They tap into the electron transport chain, becoming reduced in the process, after which they become reoxidized by transferring electrons to the fuel cell’s anode. Common electronophores include neutral red, methylene blue, and thionine. Biological fuel cells take glucose and methanol from food scraps and convert it into hydrogen and food for the bacteria. Complete oxidation of one mole of glucose to carbon dioxide would liberate 24 mol of electrons:

eÀ þ C6H12O6 þ 6H2O ! 6CO2 þ 24 þ 24H

Therefore, a total of 2.32 Â 106 C of charge per mole of glucose is potentially available to be channeled through an electrical circuit. The magnitude of the current 4.2 Fuel Cell Versus MFC 189 generated from this oxidative process would depend largely on the rate of metabolism and the efﬁciency of electron transfer to the electrode. Some success has been achieved in the conversion of the biochemical energy involved in glucose metabolism into electrical energy. The energy output in an MFC mainly depends on organic substances’ metabolism which involves glycolysis, the citric acid cycle, and the electron transport chain. Electronophores intervene during the electron transport process, carrying electrons from the bacterial plasma membrane to the anode (negative electrode). These electrons travel through an electric circuit and subsequently reduce ferricyanide to ferrocyanide at the cathode (positive electrode). Protons pumped from the bacteria into the anode environment traverse the PEM into the cathode compartment. Ferrocyanide is reoxidized to ferricyanide, while the hydrogen ions combine with oxygen to form water:

ðÞÀ3 þ À ! ðÞÀ3 4Fe CN 6 4e 4Fe CN 6 ðÞÀ4 þ þ þ ! ðÞÀ3 þ 4Fe CN 6 4H O2 4Fe CN 6 2H2O

All bacteria utilize organic materials, mostly different types of carbohydrates to generate energy in the form of nucleoside triphosphates (ATP and GTP), reduced coenzymes (NADH, QH2), and ion concentration gradients to support their own growth and development. In bacterial cells when glucose is oxidized to carbon dioxide (CO2) and water (H2O) release about 2800 kJ/mol of energy. However, the energy sources that different bacteria use depend on the speciﬁc enzymes those bacteria produce. Heterotrophic bacteria often use carbohydrates as energy sources. Many bacteria use glucose, a monosaccharide or simple sugar, because many bacteria possess the enzymes required for the degradation and oxidation of this sugar. Fewer bacteria are able to use complex carbohydrates like disaccharides (lactose or sucrose) or polysaccharides (starch). Disaccharides and polysaccharides are simple sugars that are linked by glycosidic bonds; bacteria must produce enzymes to cleave these bonds such that the simple sugars that result can be transported into the cell. If the bacteria cannot produce these enzymes, then the complex carbohydrate is not used. For example, lactose is a disaccharide consisting of monomeric glucose and monomeric galactose linked by a glycosidic bond. Bacteria that use lactose must ﬁrst produce the enzyme lactase (beta-galactosidase) to break the glycosidic bond between these monomers. Starch is a large polysaccharide consisting of long chains of monomeric glucose linked by glycosidic bonds. Bacteria that use starch produce an exoenzyme, alpha-amylase, that breaks these bonds such that free monomeric glucose is produced. In order to commercialize the MFCs, maximum efforts are in trial based on the following factors: (1) By the selection of more potential bacteria [21, 23, 27] in combination with affective electron mediators [9, 24, 36]. (2) By the use of an anaerobic (non-oxygenated) atmosphere at the anode [20, 23, 24, 27, 34, 37]. 190 4 Microbial Fuel Cell (MFC)

(3) By increasing feeding rates of fuel (sugars) [26, 29]. (4) By the modiﬁcation of electrodes such as immobilization of electronophores [37–39] and the use of conductive polymers [29, 35]. (5) By bubbling oxygen through the cathode compartment [21, 37]. Current densities as high as 1.5 mA cm2 have been reported from microbial fuel cells [26, 28] and power outputs of up to 3.6 Wm2 [9].

4.3 Generation Energetic Molecule and Ions Gradient in MFCs

4.3.1 History of the MFC

Scientists have long known about the connection among chemistry, biology, and electricity. In the late 1700s, Luigi Galvani [40], an Italian anatomist, noted that a detached frog’s leg twitched due to the electrical charges in the atmosphere. His findings helped create the field of electrochemistry. In 1911, scientists in England published one of the first papers on electricity generation by bacteria. Today MFCs are receiving more attention because they are a potential part of the solution to our energy demands and could provide a clean and renewable source of energy. Nature has been taking organic substrates and converting them into energy for billions of years. Cellular respiration is a collection of metabolic reactions that cells use to convert nutrients into adenosine triphosphate (ATP) which fuels cellular activity. The overall reaction can be considered an exothermic redox reaction, and it was with this in mind that an early twentieth-century botany professor at the University of Durham, M. C. Potter [38], first came up with the idea of using microbes to produce electricity in 1911. There was very little interest or advances made in the technology from 1911 to 1967. While Potter succeeded in generating electricity from E. coli, his work went unnoticed for another two decades before [27] Barnet Cohen created the first microbial half fuel cells in 1931. By connecting his half cells in series, he was able to generate a meager current of 2 milliamps. More work on the subject came with a study by Del Duca et al. (1963). But, due to the unstable nature of hydrogen production by the microbes, it was found to be unreliable [37]. Fortunately, this problem was later resolved by Suzuki et al. in 1976 [41]. After a year, in 1977 Suzuki and his coworkers modified the design of MFC, which is being a reference model for the development of new MFC [36, 42]. By the time of Suzuki’s work in the late 1970s, little was understood about how microbial fuel cells functioned; however, the idea was picked up and studied later in more detail first by M. J. Allen [3, 37, 43] and then later by H. Peter [44] Bennetto, both from King’s College London. People saw the fuel cell as a possible method for the generation of electricity for developing countries. His work, starting in the early 1980s, helped build an understanding of how fuel cells operate, and until his retirement, he was seen by many as the foremost authority on the subject. 4.4 Microbiology of Fuel Cell 191

It is now known that electricity can be produced directly from the degradation of organic matter in a microbial fuel cell. Like a normal fuel cell, an MFC has both an anode and a cathode chamber. The anoxic anode chamber is connected internally to the cathode chamber via an ion exchange membrane with the circuit completed by an external wire. In May 2007, the University of Queensland, Australia, in joint scientific and technology collaboration with Foster’s Brewing developed a pilot level 10 L to convert brewery wastewater into carbon dioxide, clean water, and electricity. On the basis of data obtained, later on they could successfully install a 660 gal version for the brewery, which is estimated to produce 2 kW of power [9]. While this is a small amount of power, the production of clean water is of utmost importance to Australia, for which drought is a constant threat [39, 45]. The first patent to describe microbial fuel cell technology was issued to John Davis in 1967 [46]. It was not until the 1990s that research truly began on microbial fuel cell technology and possible applications. Since 1967, there have been very few patents issued, most being given in the 2000s. The field has chosen to publish research, methods, and designs in scientific journals rather than apply for a large number of patents [39].

4.4 Microbiology of Fuel Cell

4.4.1 Types of Microbes in FMCs

The MFCs use microorganisms as the catalysts for directly converting the chemical energy available in the biomass into electricity. Only those microorganisms capable of transferring electrons outside the cell to insoluble electron acceptors (such as iron and other metal oxides or to solid electrodes), called “exoelectrogens,” contribute to electricity generation in MFCs [47]. The microbial community noticed that MFCs implanted in natural conditions are nonspeciﬁc in nature. Currently, the most studied exoelectrogens belong to the α-, β-, γ-, and δ-proteobacteria (e.g., Geobacter sulfurreducens, Geobacter metallireducens, Shewanella oneidensis, Escherichia coli, Rhodopseudomonas palustris)[3, 20, 41, 48, 49], while some non-proteobacteria (e.g., Geothrix fermentans [50]) and yeasts (e.g., Saccharomy- ces cerevisiae [43]) are also capable of exocellular electron transfer. Among all types of bacteria associated with the function of fuel cell, certain Shewanella sp. (S. oneidensis) and several members of the family Geobacteraceae, including Geobacter sulfurreducens and G. Metallireducens, have been shown to be more capable of transferring electrons to an anode through direct contact, compared to others [43, 46]. Shewanella species (Fig. 4.5) can be found in almost any soil on earth. These soil bacteria eat what is in the soil, such as microscopic nutrients and sugars, and in turn produce electrons that are released back into the soil. Electrons are subatomic 192 4 Microbial Fuel Cell (MFC)

Fig. 4.5 This is a high magnification image of Shewanella bacteria, specifically the species S. oneidensis. The bacteria are the cylindrical-shaped rods scattered in this image. (The other parts of the image are ice pieces that the bacteria were submerged in to take this picture.) (Image credit: PLoS Biology) particles that have a negative charge. These electrons can be harnessed and used to create electricity, which is a form of energy. Certain Shewanella spp. (S. oneidensis) produce endogenous electron mediators, which are reversibly reduc- ible molecules that function similarly to exogenous mediators, except that the bacteria biosynthesizes them [51]. The use of mediators confers an advantage by facilitating and allowing access to large insoluble electron acceptors, such as Fe(III) deposits or anodes, without having to directly contact the surface of such molecules. This means that the number of cells in a given surface area of an electron acceptor that can oxidize is effectively increased and that cells can have access to the electron acceptor from farther away. However, there is a significant metabolic cost associated with the production of these mediators, and in open systems the mediators can be lost and therefore ineffective [30]. In an MFC using these soil bacteria, one electrode (specifically the anode)is buried down in damp soil. Down there, the bacteria multiply and cover the electrode (creating a biofilm on it), supplying it with a lot of electrons (Fig. 4.8). At the same time, the other electrode (called the cathode) is placed on the top of the soil, leaving one of its sides completely exposed to the air. Electrons from the bottom electrode travel up a wire to the top electrode, and once there, they react with oxygen (from the air) and hydrogen (made by the bacteria as it digests nutrients in the soil) to create water. Some organisms have been shown to have developed physiological processes allowing them to utilize an anode as a terminal electron acceptor without the addition of an exogenous mediator [31]. The organisms described here do not, however, provide a comprehensive list of potentially electricigenic bacteria and are meant simply to provide examples of key strategies of electron transfer. 4.4 Microbiology of Fuel Cell 193

Fig. 4.6 This is a high magniﬁcation image of Geobacteraceae species S. oneidensis. The bacteria are the cylindrical-shaped rods scattered in this image. (The other parts of the image are ice pieces that the bacteria were submerged in to take this picture.) (Image credit: PLoS Biology)

Several members of the family Geobacteraceae, including Geobacter sulfurreducens and G. Metallireducens, have been shown to be capable of transferring electrons to an anode through direct contact. The exact mechanism by which this transfer occurs is not fully understood, but it is thought that they use some form of electrochemically active protein present on outer surface of the cell, and there is some evidence to suggest that electrically conductive pili may be involved in the process of electron transfer as well [52]. Although there is no known evolutionary pressure for these bacteria to be able to generate an electrical current, it is likely that the mechanism used by Geobacteraceae cells to transfer electrons is the same that they use to reduce insoluble extracellular deposits of Fe(III) and Mn(IV), both of which Geobacteraceae spp. have been shown (Fig. 4.6) to be well adapted to utilize as an electron acceptor [53]. There is an ongoing debate in MFC literature as to the beneﬁts of using a mixed community of bacteria versus a single strain of bacteria that is known to have efﬁcient extracellular electron transfer. However, the option exists to genetically modify bacteria to create an ideal exoelectrogen strain of bacteria [23]. Both Rhodopseudomonas palustris DX-1 and G. sulfurreducens were recently shown to produce larger voltages than mixed cultures of bacteria in MFCs [41, 51].

4.4.2 Bacterial Metabolism and Electron Transfer in MFC

There are two ways the electric energy can be generated, i.e., batteries [18, 19]or fuel cells [54, 55]. In MFCs, bacteria act as living catalysts to convert organic substrates into electricity. While this technology may sound like an answer to our energy crisis, MFCs are not yet viable for its commercial applications. Still, research is committed to optimizing their performance [46, 56]. In a biofuel cell through microbial metabolism, direct electricity is produced by using conventional 194 4 Microbial Fuel Cell (MFC)

Fig. 4.7 Schematic diagram of two-chambered MFC. A typical two-compartment MFC has an anodic chamber and a cathodic chamber connected by a PEM, or sometimes a salt bridge, to allow protons to move across to the cathode while blocking the diffusion of oxygen into the anode (www. cell.com) electrochemical technology [32, 57]. In MFCs the biochemical energy is converted to electric energy by coupling the biocatalytic oxidation of organic compounds to the chemical reduction of an oxidant at the interface between the anode and cathode (Fig. 4.7). For bacteria to produce electricity in MFCs, the cells need to transfer electrons generated along their membranes to their surfaces. Yet, very little is known about bacterial interactions with electrodes. While anodes and cathodes can function in bacterial respiration, research has been focused on understanding microbial anodic electron transfer. Anode-respiring bacteria catalyze electron transfer in organic substrates onto the anode as a replacement for natural extracellular electron acceptors (e.g., ferric oxides or humic substances) by a variety of mechanisms. On the basis of electron release and transfer, the bacteria associated with fuel cells can be grouped into three types, i.e., (1) bacteria that directly transfer electrons to anode using anode as terminal electron acceptors, (2) bacteria that use both natural and chemical mediators to transfer electrons to anode, and (3) those who can accept electron from cathode. In the first category the bacteria require physical contact with the electrode for current production. The contact point between the bacteria and the anode surface requires outer membrane-bound cytochrome complex. There are also several microorganisms reported which can transfer electrons across the membrane by themselves to anode [23]. These microorganisms are stable and have high coulombic efficiency. Shewanella putrefaciens, Geobacteraceae sulfurreducens, Geobacter metallireducens, and Rhodoferax ferrireducens [20, 23, 48, 58] are all effective and form film (Fig. 4.8) on the anode surface and transfer electrons directly to electrode across the membrane. These microbes are able to form a 4.4 Microbiology of Fuel Cell 195

Fig. 4.8 Metal-reducing bacterial bioﬁlm (microbewiki.kenyon.edu, 400 Â 287 Search by image)

biofilm on the anodic surface and transfer electrons via the cell membrane or special conductive pili (also known as nanowires). Since mediators are not needed in this kind of MFC, the operational cost can be reduced, and the concern for environmental pollution caused by artificial mediators is eliminated [59]. Furthermore, this form of MFC is operationally more stable and yields a high CE (coulombic efficiency) (Du et al., 2007 Repeated ref). Hence, mediator-less MFCs are considered to be more suitable for wastewater treatment and power generation (Fig. 4.8). In the second category, the bacteria mediators in oxidized state are easily reduced by capturing electrons from within the membrane of microorganisms. A lot of reports are available [10, 24, 51, 53, 59] on metabolic reducing power produced by Proteus vulgaris or Escherichia coli which can be converted to electricity by using electron mediators, such as thionine or 2-hydroxy-1,4- naphthoquinone (HNQ). Tanaka et al. [60, 61] reported that light energy can be converted to electricity by Anabaena variabilis when HNQ is used as the electron mediator. Park et al. [62] confirmed that with viologen dyes [9, 44] cross-linked with carbon polymers and absorbed on Desulfovibrio desulfuricans, cytoplasmic membranes can mediate electron transfer from bacterial cells to electrodes or from electrodes to bacterial cells. The electron transfer efficiencies in microbial fuel cells could be improved if more suitable electron mediators were used. Good mediators should have the following characteristics [63]: (a) it should be cell membrane permeable; (b) it should have electron affinity more than the electron carries of the electron transport chains; (c) it should possess a high electrode reaction rate; (d) it should be well soluble; (e) it should be completely nonbiodegradable and nontoxic to microbes; and (f) it should be of low cost. These characteristics describe the efficiency of mediators. Contrary to lower redox potential mediators being theoretically better, the higher redox potential mediators for high affinity for electrons absorbing from electron carriers in cell are the best. Methylene blue, neutral red, thionine, Meldola’s blue, and Fe(III)EDTA are synthetic mediators, but the problem is their toxicity which limits their use in MFCs [46, 58, 64]. Microbes are also reported to use naturally occurring compounds including microbial 196 4 Microbial Fuel Cell (MFC) metabolites (endogenous mediators) such as humic acids, anthraquinone, and oxyanions of sulfur (sulfate and thiosulfate [65]). All of them can transfer electrons from inside the cell membrane to the anode. In the third category of microbes such as Thiobacillus ferrooxidans, the cathode acts as electron donor. These organisms cause a difference in cathode, driving a suitable reaction at anode by anodophillic microorganisms to produce the electricity [66]. An ideal electron mediator for converting metabolic reducing power into electricity should form a reversible redox couple at the electrode, and it should link to 0 NADH and have a high negative E0 value in order to maximize electrical energy generation. It should also be stable in both the oxidized form and the reduced form and should not decompose during long-term redox cycling. The mediator polarity should be such that the mediator is soluble in aqueous systems (near pH 7.0) and can pass through or be absorbed by the microbial cytoplasmic membrane. The amount of free energy produced either by normal microbial metabolism or by microbial fuel cell systems is determined mainly by the potential difference (ΔE) between the electron donor and the acceptor according to the following equation:

ÀΔG ¼ nFΔE where: ΔG is the variation in free energy. n is the number of electron moles. F is the Faraday constant (96,487 J/V) [20]. The coupling of metabolic oxidation of the primary electron donor (NADH) to reduction of the ﬁnal electron acceptor (such as oxygen or fumarate in bacterial respiration systems) is very similar to the coupling of the electrochemical half reaction of the reductant (electron donor) to the half reaction of the oxidant (electron acceptor) in a fuel cell or battery system [67]. Biological reducing 0 power sources with low redox potentials, such as NADH (E0 ¼À0.32 V), reduced 0 ferredoxin (FdH2)(E0 ¼À0.42 V), or reduced ﬂavin adenine dinucleotide 0 (E0 ¼À0.19 V), can act as reductants for fuel cells, but they are not easily converted to electricity because the cytoplasmic membrane has to be nonconductive to maintain the membrane potential absolutely required for free energy (i.e., ATP) production [68].

4.4.3 Factors Affecting Performance of MFCs

There is limited information about the factors that affect the power generation of microbial fuel cells. Organic material load in soil enhances the availability of more and diversified microflora in the soil. Industrial and domestic wastewater [28], sewage sludge [69], marine sediment [24, 70], garden compost [71, 72] content 4.4 Microbiology of Fuel Cell 197 bacterial population of approximately 109 cells gÀ1 [73], and organic matter content of within 100 mg gÀ1 [74]. Such resources are rich in electrogenic bacteria and have the ability to produce electrons to generate electricity. Among different wastewaters [75, 76] that were used in MFCs, dairy wastewater contains complex organics, such as polysaccharides, proteins, and lipids, which on hydrolysis form sugars, acids, and fatty acids. Dairy wastewater treatment in MFCs produces lower power density in comparison with other wastewater [66, 77]. Besides the organic materials and microflora, various other factors also influence the electrical energy production in MFCs. Ishii et al. [78] noticed that when the anode chamber was filled with methane gas emitted from soil, energy generation activity of MFC was continued till the exhaustion of methane supplied to anode chamber. The reason could be due to reduction of soil organic carbon to generate electrical power rather than methane. Like methane, when the anode chamber of a two-chambered MFC is filled with phenolic waste, the activity of power generation was continued well till 90 % of phenol was removed from soil, compared with 13 % in non-MFC control [79]. Several physicochemical factors are noticed to be influenced over power generation in MFCs. The distance between electrodes was noticed to be direct influence over the internal resistance of MFCs [77]. The presence of dissolved oxygen impaired the anaerobic conditions at anode and decreased power output [61]. Min et al. [11] observed that the influence of temperature dependence of microbial activity was a major factor that influenced soil-based MFCs. Lorenzo et al. [80] reported the effect of three-dimensional anode surface areas on power performance of single-chambered microbial fuel cells for generation of electric energy and observed direct correlation of anode surface area with MFC power generation performance. Some interesting data are available on performance of MFCs in several types of sediment soil (SMFC), by adding organic matters leaching from plants into anode fuel cell (PMFC). When electrodes were placed on bottom of the sediment (anode), on top of the sediment, or suspended in the water phase (cathode) [81, 82], the overall performance of SMFC was noticed to be well. Organic materials excreted from plants give good performance of PMFC [5, 17, 65, 83]. SMFCs and PMFCs have great promise, not only for sustainable electricity recovery from the environment but also for their potential application in supplying electricity in self-powered devices. Such applications include devices that monitor environmental parameters [84], sensors to monitor the maturity of plants [85], and applications in bioremediation, and recovery of heavy metals from contaminated environments [86]. A number of factors, such as the speed of substrate oxidation, electron transfer to the anode, proton transfer from the anode to the cathode, and oxygen reduction at the cathode, were noticed to be affective in the overall function of SMFCs and PMFCs [4, 5, 7, 33, 84–89]. The quality and quantity of microbes associated with SMFCs and PMFCs were noticed to be major monitoring factors to improve the productivity of power generation [88–91]. In addition, the physicochemical nature of these fuel cells plays an important factor. In this connection, it has been observed that modification of the anode is also one of the most important strategies for improving the performance of SMFCs and PMFCs. Several materials have been 198 4 Microbial Fuel Cell (MFC) used to increase the surface area to allow for the attachment of a greater number of microorganisms. These materials include graphite felt, graphite granules [89], biochar [92, 93], active carbon [94], and graphite grains [7]. Interestingly, it has been noticed that members of the genus Shewanella are having high potential of reducing graphene oxide (GO) to electrically conductive graphene [95]. GO is an intermediate product of graphene, obtained by chemical exfoliation of graphite. Microbial GO reduction occurred via respiratory extracellular electron transfer (EET), where GO was used as the terminal electron acceptor [95]. It has been shown that GO can enhance bacteria capable of EET and the resultant reduced form of GO functions as an electrode with the attached bacteria. Exogenous supply of GO was found to improve power generation both the anode and the cathode in MFC when microbial cultures were used [96]. The positive aspect of using graphene oxide is a good absorbent of toxic chemicals, including heavy metals and organic pollutants [97], although both GO and graphene have recently been reported to have toxic effects on a number of living systems [98]. The presence of dissolved oxygen in anode chamber plays a critical role in functioning MFCs [12, 13, 20, 23–25, 28, 33, 81, 82, 99–116]. An anaerobic atmosphere was usually provided by passing nitrogen and carbon dioxide gas through the anode compartment. Eliminating the presence of oxygen would lead to increased transfer of electrons from the bacteria to the electrode, rather than to oxygen. On the other hand, it has been observed that bubbled oxygen through the cathode compartment [21, 112] enhanced the production of power.

4.4.4 Power Generation in MFCs

The power production in MFCs can be well monitored by various physicochemical factors surrounding the MFCs’ implanted location (Sect. 4.4.3). Generally, a current output amounting to 1.5 mA cm2 has been observed in MFCs [26, 45]. Greater efﬁciency in electron transfer and thus an increase in current generation and power output have been attained by modifying graphite electrodes [111], using alternative electrodes [45], by employing a mixed bacterial culture [23, 26, 117], and by using bacteria with higher metabolic rates. Different electron mediators have also been used to this end [117]. Park et al. [21, 24] reported that a tenfold more current was produced in their microbial fuel cell when neutral red (NR) was the electron mediator than when thionine was the electron mediator.

4.5 Types of Microbial Fuel Cells and Components

There are a large variety of microbial fuel cell designs that have been utilized in research. MFC design is controlled by the application that it will be used in. Basic MFC designs can be used to study small aspects of the microbial fuel cell system or 4.5 Types of Microbial Fuel Cells and Components 199 to test one specific parameter. However, these systems will generally not result in power densities comparable to the most efficient systems that have been constructed. These highly efficient systems have been designed solely for the purpose of maximizing power production of the microbial fuel cell system. Each group of researchers has a preferential design and materials that are used in testing.

4.5.1 Two-Chambered Microbial Fuel Cell

Earlier, two-chambered MFCs were for laboratory experiments by using chemical medium such as glucose or acetate solution to generate energy. A typical two-chambered MFC (Fig. 4.9) consists of an anodic chamber and a cathodic chamber linked by a PEM, or occasionally a salt bridge, to permit protons to move across to the cathode while obstructing the diffusion of oxygen into the anode. At present, they are run in batches and can be used for producing higher power output and can also be utilized to give power in much inaccessible conditions. It can be suitably designed to scale up to treat large volume of wastewater and other sources of carbon. They practically fall between the classiﬁcation of single- chambered and double-chambered microbial fuel cells. Reports are available on application of two-chambered MFCs for large-scale production of electricity from the wastes [45, 118].

Fig. 4.9 A schematic presentation of two-chambered MFC and showing how microbial ﬁlm attached to anode donates electron to cathode and generates electricity ([36] Rabaey, K.; Verstraete, W. Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol. 2005, 23, 291–298. Ref [110]) 200 4 Microbial Fuel Cell (MFC)

Fig. 4.10 Schematic diagram of a single-cell MFC. The single-cell MFC consists of an anode, a cathode, a proton or cation exchange membrane, and an electrical circuit. Anode- respiring bacteria cling to the anode of the MFC. In the course of their metabolic activity, these bacteria strip electrons from organic waste. The electrons then ﬂow through a circuit to the cathode, producing electricity in the process, in addition to CO2 and water. Hydroxide or OH- ions are transported from the cathode into the surrounding electrolyte (Richard Harth Energy TechWaste to Watts: Improving Microbial Fuel Cells, www.spxdaily.com)

4.5.2 One-Chambered Microbial Fuel Cell

Due to their intricate designs, two-chambered MFCs are tough to scale up even though they can be functioned also in batch or continuous mode. One-chambered MFCs offer simpler designs and are cost-effective (Fig. 4.10). They normally possess only an anodic chamber without the requisite of aeration in a cathodic chamber. Park and Zeikus [116] designed a one-chambered MFC comprising of an 4.5 Types of Microbial Fuel Cells and Components 201 anode in a rectangular anode section linked with a porous air cathode that is open directly to the air. Protons are transported from the anode solution to the porous air cathode [13]. Liu and Logan [104] designed an MFC containing an anode positioned inside a plastic cylindrical chamber and a cathode located outside. The anodes are normal carbon electrodes, but the cathodes are either porous carbon electrodes or PEM bonded with ﬂexible carbon cloth electrodes. Cathodes are often covered with graphite in which electrolytes are poured in steady fashion which behaves as catholytes and prevents the membrane and cathode from drying. Thus, water management or better ﬂuid management is an important issue in such single- chambered fuel cells. A tubular MFC with an exterior cathode and an interior anode using graphite granules was constructed by Rabaey et al. [105]. In the nonexistence of a cathodic chamber, cathode solution is supplied to the cathode by soaking an electrolyte over the outer woven graphite mat to keep it from drying up.

4.5.3 Upﬂow Microbial Fuel Cell

Upﬂow dual-chambered microbial fuel cell (Fig. 4.11) consists of two-chambered rectangular or cylindrical glass equipment made of transparent acrylic or glass materials. The Plexiglas cylinder is separated into two segments by glass wool and glass bead sheets. These two units helped as anodic and cathodic chambers

Fig. 4.11 Schematic diagram of an upﬂow dual-chambered microbial fuel cell 202 4 Microbial Fuel Cell (MFC) correspondingly. The disk-shaped graphite felt anode and cathode are positioned at the end and the ﬁrst of the reactor, correspondingly. There are no distinct anode solution and cathode solution. The diffusion barricades concerning the anode and cathode offer a DO gradient for appropriate action of the microbial fuel cells [53].

4.5.4 Stacked Microbial Fuel Cell

A stacked MFC is a combination of many MFCs connected in parallel or series manner to increase the output of electric power [28, 82]. In such complex system (Fig. 4.12), no noticeable adverse effect on the maximum power output per MFC unit was observed. It has been suggested that to get more benefit from MFC technology, it has to meet with the established methanogenic anaerobic digestion technology [108] because they can utilize the same biomass in many cases for electricity generation. MFCs are proficient of transforming biomass at temperatures under 20 C and with little substrate concentrations, both of which are difficult for methanogenic digesters [101]. But the only key drawback of MFCs is their dependence on biofilms for electron transport [109]. It is likely that the MFC technology will coexist with the methanogenic anaerobic digestion technology in the future. To improve the power density output, new anodophilic microbes that vastly improve the electron transport rate from the biofilm covering an anode to the anode are much needed [8]. Lovley pleaded that an MFC electron flow could increase by four orders of magnitude if Geobacter transports electrons to the anode at the same rate as it does to its usual electron acceptor that is ferric iron [108]. It is expected in the near future more potential microbial group can be identified to operate an MFC without any mediators or biofilms while attaining greater mass transfer and electron transfer rates.

Microorganism loaded Rubber Granular Anode Gasket

Fastening Bolt

Cathode Proton Exchange Membrane

Fig. 4.12 Schematic design of stacked-type microbial fuel cell (as proposed [27]) 4.5 Types of Microbial Fuel Cells and Components 203

Fig. 4.13 Schematic diagram of plant fuel cell (Darren Quick 2012 Plant-Microbial Fuel Cell generates electricity from living plants. Science. November 25, 2012) (Ref. [119])

4.5.5 The Plant Microbial Fuel Cell (PMFC)

The plant microbial fuel cell (PMFC) is a technology (Figs. 4.13 and 4.14) that can potentially be used to exploit wetland for renewable energy generation. About six percent of the earth surface covers with wetland, as sustainable energy source. The PMFC technology is a low-cost project and would not compete with any food or 204 4 Microbial Fuel Cell (MFC)

Fig. 4.14 Diagrammatic presentation of a ﬂat-plate plant microbial fuel cell showing the effect of a new design on internal resistances (Helder et al. The ﬂat-plate plant- microbial fuel cell: the effect of a new design on internal resistances. Helder et al. Biotechnology for Biofuels 2012, 5:70. http:// www. biotechnologyforbiofuels. com) (Ref. [120])

feed production with simple management system [81, 105, 108–111]. The technology is still in its infancy; however, a lot of efforts have been going on to make the project economically viable. Presently, the power output is noticed to be low; however, theoretical power output is estimated at 3.2 W/m2 geometric planting area [112]. On the basis of reports available, maximum power output was noticed to be improved from 65 mW/m2 to 220 mW/m2 [28, 113] by considering plants as sole organic matter source. Current biomass-electricity systems, like anaerobic digestion, produce the same amount of electricity per geometric planting area as the maximum that was achieved in PMFCs, 220 mW/m2 [112]. So even at current power output, the PMFC could compete with anaerobic digestion based on electricity production per geometric planting area. But maximum power outputs of the PMFCs have been achieved in short-term tests like polarization curves and were not sustained for longer periods of time. Over longer periods of time, average power output is limited to maximally 50 mW/m2 geometric planting area [114]. In some experiments, a decrease in power output during runtime of an experiment was observed, due to an increase of membrane resistance and buildup of ion-transport resistances [114]. In one recent publication, however, it is shown that average power output increases with runtime of the experiment. This latter experiment was done with a flat-plate PMFC [114]. Power density of a PMFC is determined by several aspects of the system: solar radiation, photosynthetic efficiency of the plant, organic matter allocation from plant to soil, and efficiency of the microbial fuel cell (MFC) [114]. Timmers et al. [118] identified that the PMFC has a high internal resistance, which limits the power density [116, 121]. In order to increase the power density, internal resistance should be decreased. To increase power output of the PMFC, internal resistances need to be reduced. With a flat-plate PMFC design, one can minimize internal resistances compared to the previously used tubular PMFC design. Marjolein Helder et al. [121] developed a flat-plate design PMFC (Fig. 4.10)in 4.5 Types of Microbial Fuel Cells and Components 205 which current and power density per geometric planting area were increased (from 0.15 A/m2 to 1.6 A/m2 and from 0.22 W/m2 to 0.44 W/m2) as were current and power output per volume (from 7.5 A/m3 to 122 A/m 3 and from 1.3 W/m3 to 5.8 W/ m3). Internal resistances times volume were decreased, even though internal resistances times membrane surface area were not. Since the membrane in the flat-plate design is placed vertically, membrane surface area per geometric planting area is increased, which allows for lower internal resistances times volume while not decreasing internal resistances times membrane surface area. Anode was split into three different sections on different depths of the system, allowing calculating internal resistances on different depths. Most electricity was produced where internal resistances were lowest and where most roots were present in the top section of the system. By measuring electricity production on different depths in the system, electricity production could be linked to root growth. This link offers opportunities for material reduction in new designs. Concurrent reduction in material use and increase in power output bring the PMFC a step closer to usable energy density and economic feasibility.

4.5.6 Tubular MFC

This type of MFC is either single chambered or double chambered, and oxygen or chemical catholytes are used as proton acceptors [81] and wastewater as substrate. This MFC was constructed by hot pressing a PEM to a carbon cloth with Pt catalyst and placing it around a tube with holes drilled into it to allow oxygen transfer. Eight graphite rods were placed around this tubular cathode, and the system was enclosed by a plastic chamber (Fig. 4.15). The tubular cathode was open on each end to allow oxygen diffusion to the cathode, while the wastewater was fed continuously to the system. A maximum power density of 26 mW/m2 was produced in this continuous ﬂow system. When air was forced through the cathode, power production was reduced, reiterating the problems caused by increased levels of oxygen diffusion into the anode compartment of an MFC [123]. Glass beads and wool were also used as separation in a two-chambered column MFC lacking a membrane. Artiﬁcial wastewater was fed through an anode chamber with graphite felt, through the glass beads and wool, and through the cathode chamber containing graphite felt and sparged with air. This system only produced 1.3 mW/m2, with the limiting factor most likely being the mass transfer of protons between the two electrodes [77]. A similar system was constructed using graphite rods in place of graphite felt for both the anode and cathode. A maximum power density of 10.9 mW/m2 was reported [45]. The glass beads and wool have also been replaced with a perforated polyacrylic plate in other MFC designs [102, 117, 124]. Tubular systems using graphite granules have been used by multiple researchers in the literature as well. These systems generally use the graphite granules for the anode with a PEM/carbon cloth system encapsulating the granules [125, 126]. A catholyte can be applied to the outside of these cathodes to improve power production [28]. 206 4 Microbial Fuel Cell (MFC)

Fig. 4.15 Elements of tubular air cathode microbial fuel cells (MFCs) and solid substrate leach bed bioreactors were integrated to develop a new MFC in which cellulose hydrolysis, fermentation reactions, and anode respiration were achieved in the anode compartment. Electricity production from a minimally processed lignocellulosic crop residue (corncobs) was sustained for >60 d [122])

4.6 Components and Architecture of Microbial Fuel Cell

4.6.1 Anode

An efficient anode should possess the following characters, i.e., noncorrosive, non-fouling, porous, inexpensive, easy to make, applicable to larger-size systems, and contain a large surface area and high conductive property. Although, most of the good anodes are having above quality but are not in use due to their corrosive nature and lack of a suitable surface for bacteria colonization. So, the use of carbon- based electrodes is very frequent in MFC research and application due its above- stated quality [127]. Generally, the anodes are noncorrosive stainless steel mesh having perfect biocompatible and good conductive in nature [102]. Copper material is not used for anode fabrication as it is toxic in nature to bacteria. Caron electrode is proven as the most versatile and easily available as compact graphite plates, rods, or granules; as fibrous material (felt, cloth, paper, fibers, foam); and as glassy carbon. There are numerous carbon suppliers worldwide, for example, ETEK and Electrosynthesis Co. Inc. (USA); GEE Graphite Limited, Dewsbury (UK); Morgan, Grimbergen 4.6 Components and Architecture of Microbial Fuel Cell 207

(Belgium); and Alfa Aesar (Germany). Generally, graphite plates are inexpensive, are easy to handle, and have a defined surface area [103, 105]. Carbon fiber, paper, foam, and cloth (Toray) have been extensively used as electrodes [64]. Substantially higher surface areas are achieved either by using a compact material like reticulated vitreous carbon (RVC; ERG Materials and Aerospace Corp., Oakland, CA) [13, 128] which is available with different pore sizes or by using layers of packed carbon granules (Le Carbone, Grimbergen, Belgium) or beads [28, 105]. In order to enhance anode performance, different chemical and physical strategies have been followed [24]. The graphite felt is having more advantage over graphite rod as it is having three times more surface area and generation of large power densities [81]. However, when current production was normalized to surface area, the felt and rod had comparable values of 28 mA/m2 and 31 mA/m2, respectively. But it has been observed that the graphite foam produced the greatest power density of 74 mA/m2 and is attributed to more cells attaching to the foam because of its structure [74]. Interesting results were also observed by using graphite granules as anode [8] in tubular MFC. Granules are small pieces of graphite, usually 1.5–5 mm in diameter, that can be used for both the anode and cathode. Although the granules are electrically conductive, there must be consistent contact between all the pieces for current to be efficiently transported through them to the collection point. A graphite rod can be placed within the granules to help maintain this contact as well as provide a connection for the wiring of the MFC [111]. Graphite granules have been used in multiple-packed bed reactor MFC architectures in the literature [102, 103, 129]. A graphite fiber brush having the size of 2.5 cm outer diameter and 2.5 cm length was first used by Logan et al. (2007) [13] to achieve a surface area of 0.22 m2.By using normal industrial brush with 7.2 μm in diameter fibers, and wrapped with titanium, a noncorrosive metal, the overall performance of MFS was noticed to be excellent. A brush having 5 cm in diameter and 7 cm long size can provided 1.06 m2 of surface area. Because these brushes have a porosity of 95 % and 98 %, respectively, minimal volume is used to house them within the MFC. In a cylinder MFC (Sec. 4.6), these brushes produced a 2400 mW/m2 power density, normalized to cathode surface area [13]. Better and faster bacterial colonization and rate of electron transport can be achieved by treating this brush with ammonia prior to its use [104]. The ammonia treatment is carried by heating the brush materials in ammonia environment at 700 C for an hour in a 5 % NH3 helium gas. The positive surface charge of the material is increased due to the formation of nitrogen containing and surface functional groups. Acclimation time was reduced by 50 % and power density increased from 1640 mW/m2 to 1970 mW/m2 after treatment. This ammonia treatment provides new material characteristics that result in better and faster bacterial adhesion and improved electron transfer at the anode [104]. By suitable metal coating on anode materials, the performance of MFC was noticed to be improved. Iron oxide-coated electrodes (30 mM/m2) give better results as compared to regular carbon paper having 8 mW/m2 size. It has been observed that an electrode with a biofilm transplanted from a working MFC 208 4 Microbial Fuel Cell (MFC) produced 40 mW/m2. Instead of increasing power supply, an iron oxide-coated anode decreases the acclimation time for the bacteria [115]. An anode utilizing Mn 4+ and a substrate of sewage sludge with the bacteria, E. coli, outperformed plain graphite 1000-fold (788 mW/m2)[24, 105]. Various other metals like stainless steel, titanium, tungsten, and aluminum oxide have also been tested for anode applications. Tungsten was noticed to be the only material to decrease acclimation time, as Se in iron oxide-coated electrodes, and also had little effect on power production. The other materials taken in trial were proven to have negative effects on the power production of the MFCs [13]. It can be difficult to directly compare anode materials when trying to determine what materials are ideal for use in an MFC. For example, if the overall internal resistance of the system is too high, increasing the anode surface area or changing the material may not affect the power output of the cell at all because the internal resistance is controlling the system [13]. Caution must be used in drawing conclusions on the applicability of materials and designs in MFC systems. Covalently linked neutral red, Mn(IV), and Fe(III) were noticed to be used as mediators to enhance the power-generating capacity of MFC [106]. Polyanilines/Pt composites like electrocatalytics were noticed to improve the current generation through assisting the direct oxidation of microbial metabolites [106, 129, 130]. Modification of anode electrode could be useful in promoting the performance of MFCs. In this regard, recently, several researchers have started to modify anode using different nano-engineering techniques that are able to make easier the electron transfer [131]. Moreover, enhancing the power density and enlarging the capability of electron accepting heterogeneous fabrication methods and modification manners involving nanomaterials have been tried [132]. Qiao et al. illustrated that carbon nanotubes (CNTs) could amplify the electron transfer feasibility and electrode surface area with utilizing carbon nanotube/polyaniline nanostructure composite as anode materials.

4.6.2 Cathode

Continuous efforts have been going on to bring modification in design of cathode for better performance. The use of graphite brush technology in improving the efficacy of cathode has brought revolution in MFC for its commercialization. The role of cathode is supposed to be highly significant in functioning MFC because it is the site of interaction of electrons, protons, and oxygen. All of the carbon-based materials discussed in the previous section for applications as an anode can also be used as cathode material, with the addition of a catalyst. Carbon paper or carbon cloth is most commonly used materials for cathode fabrication. When used in a single-chambered MFC, these materials will be wet-proofed due to the structural design of the system. For better performance, pre-coated carbon cloth and carbon paper with carbon/Nafion paste can be used. By 4.6 Components and Architecture of Microbial Fuel Cell 209 this technique, an increase in power production by 68 % was noticed, over a system of direct use of commercial carbon cloth and carbon paper. As the use of platinum is a cost-effective, cobalt tetra methoxyphenylporphyrin (CoTMPP) has been noticed to be a money-saving option in place of to platinum as a catalyst on the cathode. By such modification Pt loadings can be reduced to 0.1 mg/cm2 with only a 19 % reduction in power production, but the overall fabrication cost of cathode was noticed to be reduced [133]. It has also been noticed that by using iron phthalocyanine on Ketjenblack carbon as a catalyst at the cathode, the power output was increased, in a cylinder single-chambered MFC design [134]. Research will need to continue into the future to find more sustainable and cost-effective catalysts that can be used at the cathode. As stated earlier, cathode is also made of porous carbon electrode or PEM bonded with flexible carbon cloth electrode. In order to improve better performance, cathodes are often covered with graphite in which electrolytes are continuously supplied as catholytes to keep the membrane and cathode in wet condition. The physicochemical nature, the catalyst on the electrode, and the cathodic electron acceptor play an important factor on electricity generation of MFCs [82, 108–110]. Dissolved oxygen, ferricyanide, potassium permanganate, or manganese dioxide has often been used as cathodic electron acceptors in two-chambered MFCs. Among the various catholytes used, potassium ferricyanide (K3[Fe(CN)6]) was noticed to be most prominent, especially in a two-chambered MFC. The main advantage of ferricyanide is the low overpotential using a plain carbon cathode, resulting in a cathode working potential close to its open circuit potential. However, the disadvantage is the insufficient reoxidation by oxygen, which requires the catholyte to be regularly replaced [110]. In addition, the long- term performance of the system can be affected by diffusion of ferricyanide across the CEM and into the anode chamber. Oxygen is noticed to be a most suitable electron acceptor due to its high oxidation potential, availability, low cost (it is free), sustainability, and the lack of a chemical waste product (water is formed as the only end product). An efficiently performed cathode would use dissolved oxygen as the electron acceptor. Due to low concentration of dissolved oxygen inside water, this kind of cathode will not produce a very large driving force in comparison to other types of cathodes. On the other hand, this type of cathode does not need replacement, since the only element it consumes is oxygen, allowing it to perform for a long period of time. Performance of cathodes mainly depends on its purpose of use in different places. For saline sediment soil, graphite disk electrodes were noticed to be most suitable [123]. Generally, in seawater oxygen reduction depends on the quality and quantity of microflora present [105, 106]. Such microbial-assisted reduction has also been observed for stainless steel cathodes which rapidly reduce oxygen when aided by a bacterial biofilm [107]. Pt catalysts are usually used to increase the rate of oxygen reduction for dissolved oxygen [28] or open-air (gas diffusion) cathodes [135]. In order to reduce the costs for the MFC, the Pt load can be kept as low as 0.1 mg cm–2 [136]. The long-term stability of Pt needs to be more fully investigated, and there remains a need for new types of inexpensive catalysts. It has been 210 4 Microbial Fuel Cell (MFC) observed that some noble metals like pyrolyzed iron(II) phthalocyanine or CoTMPP are having high potential for generating electric power in MFC cathodes [115, 137].

4.6.3 Ion Exchange Membrane

Microbial metabolism in an MFC brings imbalance in ions across the anode and cathode. The proton produced as a result of bacterial activity increases the positivity of anode, and the reduction of oxygen at anode site results in negativity at cathode site. An ion exchange membrane helps in bringing balance in the gradient of charge created between anode and cathode, by moving the ions driven by the charge gradient. The main problem with membrane is it gives resistance to proton membrane which may hamper power-generating performance in MFCs. In addition, membrane cannot be exposed to air, because the anode side has to stay anaerobic [137]. The most economical membrane would be sea sediments. It has been shown that MFCs can be made on the seafloor, where the membrane is only the sea sediments. This kind of MFC is called sediment microbial fuel cell (SMFC). Unfortunately, SMFCs have very low power densities, because of their high internal resistances. Some of their large internal resistance is due to the inefficient performance of the membrane and the large distance between the anode and the cathode [137]. Exceptions are naturally separated systems such as sediment MFCs [116]or specially designed single-compartment MFCs [112, 138]. Nafion (DuPont Co., USA) is the most popular CEM easily available in the market (e.g., Aldrich and Ion Power, Inc.). Alternate option for Nafion such as Ultrex CMI-7000 (Membranes International Inc., Glen Rock, NJ) is also easily available in the common market [7]. When a CEM is used in an MFC, one has to be sure that it must be permeable to chemicals such as oxygen, ferricyanide, and other ions or organic matter used as the substrate. The market for ion exchange membranes is constantly growing, and more systematic studies are necessary to evaluate the effect of the membrane on performance and long-term stability [82].

4.7 Applications of Microbial Fuel Cell Technology

A diversified rage of sustainable resources can be used for direct power generation by using MFCs. Generally, for laboratory purpose simple organic substrates like acetate and glucose are used for various experimental purpose; however, many complex organic substrates can also be used. Effluent from industries, wastewater from food-processing plants, landfill leachate, and wetland are proven to be good resources for operating MFCs and meet the local area energy demand in near 4.7 Applications of Microbial Fuel Cell Technology 211 feature. Although MFCs have been studied as an alternative energy source, their application is presently limited to certain niche areas. With further improvements in design, cost-effectiveness, and performance efficiency based on these near-term applications, it would be possible to scale up and use MFCs as a renewable energy resource. Fuel cells are a novel addition to the inventory of alternate energy sources having minimal or no net CO2 emission. Electricity production using microbial cultures was first reported early in the last century [45, 108]. Microbial fuel cells (MFCs) have been described as “bioreactors that convert the energy in the chemical bonds of organic compounds into electrical energy through catalytic activity of micro-organisms under anaerobic conditions.” MFC technology represents a novel approach of using bacteria for generation of bioelectricity by oxidation of organic waste and renewable biomass [53]. MFC technology represents a multidisciplinary approach to the quest for alternate sources of energy. It symbolizes the confluence of the chemical, physical, and life sciences and is a meeting point of basic and applied research. The principle of working of MFCs is based on the tenets of microbial physiology coupled with electrochemistry. Structural design of MFCs brings the nuances of electrical and materials engineering to the fore. The applications of this technology come under the ambit of environmental engineering and bioremediation. MFC technology is thus multidisciplinary, in the true sense of the term, and provides scope for strengthening research across disciplines. In general, the MFC technology can be applicable for various purposes as described below.

4.7.1 Wastewater Treatment

Wastewater contains a diversiﬁed organic material [81, 109, 110] and supposed to be a sustainable source of direct electricity production. Different kinds of wastewaters such as sanitary wastes, food-processing wastewater, swine wastewater, and corn stover contain biodegradable organic wastes which are, generally, used in bacterial metabolism and ultimately helpful in production of ions responsible for energy generation in MFCs [110]. MFC technology that was considered to be used for wastewater treatment early in 1991 [81] is favorable as a completely different method because of capturing energy in the form of electricity or hydrogen gas [69]. For an efﬁcient treating system, high operational sustainability and low material costs are worthwhile characteristics [111]. Scientists have reported that to remove nitrogen and organic matters from leachate, biological treatment is prevalently used as a credible and highly cost-effective method [105, 112]. Liu et al. [113] could successfully use domestic wastewater in multiple MFC systems for generating electric power. With a basic cylinder MFC design, 146 mW/ m2 of electric energy was produced using wastewater with 200–300 mg/L chemical oxygen demand (COD) load. In this same cylinder design, swine wastewater with 8320 Æ 190 mg/L soluble COD load produced a power density of 261 mW/m2 with 212 4 Microbial Fuel Cell (MFC) the removals of both ammonia and soluble COD [139]. Aelterman et al. [105] used wastewater from a potato-processing factory and a hospital (600–2300 mg/L COD) as fuel source in a two-chambered MFC system with a chemical catholyte to generate electrical energy. A brewery wastewater having COD load of 2250 Æ 418 mg/L was used as fuel source in an MFC to study its performance and received encouraging results. Limited data are also available on the use of MFCs using bovine serum albumin (354 Æ 10 mW/m2), peptone (269 Æ 14 mW/m2), and meat-packaging wastewater (80 Æ 1mW/m2) in a single-chambered system. Zuo, Maness, and Logan (2006) [137] reported that encouraging steam-exploded corn stover biomass has been used in MFCs and produced power densities from 367 to 371 mW/m2 in conjunction with high biological oxygen demand (BOD) and COD removals. A synthetic acid mine drainage water was tested in the cylinder MFC design as well and produced about 48 % of the power that can be produced with acetate [116]. Another interesting application of MFC technology was in a sediment MFC, utilizing soil for the bacteria and organic source. This design was operated using rice plants and the rhizo-deposits from the plant to produce power and increase power production to seven times what it had been without the presence of plants [121]. Reed manna grass was also used in a similar design with promising results as a method of nondestruc- tive harvesting of bioenergy that is carbon neutral. Research has begun to study algae as an organic substrate for MFCs with a maximum power density of 110 mW/m2 reported [45].

4.7.2 MFC for Landﬁll

Landfill leachate is liquid that infiltrates the landfill system or is produced by the waste within the system. Leachate must be collected and managed to protect the surrounding environment. Leachate can also be recirculated in an operating landfill to manage the leachate as well as increase biodegradation of waste within the landfill and increase landfill gas production. While many organic constituents in the leachate can be treated through the biological processes within the landfill, ammonia can accumulate and resist treatment [140]. Ammonia can be toxic to bacteria and other organisms and inhibit accelerated biodegradation that can result from recirculation. MFCs could be used as a pretreatment for the leachate prior to recirculation to lower concentrations of constituents while producing electricity and limiting energy inputs into the system. Leachate is a well-matched substrate for use in a microbial fuel cell because of its relatively high amount of organics, conductivity, and buffering capacity, yet minimal solids. All of these characteristics, with the exception of high organics, can limit the utilization of other wastewaters for use in MFCs. Even though substrates such as wastewater and leachate will not provide equal power production to MFCs utilizing pure substrates such as acetate, the use of a substrate like leachate mimics more realistic conditions. MFCs utilizing landfill leachate also need no outside source of inoculation due to the presence of the appropriate mixed bacterial community. 4.7 Applications of Microbial Fuel Cell Technology 213

4.7.3 Generation of Electricity Directly from PMFC

One new source is the plant microbial fuel cell (2001), which produces bioelectricity using photosynthesis from solar radiation without harvesting the plants (Figs. 4.13 and 4.14). PMFC requires the anaerobic conditions, for example, wetlands (Æ800,000,000 ha worldwide). Wetlands are often unsuitable for crop growth, and thus PMFC is not competing for arable land. Even though PMFC is based on photosynthesis, it can deliver electricity 24 hours per day and year-round. This is especially interesting in remote areas without electricity or where electricity is delivered by irregular and unreliable variations in solar energy [7, 10, 32, 45, 118, 141, 142]. Researchers at Wageningen University have developed a fuel cell that runs off electrons present in the soil around living plants’ roots (Fig. 4.10). During photosynthesis, organic material is produced that the plant cannot use, which is then secreted through the roots. Bacteria in the soil break down this organic material, which releases electrons. All that is needed is an electrode to capture those electrons and you’ve got electricity. The university says, “Plant-Microbial Fuel Cells can be used on various scales. Initially on flat roofs or in remote areas in developing countries and later, when larger effective surface areas become feasible, central grids can be realized in areas of marshland. The researcher thinks that green energy- producing roofs will become a reality within a few years and production on a larger scale will follow suit soon after 2015” [143]. Right now the technology is only able to produce about 0.4 W per square meter of plant growth, but the researchers think that soon they will boost the output up to 3.2 W per square meter, which would allow a 100-square-meter green roof to power a household with an average energy consumption of 2800 kWh per year [144]. The plants that can be used include any common grasses as well as rice in warmer climates. The major benefit of the plant microbial fuel cell over other renewable energy technologies is that it doesn’t compete for land use like solar panels, wind farms, and biofuel production do since the electricity production is largely happen- ing underground. Meanwhile, the researchers have created a company called Plant-e that plans to have a product on the market by next year that could be used to power things like LED lights, laptops, and cell phones. On a larger scale, the company has installed the first proof-of-concept electricity-generating green roof at the Netherlands Insti- tute of Ecology building [100].

4.7.4 Microbial Fuel Cell for Health Care

Microbial fuel cell is an energy-generating system using body glucose as fuel. Eugenii Katz, Hebrew University of Jerusalem, reported that biofuel cells could potentially be used in various biomedical applications, for example, to power a 214 4 Microbial Fuel Cell (MFC) pacemaker. As medicine comes to rely increasingly on implantable electronic devices for treating a number of conditions, there will be a growing demand for the reliable power that a fuel cell can supply. Other than avoiding any maintenance that might require surgery, a biofuel cell could use fuel that is readily available in the body, for example, glucose in the bloodstream, and it would ideally draw on this power for as long as the patient lives, explains Katz [111]. A basic biofuel cell can operate in two ways. It can use biological catalysts— enzymes extracted from biological systems—to oxidize fuel molecules at the anode and to enhance oxygen reduction at the cathode of the biofuel cell. Alternatively, whole microbial cells can be used to supply the biofuel cells with the fuel. In both cases the main technical problem is the electrical coupling of the biological components of the system with the fuel cell’s electrodes. Molecules known as electron transfer mediators can provide efficient transport of electrons between the biological components, enzymes or microbial cells, and the electrodes of the biofuel cell. Integrated biocatalytic systems that include biocatalysts, electron transfer mediators, and electrodes have been recently developed and applied in biofuel cells. Eugenii Katz, Associate Professor at the Hebrew University of Jerusalem, Israel, believes that biofuel cells could potentially be used in various biomedical applications, for example, to power a pacemaker. Development of hydrogen fuel cells has been driven by the need for clean methods of producing electricity from renewable fuel sources. In a hydrogen fuel cell, electricity is generated efficiently from the oxidation of hydrogen, coupled to the reduction of oxygen, with water as the only waste product. The catalysts in commercial fuel cells are typically precious metals such as platinum [14]. The ability of a hydrogenase-coated electrode to catalyze efficient oxidation of hydrogen suggests exciting possibilities for use of such an electrode in a biofuel cell, in which the conventional platinum fuel cell anode is replaced by a precious metal-free hydrogenase electrode. Coupled to a cathode incorporating the fungal enzyme, laccase, which catalyzes reduction of oxygen to water, the hydrogenase biofuel cell produces a small but measurable power output. Work on the hydrogenase biofuel cell is still at a very early stage but poses fascinating intellectual challenges.

4.7.5 Power Supply to Microelectronic Devices

With the increase in research, application of MFC technology has been diversiﬁed to operate microelectronic devices by replacing chemical battery cells. Generally, batteries are used to power chemical sensors and telemetry systems and noticed to be cost-effective due to its regular replacement after certain stipulated time interval. In addition, maintenance of such microelectronic instruments is expensive, too. A possible solution to this problem is to use self-renewable power supplies, such as MFCs, which can operate for a long time using local resources. Extensive research 4.7 Applications of Microbial Fuel Cell Technology 215 toward developing reliable MFCs to this effect is focused mostly on selecting suitable organic and inorganic substances that could be used as sources of energy [145, 146]. MFC technology can be well applicable to operate sensor for pollutant analysis and in situ process monitoring and control. Biological oxygen demand (BOD) is the amount of dissolved oxygen required to meet the metabolic needs of aerobic organisms in water rich in organic matter, such as sewage. The proportional correlation between the coulombic yield of MFCs and the concentration of assim- ilable organic contaminants in wastewater makes MFCs possibly usable as BOD sensors. An MFC-type BOD sensor can be kept operational for over 5 years without extra maintenance, far longer in service life span than other types of BOD sensors reported in the literature [53]. Data on the natural environment can be helpful in understanding and modeling ecosystem responses, but sensors distributed in the natural environment require power for operation. MFCs can possibly be used to power such devices, particularly in river and deep-water environments where it is difﬁcult to routinely access the system to replace batteries. Sediment fuel cells are being developed to monitor environmental systems such as creeks, rivers, and oceans. Power densities are low in sediment fuel cells because of both the low organic matter concentrations and their high intrinsic internal resistance. However, the low power density can be offset by energy storage systems that release data in bursts to central sensors.

4.7.6 Hydrogen Production

Now, it is possible to generate hydrogen by combining MFC and photoelectrochemical cell depending on photosynthesis process. By this hybrid device, it could be possible to generate hydrogen from wastewater using sunlight as source of energy [14, 17]. In the MFC component, bacteria degrade organic matter in the wastewater, generating electricity in the process. The biologically generated electricity is delivered to the PEC component to assist the solar-powered splitting of water (electrolysis) that generates hydrogen and oxygen. Interestingly, it has been observed that when the system is fed with wastewater and illuminated in a solar simulator, the PEC-MFC device showed continuous production of hydrogen gas at an average rate of 0.05 m3/day. At the same time, the turbid black wastewater became clearer. The soluble chemical oxygen demand—a measure of the amount of organic compounds in water, widely used as a water quality test—declined by 67 % over 48 h. The hydrogen generation declined over time as the bacteria used up the organic matter in the wastewater. Replenishment of the wastewater in each feeding cycle led to complete restoration of electric current generation and hydrogen gas production [100, 147]. 216 4 Microbial Fuel Cell (MFC)

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One last important renewable energy is hydrogen. Elemental hydrogen occurs freely on earth in only negligible quantities but is chemically active and found in many compounds in combination with other elements. Hydrogen can be produced from a variety of feedstocks. These include fossil resources, such as natural gas and coal, as well as renewable resources, such as biomass and water with input from renewable energy sources (e.g., sunlight, wind, wave, or hydropower). A variety of process technologies can be used, including chemical, biological, electrolytic, photolytic, and thermochemical for hydrogen production. Each technology is in a different stage of development, and each offers unique opportunities, beneﬁts, and challenges. Local availability of feedstock, the maturity of the technology, market applications and demand, policy issues, and costs will all inﬂuence the choice and timing of the various options for hydrogen production. In general, all these process routes developed and available right now are not attractive from commercial point of view. Photosynthesis can be simply represented by the equation:

CO2 þ H2O þ light ! 6CHðÞþ2O O2

Approximately 114 kcal of free energy are stored in plant biomass for every mole of CO2 ﬁxed during photosynthesis. Solar radiation striking the earth on an annual basis is equivalent to 178,000 terawatts, i.e., 15,000 times that of current global energy consumption. Although photosynthetic energy capture is estimated to be ten times that of global annual energy consumption, only a small part of this solar radiation is used for photosynthesis. Approximately two thirds of the net global photosynthetic productivity worldwide is of terrestrial origin, while the remainder is produced mainly by phytoplankton (microalgae) in the oceans which cover approximately 70 % of the total surface area of the earth. Since biomass originates from plant and algal photosynthesis, both terrestrial plants and microalgae are appropriate targets for scientiﬁc studies relevant to biomass energy production. Recent research has focused on photosynthetic production of hydrogen by marine

© Springer International Publishing Switzerland 2016 223 B. Kumara Behera, A. Varma, Microbial Resources for Sustainable Energy, DOI 10.1007/978-3-319-33778-4_5 224 5 The Microbiological Production of Hydrogen

Table 5.1 Hydrogen production in fermenting bacteria, photosynthetic bacteria, and algae (Refs. [1, 84–93, 120, 144, 160, 230–262]) Name of microbes Hydrogen production Fermenting bacteria Escherichia coli 1.0 mole/mol substrate Citrobacter intermedius 0.85 mol/mol substrate Aerobacter sp. 0.36 mol/mol substrate Serratia sp. 0.6 mol/mol substrate Clostridium botulinum 2.33 mol/mol substrate C. acetobutylicum 1.4 mol/mol substrate Lactic acid bacteria 0.74 mol/mol substrate Spirulina maxima 2.3 mol/mol substrate Bacillus macerans 1.35 mol/mol substrate B. polymy 0.75 mol/mol substrate Ruminococcus 2.6 mol/mol substrate Vitro succinogenes 4.0 mol/mol substrate Photosynthetic Rhodospirillum rubrum 1.7 mg protein -do- 1.68 mg protein -do- 0.96 mg protein R. rubrum mutant 4.0 mg protein Rhodopseudomonas capsulata 40 mg protein R. palustris 0.78 mg protein Chromatium sp. 1.2 mg protein Thiocapsa roseopersicina 1.84 mg protein Chromatium sp. Miami 67 ml dry wt hr Chromatium sp. Miami 1071 0.2 Ml mg dry wt Rhodospirillum rubrum 1.7 μl moles mg prot hr -do- 1.8 μl moles mg prot hr Rhodopseudomonas capsulata mutant 4.8 μl moles mg prot hr Rhodopseudomonas sp. 0.54 μl moles mg prot hr

Green algae μmoles of H2 produced hr Ankistrodesmus braunii 1.1 mg chl Chlorella protothecoides 16.4 mg chl Coelastrum proboscideum 24.6 mg chl Selenastrum sp. 11.8 mg chl Scenedesmus obliquus 20.2 mg chl Kirchneriella lunaris 31.2 mg chl Chlamydomonas reinhardtii 17.0 mg chl Chlorella fusca 0.49 mg chl Chlorella vulgaris 11.0 mg chl Chlorella sp. 11.0 mg chl Blue-green algae (cyanobacteria) Anabaena ﬂos-aquae 0.17 mg dr wt A. cylindrica (continued) 5 The Microbiological Production of Hydrogen 225

Table 5.1 (continued) Name of microbes Hydrogen production Anabaena sp. PCC7120 2.6 mmol/mg chl a/h Anabaena cylindrica IAMM-1 2.1 mmol/mg chl a/h Anabaena cylindrica IAMM-58 4.2 mmol/mg chl a/h Anabaena cylindrica UTEX B-629 0.91 mmol/mg chl a/h Anabaena ﬂos-aquae UTEX 1444 1.7 mmol/mg chl a/h Anabaena ﬂos-aquae UTEX LB 2558 3.2 mmol/mg chl a/h Anabaenopsis circularis IAM M-13 0.25 mmol/mg chl a/h Calothrix scopulorum mg dr wt Nostoc muscorum mg dr wt -do- mg dr wt Nostoc muscorum IAM M-14 0.60 mmol/mg chl a/h Nostoc linckia IAM M-30 0.17 mmol/mg chl a/h Nostoc commune IAM M-13 0.25 mmol/mg chl a/h Anabaena variabilis AVM13 68 mmol/mg chl a/h Anabaena variabilis PK84 0.11 mmol/mg chl a/h Anabaena variabilis PK84 1 67.6 mmol/mg chl a/h Anabaena variabilis PK84 0.11 mmol/mg chl a/h Anabaena variabilis PK84 1 67.6 mmol/mg chl a/h Anabaena variabilis PK84 0.11 mmol/mg chl a/h Anabaena variabilis ATCC 29413 45.16 mmol/mg chl a/h Anabaena variabilis ATCC 29413 0.05 mmol/mg dry wt/ Anabaena variabilis ATCC 29413 39.4 mmol/mg chl a/h Anabaena variabilis 1403/4B 20 mmol/mg chl a/h Anabaena azollae 38.5 mmol/mg chl a/h Anabaena variabilis SPU 003 20 mmol/mg chl a/h Oscillatoria brevis 0.17 mg dr wt Anabaena variabilis PK17R 59.18 mmol/mg chl a/h 11 mg dr wt Oscillatoria sp. Miami bg7 0.18 mg dr wt 230 mg chl Synechococcus sp. Miami 3200 n mol mg dr wt Synechococcus PCC 6830 0.26 mmol/mg chl a/h Synechococcus PCC 602 0.66 mmol/mg chl a/h Synechococcus PCC 6307 0.02 mmol/mg chl a/h Synechococcus PCC 6301 0.09 mmol/mg chl a/h Microcystis PCC 7820 0.16 mmol/mg chl a/h Gloeobacter PCC 7421 1.38 mmol/mg chl a/h Chroococcidiopsis thermalis CALU 758 0.7 mmol/mg chl a/h Microcystis PCC 7806 11.3 nmol/mg prot/h Microcoleus chthonoplastes 1.7 nmol/mg prot/h Anabaena cylindrica B-629 0.103 mmol/mg dry wt/h Oscillatoria brevis B-1567 0.168 mmol/mg dry wt/h (continued) 226 5 The Microbiological Production of Hydrogen

Table 5.1 (continued) Name of microbes Hydrogen production Calothrix scopulorum 1410/5 0.128 mmol/mg dry wt/h Calothrix membranacea B-379 0.108 mmol/mg dry wt/h Oscillatoria sp. Miami BG7 0.250 mmol/mg dry wt/h Oscillatoria limosa 0.83 mmol/mg chl a/h and nonmarine bacteria and algae (Table 5.1). The basic aim is to use water and sunlight to generate hydrogen gas in a biological system (analogues to the electrolysis of water) utilizing chloroplast membrane and the enzyme hydrogenase as catalyst. In this system, electron donated by the splitting of water or electron donor substances are excited to a more negative redox potential state, and then a portion of these are transferred to H+ to form hydrogen. Although, most of the early experiments with this phenomenon utilized freshwater species as subjects, the marine environment contains many species of photosynthetic bacteria and algae which are capable of the same function. In fact, from a quantitative point of view, the marine environment is perhaps better suited for studying such systems since salt water is more ubiquitous than freshwater. In tropical and subtropical marine environment, photosynthetic organisms (bacteria and algae) can be found in abundance year around. The intact cell contains many substances which complete the hydrogen- producing electron. Each of electron flow systems decreases the efficiency of hydrogen production. To overcome this problem, the cell can be dissected to yield only the essential elements and thereby eradicate all possible competitors. So, now attempts have been going on to understand the mechanism of cell-free system for hydrogen production. During the past decades, the progress in cell and enzyme immobilization in hallow fiber bioreactors has aroused considerable hope and interest to produce hydrogen, massively.

5.1 Introduction

Hydrogen is regarded as a potential energy source of the future, since it is easily converted to electricity and burns cleanly, as a highly efﬁcient energy carrier [1]. It is renewable, does not evolve the “greenhouse gas” CO2 in combustion, liberates large amounts of energy per unit weight in combustion, and is easily converted to electricity by fuel cells. In addition, biological hydrogen production has several advantages over hydrogen production by photoelectrochemical or thermochemical processes. Biological hydrogen production by photosynthetic microorganisms, for example, requires the use of a simple solar reactor such as a transparent closed box, with low-energy requirements. Electrochemical hydrogen production via solar battery-based water splitting on the hand requires the use of solar batteries with high-energy requirements. 5.1 Introduction 227

Hydrogen is currently produced by fossil fuel-based processes which emit large amounts of CO2 and relatively smaller amounts of other air pollutants such as sulfur dioxide and nitrogen oxides. Biological H2 production has thus recently received renewed attention owing to urban air pollution and global warming concerns [2]. Biological hydrogen production methodologies incorporating artificial recon- stitution systems with chloroplast, ferredoxin, and hydrogenase, a heterocystous cyanobacterial system with oxygen scavengers, and an algal system in a day-and- night cycle have been studied in Japan. From an engineering point of view, however, bacterial fermentation mechanisms for hydrogen production under either dark or light conditions are currently of importance in terms of environmental issues and the utilization of organic wastes such as waste effluent of the food and fermentation industries, pretreated sewage sludge, and market garbage. The use of microalgae as source of liquid fuels is an attractive proposition from the point of view that microalgae are photosynthetic renewable resources, are of high lipid content, have faster growth rates than plant cells, and are capable of growth in saline waters which are unsuitable for agriculture. While the lipid content of microalgae on a dry cellular weight basis varies between 20 and 40 %, lipid contents as high as 85 % have been reported for certain microalgal strains. Botryococcus braunii is a unique microalgal strain, having a long-chain hydrocarbon content of between 30 and 40 % (dry weight basis), which is directly extract- able to yield crude oil substitutes. Both physical and chemical processes are applicable in the production of liquid fuels from algal strains of high lipid content. These processes include direct lipid extraction in the production of diesel–oil substitutes, transesterification in the formation of ester fuels, and hydrogenation in the production of hydrocarbons [3]. Oily substances are also produced via liquefaction of microalgal biomass through thermochemical reactions under conditions of high pressure and temperature. The future widespread use of hydrogen is likely to be in the transportation sector where it will help reduce pollution. Vehicles can be powered with hydrogen fuel cells, which are three times more efficient than a gasoline-powered engine. As of today, in all these areas, hydrogen utilization is equivalent to 3 % of the energy consumption, but it is expected to grow significantly in the future [4]. Recently, many new alternatives through biological hydrogen production have proven to be encouraging to think of the possibility of hydrogen as pollution-free and low-cost biofuel. Biological hydrogen production is the most challenging area of biotechnology with respect to environmental problems. The future of biological hydrogen production depends not only on research advances, i.e., improvement in efficiency through genetically engineering microorganisms and/or the development of bioreactors, but also on economic considerations (the cost of fossil fuels), social acceptance, and the development of hydrogen energy systems. As said earlier, hydrogen fuel is the cleanest fuel available. It can be generated using surplus renewable energy supply and after using an ITM Power electrolyzer. This offers a low-cost renewable clean fuel, which can be made on-site at the point of use, eliminating the need for transported fuel deliveries. 228 5 The Microbiological Production of Hydrogen

The main area for hydrogen fuel use is for road transportation, which makes up a huge percentage of transportation emissions worldwide. The UK has a target to reduce carbon in transportation by 90 %, and in the USA, California was the ﬁrst to pass legislation on renewable energy mobility with funds being made available to support the deployment of a hydrogen infrastructure. Due to its zero carbon offering, hydrogen fuel for fuel cell electric vehicles (FECVs) is now high on the agenda in a number of countries with signiﬁcant government projects enabling the rollout of hydrogen mobility program. These programs are supporting the availability of fuel cell electric vehicles (FCEVs) to the public while at the same time ensuring that there is a hydrogen infrastructure in place to refuel. The vehicles provide a range of over 400 miles from one tank of hydrogen, and refuel in less than 5 min, and the only emission is water vapor.

5.2 History and Milestone of Microbial Hydrogen Production

In the first half of the last century, it was observed that the unicellular green alga Scenedesmus obliquus, after adaptation to anaerobic conditions, develops hydrogen metabolism. Hans Gaffron, University of Chicago, in 1939 had taken the first credit of observing the phenomenon of switch from the production of oxygen to the production of hydrogen [5]inChlamydomonas reinhardtii (a green alga). Soon after a gap of 3 years, in 1942 Hans Gaffron and Jack Rubin observed that the green alga Scenedesmus obliquus, when subjected to anaerobic conditions, produces hydrogen in the absence of carbon dioxide or uses hydrogen as the electron donor to fix carbon dioxide when the latter is present. Other green algae, including Chlorella fusca and Chlamydomonas reinhardtii, behave similarly when incubated anaerobically. Unfortunately, neither he nor his fellow scientists could able to find out the scientific cause of such incident, for a long period. In 1949, Howard Gest and Martin Kamen [6] at Washington University in St. Louis, Mo., concluded that the photosynthetic bacterium Rhodospirillum rubrum produced hydrogen through a side reaction of nitrogenase, the enzyme responsible for converting nitrogen into ammonia. Nitrogen-fixing cyanobacteria contain nitrogenase in specialized cells called heterocysts; in sunlight, several Anabaena species produce hydrogen via nitrogenase in heterocysts (Fig. 5.8). Because nitrogenase requires energy from adenosine triphosphate (ATP) molecules, this metabolic route to hydrogen production is less efficient than that for hydrogenase. Other investigators observed photosynthetic hydrogen evolution in anaerobic cultures of Chlorella fusca [7] and Chlamydomonas moewusii [8]. In 1974, it was concluded that many species of unicellular green algae have a hydrogen metabolism [9]. One of the most efficient hydrogen producers was C. reinhardtii [10, 11]. In 1981, Jeffrey Houchins and Robert Burris at the Univer- sity of Wisconsin–Madison reported the discovery of a reversible, or bidirectional, 5.2 History and Milestone of Microbial Hydrogen Production 229 hydrogenase in the cyanobacterium Anabaena 7120 (ATCC Nostoc muscorum) that produced hydrogen when ammonia repressed the nitrogenase. Although not universal, bidirectional hydrogenases are found in many cyanobacteria, including various species of Nostoc, Anabaena, Synechocystis, and Synechococcus. During the 1980s, Paul Roessler and Stephen Lien at the Solar Energy Research Institute, now the National Renewable Energy Laboratory (NREL), purified a hydrogenase from C. reinhardtii and determined that ferredoxin is its direct electron donor. The reaction direction of the bidirectional hydrogenase is dictated by the partial pressure of hydrogen and the presence of alternative electron sinks such as carbon dioxide. Hydrogen production increases considerably under either anaerobic or microaerobic conditions, as is the case with green algal hydrogenases. The physiological function of the bidirectional hydrogenase is still under debate, with proposed roles in fermentation and in serving as an electron valve during photosynthesis. The bidirectional hydrogenase in the genetically manipulatable Synechocystis sp. PCC 6803 is constitutively transcribed, making this strain attractive for further study of photobiological hydrogen production. In the late 1990s, Professor Anastasios Melis, a researcher at the University of California at Berkeley, discovered that if the algae culture medium is deprived of sulfur, it will switch from the production of oxygen (normal photosynthesis) to the production of hydrogen. He could notice that the change in oxygen to hydrogen production is enzyme (hydrogenase) dependent, highly oxygen sensitive. Melis [12] found that depleting the amount of sulfur available to the algae interrupted its internal oxygen flow, allowing the hydrogenase an environment in which it can react, causing the algae to produce hydrogen [13]. Chlamydomonas reinhardtii is also a good strain for the production of hydrogen [14]. Subsequently, after a long gap, in 1997, Professor Anastasios Melis could find out that the algal culture deprived of sulfur induced hydrogen production, instead of oxygen generation. This is being regulated by the enzyme hydrogenase [15, 16]. After about a decade, in 2006, researchers from the University of Bielefeld and the University of Queens- land developed genetically modified strain of Chlamydomonas reinhardtii having the quality of large amount of hydrogen production [17]. In 2007, the role of copper was observed in blocking oxygen production and switching onto hydrogen generation [18]. In the same year, Anastasios Melis studying solar-to-chemical energy conversion efficiency in tlaX mutants of Chlamydomonas reinhardtii achieved 15 % efficiency, demonstrating that truncated Chl antenna [19] size would minimize wasteful dissipation of sunlight by individual cells [20]. This solar-to-chemical energy conversion process could be coupled to the production of a variety of biofuels including hydrogen. In the subsequent year, Anastasios Melis could be able to increase the hydrogen to about 25 % over the theoretical value of 30 % in efficiency in tlaR mutants of Chlamydomonas reinhardtii [21]. Interestingly, it was noticed that by increasing the temperature, the hydrogen production rate was increased to tenfold over the previous value [22]. By addition of bioengineered enzyme, the hydrogen production was increased to 400 % over the control value [23]. In 2011, a team at Argonne’s Photosynthesis Group demonstrated how platinum nanoparticles can be linked to key proteins in algae to produce hydrogen 230 5 The Microbiological Production of Hydrogen fuel five times more efficiently [24, 25]. A group of scientists from Uppsala University observed that in Chlamydomonas reinhardtii, the photosystem II produces direct conversion of sunlight 80 % of the electrons that end up in the hydrogen gas [26]. Recently, researchers at Ruhr University and Max Planck Institute also tried to utilize the electrons from photosystem I for hydrogen production [27]. Researchers at the US Department of Energy’s Argonne National Labo- ratory are currently trying to understand how the part of the hydrogenase enzyme creates the hydrogen gas and introduced it into the photosynthesis process. By this process, hydrogen gas equivalent to oxygen produced by microalgae can be produced [28, 29]. Chlamydomonas moewusii is also a good strain for the production of hydrogen. It would take about 25,000 km2 to be sufficient to displace gasoline use in the USA. To put this in perspective, this area represents approximately 10 % of the area devoted to growing soya in the USA [12]. The US Department of Energy has targeted a selling price of $2.60/kg as a goal for making renewable hydrogen economically viable. One kilogram is approximately the energy equivalent to a gallon of gasoline. To achieve this, the efficiency of light-to-hydrogen conversion must reach 10 %, while current efficiency is only 1 % and selling price is estimated at $13.53/kg [30]. According to the DOE cost estimate, for a refueling station to supply 100 cars per day, it would need 300 kg. With current technology, a 300 kg per day stand-alone system will require 110,000 m2 of pond area, 0.2 g/l cell concentration, a truncated antennae mutant, and 10 cm pond depth [25]. Areas of research to increase efficiency include developing oxygen-tolerant FeFe-hydrogenases [31] and increased hydrogen production rates through improved electron transfer [32].

5.3 Hydrogen Production Process

Hydrogen can be produced using a number of different processes. Thermochemical processes use heat and chemical reactions to release hydrogen from organic materials such as fossil fuels and biomass. Water (H2O) can be split into hydrogen (H2) and oxygen (O2) using electrolysis or solar energy. Microorganisms such as bacteria and algae can produce hydrogen through biological processes.

5.3.1 Sustainable Hydrogen from Water

5.3.1.1 Water Electrolytic Processes

Electrolyzers use electricity to split water into hydrogen and oxygen. This reaction takes place in a unit called an electrolyzer. Electrolyzers can range in size from small, appliance-size equipment that is well suited for small-scale distributed hydrogen production to large-scale, central production facilities that could be tied 5.3 Hydrogen Production Process 231

Fig. 5.1 Schematic Cathode Anode diagram of an electrolyzer Power uses electricity to split water Supply into hydrogen and oxygen (Ref. [33]) e- e-

Hydrogen Oxygen

Hydrogen Oxygen Bubbles Bubbles Membrane e- e-

H+ H+ + - → → +++ - 4H + 4e 2H2 2H2O O2 4H 4e Cathode Reaction Anode Reaction

directly to renewable or other non-greenhouse gas-emitting forms of electricity production (Fig. 5.1). This technology is well developed and available commercially, and systems that can efﬁciently use intermittent renewable power are being developed [33].

5.3.1.2 Thermochemical Process

Hydrogen can be generated from water thermally by using high temperature. Thermal processes are not encourageable as this process involves high cost. Besides this, engineering problems like development and maintenance of high-temperature- resistant (2500 k) vessels for decomposing water into hydrogen are the main causes of tussle for development of such project. Hydrogen can be produced at relatively lower temperature through decomposition of water in a series of chemical reactions in a close cycle, and the chemical intermediaries are generated. Among the various thermochemical processes, the Sulfur-Iodine thermochemical water-splitting cycle is an effective and reasonable way for hydrogen (H2) producing [34]. But this reaction requires large quantity of heat and chemicals which may cause problems like corrosion and toxicity [35]. The former requirement can be managed through nuclear energy as an inexpensive heat source. However, no adequate scientiﬁc attention has yet been given on techno- economic feasibility on the aforesaid project. It is believed that electrolytic water- 232 5 The Microbiological Production of Hydrogen splitting techniques are more encourageable as compared to thermochemical process.

5.3.1.3 Direct Solar Water-Splitting Processes

Direct solar water-splitting, or photolytic, processes use light energy to split water into hydrogen and oxygen. These processes are currently in the very early stages of research but offer long-term potential for sustainable hydrogen production with low environmental impact. Among various interesting reactions, the splitting of water into molecular hydrogen and molecular oxygen by visible light (reaction 5.1)is potentially one of the most promising ways for the photochemical conversion and storage of solar energy [34–37]:

visible light H2O ! H2 þ 1=2O2 ð5:1Þ Visible light

The water-splitting process has two other advantages. First, the raw material, i.e., water, is abundant and cheap. Second, the combustion of H2 in air (reverse of reaction) [36] again gives water. In other words, the overall process is cyclic and nonpolluting. The main disadvantage is that H2 can react explosively with O2. However, the explosive limit of H2 in air is 4.00 %, less than that of butane: 1.86 %. Hence, the utilization of hydrogen is no more dangerous than that of natural gas, and like natural gas, hydrogen can be quite easily stored and transported. Since water does not absorb visible light, intermediates are needed to achieve the water photocleavage via a cyclic pathway (reaction 5.1). Numerous strategies have been described, and several model systems capable of producing separately hydrogen and oxygen have been proposed [38, 39].

5.3.1.4 Hydrogen Production Through Photoelectrochemical Processes (PEC)

One extremely attractive way of producing hydrogen is by dissociating water on a semiconductor substrate using sunlight (Fig. 5.2). The efﬁciency of this process is mainly determined by the photo-physical properties and morphology of the semiconductor material used. In the current state of the art of this technology, the commercial application of hydrogen production by means of visible spectrum photon energy requires signiﬁcant development of the science and engineering involved to achieve active and stable photocatalysts in the dissociation reaction. Issues such as the transfer of charge between the semiconductor and the cocatalyst and its dependence on structural and electronic factors in the interface remain unresolved. These areas represent excellent opportunities to improve the photocatalysts used in the photochemical dissociation of water. Innovative processes to control the morphology of the catalyst at the nanometric scale represent 5.3 Hydrogen Production Process 233

Fig. 5.2 Two different approaches to PEC solar hydrogen production reactors: (a) electrode systems similar to ﬂat-plate photovoltaic panels and (b) particle systems comprised of slurries of PEC semiconductor particles. (Ref. [46]) http://energy. gov/eere/fuelcells/ hydrogen-p

another avenue of research that will enable modulation of photocatalyst morphology and reactivity.

5.3.2 Hydrogen from Fossil Fuel

Hydrogen can be produced using diverse, domestic resources including fossil fuels, such as natural gas and coal (with carbon sequestration), nuclear energy, and other renewable energy sources. Natural gas, methane, is a rich source of hydrogen and is being processed by steam reforming of methane. In this process, methane and water vapors are combined to produce hydrogen and carbon monoxide:

CH4 þ H2O ! 3H2 þ CO

The reason for popularization of this process is availability of natural gas in abundant quantity in most parts of the world. In addition, natural gas is having highest hydrogen content of all fossil fuels and is of low cost. In terms of production 234 5 The Microbiological Production of Hydrogen capacity, the process second to steam reforming of methane for hydrogen production is the partial oxidation of petroleum oil. Hydrogen can also be produced from petroleum using a partial oxidation reaction that producesCO2,H2, and CH4. Direct gasiﬁcation of coal and biomass is also a well-established process for the production of hydrogen but with CO.

5.3.3 Hydrogen from Cellulose Biomass

Hydrogen can be obtained from a renewable source such as cellulose biomass. The cellulose can be turned into H2 via various thermochemical processes such as combustion, liquefaction, pyrolysis, and gasification. Lignocellulose material is partially oxidized at temperatures of over 1000 K, producing a gaseous fraction together with a carbon residue that is subsequently reduced to form H2, CO, CO2, and CH4. Gasification of biomass in the presence of O2 generates a gas stream rich in hydrogen which is reformed with water vapor at the exit from the gasifier to produce additional hydrogen. The main drawback of the gasification of biomass is tar formation. The heavy residues polymerize and form more complex structures that are not suitable for the production of hydrogen by steam reforming. Tar formation can be minimized by an appropriate design of gasifier, incorporating catalytic additives, and by controlling the operating parameters. The catalysts reduce the tar content but are particularly effective at improving the quality and enhance conversion of the gas fraction produced. Another problem inherent in the gasification of biomass is the formation of ash, which can cause a buildup of solids, plugging, and deactivation. These problems can be minimized by extraction and fractioning.

5.3.4 Hydrogen Production by Bioethanol Reforming

The most convenient way of transporting hydrogen is to use renewable precursors, such as ethanol (C2H5OH) and sugars (C6H12O6) in the liquid phase. These precursors are then transformed into hydrogen by means of reforming processes with steam or under pressure in the liquid phase at the point of hydrogen consumption. Bioethanol is a prosperous renewable energy carrier mainly produced from biomass fermentation. Reforming of bioethanol provides a promising method for hydrogen production from renewable resources. Besides operating conditions, the use of catalysts plays a crucial role in hydrogen production through ethanol reforming. Rh and Ni are so far the best and the most commonly used catalysts for ethanol steam reforming toward hydrogen production. The main difﬁculty these reactions face is that they are not selective, given that the reaction conditions encourage other side reactions that give rise to undesirable by-products (carbon monoxide, methane, acetaldehyde) and, therefore, a reduction 5.4 Microbial Resources for Hydrogen 235

in the H2 selectivity. Moreover, the catalysts used suffer deactivation as a result of carbon deposits building up, which makes it difﬁcult to put this technology into practice. The challenge is to develop catalytic systems that operate at lower temperatures in order to minimize deactivation processes.

5.3.5 Hydrogen Production by Water Biophotolysis

Biological hydrogen evolution provides a sustainable and environmentally friendly way to produce clean energy from renewable resources. Cyanobacteria and green microalgae split water into molecular hydrogen and oxygen using sunlight under special conditions [40, 41]. Progress has been made in solving the intrinsic incom- patibility of the simultaneous evolution of hydrogen and oxygen gas in photoautotrophic cells, particularly the adverse impact on the key hydrogen enzymes (hydrogenase and nitrogenase). Hydrogen can also be produced by biological systems. Some photosynthetic microorganisms are able to break water molecules down into their components (H2 and O2). Certain algae, such as the Scenedesmus green algae, produce H2 when they are illuminated with visible light or kept in anaerobic conditions in the dark. Green algae are also employed in another method of H2 production. The Scenedesmus species produces hydrogen not only under irradiation with light but also by fermentation under anaerobic conditions, using starch as a reducing source. Although the rate of production of H2 per unit weight by fermentation is lower than that obtained by irradiation with light, the output is kept stable by the absence of oxygen. Cyanobacteria also produce hydrogen by fermentation in the absence of light under anaerobic conditions. Of the various cyanobacteria tested, the Spirulina species has shown the highest level of activity [42–45].

5.4 Microbial Resources for Hydrogen

5.4.1 Fermentative Hydrogen Production

Anaerobic microbes are proven to be best biocatalysts for hydrogen production in dark condition by using organic wastes as cheap substrate. This process otherwise known as “dark fermentation” has drawn attention because of its ability to produce an environmentally friendly energy source while simultaneously stabilizing waste [46–51]. The theoretical maximum yield of hydrogen fermentation is reported to be four moles of hydrogen per mole of glucose [52] or eight moles of hydrogen per mole of sucrose [47], if all of the substrate would be converted to acetic acid. If all the substrate would be converted to butyric acid, these values are two and four moles of hydrogen per more of glucose and sucrose, respectively. However, in 236 5 The Microbiological Production of Hydrogen practice, a fraction of biomass is only diverted for hydrogen production, and the remaining is utilized for other metabolic activity. Keeping this in view, attempts have been made to regulate hydrogen production by regulating the physical conditions of bioreactor [53, 54]. The presence of methanogens like bacteria in the bacterial consortium used for fermentation consumes hydrogen and reduces the quantity of hydrogen in the fermenter tank. In order to overcome with such problem, attempts have been made to preheat the inoculum used to seed the reactors as a method to inactivate or eliminate these microorganisms. Lay [48] and Okamoto [55] used wet heat treatment (boiling for 15 min) of anaerobic digester sludge, whereas Van Ginkel et al. [56] used dry heat treatment of compost and soils. The main reason being for such practice is to arrest the activity of hydrogen-consuming microorganisms and to select for hydrogen-producing bacteria. Besides this, the endospores of the hydrogen-producing bacteria (e.g., Clostridium and Bacillus species) survive due to high-temperature-resistant nature of bacterial endospore. When favorable conditions return, the spores germinate and become vegetative cells [57, 58]. A similar idea (heat application at 80 C for 10 min) has been used to eliminate nonspore- forming bacteria during isolation procedures of spore-forming bacteria, such as Clostridium species [58, 59]. It has been reported that Clostridium botulinum and Clostridium perfringens (strains T-65 and S-45), two human pathogens [60], can tolerate 80 C/10 min and 75 C/120 min, respectively [61]. The spores of some heat-resistant strains of C. perfringens can be activated by heat treatment at 100 C[60]. In most cases, heat activation at 75–80 C for 15–20 min is used to inactivate vegetative cells and activate germination of spores [58]. Generally, spore germination in bacteria is a rapid process and lasts for 60–90 min. As the spore germination is sensitive to pH, by increasing the bacterial inoculums to 9.5, about most of the hydrogen- consuming bacteria are killed [58]. Lowering incubation temperature from 45 to 7 C resulted in a decrease in the germination rate from 85 to 75 % (at a pH of 6.0). Moreover, oxygen is known to inhibit germination. The addition of sodium bicarbonate and CaCl2 enhanced germination of C. perfringens spores. Finally, the presence of lysozyme, a protein found in hen egg white, mucus, tears, and blood, helps germination of endospores [60]. So, sincere attempts have been going on for biohydrogen production from organic wastes, particularly wastewater and agricultural residues, which are abun- dantly available in all over the world. Interesting impressive reports are available with a focus on hydrogen-producing bacteria, fermentation processes (dark fermentation and photofermentation), and bioreactor conﬁgurations. Although dark fermentation is more efﬁcient for hydrogen production, by-products generated during the fermentation not only compromise hydrogen production yield but also inhibit the bacteria. Two strategies, combination of dark fermentation and photofermentation and coupling of dark fermentation with a microbial electrolysis cell (MEC) (Fig. 5.3)[62], are expected to be most encouraging from commercial point of view. 5.4 Microbial Resources for Hydrogen 237

Fig. 5.3 A schematic model for hydrogen production from organic waste through dark fermentation and photofermentation (Keywords: biohydrogen dark fermentation microbial electrolysis cell (MEC), microbial fuel cell (MFC), organic wastes photofermentation [62])

5.4.2 Hydrogen from Rhodopseudomonas palustris

Rhodopseudomonas palustris is a rod-shaped gram-negative purple nonsulfur bacterium (PNSB) and chemoheterotrophic [63]. These bacteria can be used for bioplastic synthesis and hydrogen production. R. palustris has the unique characteristic of encoding for a vanadium-containing nitrogenase. It produces, as a by-product of nitrogen fixation, three times more hydrogen than do molybdenum- containing nitrogenases of other bacteria. R. palustris is but one among diverse microbes that release H2 during growth, ridding themselves of excess electrons that they generate. These electrons combine with dissolved protons that are freely available within the cells. The potential to manipulate R. palustris to be used as a reliable hydrogen production source or for biodegradation still lacks detailed knowledge of its metabolic pathways and regulation mechanisms [63]. In addition, PNSB have certain plus points over cyanobacteria and algae. These bacteria produce H2 under anaerobic conditions; their H2-generating enzymes are not exposed to oxygen molecules. Moreover, because they obtain energy from light, H2 yields from electron-donating compounds are very high. However, the electron donors that PNSB use to produce H2 are not as abundant as water. Besides this, PNSB metabolize diversified organic compounds, including agricultural and food wastes [54–68]. With the depletion of oxygen, R. palustris cells turn to deep purple color and start synthesizing light-absorbing pigments that help to carry out photosynthesis. R. palustris generates ATP anaerobically by a photosynthetic process that differs from that of green plants, algae, and cyanobacteria in that it does not produce oxygen. It has been observed that the genetic code of R. palustris is having the Calvin cycle for fixing CO2, enabling it to grow photoautotrophically with CO2 as its sole carbon source. However, it prefers to grow photoheterotrophically on organic compounds, especially acetate and lignin. Under anaerobic condition, and in the presence of light, R. palustris and other PNSB convert atmospheric nitrogen 238 5 The Microbiological Production of Hydrogen to ammonia with the evolution of hydrogen also. The reason being of hydrogen production is to allow it to dispose of electrons that generate during metabolism. Nitrogenase diverts about 25 % of its electron to produce hydrogen, even in the presence of 50 % atmospheres of nitrogen. In 1949, Martin Kamen at Washington University could first observe that in the PNSB Rhodospirillum rubrum, grown in glutamate containing medium, the hydrogen production gets reduced in the presence of nitrogen gas. This observation led Gest to hypothesize, test, and prove that the enzyme responsible for producing H2 also fixes N2. This is because ammonium completely represses nitrogenase activity, and thereby H2 production, whereas glutamate does not. The nitrogenase enzyme protein content in PNSB bacteria is about 2 % of total cellular protein but having slow catalyst in nature. Moreover, making this enzyme is complicated in itself. Specifically, the enzyme contains an iron–molybdenum cofactor that must be synthesized separately and then inserted into the apoenzyme by a series of auxiliary proteins. Finally, the nitrogenase + reaction uses 16 ATP molecules to fix a single molecule of N2 to make 2NH4 . For these reasons, microbes tightly regulate their synthesis of nitrogenase and its activity. In R. palustris, nitrogenase regulation is much complicated, as compared to other nitrogen-fixing bacteria. In this connection, James B. McKinlay [50] from the Indiana University and Caroline S. Harwood from University of Washington from their gene expression experiments could conclude that the nitrogenase activity mainly depends on cellular levels of the metabolite α-ketoglutarate and the amino acid glutamine. Levels of α-ketoglutarate are high in cells that are starving for nitrogen and therefore must resort to nitrogen fixation, whereas high levels of glutamine are found in cells with access to a rich nitrogen source such as ammonium. For R. palustris to produce H2, researchers are deeply involved in increasing its hydrogen production potential by regulating the dual function of nitrogenase activity through new mutants and increasing the functional efficiency of nitrogenase isozymes—the most commonly found molybdenum-containing nitrogenase and the alternative vanadium- and iron-containing nitrogenases [69–71].

5.4.3 Hydrogen from MEC

Researchers from China successfully demonstrated biological production of hydrogen from organic matters using bacteria at below 25 C. Recently, Defeng Xing and his team at the Harbin Institute of Technology could be able to achieve signiﬁcant amount of hydrogen from organic wastes between 4 and 9 C by using a MEC (Fig. 5.4). This eliminates the cost of heating and could enable hydrogen production to be carried out at high latitudes and mountainous regions where the air temperature is below 10 C. In MECs, direct electric current is applied to bacteria for hydrogen production. By bacterial fermentation of organic matters or decomposed plant residues, acetic acid is formed and subsequently consumed by bacteria, releasing proton, electron, and CO2. Addition of an electric current enables the 5.4 Microbial Resources for Hydrogen 239

Fig. 5.4 An MEC allows hydrogen production from organic materials at low temperatures [223](Source: http://www.rsc.org/ chemistryworld/News/ 2011/February/11021103. asp, Ref. [223])

protons and electrons to join together to make hydrogen gas, and the higher the current, the more hydrogen is produced. Methanogenesis, or methane formation, is a common problem in MECs, which occurs at higher temperatures as a result of bacterial anaerobic respiration. This can reduce the efﬁciency of electron transfer to the cathode, reducing the overall output of hydrogen. However, at temperatures below 10 C, no methane was produced since the growth of the methane-producing organisms was inhibited and the yield of hydrogen produced is comparable to that at temperatures above 25 C.

5.4.4 Hydrogen Production in Extreme Bacterium

Recently, Melanie Mormile, professor of biological sciences at Missouri S and T, and her team discovered the bacterium Halanaerobium hydrogeninformans in Soap Lake, Washington [72]. This extremophile bacterium produces hydrogen under saline and alkaline conditions [72]. An extremophile is a microorganism that lives in conditions of extreme temperature, acidity, alkalinity, or chemical concentration. Living in such a hostile environment, Halanaerobium hydrogeninformans has metabolic capabilities under conditions that occur at some contaminated waste sites. It is supposed to be an iron-reducing bacterium and describe a new species. This bacterium can produce hydrogen and 1,3-propanediol under high pH and salinity conditions that might turn out to be valuable industrially. An organic compound, 1,3-propenediol, can be formulated into industrial products including composites, adhesives, laminates, and coatings. It’s also a solvent and can be used as antifreeze. It is too early to predict the possibility of this bacterium to be used as feature source for hydrogen production and to exploit the vast saline ecosystem for hydrogen production. 240 5 The Microbiological Production of Hydrogen

5.4.5 Hydrogen Production by Clostridium sp.

Clostridium thermocellum is a thermophilic, cellulolytic bacterium that has been extensively studied for its efficient depolymerization of crystalline cellulose into hydrogen, ethanol, and organic acids as fermentation end products [73–76]. Hydro- gen production rates and yields are regulated by the carbon-flow distribution among various metabolic pathways, which are greatly influenced by the culture environment. Growth nutrient composition, a crucial component of the microbial environment, needs to be designed with care as it determines the economy and efficiency of a process to a great extent. To date, considerable research efforts have been directed toward optimization of biohydrogen production [77–79] from soluble feedstock, but none involved direct fermentation of cellulose. The bacterium Clostridium kluyveri metabolizes ethanol and acetate to butyrate and caproate though it is formed during fermentation (Fig. 5.5)[80]. During this process, ATP is generated by the acetate kinase reaction. The fermentation of glucose by Clostridium kutricum and C. acetabutylicum can be regarded as also prototype clostridial fermentation. Their end products are butyrate, acetate, butanol, ethanol, acetone, 2-propanol, CO2, and H2.

5.4.6 Hydrogen Production by Acetogenic Bacteria

Acetogenic bacteria are a specialized group of strictly anaerobic bacteria that are ubiquitous in nature. Together with the methane‐forming archaea, they constitute the last limbs in the anaerobic food web that leads to the production of methane from polymers in the absence of oxygen. The acetogenic bacteria convert CO2 in sugars into acetate using hydrogen as a source of energy. They are obligate in nature and produce hydrogen as an intermediate metabolite. On the basis of physiology and biochemistry, a narrow association exists between the H2-producing acetogenic bacteria and the H2-consuming methanogenic bacteria, thereby regulating the H2 level in their environment. Methanogenic bacteria utilize molecular hydrogen in the usual anaerobic digester so rapidly that the hydrogen partial pressure can be kept as low as 10À4 atm which is enough to ensure the active performance of the hydrogen- producing acetogenic bacteria. This is the reason that the degradation of higher fatty acids depends largely on the activity of methanogenic bacteria. Microbial association in which a H2-producing organism can grow only in the presence of H2-consuming organism are called syntrophic association. The coupling of formation and use of H2 is called interspecies hydrogen transfer (HTS). Hydrogenase has been demonstrated in C. aceticum, A. woodii, C. thermoaceticum with C. aceticum, and A. woodii. It has been noticed that hydrogen is produced when fermentation of fructose, glucose, and lactate (Fig. 5.6)[81] is carried out. Winter and Wolfe [82] clearly demonstrated the production of hydrogen when A. woodii is cultured with a methanobacterium. In 5.4 Microbial Resources for Hydrogen 241

Fig. 5.5 Production of hydrogen from ethanol and acetate. Ethanol-acetate fermentation of C. kluyveri. For simpliﬁcation purposes, it was assumed that only butyrate, acetic acid, and H2 rather than caproate are formed as fermentation products. Inset shows the two membrane- associated, energy-converting enzyme complexes involved in the fermentation: ferredoxin, NAD oxidoreductase (RnfA-E) and ATP synthase (AtpA-I). The clostridial ferredoxin with two [4Fe4S] clusters accepts two electrons upon reduction. In the scheme, it is assumed that seven of the ten NADH required for the reduction of 5 crotonyl-CoA to 5 butyryl-CoA are generated during ethanol oxidation to acetyl-CoA (gray background) and that three are generated via NAD+ 2À 2À reduction with Fdred (orange background). It has been omitted that some of the Fdred generated during crotonyl-CoA reduction is also used for the regeneration of NADPH, which is required for acetoacetyl-CoA reduction. The yellow area indicates the microcompartment, and the shaded area highlights the steps proposed to be involved in the catalysis of reaction 5.5 by the butyryl-CoA dehydrogenase complex composed of butyryl-CoA dehydrogenase (Bcd) and the electron transport ﬂavoproteins (EtfAB). Bcd, EtfA, and EtfB each contain FAD (Ref. [224]) this experiment, interspecies hydrogen transfer was clearly demonstrated. It has also been shown that H2 cycling system is present in Desulfovibrio gigas. In such a system, hydrogen is generated inside the cell by a cytoplasmic hydrogenase. The molecular hydrogen passes through the cell membrane to the outside, where a second hydrogenase oxidizes it to proton. The oxidation generate electrons which are ultimately transferred back into cell and used to reduce SO4. The proton generated on the outside of the membrane may also enter the cell through a reverse ATPase enzyme with concomitant of ATP. During acidogenesis, the short-chain fatty acids, other than acetate produced in anaerobic fermentation, are further converted to acetate, hydrogen gas, and carbon dioxide by the acetogenic bacteria. β-Oxidation is the mechanism of anaerobic oxidation of long-chain fatty acids with products hydrogen and acetate. The available H2 and CO2 are partly converted into acetate by the homoacetogenic bacteria. 242 5 The Microbiological Production of Hydrogen

Fig. 5.6 Schematic presentation of PSI and PSII, showing splitting of water and movement of electron through PSII to PSI, and harvesting high-energetic biological compound. Engineering of the hydrogen-producing enzyme to create an Fd-H2ase fusion changes the composition of the hydrogen production circuit to include both direct (box 1) and indirect (box 2) H2 production modes. The CO2 ﬁxation circuit (box 3) remains open but operates at a reduced level. Credit: Image provided by Paul King, NREL

Both propionate and butyrate are important intermediates in anaerobic digestion and then are converted by the hydrogen-producing acetogenic bacteria into acetate and hydrogen (Table 5.2)[83].

5.4.7 Hydrogen Production by Desulfurizing Bacterium (Desulfovibrio vulgaris)

The sulfate-reducing bacteria occur predominantly in decomposing sediment and black mud where organic material is under anaerobic degradation. The desulfurizer seems to be specially adapted to the products of incomplete carbohydrate metabolism, i.e., fatty acid, oxyacids, alcohols, and hydrogen from fermentations (Fig. 5.7). Some of the desulfurizing bacteria are also able to metabolize lactate or pyruvate in the absence of sulfate:

4CH2 þ COCOOH þ H2SO4 ! 4CH3 À COOH þ 4CO2 þ H2S

And the product is replaced by a fermentative reaction and simultaneous evolution of hydrogen:

CH2 þ COOH þ H2O ! CH2 À COOH þ CO2 þ H2 5.4 Microbial Resources for Hydrogen 243

Table 5.2 Some acetogenic reactions (propionate and butyrate degradation) and the corresponding free energy change—ΔGo Reactions ΔGo (KJ/mol) À CH3–CH2–COO + 3H2OCH3COO + HCO3- +2H+ + 2H2 +76 1 À À + CH3–CH2–CH2–COO +3H2O 2CH3COO +2H +2H2 +48.1 Source: acetogenesis—ILIAS 3 http://cgi.tu-harburg.de/~awwweb/wbt/emwater/lessons/lesson_ b3/lm_pg_1292.html Ref. [83]

Fig. 5.7 Schematic representation of two proposed pathways for electron transport during sulfate reduction in Desulfovibrio vulgaris Hildenborough. The hydrogen cycling model describes the ﬂow of reducing equivalents from the electron donor to oxidized sulfur species through hydrogen metabolism. This ﬂow can be mediated by hydrogenases and other electron carriers, including cytochromes. The second pathway can transfer electrons to the membrane-associated menaquinone pool. Dark-gray ovals indicate enzymes and electron carriers deleted in the mutant strains used in this study. Abbreviations: LacP, lactate permease; Ldh, lactate dehydrogenase; Por, pyruvate-ferredoxin oxidoreductase; Hase, hydrogenase; Cyt. c, cytochrome c; Mq, menaquinone pool; APS, adenosine 50-phosphosulfate. Dashed lines and the question mark indicate currently hypothetical pathways and components. Published online 2013 Jun 25 [225]

5.4.8 Hydrogen Production by Cyanobacteria

Cyanobacteria (also called blue-green bacteria, blue-green algae, Cyanophyceae, or cyanophytes) are a large and widespread group of photoautotrophic microorganisms, which originated, evolved, and diversiﬁed early in earth’s history [84–98]. The earliest forms attributed to this group were found in sedimentary rocks formed 3.5 billion years ago, and it is commonly accepted that cyanobacteria played a crucial role in the Precambrian phase by contributing oxygen to the atmosphere [99]. 244 5 The Microbiological Production of Hydrogen

These bacteria are quite a large number of diversified algae found in a wide range of variable habits including aquatic (salt water and freshwater), terrestrial, and extreme environments (like frigid lakes of the Antarctic or hot springs). Besides blue-green pigments, this group of algae is having a variety of other pigments like chlorophyll a, carotenoids, and phycobiliproteins. The presence of chlorophyll a and physiological nature of oxygenic photosynthesis differentiate from other photosynthetic bacteria, such as purple and green bacteria. Some cyanobacteria are having the quality of anoxygenic photosynthesis by using hydrogen sulfide (H2S) as the electron donor. Cyanobacteria are also having unique quality of fixing atmospheric nitrogen into ammonia (NH3) which eukaryotes do not have. Alike, possessing physiological and metabolic diversity, the members of cyanobacteria are varying in morphological appearance with unicellular, filamentous, and colonial forms. Within the filament, vegetative cells may develop into structurally modified and functionally specialized cells: the akinetes (resting cells) or the heterocysts (cells specialized in nitrogen fixation (Fig. 5.8)). For more detailed information on cyanobacteria, see reference [100]. Both the unicellular and filamentous cyanobacteria are also having symbioses and nitrogen-fixing ability with a diversified host. In symbiosis, some cyanobacteria perform both photosynthesis and nitrogen fixation, while others exhibit only one of these properties [101–105]. So, it is assumed that ancestors of cyanobacteria evolved to become plastids after a long period of endosymbiosis. In biochemical and structural detail, cyanobacteria are especially similar to the chloroplasts of red algae [106, 107].

Fig. 5.8 Heterocyst outline of H2-related metabolic routes in heterocyst-forming cyanobacteria. Vegetative cells synthesize saccharides (CH2O) by ordinary photosynthesis with accompanying evolution of O2 and uptake of CO2. Heterocysts receive the saccharides and use them (accompa- À nied by CO2 evolution) as the sources of e for N2ase reaction. For efﬁcient net production of H2, H2ases (uptake H2ase Hup and bidirectional H2ase Hox) have been inactivated. C6P, hexose phosphate; Fdox and Fdred, ferredoxin oxidized and reduced, respectively; OPPP, oxidative pentose phosphate pathway; PSI and PSII, photosystems I and II, respectively (adapted from [158] with modiﬁcation) 5.4 Microbial Resources for Hydrogen 245

The classification of cyanobacteria is still a matter of debate on its botanical status [108–112] and bacterial nature [113–115]. In spite of having such differential opinion, cyanobacteria are classified into four or five major subgroups. However, Rippka et al. [113] proposed five sections coincide broadly with orders of other classifications: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, and Stigonematales [100, 116]. With the advancement of genetic engineering, different molecular methods are being used and developed to infer phylogenetic relationships [116, 117]. A polyphasic taxonomy of cyanobacteria, integrating both phe- notypic and genotypic characters, is currently being assembled [100, 101, 110, 118, 119]. The blue-green algae have two photosystems, PSI and PSII, and are capable of generating reducing potential from water (Fig. 5.6). The capacity of algae to produce hydrogen gas and effect of light on the process (at low light intensity) were first reported by Gaffron and Rubin [15]. Nakumura [14] had taken first credit to explain hydrogen production under dark anaerobic condition by photosynthetic bacteria. The photoproduction of hydrogen in blue-green algae mainly depends on the type of species (Table 5.1). Besides this, other physical and physiological factors are also responsible for regulating the production of hydrogen.

5.4.8.1 Enzymes Associated with Hydrogen Production

In cyanobacteria, hydrogen production is accomplished by the catalytic action of two distinct complex enzymes (Fig. 5.9): (i). Nitrogenase This enzyme is found in the heterocysts of filamentous cyanobacteria when they grow under nitrogen-limiting conditions, catalyzing the production of hydrogen concomitantly with the reduction of N2 to NH3. (ii). Hydrogenase (a). An uptake hydrogenase, catalyzing the consumption of hydrogen produced by the nitrogenase. (b). A bidirectional hydrogenase, which has the capacity to both take up and produce hydrogen [120–127]. In N2-fixing strains, the net H2 production is the result of H2 evolution by nitrogenase and H2consumption mainly catalyzed by the uptake hydrogenase. Nitrogenases The nitrogenase complex is having two protein entities, namely, dinitrogenase (MoFe protein or protein I) and the dinitrogenase reductase (Fe protein or protein II). The earlier one is an α2β2 heterotetramer of about 220 to 240 kDa, and the α- and β-subunits are encoded by nifD and nifK, respectively. The nifH, a homodimer of about 60 to 70 kDa, is encoded with dinitrogenase and mediates the transfer of electrons from the external electron donor (a ferredoxin or a flavodoxin) to the dinitrogenase [123, 128, 129], specifically. In addition to the three structural nif 246 5 The Microbiological Production of Hydrogen

Fig. 5.9 Schematic presentation of two groups of enzymes associated with hydrogen production in cyanobacteria. Enzymes directly involved in hydrogen metabolism in cyanobacteria. While the uptake hydrogenase is present in all nitrogen-fixing strains tested so far, the bidirectional enzyme is distributed among both nitrogen-fixing and non-nitrogen-fixing cyanobacteria (Ref. [158]) genes, many other genes are involved in the nitrogen fixation process and its regulation [130]. Recently, following the completion of several cyanobacterial genome projects, it became evident that some strains contain multiple copies of certain nif genes; for example, N. punctiforme (Nostoc strain PCC 73102) appears to have three copies of nifH [131]. The reduction of nitrogen to ammonia, catalyzed by nitrogenase, is a highly endergonic reaction requiring metabolic energy in the form of ATP. Generally, when atmospheric nitrogen is available freely, about 75 % of the electrons are diverted for nitrogen reduction process, and the rest of 25 % are used 5.4 Microbial Resources for Hydrogen 247 for proton reaction. Since two ATP molecules are required for each electron transferred from dinitrogenase reductase to dinitrogenase, the reaction requires a minimum of 16 ATP molecules until the dinitrogenase has accumulated enough electrons to reduce nitrogen to ammonia. The overall reaction can be written as follows:

þ eÀ N2 þ 8H þ 8 þ 16 ATP ! 2NH3 þ H2 þ 16 ADP þ 16Pi

Due to extreme oxygen labile nature of nitrogenase, all diazotrophs must protect the enzymatic complex from the deleterious effects of oxygen (O2). Oxygenic photosynthesis is only noticed in cyanobacteria and prochlorophytes [123, 132]. Cyanobacteria are having high adaptability nature, ranging from fixing nitrogen only under anoxic conditions to temporal or even spatial separation of nitrogen fixation and oxygen evolution to protect their nitrogen-fixing machinery not only from atmospheric oxygen but also from the intracellularly generated oxygen [102, 130, 133–137]. In nonheterocystous cyanobacteria, temporal separation between photosynthetic oxygen evolution and nitrogen fixation seems to be the most common strategy adopted by nonheterocystous cyanobacteria [99, 138, 139]. Spatial separation of the two processes is achieved in many filamentous cyanobacteria by differentiation of vegetative cells into cells specialized in nitrogen fixation, i.e., the heterocysts. In the marine filamentous nonheterocystous Trichodesmium, a spatial separation probably occurs between the two processes without any obvious cellular differentiation; only a small fraction of cells within a colony or along the filament are capable of nitrogen fixation. In contrast to the irreversible changes occurring during heterocyst differentiation, those occurring in nonheterocystous cyanobacteria can be reversed [122, 140, 141]. It has been noticed that some filamentous cyanobacteria are having about 10 % heterocyst cells differentiated from vegetative cells (Fig. 5.5). The phenomenon of formation of heterocyst cells is not random but due to the filament senses to nitrogen status in the culture medium, as observed in Anabaena variabilis [131]. However, this does not exclude cell-to-cell signaling in the maintenance of heterocyst pattern [130, 131, 136], and it has been shown that the small diffusible peptide PatS and products of nitrogen fixation control heterocyst differentiation in Anabaena strain PCC 7120 [142, 143]. The heterocyst provides a micro anaerobic environment suitable for the functioning of nitrogenase since (1) it lacks photosystem II activity, (2) it has a higher rate of respiratory oxygen consumption, and (3) it is surrounded by a thick envelope that limits the diffusion of oxygen through the cell wall [133, 137]. Heterocysts import carbohydrates and in return export glutamine to the vegetative cells. An interesting feature of heterocyst differentiation in cyanobacteria is the occurrence of developmentally regulated genome rearrangements [144–153]. These rearrangements occur late in heterocyst differentiation, at about the same time as the nitrogenase genes are transcribed. In vegetative cells of Anabaena strain PCC 7120, the fdxN, nifD, and hupL genes are interrupted by DNA elements that are excised, during heterocyst differentiation, by site-specific recombinases encoded by xisF, xisA, and xisC, respectively. In 248 5 The Microbiological Production of Hydrogen addition, the excision of the fdxN element requires the products of the two genes xisH and xisI [148]. Bidirectional Hydrogenase The bidirectional hydrogenase of cyanobacteria is having pentameric enzyme structure and uses NADPH as electron donor for the production of hydrogen molecule and is found in the cytoplasmic member. In Synechocystis sp. PCC 6803, all five genes that encode hydrogenase subunits (hoxEFUYH) are localized in one cluster and co-expressed. Maturation of bidirectional hydrogenase requires several auxiliary Hyp proteins [149]. It is possible to increase hydrogen production in Synechocystis sp. PCC 6803 (Table 5.1) by overexpression of respiratory complex [150, 151]. The HoxH and HoxY protein entities of this enzyme do the catalytical activity, whereas the remaining three proteins form a diaphorase unit [152]. While hox genes are constitutively expressed in the presence of oxygen, hydrogen is produced until oxygen is accumulated during photosynthetic reactions in the quantities that inhibit bidirectional hydrogenase [153]. This process is reversible: hydrogen production by the existing hydrogenase resumes once oxygen is removed or consumed by respiration. Reactivation of enzyme can be sped up by NADH or NADPH at catalytic concentrations [151]. Targeted mutagenesis of cyanobacterial hydrogenases is currently exploring this enzyme end to create less oxygen-sensitive enzymes. Alternatively, inhibition of oxygen evolution by PS II can be induced to prevent oxygen poisoning of hydrogenase in vivo. With a little exception, this enzyme is common to all non-nitrogen-fixing cyanobacterial strains [154]. Presently, the site of availability and physiological significance of this enzyme are matters of debate. However, it is assumed to be cytoplasmic in nature and as loosely associated with cytoplasmic and thylakoid membranes [155, 156]. Bidirectional hydrogenase may serve as a valve for low potential electrons that are generated in light reactions of photosynthesis [153]; it may facilitate oxidation of molecular hydrogen in periplasmic space and shuttle electrons to the respiratory electron transport chain [151]; it may help reoxidize excessive NADH formed during glycolysis [156]. The fact that amino acid sequence of this enzyme bears conserved regions that are similar to respiratory complex I subunits [155] may indicate that bidirectional hydrogenase might function as a NADPH: plastoquinone oxidoreductase. It is possible that different cyanobacterial strains use this enzyme in different ways or not at all. Uptake Hydrogenase Generally, most of the nitrogen-fixing cyanobacterial [157] strains are having uptake hydrogenase. This enzyme is supposed to be cytoplasmic in nature or attached on thylakoid body having lack of N-terminal signal sequence in the primary structure. This protein consists of HupL polypeptide that binds molecular hydrogen and HupS polypeptide that plays a role in hydrogen oxidation. Uptake hydrogenase is less sensitive to molecular oxygen, compared to bidirectional hydrogenase [158, 159]. 5.4 Microbial Resources for Hydrogen 249

The molecular hydrogen generated by nitrogenase is being oxidized and further utilized. So, uptake hydrogenase counteract to the production of molecular hydrogen by nitrogen-ﬁxing cyanobacteria. By means of gene manipulation, the activity of uptake hydrogenase is either reduced or eliminated to enhance the production of hydrogen [160–164] (Table 5.2). Meanwhile, attempts have been going to induce the possibility of hydrogen production capacity in strains that naturally lack uptake hydrogenase (such as Synechocystis sp. PCC 6803). While bringing this idea into practice, it is important to evaluate the overall hydrogen yield as the lack of uptake hydrogenase a priori does not result in higher hydrogen production by cyanobacteria. For instance, hydrogenase-deﬁcient Cyanothece 7425 produces less hydrogen compared to a hydrogenase-containing Cyanothece 51142 [165].

5.4.9 Hydrogen Production by Green Algae

The discovery of hydrogen production in unicellular green algae by Gaffron and his coworker [166] stimulates curiosity in researchers to understand biological production of hydrogen deeper. Some unicellular green algae produce hydrogen on prior anaerobic incubation in dark [167–170]. Such pretreatment induces hydrogen enzyme activation with high specific activity of hydrogen evolution. The hydrogenase present in green algae is a monomeric form of the enzyme, reported to belong to the class of Fe hydrogenases [171–174], and is encoded in the nucleus of the unicellular green algae. Mostly, it is located in the chloroplast stroma [174]. The light energy facilitates the oxidation of water molecules, and as a result, electrons and protons transport to ferredoxin occur. The photosynthetic ferredoxin (PetF) serves as the physiological electron donor to the Fe hydrogenase and, thus, links the Fe hydrogenase to the electron transport chain in the chloroplast of the green algae [175]. The activity of hydrogenase is only transient (it lasts from several seconds to a few minutes) because, in addition to electrons and protons, the light-dependent oxidation of water entails the release of molecular O2. Oxygen inhabits Fe hydrogenase [176]. Thus, the physiological significance and role of the Fe hydrogenase in green algae, which normally grow under aerobic photosynthetic conditions, has long been a mystery. So, it has been a matter of curiosity about the critical relationship between O2 sensitivity of the Fe hydrogenase and the oxidative environmental condition on earth. Henceforth, the nature of green algae to photosynthetically generate H2 has been amazing since few decades. Upon illumination of the unicellular green alga Chlamydomonas reinhardtii after adaptation to anaerobic conditions, electrons originating from water splitting at PSII are driven by the photosynthetic electron transport chain to ferredoxin and to a reversible iron hydrogenase, thereby enabling the production of molecular hydrogen from water and solar energy. This process lasts for a short interval of time as hydrogenase activity and expression are highly sensitive to the presence of O2 [174, 177, 178], and because O2 is produced at PSII, hydrogen photoproduction 250 5 The Microbiological Production of Hydrogen stops after a few minutes of illumination, proposed an experimental protocol based on sulfur (S) deprivation, allowing long-term hydrogen production [179]. This process happens in two sequence stages. At first stage, oxygenic photosynthesis drives production of biomass and carbohydrate stores, and during a second anaerobic stage, the hydrogenase is induced, and hydrogen is produced. Sulfur depletion from culture medium mainly causes two problems: (1) huge accumulation of starch that defines a common response to nutrient starvation and (2) a gradual drop in PSII activity [180]. Once the rate of photosynthetic O2 evolution drops below the rate of respiration, anaerobic conditions are reached, enabling the induction of hydrogenase and the production of significant amounts of hydrogen for several days. In parallel to hydrogen production, starch is degraded [179, 181]. The role of starch metabolism has been recognized since long back with the pioneering work of Gibbs and coworkers [182, 183]onC. reinhardtii. In the year 2004, Posewitz et al. reported the importance of starch metabolism in two mutants of C. reinhardtiista6 and sta7.; two different pathways are supposed to provide reductants (i.e., reduced ferredoxin) for hydrogen production in the light, a direct pathway involving PSII and an indirect PSII-independent pathway that relies on a nonphotochemical reduction of plastoquinones [184]. Starch catabolism was proposed to play a role in both pathways [181] by (1) sustaining mitochondrial respiration and allowing the maintenance of anaerobic conditions for the PSII- dependent direct pathway and (2) by supplying electrons to the chlororespiratory pathway and to the hydrogenase through a PSI-dependent process during the indirect pathway [181, 184, 185]. Such a dual role of starch was first confirmed by the study of a Rubisco-deficient mutant (CC2653), unable to accumulate starch and to produce hydrogen in conditions of S deprivation [186], but was later on challenged by the study of another Rubisco-less mutant (CC2803), which was reported to produce significant amounts of hydrogen in S starvation conditions, although not accumulating starch [187]. The contribution of the decomposition of organic compounds to hydrogen production is dependent on the algal species concerned and on culture conditions. Even when organic compounds are involved in hydrogen production, an electron source can be derived from water, since organic compounds are synthesized by oxygenic photosynthesis. The reason for hydrogenase inactivity in green algae under normal photosynthetic growth conditions is unclear. Hydrogenase is thought to become active in order to excrete excess reducing power under specific conditions, such as anaerobic conditions. The oil crisis in 1973 prompted research on biological hydrogen production, including photosynthetic production, as part of the search for alternative energy technologies. Green algae were known as light-dependent, water-splitting catalysts, but the characteristics of their hydrogen production were not practical for exploitation. Hydrogenase is too oxygen labile for sustainable hydrogen production: light- dependent hydrogen production ceases within a few to several tens of minutes since photosynthetically produced oxygen inhibits or inactivates hydrogenases. A continuous gas flow system designed to maintain low oxygen concentrations within the 5.4 Microbial Resources for Hydrogen 251 reaction vessel was employed in basic studies [188] but has not been found practically applicable. Greenbaum and coworkers reported very high (10–20 %) efficiencies of light conversion to hydrogen, based on PAR (photosynthetically active radiation which includes light energy of 400–700 nm in wavelength). These authors recently reported what may represent a “short circuit” of photosynthesis, whereby hydrogen production and CO2 fixation occurred by a single photosystem (photosystem II only) of a Chlamydomonas mutant [189]. Green algae are applicable in another method of hydrogen production. The work of Gaffron and Rubin [166] demonstrated that Scenedesmus produced hydrogen gas not only under light conditions but also produced it fermentatively under dark anaerobic conditions, with intracellular starch as a reducing source. Although the rate of fermentative hydrogen production per unit of dry cell weight was less than that obtained through light-dependent hydrogen production, hydrogen production was sustainable due to the absence of oxygen. On the basis of experiments conducted on fermentative hydrogen production under dark conditions, Miura and Miyamoto’s group [190] proposed hydrogen production in a light–dark cycle. According to their proposal, CO2 is reduced to starch by photosynthesis in the daytime (under light conditions), and the starch thus formed is decomposed to hydrogen gas and organic acids and/or alcohols under anaerobic conditions during nighttime (under dark conditions). The technological merits of this proposal include the fact that oxygen inactivation of hydrogenase can be prevented through maintenance of green algae under anaerobic conditions, nighttime hours are used effectively, temporal separation of hydrogen and oxygen production does not require gas separation for simultaneous water splitting, and organic acids and alcohols can be converted to hydrogen gas by photosynthetic bacteria under light conditions. A pilot plant using a combined system of green algae and photosynthetic bacteria was operated within a power plant of Kansai Electric Power Co. Ltd. (Nankoh, Osaka, Japan). Miyamoto and coworkers [191] recently proposed chemical digestion of algal biomass as a means of producing substrates for photosynthetic bacteria, thus improving the yield of starch degradation.

5.4.9.1 Physiology of H2 production

Hydrogen evolution in certain green algae occurs when dark-adapted anaerobically cells are exposed to light ([166] Gaffron, 1939). Interestingly, the same group of scientists, also, reported the reverse reaction, e.g., hydrogen production in the light was ﬁrst reported with the green alga Scenedesmus obliquus (the production of hydrogen was noticed to be for a short period). Electrons were generated either upon the photochemical oxidation of water by PSII, which results in the simultaneous production of O2 and H2 [7, 192], or upon the oxidation of endogenous substrate, feeding electrons into the thylakoid membrane with the simultaneous release of CO2 to the medium [193, 194]. It has been noticed that C. reinhardtii can photoproduce hydrogen when PSII is blocked by DCMU [10] as the function of 252 5 The Microbiological Production of Hydrogen

Fig. 5.10 The scheme of solar-powered H2 production during oxygenic photosynthesis and subsequent formation of carbohydrates in microalgae. The oxygenic “light reactions” of photosynthesis are driven by the solar energy captured by the light-harvesting complexes of PSI and PSII. Electrons extracted from H2O by the oxygen-evolving complex of PSII are passed along to the photosynthetic electron transport chain via plastoquinone (PQ), the cytochrome b6f complex (Cyt b6f ), plastocyanin (PC), photosystem I (PSI), and ferredoxin (Fd) and then by ferredoxin- NADP+ oxidoreductase to NADP+ with final production of NADPH. H+ are released into the thylakoid lumen by PSII and the PQ/PQH2 cycle and used for ATP production via ATP synthase. The ATP and NADPH generated during primary photosynthetic processes are consumed for CO2 fixation in the Calvin–Benson cycle, which produces sugars and ultimately starch. Under anaerobic conditions, hydrogenase can accept electrons from reduced Fd molecules and use them to + reduce protons to H2. Certain algae under anaerobic conditions can use starch as a source of H and À e for H2 production (via NADPH, PQ, cyt b6f, and PSI) using the hydrogenase. In cyanobacteria, + À the H and e derived from H2O can be converted to H2 via a nitrogenase or fermentation. During fermentation process, carbohydrate stores such as starch can be converted to sugars and subsequently pyruvate via glycolysis, before producing H2 and organic acids (e.g., formate, acetate, and butyrate). Thylakoid membrane is denoted with green color. Electron transfer is shown with blue color and protons with brown color. Adapted from Refs. [179, 226–229] cytochrome b-f complex is blocked. However, under anaerobic conditions in the presence of DCMU, accumulated reducing equivalents from the fermentative catabolism of the algae cannot be oxidized via respiration because the terminal electron acceptor O2 is absent. The complexity of functioning of two photosystems (PSI and PSII) and hydrogen production phenomenon vary with the variation of algal species. However, a general process of hydrogen production linked to photosynthesis is depicted in the following schematic diagram (Fig. 5.10). An NAD(P)H reductase protein complex that feeds electrons into the plastoquinone pool has been identified in many vascular plant chloroplasts [195–197] but so 5.5 Bioreactors for Hydrogen Production 253 far only from the green alga Nephroselmis olivacea [198]. Nevertheless, inhibitor experiments have yielded evidence in support of a thylakoid membrane-localized NAD(P)H reductase in C. reinhardtii [199], suggesting that electrons derived upon the oxidation of endogenous substrate may feed into the plastoquinone pool. Thereafter, electrons are driven upon light absorption by PSI to ferredoxin. The latter is an efficient electron donor to the Fe hydrogenase, which efficiently com- bines these electrons with protons to generate molecular H2 [175]. The physiology of H2 production upon S deprivation has many similarities and some distinct differences from the process described above. Sulfur-deprived and sealed cultures of C. reinhardtii become anaerobic in the light due to a significant and specific slowdown in the activity of the O2-evolving PSII, which is followed by automatic induction of the Fe hydrogenase and by photosynthetic H2 production. Biochemical analyses revealed that, concomitant with the H2 production process, starch and protein content of the cells gradually declined. Such catabolic pathway (s) could be generating reductant that feeds electrons into the thylakoid membrane, perhaps via a chloroplast NAD(P)H-dependent process [182]. Mitochondrial respiration scavenges the small amounts of O2 that evolve due to the residual activity of photosynthesis and thus ensures the maintenance of anaerobiosis in the culture (Fig. 5.11).

5.5 Bioreactors for Hydrogen Production

Various physicochemical methods, as discussed earlier, for H2 production are costly, and most of them seem to be un-eco-friendly. However, some microalgae are noticed to produce hydrogen either from organic materials (i.e., sugar or biomass) or from water in reaction catalyzed by nitrogenase or hydrogenase enzymes [200]. Microbial production of hydrogen has a number of advantages, and it could be a cost-effective alternative to the current industrial methods of H2 production [201]. Bioreactors are absolutely essential for industrial, large-scale H2 production by microorganisms [202]. The bioreactor is a devise for cell growth (microbial, plant, or animal cells) with practical purpose under controlled conditions. Bioreactors are closed systems and vary in size from the small-type desktop laboratory scale to the larger one having 5KL capacity. Based on the nature of H2-evolving reactions, the bioreactors can mainly be grouped into two: (i). Bioreactors based on dark anaerobic hydrogen production a. Bioreactors based on “Water–gas shift reaction” b. Bioreactors based on fermentation (ii). Photobioreactors based on hydrogen production General aspects of photobioreactors for microbial algae culture are discussed elsewhere in this book (Chap. 3), in detail. 254 5 The Microbiological Production of Hydrogen

Fig. 5.11 Coordinated photosynthetic and respiratory electron transport and coupled phosphorylation during H2 production. Photosynthetic electron transport delivers electrons upon photo- oxidation of water to the hydrogenase, leading to photophosphorylation and H2production. The oxygen generated by this process serves to drive the coordinate oxidative phosphorylation during mitochondrial respiration. Electrons for the latter ([4e]) are derived upon endogenous substrate catabolism, which yields reductant and CO2. Release of molecular H2 by the chloroplast enables the sustained operation of this coordinated photosynthesis-respiration function in green algae and permits the continuous generation of ATP by the two bioenergetic organelles in cell. Source: http://dx.doi.org/10.1104/pp.010498

(i) Bioreactors Based on Dark Anaerobic Hydrogen Production (a) Bioreactors based on “water–gas shift reaction (WGS)” In the water–gas shift (WGS) reaction, CO is oxidized to CO2, while water is reduced to hydrogen:

CO þ H2O $ CO2 þ H2 ð5:2Þ

This process is used industrially to produce additional H2 from synthesis gas streams using a two-stage catalytic reactor (FeO/Cr at 450 C, CuO/ZnO at 250 C). Several strains of photosynthetic bacteria, isolated from the natural environment, are able to perform the WGS reaction at ambient temperatures [1–5]. This may be an attractive alternative to conventional processes. The WGS bioreactors are based on unique type of H2-producing activity originally found in a strain of purple bacteria by Uffen [203]. Cultures of Uffen’s strain in a complex organic medium carry out a water–gas shift reaction in darkness to produce H2 according to Eq. (5.2) During the shift reaction, the bacterial cells produce hydrogen from water as veriﬁed by experiments with Н2O[204]. Since the time of Uffen’s discovery, a strain of nonsulfur purple bacterium Rubrivivax gelatinosus CBS has been isolated from the mud water in Colorado (USA) by scientists from the National Renewable Energy Laboratory (NREL) [205, 206]. This bacterium utilizes CO in darkness and does not require complex organic substrates. 5.5 Bioreactors for Hydrogen Production 255

The organism Rubrivivax gelatinosus CBS is a nonsulfur, purple photosynthetic bacterium that is capable of performing the same water–gas shift reaction under anaerobic conditions, atmospheric pressure, and an ambient temperature of 25 C. Wolfrum (2003) [207] has shown that the organism can perform the shift reaction at pressures up to 4 atmosphere absolute pressure. It is believed that the organisms can be effective at even higher pressures with increased cell loading to the reactor, but these tests have not been performed. The organism uses the biological WGS reaction as a means to obtain energy to maintain metabolic processes and grow. In the wild, Rx. gelatinosus CBS can obtain energy through photosynthesis, aerobic heterotrophic metabolism (e.g., acetate or malate), and anaerobic fermentation pathways. The biological WGS represents an anaerobic, dark-phase pathway. This pathway produces much less energy for metabolic activities as compared with either the photosynthetic or aerobic pathways. This lower energy production results in a slower cellular growth rate which increases the time needed to reach steady state but reduces the amount of waste cell mass produced by the biological WGS process once it is operating. The beneﬁt of the dark-phase reaction is that the biological WGS system can be operated in a conventional closed reactor, similar to trickling ﬁlters used for wastewater treatment—no expensive photobioreactor is required. The energy obtained through the biological WGS is obtained by transferring electrons from CO to H2O in the following coupled reactions:

À þ CO þ H2O ! CO2 þ 2e þ 2H ð5:3Þ þ À H þ 2e ! H2 ð5:4Þ

CO þ H2O ! CO2 þ H2 ð5:5Þ

This reaction produces 4.46 kcal/mol CO reacted. For comparison, the aerobic conversion of 1 mol of CO to CO2would produce 61.1 kcal/mol CO:

CO þ O2 ! CO2 ð5:6Þ

The aerobic reaction would result in more energy to the organism, resulting in more cell growth per mol CO, but no H2 would be produced under aerobic conditions. It is desirable to minimize cell growth so less excess cell waste is produced, but if the growth rate is less than the natural death rate, the reactor operation will not be stable. In any biological system, cells are continually dying. Many of the nutrients in the dead cells are recycled when the cells lyse or split, but a portion of the cell remains as refractory or inert material that cannot be “redigested” and used for new cells. (This can be thought of as the “teeth and bones” of the cells—insoluble minerals that do not dissolve). In many batch systems, not enough refractory material builds up to affect operation of the system, but in continuous systems, the accumulation of this refractory material must be removed. Cell growth rates are for reactor start-up and recovering from process upsets. Initial reactors can be “seeded” or inoculated with a sterile culture, but most of the cells used to convert the CO to H2 come from new cell growth after the inoculation. The higher the growth rate, the faster the reactor will reach full production capacity. Upsets in pH 256 5 The Microbiological Production of Hydrogen or temperature can also result in a loss of biological activity due to damage to or death of the cells. Higher growth rates would allow quicker recovery in the reactor. One possibility during start-up is to provide the Rx. gelatinous CBS with “food” in the form of acetate, malate, or a low-cost sugar source (i.e., corn steep liquor) and provide oxygen to temporarily boost the energy production and growth rate of the cells. Once the oxygen is removed, there will be some delay, while the cells produce new enzymes for their anaerobic pathway, but the cells will eventually switch back to using CO for generating energy and H2. Even without knowing the exact metabolic pathways and enzymes involved in the biological WGS reaction, the overall free energy change of the chemical reactions can be used to determine the maximum amount of energy the Rx. gelatinous could obtain from performing the biological WGS. By assuming an average energy efficiency of 60 % for biological processes [208], it is possible to estimate the amount of cells produced per gram of CO fed to the bacteria. Under anaerobic conditions, Rx. gelatinous should produce 1.4 g of cells per 1 mol of CO. When growing aerobically on O2 and acetate, the cell yield would be 2.0 g of cell mass per mol of acetate. The WGS technology for biological hydrogen production is still at incipient stage of development. The future would predict some positive and encouraging result on the industrial application of this technology for large-scale production of hydrogen. (b) Bioreactors based on fermentation Anaerobic fermentation, i.e., [CH2O]n ! Ferredoxin ! Hydrogenase ! H2 means hydrogen production by using organic matters is common in bacterial hydrogen production process. Due to good growth rate of bacteria, this process has many advantages, even from commercial point of view [209]. Traditional types of bioreactors with minor modification can be used for hydrogen production by fermentation process, as practices in ethanol manufacturing process. Both continuous and batch fed mean practices can be well adapted for microbial hydrogen production, without much difficulties. The main limiting factor for dark hydrogen production is the low substrate conversion efficiency of bacteria. Here, substrate means both externally added and internal-stored organic acids and other products resulted from bacterial metabolism, during growth and development [209]. Moreover, from commercial point of view, the use of organic substrate is a cost-effective process. It has been seen that the availability of glycerol is abundant as a by-product in biodiesel manufacturing [210] and can be a cheap substitute for organic matters in hydrogen production through fermentation. World biodiesel production was expected to reach 12 billion liters by the end of 2010 [211], with about one pound of glycerol created for every 10 pounds of produced biodiesel. At present, a surplus of glycerol is destroyed by incineration [210]. Although, burning glycerol produces nitrogen oxide as well as carbon dioxide (CO2), the primary greenhouse gas. Exact metabolic pathways of glycerol are not known for E. aerogenes. It is well known that this bacterium can ferment glucose via 2,3-butanediol fermentation with main products such as 2, 3-butanediol, H2 and CO2, and minimal amounts of ethanol. Both 2,3-butanediol and ethanol are valuable industrial 5.5 Bioreactors for Hydrogen Production 257

Fig. 5.12 Outline of H2 production by mariculture-raised cyanobacteria. Step 1: Cell growth in a transparent plastic bag floating on the sea surface. The bioreactor is filled with air containing CO2; Step 2:H2 production in a photobioreactor composed of three bags (at least one layer is a H2 gas barrier membrane) floating on the sea surface. The spent cells can be used as fish feed [48] commodities. Hydrogen production rates and the glycerol uptake efficiency (65 %) by E. aerogenes were exactly the same in the bioreactor and test tubes. This indicates the possibility that bioreactor for glycerol conversion into H2 can be operated without agitation, which may offer a significant saving of energy for industrial applications of this technology [210]. (ii) Photobioreactors Presently, the use of photobioreactors is a common practice in commercial-level hydrogen production from variety of microalgae. This topic has been extensively discussed earlier in Chap 3 (3.2.2). Floating-Type Flexible Plastic Bioreactors Hidehiro Sakurai et al. (2015) proposed flexible type of floating bioreactor made of plastic bags (Fig. 5.12). These plastic bags can be kept on floating state where the surface water of sea or ocean remain calm without any waves (e.g., the calm belt about 30 north or south, such as the “mysterious Bermuda Triangle” region) [212, 213]. The culture medium is based on freshwater, and the bioreactors would spread over the sea surface because of the difference in density between 258 5 The Microbiological Production of Hydrogen the culture medium and the surrounding seawater. Salt lakes can also be used as the fields for cultivation. The size of the bags is flexible; for large-scale H2 production, for example, 20 bags of 25 m Â 200 m in surface area could cover the surface of 1km2. As proof of this concept, Kitashima et al. [214] demonstrated that transparent flexible H2-barrier plastic bags are usable for studies of photobiological H2 production by cyanobacteria in the laboratory. From the cost point of view, the floating-type plastic bags would be less costly as compared to closed hard panel or tubular photobioreactors that are described for laboratory or pilot-scale studies (e.g., [213, 215–217]). The bioreactor is having three layers of plastic bags (Fig. 5.12) for a total of six layers (sunny side and shady side) of transparent plastic film. The innermost bag holds the cyanobacteria culture, the middle bag has very low permeability to H2, and the outermost bag serves to mechanically protect the inner bags. The thickness of each film is 0.08 mm, and 480 cm3 of plastic per m2 of the bioreactor’s sunny side surface is required. The above assumptions result in the cost of the bioreactor being about 24–48 cents per m2 of bioreactor surface per year, assuming a renewal cycle of every 2 years. The plastic films of 480 cm3 are assumed to be produced by consuming 360 mL of crude oil for processing that is equivalent to 3.92 kWh Á per m2 per year. The plastic film presently available in the market is much cheaper than the other materials being used for making tubular types or other types of photo bioreactors. The plastic bags can be recycled at an energy cost of 20 % of the feedstocks (0.78 kWh). The amount of energy in feedstocks derived from fossil fuels can be further decreased because currently H2 generated from fossil fuels is used as a part of the feedstocks for plastic film production and photobiologically produced H2 can replace some part of it.

5.6 Future Prospects of Microbial Hydrogen Production

Hydrogen, a valuable commodity gas, is increasingly recognized as an important fuel and energy storage vector of the future. Hydrogen has very special properties as a transportation fuel, including a rapid burning speed, a high effective octane number, and no toxicity or ozone-forming potential. It has much wider limits of flammability in air (4–75 % by volume) than methane (5.3–15 % by volume) and gasoline (1–7.6 % by volume) [217]. Hydrogen can be produced from a variety of feedstocks. These include fossil resources, such as natural gas and coal, as well as renewable resources, such as biomass and water with input from renewable energy sources (e.g., sunlight, wind, wave, or hydropower). A variety of process technologies like chemical, biological, electrolytic, photolytic, and thermochemical are involved in hydrogen production. Each technology is in a different stage of development, and each offers unique opportunities, benefits, and challenges. Capacity of annual H2 supply was estimated to be 12–18 billion Nm3 in 2015 and 21–27 billion Nm3 in 2030 [218]. Technavio’s analysts forecast the Global Hydrogen Gas Market to grow at a CAGR of 6.84 % over the period 2012–2016. The hydrogen generation 5.6 Future Prospects of Microbial Hydrogen Production 259 market will grow from an estimated $103.5 billion in 2014 to $138.2 billion by 2019, with a CAGR of 5.9 % [219, 220]. Demand for hydrogen as a fuel for fuel cells, in both transport and stationary applications (the power-to-transport and power-to-power vectors, respectively) will continue to grow, alongside hydrogen for energy storage (the power-to-gas vector). With increased energy demand, requirements to use curtailed renewable energy, growth in the clean tech backup power market, and deployment of a growing number of fuel cell-powered vehicles in the transport sector overall demand for hydrogen as a fuel is projected to grow. Traditionally, the hydrogen energy market has been centered on the petroleum refinery and chemical manufacturing sectors. However, interest in hydrogen for the power-to-gas market, combined with the rise in usage of hydrogen in the fuel cell sector, has seen greater usage of hydrogen in other areas. The increasing availability of distributed electrolysis, low off-peak electricity prices, and cheap natural gas will see the economics of the hydrogen market grow. Along with a step-change increase in interest in the power-to-gas market, this will result in the overall hydrogen market becoming an increasingly important commercial commodity. Navigant Research forecasts that global demand for hydrogen from the power-to-power, power-to-transport, and power-to-gas sectors will reach 3.5 billion kg annually by 2030. Today’s capital costs for physicochemical-based hydrogen production technologies are substantially higher than those for other fuels. Developers are working to reduce these costs by applying the principles of “design for manufacture,” identi- fying better materials, decreasing the number of necessary parts, designing simpli- fied systems, and moving into mass production. Operating costs should also decline as equipment developers identify improved materials, consolidate processing steps, reduce maintenance and labor requirements, and otherwise enhance equipment performance and integration. Biological hydrogen production process is noticed to be a better option over the physicochemical technologies. Through biological process variety of bio-liquid feedstocks, such as sugars, sugar alcohols (like ethanol), bio-oils, and less-refined sugar streams (such as cellulose from nonedible plants), can be used. These feedstocks are comparatively cheap and sustainable in nature. Researchers are trying to find better catalysts to improve the hydrogen yield of these technologies. Photosynthetic bacterial production of hydrogen is supposed to be noble among all the options available for hydrogen production. Sunlight is the driver for bacteria to break down organic material, thus releasing hydrogen. Purple, nonsulfur bacteria with a specialized enzyme can produce hydrogen gas when exposed to near- infrared light energy. Even in the absence of sunlight through dark fermentation by anaerobic bacteria, organic materials are decomposed into hydrogen and other by-products. Researchers are working to identify specific strains of bacteria that can directly and efficiently ferment organic material (or cellulose) to hydrogen. Researchers will then develop mutations that selectively block the generation of waste acids and solvents that reduce ongoing hydrogen productivity. 260 5 The Microbiological Production of Hydrogen

MEC is also a promising technology for bacterial hydrogen production. As the bacteria decompose the organic materials, they produce a low voltage at the anode. Hydrogen is produced at the fully submerged cathode with the input of a tiny amount of additional energy. Optimizing the environment to expedite this natural process could potentially produce hydrogen with much greater efficiency than standard electrolysis. Perhaps the most promising biological production pathway incorporates some or all of these technologies into a single system. This integrated approach could alleviate the need to overcome all barriers to individual technologies, as long as the overall system is cost competitive. Integrated systems may use the by-products of some production pathways as inputs to others in a nearly closed- loop system that produces hydrogen at each stage. Biological hydrogen production is the most challenging area of biotechnology with respect to environmental problems. The future of biological hydrogen production depends not only on research advances, i.e., improvement in efficiency through genetically engineering microorganisms and/or the development of bioreactors, but also on economic considerations (the cost of fossil fuels), social acceptance, and the development of hydrogen energy systems. As is evidenced by several funded programs from many national government agencies all over the world, hydrogen is being promoted as the fuel for the future [221]. In order to have photobiologically produced H2 and to make meaningful contributions to the mitigation of global warming problem, economical production of H2 is essential. The technologies required for the practical application of large-scale photobiological H2 production are in the development stages. However, Sakurai et al. [213] presented a very À1 preliminary estimate of 26.4 cents kWh of H2 produced on the sea surface using many presumptive assumptions, for example, assuming the solar energy conversion efficiency of 1.2 %. Currently, the maximum energy conversion efficiency of cyanobacterial H2 production under outdoor conditions is about 0.2 %. There is an urgent need to demonstrate higher efficiencies to policymakers and developers in order to convince them of the potential benefits of photobiological H2 production by cyanobacteria. Increasing the solar energy conversion efficiency with improvements in technologies for purifying and transporting H2 will decrease the total cost. Public acceptance of renewable fuels very much depends upon the affordability of the price and will go a long way toward convincing consumers and government leaders to adopt technologies that will ultimately mitigate global climate change. Future cost estimates will be more accurate as the technologies in cyanobacterial H2 production and its related processes advance. It is also expected that more refined cost estimates will allow for additional targeted improvements in the technologies, ultimately resulting in an overall cost reduction. On hypothetical concept, it has been estimated that if the hydrogen production would be at 1.2 % of total radiation (18 kWh mÀ2 per year) on the sea surface and purified H2 is delivered to the end users with a final energy yield of 0.5, the net energy obtained is calculated to be 9 kWh mÀ2 per year or 32.4 Â 106 JmÀ2per year [213]. The current world fuel energy consumption is estimated to be 460 Â 1018 J per year [222]. It follows that by using a surface of the sea equivalent to 1 % of the global surface (1.36 Â 106 km2, about 2.3 times the area of the island of Tasmania References 261 or 3.1 times the area of the island of Great Britain), it can be able to replace 19 % of the current world fossil fuel consumption. In order to make a meaningful contribution to the mitigation of the hazards to the human population that are predicted to occur with global climate change, the promotion of R&D efforts for economical large-scale photobiological biofuel production and the advancements of biological and other needed technologies should be encouraged.

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A Alkali treatment, 77, 98 Acetate, 44, 45, 47–49, 55, 73, 75, 183, 184, 1-Allyl-3-methylimidazolium chloride 199, 210, 212, 237, 240–242, 252, (AMIMCI), 99 255, 256 American Recovery and Reinvestment Act, 20 Acetic, 44 Amino acids, 8, 41, 44, 81, 238, 248 Acetic acid, 44, 45, 48, 49, 78, 94, 165, 235, Amoco Oil Company, 4 238, 241 Anabaena sp., 225 Acetivibrio cellulolyticus, 41 Anaerobic bafﬂe reactor, 104 Acetobacterium, 44 Anaerobic decomposition, 35 Acetogenic bacteria, 44, 55, 75, 240–242 Anaerobic digester (AD), 37–39, 49, 51, 73, 74, Acetone, 9, 48, 49, 99, 240 78, 94, 104, 236, 240 Acetone–butanol–ethanol (ABE), 9, 49 Anaerobic fermentation, 60, 61, 70–71, 92, Acid hydrolysis, 98, 165 100, 155, 165, 241, 255, 256 Acidogenesis, 44, 45, 50, 64, 241 Ancient Egypt, 3 Adenosine triphosphate (ATP), 49, 51, 181, Ancyllostoma duodenale (hook worm), 62 185, 187, 189, 190, 196, 228, 237, 238, Animal fat, 6, 10, 17, 123 240, 241, 246, 247, 252, 254 Ankistrodesmus braunii, 224 Aerobacter sp., 224 Anode chamber, 10, 184, 191, 197, 198, AGATE, 25 205, 209 Agricultural residues, 35, 36, 98, 236 Antioxidants, 8, 126 Alcohols, 3, 4, 11–14, 21, 35, 44, 47, 49, 67, Aphrodisiac, 3 154, 158, 161, 242, 251, 259 Apoenzyme, 238 Alexander, 3 Aquaﬂow Bionomic Corporation, 10 Alexandria, 33 Aquatic plants, 36 Algae Biomass Organization, 7 Aquatic Species Program (ASP), 6, 69 Algae cultivation methods, 134–148 Argonne National Laboratory, 9, 230 Algae.Tec, 131 Arthrospira platensis (Spirulina), 128, 136, Algal biodiesel supply chain, 131–133 141, 156, 162, 165 Algal biomass, 6–8, 10, 17, 69, 70, 126, 129, Ascaris lumbricoides (round worm), 62 133, 136, 137, 142, 146, 148–150, 152, Ashland, 4 154–156, 158, 163, 251 ATP. See Adenosine triphosphate (ATP) Algal fermenter, 135, 139, 145–147 Autotrophic growth, 124 Algenol, 7, 23, 24, 143, 144, 166, 167, 169 A. woodi, 240 Algenol Biofuels Inc, 130

B 185, 188, 189, 191, 198, 200, 211, 223, Bacillus macerans, 224 226–229, 234, 237, 238, 240–242, 244, Bacterial metabolism, 193–196, 211, 256 251, 252, 254–257 Bacteroides, 44 Carbon nitrogen ratio (C/N ratio), 58, 60, 73, Batch reactor, 103 79, 92, 94 Battery, 185, 193, 196, 214, 215, 226 Carbon paper, 207–209 Bidirectional hydrogenase, 229, 245, 248 Carotenoids, 128, 154, 163, 244 Bifuel, 1–28, 35, 60, 69, 70, 123, 124, 126, Cassava, 123, 162, 163 130–134, 137, 148, 154–164, 166, 167, Cathode, 182, 185, 187–190, 192, 194, 170, 193, 204, 213, 214, 227, 229, 261 196–202, 205–210, 214, 239, 260 Biodiesel, 1, 3, 5–8, 10–25, 35, 38, 39, 68, Cathode chamber, 10, 191, 205 123–134, 137, 139, 141, 146, 151, 154, Catholytes, 201, 205, 209, 212 155, 157–161, 163, 164, 167, 169, 256 Cellulose biomass, 41, 234 Bioenergy technologies ofﬁce (BETO), 130 Centrifuge, 151 Bioethanol reforming, 234–235 Chemical oxygen demand (COD), 48, 55, 58, BioFields, 24, 139 66, 72, 75, 76, 93, 211, 212, 215 Bioﬁltration, 90–91 Chemical pretreatment, 76–77, 98 Biogas digester, 39, 61, 62, 65–66, 69, 79, Chemical scrubbing, 86–87 100–105, 108 Chlamydomonas moewusii, 9, 228, 230 Biogas storage, 109–111 Chlamydomonas reinhardtii, 9, 162, 164, 165, Biogasta˚get amanda, 9 224, 228–230, 249–251, 253 Biogas upgradation process, 79–85 Chlorella fusca, 224, 228 Biological hydrogen production, 226, 227, 229, Chlorella sp. 250, 256, 259, 260 C. fusca, 224, 228 Biological oxygen demand (BOD), 66, 67, 72, C. vulgaris, 69, 128, 136, 141, 156, 162, 74, 76, 104, 107, 212, 215 165, 224 Biomass Research and Development Act, 20 Chlorococcum sp., 156, 164, 165 Blue green algae, 6, 7, 124, 153, 166, 167, 224, Chlorophyll, 8, 154, 244 243, 245 Christmas tree, 142 Blue Marble, 25–26 Chromatium sp. Miami, 224 BOD. See Biological oxygen demand (BOD) Citrobacter intermedius, 224 Botryococcus braunii, 125, 155, 156, 162, 227 Close type photobioreactor, 135, 161 B. polymy, 224 Clostridia, 9, 44, 48 Bulk harvesting, 148–151 Clostridium sp. Butanol, 9, 13–15, 48, 49, 110, 158, 162, C. aceticum, 44, 240 188, 240 C. acetobutylicum, 41, 49, 224, 240 Butyrate, 44, 47, 48, 55, 184, 240–243, 252 C. beijerinckii, 48 Butyric acid, 44, 49, 235 C. botulinum, 224, 236 Butyrivibrio, 44 C. butyricum, 10, 48, 49 C. cellobioparum, 41, 49, 56 C. cellulolyticum, 49 C C. kluyveri, 240 Calothrix sp., 225, 226 C. kutricum, 240 C. scopulorum, 225, 226 C. pasteurianum, 48, 49 Campylobacter, 44 C. perfringens, 49, 236 Carbohydrate, 1–3, 9, 41, 49, 55, 61, 67, 68, 75, C. thermoaceticum, 240 95, 161–166, 189, 242, 247, 250, 252 C. thermocellum, 49, 240 Carbon cloth, 201, 205, 208, 209 C/N ratio. See Carbon nitrogen ratio (C/N ratio) Carbon dioxide (CO2), 1, 6–8, 10, 15–19, 24, Coagulation, 150–151 26, 36–38, 40, 44, 45, 47–51, 54, 55, Coal (with carbon sequestmaration), 233 66–71, 73, 75, 78–81, 83–92, 94, 96, Cobalt tetra methoxy phenylporphyrin 106, 109, 124, 126, 130, 132, 135–142, (CoTMPP), 209, 210 144, 145, 157, 158, 161, 167, 169, 170, Coccomyxa acidophila, 128 Index 275

COD. See Chemical oxygen demand (COD) Electron acceptors, 44, 47, 187, 188, 191–194, Coelastrella striolata, 128 196, 198, 202, 209, 252 Coelastrum proboscideum, 152, 224 Electron transfer, 181, 183, 187–189, 191–195, Coenzyme A, 49 197, 198, 202, 207, 208, 214, 230, Community biogas plant, 60 239, 252 Compressed biomethane (CBM), 109, 111 Elemental hydrogen, 223 Consortium of microflora, 61–63 Endogenous mediators, 196 Continuous biogas digester, 104 Endoglucanase, 41 Cooking gas, 1 Endospore, 236 Corn silage, 39 Energy balance, 23, 70 Cow dung, 54–57, 95 Energy Independence Security Act, 20 Cryogenic separation, 88, 89 Energy Policy Act, 4, 20 Cyanobacteria, 124, 164, 166, 167, 224, 228, Entamoeba histolytica, 62 229, 235, 237, 243–249, 252, 257, Enterobius vermicularis (pinworm), 62 258, 260 Escherichia coli, 10, 62, 67, 170, 190, 191, Cytoplasmic membrane, 195, 196 195, 208, 224 Ester fuels, 227 Ethanol, 1–7, 9, 13–25, 38, 44, 48, 49, 55, 99, D 123, 126, 129, 133, 137, 144, 155, 158, Dark anaerobic hydrogen production, 253, 254 160, 161, 163–171, 234, 240, 241, Dark fermentation, 25, 165, 235–237, 259 256, 259 Dehydration processes, 153–154 Ethanol stilage, 2 Department of Energy (DOE), 6, 7, 9, 69, 129, Eubacterium cellulosolvens, 41 147, 230 Euglena gracilis, 156, 161, 162 Department of Energy’s Argonne National Exclusive economic zone (EEZ), 170 Laboratory, 9 Exoelectrogens, 191, 193 Desulfotomaculum ruminis, 56 Exoglucanase, 41 Desulfovibrio gigas, 241 Extracellular electron transfer (EET), 181, 183, Desulfurizing bacterium, 242–243 193, 198 3-(3,4-Dichlorophenyl)-1,1-dimethylurea Extreme bacterium, 239 (DCMU), 251, 252 ExxonMobil, 7 Diesel engine, 5, 6, 12, 13, 16, 17, 24, 105 Dimethyl dioxirane (DMDO), 76 Dinitrogenase reductase, 245, 247 F Direct Ethanol from MicroAlgae (DEMA), 169 FADH2, 187 Direct gasification, 234 FAME. See Fatty acid methyl ester (FAME) Diversified Technologies Inc., 27 Farm mortality, 38 DOE. See Department of Energy (DOE) Father of Ethanol Car, 4 Dow Chemical, 167 Fatty acid ethyl esters (FAEE), 158 DuPont Dow Elastomers L.L.C., 110 Fatty acid methyl ester (FAME), 5, 24, 158 Feedstock, 1, 3, 11, 17, 18, 21, 23–25, 27, 36–38, 51–79, 93–95, 98, 106, 108, 123, E 124, 126, 129, 130, 132, 137, 145, 146, E85, 4, 5, 20, 166 160, 161, 163, 223, 240, 258, 259 Echinococcus granulosus (hydatid), 62 Fermentative hydrogen production, 235–237, EET. See Extracellular electron transfer (EET) 251 Electricity, 1, 5, 8, 10, 36, 37, 41, 54, 72, 103, Fermenting bacteria, 55, 224 105, 106, 108, 126, 129, 143, 150, 155, Ferredoxin, 196, 227, 229, 241, 243–245, 249, 181–188, 190–197, 199, 200, 202–206, 250, 252, 253, 256 209, 211–215, 226, 230, 231, 259 FFV. See Flexible-fuel vehicles (FFV) Electrode, 149, 181, 183, 185–192, 194–198, Fiat, 4 201, 205–209, 213, 214, 233 Filamentous cyanobacteria, 244, 245, 247 Electrolyzer, 227, 230 First generation biofuels, 11 276 Index

First World War, 4 Green strike group, 25 Fissionable nuclear fuel, 15 Grinding, 77 Fixed dome digesters, 101–102 Flammable gas, 8, 37 Flat-plate photobioreactor, 135, 143–144, 233 H Flat-plate PMFC, 204 Haematococcus pluvialis, 128, 139, 156 Flexible-fuel motorcycles, 5 Halanaerobium hydrogeninformans, 239 Flexible-fuel vehicles (FFVs), 4, 5, 166 Heterocystous cyanobacterial, 227 Flexible plastic bioreactors, 257–258 Heterocysts, 228, 244, 245, 247 Flex vehicles, 5 Heterotrophic growth, 124, 132 Floating drum digester, 102 High-density polyethylene (HDPE), 110 Flocculation, 148–151 High pressure homogenizer, 77–78 Flotation, 148–150 High solid agro wastes, 78–79 Flue gases, 126, 132, 138 High temperature treatment, 97 Food-processing wastes, 38, 211 Honeywell UOP, 10 Ford Model T, 4 Household digesters, 39, 100–101 Fossil fuels, 1, 5, 11–15, 17–21, 23, 35, 36, HRT. See Hydraulic retention time (HRT) 131, 132, 139, 145, 170, 227, 230, Human excreta (HE), 54, 60–67 233–234, 258, 260, 261 Hydraulic retention time (HRT), 62, 65, 92, 93 Fresh produce wastes, 38 Hydrogenase, 51, 214, 226–230, 235, 240, 241, Fuel cell, 10, 184–198, 201, 203, 211, 213, 214, 243, 245, 246, 248–254, 256 226, 228, 259 Hydrogen from MEC, 238–239 Fuel cell electric vehicles (FECVs), 228 Hydrogenotrophic, 47, 73 Fusobacterium, 44 Hydrogen-oxygen fuel cell, 184 Hydrogen production process, 230–235, 256, 259 G Hydrogen transfer (HTS), 240, 241 Garnero, Ma´rio, 4 Hydrolysis, 41–45, 50, 64, 75, 76, 79, 97, 98, Gas compressibility, 109 165, 197, 206 Gas flammability, 109 Hydropower, 223, 258 Gasification, 36, 40, 70, 129, 155, 234 2-Hydroxy-1,4-naphthoquinone (HNQ), 195 Gasoline, 4, 5, 9, 13–15, 17, 20, 22–24, 26, 54, 137, 144, 166, 230, 258 Geobacter metallireducens, 191, 193, 194 I Geobacter sulfurreducens, 187, 191, 193, 194 GHG. See Greenhouse gas (GHG) Illuminated photobioreactors (IIPBR), 141 GICON, 141, 142 Industrial wastes, 36, 38, 41, 75, 76, 181, Global emissions, 10, 18 183, 188 Global hydrogen gas market, 258 Inhibition, 9, 46, 49, 62, 73, 94, 96, 248 Glutamine, 238, 247 Inoculation, 66, 83, 95–96, 212, 255 Gracilaria, 161 In-situ biogas upgradation, 84–85 Grape wine, 3 Integrated Algal BioRefinery (IABR), 26 Graphene oxide (GO), 198 Internal-combustion devices, 185 Graphite felt, 198, 202, 205, 207 Internal combustion (IC) engine, 3, 105 Graphite grains, 198 International Energy Agency, 19 Graphite granules, 198, 201, 205, 207 Interspecies hydrogen transfer, 240, 241 Graphite mat, 201 Ion exchange membrane, 10, 191, 200, 210 Gravity sedimentation, 148, 150, 151 Ionic liquid pretreatment, 99 Green crude, 26, 137 Green diesel, 8, 10 J Greenhouse gas (GHG), 11, 17, 22, 23, 36, 47, 51, 78, 126, 133, 154, 226, 231, 256 Jatropha, 123, 160 Greenhouse gas emissions, 11, 17, 36, 51, Jet fuel, 7, 10, 23–26, 130, 131, 137, 144, 133, 154 167, 169 Green microalgae, 154 Jojoba oil, 123 Index 277

K Methanomicrobiales, 45 Kyoto protocol, 5 Methanoplanus limicola (Squaretype), 46 Methanopyrales, 45 Methanosarcina barkeri, 46, 47, 49, 50, 71 L Methanosarcina barkeri (Coccal packets), 46 Lactic acid bacteria, 224 Methanospirillum hungatei (Spirilla), 46 Landfill gas, 8, 39, 45, 80, 81, 85, 212 Methanothermobacter thermautotrophicus, 46 Landfill leachate, 210, 212 Methanothrix soehngenii (Filamentous), 46 Large-scale Microalgae Production, 132–147 Methylamine, 45 Lauric acid, 145 Methyl-coenzyme, 49, 51 LCFA. See Long chain fatty acids (LCFA) Methyldiethanolamine (MDEA), 87 Linear low density polyethylene (LLDPE), 110 MFC. See Microbial fuel cell (MFC) Lipids, 6, 44, 68, 124,–126, 128, 150, 153–166, Microalgae, 6, 24, 51, 68–70, 123–155, 197, 227 158–166, 170, 171, 223, 227, 230, 235, Liquefaction, 106, 107, 111, 129, 227, 234 252, 253, 257 Liquefied biomethane (LBM), 109, 111 Microalgae cultivation, 132–135, 148 Liquid fuels, 1, 24, 37, 123–171, 227 Microbial biofuel, 15–18 Liquid process wastes, 71 Microbial electrolysis cell (MEC), 236–239, Livestock manures, 38, 41 260 l-N-bytyl-3-methyl (tetradecyl) ammonium Microbial fuel cell (MFC), 10, 181–215, 237 chloride (BDTACI), 99 Microcoleus vaginatus, 139 Long chain fatty acids (LCFA), 44, 154, 241 Microcystis sp., 225 Long chain hydrocarbons, 10, 75, 147, 227 Microelectronic devices, 214–215 Low-density polyethylene (LDPE), 110 Mid-chain oils, 147 Lysozyme, 236 Milk house wash water, 38 Millennium development goals (MDGs), 21 Millennium Summit, 21 M Miscanthus, 53, 123 Mahua, 123 Mittelbach, Martin, 5 Maize, 1, 17, 20, 52–54, 69, 78, 106, 160 Mixotrophic growth, 124, 132 Market garbage, 227 Monoethanolamine (MEA), 87 Market organic wastes (MOW), 40, 52, 55–59 Monogalactosyldiacylglycerol (MGDG), 155 MEC. See Microbial electrolysis cell (MEC) MOW. See Market organic wastes (MOW) Mechanical pretreatment, 77, 97 Municipal wastewater solids, 41 Mediator-less, 183, 187, 195 Muradel’s GreenBlack technology, 131 Membrane separation, 84, 85 Membrane technology, 87–88, 170 Methane emission, 45, 51–79 N Methanobacteriales, 45 NADH, 185, 187, 189, 196, 241, 248 Methanobacterium bryantii (Long rod), 46 Nannochloris sp., 125, 155, 156 Methanobacterium formicum (Rods), 46 Nannochloropsis sp., 125, 128, 149, 155, 156 Methanobacterium ruminantium, 56 National Association of Automotive Vehicle Methanobacterium thermoautotrophicum, Manufacturers, 4 46, 71 National Horticulture Board, 56 Methanobrevibacter smithii (Shortrods and National Project on Biogas Development, 39 chains), 46 National Renewable Energy Laboratory Methanococcales, 45 (NREL), 229, 242, 254 Methanogenesis, 44, 45, 47, 48, 50, 55, 64, 73, Natural gas, 16, 17, 25, 38, 41, 51, 103, 109, 79, 239 223, 232, 233, 258, 259 Methanogenic archaea, 46 Navy’s Green Fleet Initiative, 130 Methanogens, 36, 45–47, 49, 50, 73, 94, Negative electrode, 185, 189 96, 236 Nephroselmis olivacea, 253 278 Index

Neutral red, 183, 187, 188, 195, 198, 208 Photosynthetically active radiation (PAR), 251 NGO. See Nongovernmental organization Photosynthetic bacteria, 68, 224, 226, 244, 245, (NGO) 251, 254, 259 Night soil, 35, 36, 40, 60, 75, 101, 112 Photosynthetic microalgae, 38 Nitrogenase, 228, 229, 235, 237, 238, 245–247, Photosystems, PSI & PSII, 168, 242, 244, 249, 252, 253 245, 252 N-methylmorpholine-N-oxide monohydrate Phytoplankton, 223 (NMMO), 99 Pigment, 8, 126, 128, 153, 166, 237, 244 Nongovernmental organization (NGO), 18, Pinworm, 62 39, 108 Plant microbial fuel cell (PMFC), 197, Nostocales, 245 203–205, 213 Nostoc sp., 229 PMFC. See Plant microbial fuel cell (PMFC) NREL. See National Renewable Energy Polyunsaturated fatty acid (PUFA), 154, 155 Laboratory (NREL) Positive electrode, 185, 189 Nutraceutical, 25, 27, 129, 137, 141 Pressure-swing-adsorption (PSA), 84–86, Nutrients concentration, 94–95 89–90 Primary electron donor, 196 Proceedings of the National Academy of O Sciences (PNAS), 2 Ocean Sunrise Project, 170 Proviron, 28 Oleic acid, 145 Prymnesium parvum, 161, 162 One-Chambered MFC, 200–201 PSA. See Pressure-swing-adsorption (PSA) Open ponds, 6, 68, 69, 135–138, 147, 163 Pulsed electric field (PEF), 27, 153 Organic industrial wastes, 36 Purple nonsulfur bacterium (PNSB), 237, 238 Organic material load, 196 Origin Oils Inc., 27 Oscillatoriales, 245 Q Oscillatoria sp., 225, 226 QH2, 187, 189 Otto, Nikolaus, 3 Oxidant Chamber, 185 Oxidative phosphorylation, 185, 254 R Oxidative pretreatment, 99 Race-way-ponds, 161 Ozone, 76, 99, 258 Rapeseed, 6, 123, 147, 160, 162 Red algae, 124, 166, 244 Refractance window drying technology, 153 P Reliance Industries Ltd., 24 Pacific Biodiesel, 6 Removal of ammonia, 80, 87 Palm oil, 6, 123, 160, 170 Removal of hydrogen sulfide, 81–83 Parasite eggs, 62 Removal of nitrogen, 80 PEC. See Photoelectrochemical (PEC) Removal of particulates, 81 Peptococcus, 44 Removal of siloxanes, 80–81 Peracetic acid (PAA), 77, 98 Removal of water, 80 Peroxymonosulfate (POMS), 76 Renewable energy directive (RED), 22 Petrodiesel, 3, 6, 7, 10–13, 16, 17, 129 Research Institute of Innovative Technology Phaeodactylum, 149, 155 for the Earth (RITE), 6 Photobioreactor, 24, 124, 132, 134–145, 147, Residual fatty acid, 55 253, 255, 257, 258 Retentiontime (RT), 63, 77, 79, 93, 97, 98, 102, Photocleavage, 232 103, 107 Photoelectrochemical (PEC), 215, 226, Rhodopseudomonas palustris, 191, 193, 224, 232–233 237–238 Photolytic, 223, 232, 258 Rhodospirillum rubrum, 224, 228, 238 Photosynthesis, 9, 15–17, 36, 124, 126, 133, Rice, 2, 40, 52, 78, 212, 213 145, 158, 167, 170, 213, 215, 223, 229, Roundworm, 62, 63, 67 230, 237, 244, 247, 248, 250–255 Rubrivivax gelatinosus, 254, 255 Index 279

Rudolph Diesel, 5 Stickland reaction, 44 Ruminococcus, 41, 224 Stone Age jugs, 2 Ruminococcus albus, 41, 56 Streptococcus, 44 Structured fat family, 147 Substrate phosphorylation, 51 S Succinate, 47, 49, 168 Saccharomyces cerevisiae, 166, 171, 183, 191 Sugar beet, 1, 9, 52, 54, 58, 59, 162 Salmonella sp., 62, 67 Sugar cane, 21 Salmon oil, 123 Sunflower seed oil, 6 Samuel Morey, 3 Sunlight, 15, 24, 26, 136–139, 143, 167, 169, Sapphire Energy Inc., 7, 10, 23, 26, 130, 215, 223, 226, 228–230, 232, 235, 134, 137 258, 259 Sapphire Energy Integrated Algal Biorefinery Superoxide dismutase (SOD), 47 (SEIABR), 130 Sura, 3 Sargassum, 161, 164 Symbiotic process, 7 Scenedesmus, 149, 152, 153, 156, 170, Synechococcus sp., 162, 164 235, 251 Synechocystis sp., 139 Scenedesmus obliquus, 125, 156, 162, 164, Synechocystis sp. PCC 6803, 169, 229, 165, 224, 228, 251 248, 249 Schizochytrium sp., 125, 128 Syntrophic acetate oxidizers (SAO), 73 Second generation biofuels, 11, 123, 124 Sedimentation, 47, 135, 148–153 Sediment microbial fuel cell (SMFC), 197, 210 T Selenomonas, 44 Taenia solium (Pork tape worm), 62 Selexol adsorption, 85 Thermal pretreatment, 76, 97–98 Serratia sp., 224 Thermoanaerobacter, 49 Sewage sludge, 38, 75–78, 80, 107, 108, 196, Thermoanaerobacterium, 49 208, 227 Thermochemical pretreatment, 76, 77 Sewage sludge, 75 Thermochemical processes, 226, 230–232, 234 Shewanella oneidensis, 183, 191–193 Thickening, 76, 148, 151–152 Shigella sp., 62, 67 Thiobacillus ferrooxidans, 196 Siloxanes, 79–81 Total organic solid (TOS), 78 Single Chambered MFC, 208, 209 Total solid (TS), 56, 58, 59, 61, 66, 67, Slaughterhouse wastes, 38, 71–74, 81 102, 148 Sludge pretreatment process, 76–78 Transesterification, 6, 12, 129, 154, 155, Solar Energy Research Institute, 229 158–160, 164, 227 Solar radiation, 103, 204, 213, 223 Transesterification process, 6, 158 Solar water splitting processes, 232 Triacylglycerides (TAGs), 154 Solazyme Inc., 7, 10, 23–25, 130, 145–147 Trichuris trichiura (whipworm), 62 Solazyme Integrated Biorefinery (SzIBR), 130 TS, 40, 55, 62, 66, 67, 73, 79 Solid process wastes, 71 Tubular photobioreactor, 134, 135, Solids retention time (SRT), 93 139–143, 258 Solvent extraction, 155, 157 Tubular PMFC, 204 Soybean oil, 6, 170 Tubular reactors, 142 Spent water, 71–72, 74 Two chambered MFC, 194, 197, 199, 200, 209 Spirulina maxima, 156, 162, 164, 224 Two-stage AD, 49, 50 Spirulina spp., 137, 141 Stacked microbial fuel cell, 202–203 Standard Oil Company, 4 U Staphylococcus sp., 62 Ultrafiltration, 63, 152 Starch, 11, 49, 59, 123, 124, 129, 133, 149, 150, Ultrasonic aggregation, 151, 153 160, 165, 170, 171, 184, 189, 235, Ultrasonic pretreatment, 77 250–253 United States Department of Energy, 6, 7, 9, Steam treatment process, 97 69, 147, 230 280 Index

Upﬂow MFC, 201–202 W Uptake hydrogenase, 245, 246, 248–249 Waste feed, 38 UV treatment, 63 Waste vegetable oil, 6 Wastewater treatment, 6, 39, 76, 150, 151, 182, 195, 197, 211–212, 255 V Water biophotolysis, 235 Vegetable oil, 5, 6, 10, 11, 23, 25, 41, 123, 130, Water electrolytic processes, 230–232 155 Water-gas shift reaction (WGS), 253–256 Veillonella opalescens, 56 Water scrubber, 83, 85–86, 91 Vertical column photobioreactor, 135, Water splitting process, 232 144–145 Wave, 223, 257, 258 VFAs. See Volatile fatty acids (VFAs) WGS. See Water-gas shift reaction (WGS) Vibrio cholerae, 62 Wheat, 1, 40, 52, 53, 78, 163 Vibrio parahaemolyticus, 62 Wind, 15, 35, 213, 223, 258 Vitamins, 8 World Health Organisation (WHO), 60 Volatile fatty acids (VFAs), 44, 49, 50, 60, 70, World War II, 6, 137 73, 78, 96