Lignocellulosic Production and Industrial Applications 9781119323686

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Lignocellulosic Biomass Production and Industrial Applications

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Lignocellulosic Biomass Production and Industrial Applications

Edited by

Arindam Kuila and Vinay Sharma

This edition first published 2017 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2017 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data Names: Kuila, Arindam, editor. | Sharma, Vinay, editor. Title: Lignocellulosic biomass production and industrial applications / edited by Arindam Kuila and Vinay Sharma. Description: Beverly, MA : Scrivener Publishing ; Hoboken, NJ : John Wiley & Sons, 2017. | Includes index. | Identifiers: LCCN 2017010812 (print) | LCCN 2017013368 (ebook) | ISBN 9781119323853 (pdf) | ISBN 9781119323877 (epub) | ISBN 9781119323600 (cloth) Subjects: LCSH: Lignocellulose--Biotechnology. | Biomass--Industrial applications. Classification: LCC TP248.65.L54 (ebook) | LCC TP248.65.L54 L5383 2017 (print) | DDC 662/.88--dc23 LC record available at https://lccn.loc.gov/2017010812 Cover image: Pixabay (background) and Arindam Kuila (foreground) Cover design by Russell Richardson Set in size of 11pt and Minion Pro by Exeter Premedia Services Private Ltd., Chennai, India Printed in 10 9 8 7 6 5 4 3 2 1

Contents Preface 1

Valorization of Lignocellulosic Materials to Polyhydroxyalkanoates (PHAs) Arpan Das 1.1 Introduction 1.1.1 What is PHA? 1.1.2 Mechanism of PHA Production 1.2 Lignocellulose: An Abundant Carbon Source for PHA Production 1.2.1 Cellulose 1.2.2 Hemicelluloses 1.2.3 Lignin 1.2.4 Pectin 1.3 Lignocellulosic Pretreatment Techniques 1.3.1 Physical Pretreatment Techniques 1.3.1.1 Milling 1.3.1.2 Irradiation 1.3.2 Chemical Pretreatment 1.3.2.1 Acid Hydrolysis Pretreatment 1.3.2.2 Alkaline Hydrolysis 1.3.2.3 Oxidative Delignification by Peroxide 1.3.2.4 Organosolv Process 1.3.2.5 Ozonolysis Pretreatment 1.3.2.6 Ionic Liquids Pretreatment 1.3.3 Physico-Chemical Pretreatment 1.3.3.1 Liquid Hot-Water Pretreatment 1.3.3.2 Steam Explosion 1.3.3.3 Ammonia Fiber Explosion (AFEX) 1.3.4 Bological Pretreatment

xv 1 1 3 3 5 5 7 7 8 8 8 8 10 10 10 11 11 11 12 12 12 13 13 13 14

v

vi Contents 1.4 Hydrolysis of Lingocellulose 1.5 Lignocellulose Biomass as Substrate for PHA Production 1.6 Conclusion References 2

3

Biological Gaseous Energy Recovery from Lignocellulosic Biomass Shantonu Roy 2.1 Introduction 2.2 Simple Sugars as Feedstock 2.3 Complex Substrates as Feedstock 2.4 Biomass Feedstock 2.4.1 Energy Crop 2.4.1.1 Miscanthus sp. 2.4.1.2 Sweet Sorghum Extract 2.4.1.3 Sugar Beet Juice 2.4.2 Algal Biomass 2.5 Waste as Feedstock 2.5.1 Municipal Solid Waste (MSW) 2.5.2 Food Waste 2.6 Industrial Wastewater 2.6.1 Dairy Industry Wastewater 2.6.2 Distillery Wastewater 2.6.3 Chemical Wastewaters 2.6.4 Glycerol 2.6.5 Palm Oil Mill Effluent 2.7 Conclusion Acknowledgments References Alkali Treatment to Improve Physical, Mechanical and Chemical Properties of Lignocellulosic Natural Fibers for Use in Various Applications Suvendu Manna, Prosenjit Saha, Sukanya Chowdhury and Sabu Thomas 3.1 Introduction 3.1.1 Composition of Natural Fibers 3.1.2 Properties of Natural Fibers 3.2 Alkali Treatment 3.2.1 General Processing 3.2.2 Steam Treatment 3.2.3 Alkali-Steam Treatment

14 16 19 19 27 27 28 32 32 33 34 34 34 35 36 37 38 38 38 39 39 40 40 40 41 41

47

48 49 51 52 52 54 54

Contents vii 3.3

Application of the Alkali-Steam-Treated Fibers 3.3.1 Biocomposite 3.3.1.1 Green Biocomposite 3.3.1.2 Bionanocomposites 3.3.2 Water Treatment 3.3.2.1 Fluoride Removal 3.3.2.2 Dye Removal 3.3.2.3 Heavy Metal Removal 3.4 Summary References 4

Biodiesel Production from Lignocellulosic Biomass Using Oleaginous Microbes S.P. Jeevan Kumar, Lohit K. Srinivas Gujjala, Archana Dash, Bitasta Talukdar and Rintu Banerjee 4.1 Introduction 4.2 Lignocellulosics Distribution, Availability and Diversity 4.2.1 Forest Trees and Residues 4.2.2 Food Crops 4.2.3 Non-Food/Energy Crops 4.2.4 Tree-Based Oils 4.2.5 Industrial Process Residues 4.3 Prospective Oleaginous Microbes for Lipid Production 4.3.1 Oleaginous Algae 4.3.1.1 Green Algae 4.3.1.2 Blue-Green Algae 4.3.1.3 Golden Algae 4.3.1.4 Red and Brown Algae 4.3.1.5 Diatoms 4.3.2 Oleaginous Yeast and Mold 4.3.3 Metabolic Engineering Approaches for LCB Utilization 4.3.3.1 Metabolic Engineering of Xylose and Co-Utilization of Substrate 4.4 Technical Know-How for Biodiesel Production from LCBs 4.5 Fermentation 4.5.1 Solid-State Fermentation (SSF) 4.5.2 Submerged Fermentation (SF) 4.6 Transesterification for Biodiesel Production 4.6.1 Biodiesel 4.6.2 Transesterification

55 55 55 56 57 57 58 59 59 60 65

66 67 67 68 68 69 70 70 70 70 71 72 72 72 72 73 74 77 78 79 79 80 80 80

viii

Contents 4.6.3 Acid/Base Transesterification 4.6.4 Enzymatic Transesterification 4.6.4.1 Lipases 4.6.5 In-Situ Transesterification 4.7 Characteristics of Fatty Acid Methyl Esters 4.8 Conclusion References

5 Biopulping of Lignocellulose Arijit Jana, Debashish Ghosh, Diptarka Dasgupta, Pradeep Kumar Das Mohapatra and Keshab Chandra Mondal 5.1 Introduction 5.2 Composition of Lignocellulosic Biomass 5.3 Pulping and its Various Processes 5.4 Biopulping – Process Overview 5.4.1 Role of Rot Fungi and its Effect in Wood Biomass 5.4.2 Role of Enzymes in Biopulping 5.5 Advantages and Disadvantages of Biopulping 5.6 Future Prospects Acknowledgment References 6

Second Generation Bioethanol Production from Residual Biomass of the Rice Processing Industry Luciana Luft, Juliana R. F. da Silva, Raquel C. Kuhn and Marcio A. Mazutti 6.1 Introduction 6.2 Residual Biomass 6.3 Rice and Processing 6.4 Pretreatment Techniques 6.4.1 Physical Pretreatment 6.4.1.1 Mechanical 6.4.1.2 Microwave 6.4.1.3 Pyrolysis 6.4.2 Physicochemical Pretreatment 6.4.2.1 Steam Explosion 6.4.2.2 Wet Oxidation 6.4.2.3 Ultrasound 6.4.2.4 Supercritical CO2 Explosion 6.4.2.5 Ammonia Fiber Expansion

81 81 82 82 83 83 84 93

93 95 97 98 100 102 104 105 105 106 111

112 112 113 115 116 116 117 118 118 119 119 120 120 121

Contents ix 6.4.3

Chemical Pretreatment 6.4.3.1 Ozonolysis 6.4.3.2 Acid Treatment 6.4.3.3 Alkaline Treatment 6.4.3.4 Organosolv Process 6.4.3.5 Ionic Liquids 6.4.4 Biological Pretreatment 6.5 Hydrolysis 6.6 Fermentation 6.7 Bioethanol Production 6.8 Concluding Remarks Acknowledgments References 7

Microbial Enzymes and Lignocellulosic Fuel Production Avanthi Althuri, Anjani Devi Chintagunta, Knawang Chhunji Sherpa, Rajiv Chandra Rajak, Debajyoti Kundu, Jagriti Singh, Akanksha Rastogi and Rintu Banerjee 7.1 Introduction 7.1.1 Enzymes for Lignocellulosic Biomass-Based Biofuel Production 7.2 Lignocellulosic Biomass as Sustainable Alternative for Fuel Production 7.2.1 Constituents of Lignocelluloses: Cellulose, Hemicellulose, Lignin and Other Biomolecules 7.3 Enzymes and Their Sources for Biofuel Generation 7.4 Microbial Enzymes towards Lignocellulosic Biomass Degradation 7.4.1 Ligninases 7.4.1.1 Lignin Peroxidase 7.4.1.2 Manganese Peroxidase 7.4.1.3 Hybrid Peroxidase 7.4.1.4 Phenol Oxidases 7.4.1.5 Other Lignin-Degrading Enzymes 7.4.2 Carbohydratases 7.4.2.1 Cellulase 7.4.2.2 Auxiliary Cellulose-Degrading Enzymes 7.4.2.3 Hemicellulase 7.4.2.4 Expansins and Swollenins

121 121 122 123 123 123 124 124 125 127 127 128 128 135

136 136 137 138 139 142 146 146 147 147 148 149 150 150 151 152 154

x Contents 7.4.2.5 Carboxyl Esterases 7.4.2.6 Zymase 7.5 Applications in Biofuel Production 7.5.1 Bioethanol 7.5.2 Biomethane and Biomanure 7.6 Conclusion References 8

Sugarcane: A Potential Agricultural Crop for Bioeconomy through Biorefinery Knawang Chhunji Sherpa, Rajiv Chandra Rajak and Rintu Banerjee 8.1 Introduction 8.2 Present Status of Sugarcane Production and its Availability 8.3 Morphology of Sugarcane 8.4 Factors Involved in Sugarcane Production 8.4.1 Climatic Conditions 8.4.1.1 Temperature 8.4.1.2 Rainfall and Relative Humidity 8.4.1.3 Sunlight 8.4.2 Soil Quality 8.4.3 Varieties of Sugarcane 8.4.4 Land Requirement 8.4.5 Propagation 8.4.6 Nutrient Management 8.4.7 Water Management 8.4.8 Weed Management 8.4.9 Biotic Factors: Pests and Pathogens 8.4.10 Crop Rotation 8.4.11 Ratooning 8.4.12 Intercropping 8.5 Major Limitations of Sugarcane Production 8.6 An Overview of Biotechnological Developments for Sugarcane Improvement 8.7 By-Products of Sugarcane Processing 8.7.1 Bagasse 8.7.2 Molasses 8.7.3 Vinasse 8.8 Applications of Sugarcane for Biorefinery Concept

156 157 159 159 161 162 163 171

171 173 174 175 175 175 175 175 176 176 177 177 182 182 183 183 183 183 185 185 186 188 188 189 189 189

Contents xi 8.9

Utilization of Sugarcane Residue for Bioethanol Production 8.10 Conclusion References 9

Lignocellulosic Biomass Availability Map: A GIS-Based Approach for Assessing Production Statistics of Lignocellulosics and its Application in Biorefinery Sanjeev Kumar, G. Lohit Kumar Srinivas and Rintu Banerjee 9.1 Introduction 9.2 Geographical Information System (GIS) 9.3 Application of GIS in Mapping Lignocellulosic Biomass 9.4 Biofuels from Lignocellulosics 9.5 Conclusion References

10 Lignocellulosic Biomass Utilization for the Production of Sustainable Chemicals and Polymers Gunjan Mukherjee, Gourav Dhiman and Nadeem Akhtar 10.1 Introduction 10.2 Lignocellulosic Biomass 10.3 Pretreatment Strategies 10.3.1 Physical Pretreatment 10.3.1.1 Physical Comminution and Extrusion 10.3.1.2 Pyrolysis, Irradiation and Pulsed Electric Field 10.3.2 Chemical Pretreatment 10.3.2.1 Acid and Alkali Pretreatment 10.3.4 Thermophysical Pretreatments 10.3.5 Thermochemical Pretreatments 10.3.5.1 Oxidation 10.3.6 Biological Pretreatment 10.4 Value-Added Chemicals from Lignocellulosic Biomass 10.4.1 Lignocellulose-Derived Sugars 10.4.2 Lignin-Derived Chemicals 10.4.2.1 Vanillin 10.4.2.2 Vanillin-Based Resins 10.4.2.3 Cyanate Ester Resins 10.4.2.4 Epoxide Resins 10.4.2.5 Benaoxazine Resins 10.4.2.6 Polyester 10.4.2.7 Polyurethanes

190 192 192

197 198 199 202 209 211 212 215 216 216 219 219 219 219 220 220 221 222 222 223 224 224 225 226 226 226 227 227 227 227

xii Contents 10.5

Sustainable Polymers from Lignocellulosic Biomass 10.5.1 Sugar-Containing Polymers 10.5.1.1 1,4-Diacid-Based Polymers 10.5.1.2 5-(Hydroxymethyl) Furfural (HMF)- and 2,5-Furandicarboxylic Acid (FDCA)-Based Polymers 10.5.1.3 3-HPA (3-Hydroxy Propionic Acid) Platform-Based Polymers 10.5.1.4 Aspartic Acid Platform-Based Polymers 10.5.1.5 Glutamic Acid Platform-Based Polymers 10.5.1.6 Glucaric Acid-Based Polymers 10.5.1.7 Itaconic Acid (ITA) Platform-Based Polymers 10.5.1.8 Levulinic Acid Platform-Based Polymer 10.5.1.9 3-Hydroxy-Butyrolactone (3-HBL) Platform-Based Polymer 10.5.1.10 Sorbitol-Based Polymers 10.5.1.11 Glycerol-Based Polymers 10.5.1.12 Lactic Acid-Based Platform 10.5.1.13 Acetone-ButanolEthanol-Based Polymer 10.5.1.14 Xylose/Furfural/Arabinitol Platform-Based Polymer 10.5.1.15 Polyhydroxyalkanoate (PHA) 10.5.1.16 Rubber Polymers 10.5.1.17 Other Lignocelluolse-Derived Polymers 10.6 Potential Challenges for a Sustainable Biorefinery 10.7 Environmental Effects of Biorefineries 10.8 Future Perspectives of Biorefineries and Their Products 10.9 Conclusion References

228 228 228

229 229 230 230 230 231 231 232 232 232 233 233 233 234 234 234 234 235 236 236 237

Contents xiii 11 Utilization of Lignocellulosic Biomass for Biobutanol Production Anand Prakash, Vinay Sharma, Deepak Kumar, Arindam Kuila and Arun Kumar Sharma 11.1 Introduction 11.2 Bioconversion of Lignocellulosic Biomass to Biobutanol 11.3 Composition of Lignocellulosic Biomass 11.4 Structure of Lignocellulosic Biomass 11.5 Biobutanol Production from Lignocellulosic Biomass 11.5.1 Pretreatment 11.5.2 Hydrolysis 11.5.2.1 Cellulases and Xylanases 11.5.2.2 The Cellulase of Trichoderma reesei RUT-C30 11.5.3 Fermentation 11.5.3.1 Development of New Fermentation Technologies 11.6 Conclusion References 12 Application of Lignocellulosic Biomass in the Paper Industry Mainak Mukhopadhyay and Debalina Bhattacharya 12.1 Introduction 12.2 Major Raw Materials Used in the Paper Industry 12.2.1 Agricultural Residues 12.2.1.1 Sugarcane Bagasse 12.2.1.2 Corn Stalks 12.2.1.3 Rice Straw, Wheat Straw and Cereal Straw 12.2.1.4 Bamboo 12.2.1.5 Sabai Grass 12.2.1.6 Jute 12.2.1.7 Ramie 12.2.1.8 Leaf Fibers 12.2.1.9 Cotton Fibers 12.2.1.10 Cotton Rags 12.3 Pulp and Papermaking Process 12.3.1 Pulping Process 12.3.1.1 Mechanical Pulping 12.3.1.2 Chemical Pulping

247

247 248 248 248 249 250 250 251 253 255 257 258 258 265 265 266 266 266 267 267 267 267 268 268 268 268 268 269 269 269 270

xiv Contents 12.3.2

Bleaching Process 12.3.2.1 Chlorine Bleaching 12.3.2.2 Elemental Chlorine Free Bleaching (ECF Bleaching) 12.3.2.3 Total Chlorine Free Bleaching (TCF Bleaching) 12.3.2.4 Hydrogen Peroxide (H2O2) Brightening 12.4 Waste Generation 12.4.1 Wastewater 12.4.2 Rejects 12.4.3 Green Liquor Sludge, Dregs and Lime Mud 12.4.4 Wastewater Treatment Sludge 12.4.5 Primary Sludge 12.4.6 Secondary or Biological Sludge 12.4.7 Organic Pollutants and Suspended Solids 12.4.8 Organochlorine Compounds 12.4.9 Inorganic Chemicals 12.4.10 Chlorophenolics 12.4.11 Dioxins and Furans 12.5 Waste to Value-Added Products 12.5.1 Biogas 12.5.2 C5 and C6 Sugar Fermentation 12.5.3 Value-Added Compounds from Lignins 12.5.4 Organic Solutions 12.6 Conclusion References Index

270 271 271 271 272 272 272 272 272 273 273 273 273 273 274 274 274 274 274 275 275 275 275 276 279

Preface Lignocellulosic materials such as agricultural residues (e.g., wheat straw, sugarcane bagasse, corn stover), forest products (hardwood and softwood), and crops such as switchgrass and salix, are becoming a potent source for generating different valuable products. Lignocellulosic biomass is mainly composed of cellulose, hemicellulose and lignin, along with smaller amounts of pectin, protein and extractives (soluble nonstructural materials such as nonstructural sugars, nitrogenous material, chlorophyll and waxes). Cellulose and hemicellulose are the main constituents of lignocellulosic biomass, occupying a major portion of the fibrous structure of plant cell walls. This book, entitled Lignocellulosic Biomass Production and Industrial Applications, describes the utilization of lignocellulosic biomass for different possible applications. Although there have been numerous reports on lignocellulosic biomass for biofuel application, there have been very few other applications reported for lignocellulosic biomassbased chemicals, polymers, etc. Therefore, this book covers all of the possible lignocellulosic biomass applications. It describes the different types of biofuel production, such as bioethanol, biobutanol, biodiesel and biogas, from lignocellulosic biomass. Also presented are various other lignocellulosic biomass biorefinery applications for the production of chemicals, polymers, paper and bioplastics. In addition, there is a discussion of the major benefits, limitations and future prospects of the use of lignocellulosic biomass. Arindam Kuila Vinay Sharma Banasthali, India February 2017

xv

1 Valorization of Lignocellulosic Materials to Polyhydroxyalkanoates (PHAs) Arpan Das Department of Microbiology, Maulana Azad College, Kolkata, West Bengal, India

Abstract Biobased products have generated great interest since sustainable development policies are expanding along with decreasing fossil fuel reserves and growing environmental concerns. Among the petrochemical products, synthetic plastic plays an important role in human daily life, but its recalcitrant properties cause pervasive environmental pollution. In this regard, Polyhydroxyalkanoates (PHAs) are very encouraging resources that might serve as an eco-friendly alternative to petrochemical plastics. But the main obstacle is the cost of that polymer material, which is used as a carbon source during the production of PHAs. Lignocellulosic biomasses represent a very promising substrate for PHA production as they are cheap, abundant and do not compete with the human food chain. Lignocellulosic hydrolysates with a wide range of sugars and organic acids can extensively influence the overall yield of PHAs. This chapter provides a glimpse into the current research focusing on the production of PHAs using lignocellulosic materials as main carbon source. Keywords: Polyhydroxyalkanoates, agrowastes, lignocellulose, cellulose, hemicellulose, lignin

1.1

Introduction

The accumulation of petrochemical polymers in our surroundings and growing awareness of environmental pollution throughout the world has

Corresponding author: [email protected] Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial Applications, (1–26) © 2017 Scrivener Publishing LLC

1

2 Lignocellulosic Biomass Production and Industrial Applications triggered the search for new biocompatible products for a safe environment. Currently, most polymer products are designed and prepared synthetically and very limited consideration is given to their ultimate disposal. However, these nondegradable plastics are building up in the environment at the rate of 25 million tons per year, which may persist for hundreds of years. Under these circumstances it is worth designing and developing appropriate biodegradable materials whose disposal ensures a better environment and ecosystem. Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible plastics that have been identified as an alternative to petroleum-based synthetic plastics. This type of polyester polymer is produced by many bacteria, archaea as well as some fungi. It accumulates as discrete granules to levels as high as 90% of cell dry weight as a response to environmental stress and nutrient imbalance (when the carbon substrate is in excess of other nutrients such as nitrogen, sulfur, phosphorus or oxygen [1]), and plays a role as a sink for intracellular energy and carbon storage. These water insoluble storage polymers are biodegradable, exhibit thermoplastic properties and can be produced from different renewable carbon sources. PHAs are high molecular mass polymers with properties similar to conventional plastics such as polypropylene. Therefore, they have a wide range of applications such as in the manufacture of bottles, packaging materials, films for agriculture and also in medical applications [2, 3]. The main advantage is that the biodegradable polymers are completely degraded to water, carbon dioxide and methane by anaerobic microorganisms in various environments such as soil, sea, lake water and sewage and, hence, are easily disposable without harm to the environment. However, the high cost of PHA production compared to cheap petrochemical polymers, prevents their use on an industrial scale. Continuous efforts are being made and several studies are going on to develop a cost-effective strategy by using inexpensive substrates as a carbon source, which can significantly affect the production of PHA and has become an important objective for the commercialization of bioplastics. Since about 45% of the total cost of PHA production are attributed to carbon sources, such as refined glucose or sucrose [4], cheap wastes from agriculture and the food industry are used as inexpensive carbon substrates, thus improving the economic feasibility of PHA production. Moreover, lignocellulosic biomasses are considered to be very promising renewable sources for the biotechnological production of fuels and chemicals, including PHA. Lignocellulose hydrolysate is a potentially inexpensive and renewable feedstock that can be processed through different physical, chemical or enzymatic processes to fermentable sugars such as glucose, galactose, xylose, and mannose. However, during the process to produce fermentable sugars, other by-products like

Valorization of Lignocellulosic Materials

3

acetic acid, 5-hydroxymethyl furfural, formic acid, phenolic compounds, etc., are released during the treatment of hemicellulose and lignin. These compounds are exceedingly toxic to microorganisms during subsequent fermentation processes. In order to increase the fermentability of the hydrolysate, a number of detoxifcation methods are also required to remove potential inhibitors. Overliming [5], activated charcoal [6], membrane filtration [7], ion exchange resins [8], and biological treatments [9] are among the most frequently used treatments.

1.1.1

What is PHA?

Polyhydroxyalkanoates (PHAs) are storage compounds that are widely produced by many microorganisms under nutrient-limited growth conditions, such as nitrogen, phosphorous or oxygen starvation, and when an excess of carbon source is present [10]. These storage materials serve as the carbon and energy reserves of the producing microorganisms. Generally, PHAs are considered as an alternative to petrochemical-based synthetic polymers. Based on the chain length of the fatty acid monomers, PHAs can be classified into three categories: short-chain-length (scl) PHAs with 3 to 5 carbon atoms, medium-chain-length (mcl) PHAs with 6 to 14 carbon atoms and long-chain-length (lcl) PHAs with more than 14 carbon atoms [11]. The difference in length and/or chemical structure of the alkyl side chain of the PHAs influences the material properties of the polymers to a great extent [12]. In general, the scl-PHAs are more crystalline than the mcl-PHAs. As such, scl-PHAs usually exhibit thermoplastic-like properties, while mcl-PHAs and lcl-PHAs behave like elastomers or adhesives. Due to their physical characteristics, scl-PHAs can be used for manufacturing items for packaging or everyday plastics commodities. However, PHAs are disadvantaged due to their significantly higher production costs, while a major portion of the final cost is represented by the price of carbon substrate (28–50%). Therefore, research has focused on inexpensive fermentable raw materials as substrates for biotechnological PHA production.

1.1.2

Mechanism of PHA Production

Polyhydroxybutyrate is the intracellular granule, synthesized by bacteria, and acts as an energy storage facility. In some Bacillus sp., it provides energy for sporulation [13]. The low molecular weight P(3HB) is a part of bacterial Ca2+ channels [14]. These granules are synthesized by the microorganisms in a limited concentration of O, N, P, S, or trace elements, e.g., Mg, Ca, Fe and high carbon concentration in the medium [15]. Generally these

4 Lignocellulosic Biomass Production and Industrial Applications nutrient sources are used for the synthesis of proteins essential for the growth in bacteria. But, nitrogen source depletion leads to the cessation of protein synthesis, which in turn leads to the inhibition of tricarboxylic acid cycle (TCA cycle) enzymes, such as citrate synthase and isocitrate dehydrogenase, and consequently slows down the TCA cycle [16]. As a result, the acetyl-CoA routes to P(3HB) biosynthesis. Both the shortening of external nutrients and internal sources, such as RNA or enzymes, facilitate the PHA synthesis. Figure 1.1 is a schematic representation of glucose and xylose metabolism for PHA production. Xylose is assimilated in bacteria by the pentose phosphate pathway through isomerization to d-xylulose by xylose isomerase, followed by a phosphorylation by xylulokinase that produces d-xylulose 5-phosphate, finally yielding glucose 6-phosphate. It has been noticed that in some bacteria like Pseudomonas Xylose Xylulose

Xylulose-5P Glucose Fructose-6P Glucose-6P

2 KDPG

Glyceraldehyde-3P

Pyruvate

Acetyl CoA

Malonyl CoA

R-3-Hydroxyacyl ACP TCA cycle R-3-Hydroxyacyl COA

PHAs

Figure 1.1 Flow diagram of polyhydroxyalkanoates (PHA) production from lignocellulose precursor molecules.

Valorization of Lignocellulosic Materials

5

putida, the enzymes responsible for converting xylose to the entry intermediate xylulose 5-phosphate of PP pathway are missing. By introducing the relevant enzymes XylA and XylB, P. putida KT2440 was able to utilize xylose [11]. From Figure 1.1 it can also be seen that PHB formation and the TCA cycle share the same precursor, acetyl-coenzyme A (acetyl-coA), indicating that when synthesizing PHAs using aerobic bacteria, the role of oxygen becomes crucial. It is also reported that when dissolved oxygen (DO) is limited to a certain degree (30–60%), the PHA production quantity changes. The best DO level for optimal PHA production has been found to be 30%. The mechanism behind this is that, under limited DO conditions, an influx of acetyl-coA will move towards PHA production and away from the TCA cycle [17].

1.2

Lignocellulose: An Abundant Carbon Source for PHA Production

Chemically, lignocellulose, the most abundant raw material on earth, is composed of two linear polymers, cellulose and hemicellulose with a nonlinear lignin polymer [18]. In addition, small amounts of other materials, such as ash, protein, pectin, etc., are present in different degrees based on the source. Lignocellulose is physically hard, dense and recalcitrant towards degradation. However, it is an extremely rich and abundant source of carbon and chemical energy, therefore, the recycling of carbon involving lignocelluloses is essential to maintain the global carbon cycle. Although the composition of lignocellulose strongly depends on the type and origin of the particular plant biomass (Table 1.1), the average proportions (w/w) are as follows: 35–50% cellulose, 20–40% hemicellulose, and 5–30% lignin [19, 20].

1.2.1

Cellulose

Cellulose, the most widespread organic material in the world, is the primary product of photosynthesis in terrestrial environments. Its regeneration occurs rapidly, and it does not represent a direct food resource for humans [21]. Cellulose naturally occurs in wood, hemp and other plant-based materials and serves as the dominant reinforcing material in plant structures. This biopolymer is also synthesized by algae, tunicates, some fungi, invertebrates and certain bacteria belonging to the genera Acetobacter, Agrobacterium, Alcaligenes, Pseudomonas, Rhizobium or Sarcina. Even some amoeba (protozoa, for example, Dictyostelium

6 Lignocellulosic Biomass Production and Industrial Applications Table 1.1 Variations in cellulose, hemicellulose and lignin composition in different lignocellulosic materials. Lignocellulose

Cellulose

Hemicellulose

Lignin

Ref.

42

25

20

[66]

32.1

24

18

[67]

Wheat straw

29–35

26–32

16–21

[68]

Newspaper

40–55

25–40

18–30

[69]

Agricultural residues

37–50

25–50

5–15

[70]

Hardwood

45–47

25–40

20–25

[71]

Softwood

40–45

25–29

30–60

[71]

Grasses

25–40

35–50

–

[70]

Sugar cane bagasse Rice straw

discoideum) can synthesize cellulose [22, 23]. Since its discovery in 1838 by Payen, the chemical and physical properties of cellulose have been extensively investigated. A number of efforts of scientists from very diff ferent fields have been dedicated to understanding and controlling its biosynthesis, assembly and structural features. It is a linear condensation polymer consisting of D-anhydroglucopyranose joined together by β-1,4glycosidic bonds with a degree of polymerization (DP) from 100 to 20,000 [24]. It also has a technical name, 1,4-β-polyanhydroglucopyranose. Every d-glucose unit is corkscrewed at 180° with respect to its neighbors, and the repeated segment is frequently treated as a dimer of glucose, known as cellobiose. Each cellulose chain possesses a directional chemical asymmetry with respect to the terminus of its molecular axis: one end is a chemical reducing functionality (hemiacetal unit) and the other is a hydroxyl group, known as the non-reducing end. Coupling of adjacent cellulose chains and sheets of cellulose by hydrogen bonds and van der Waals forces results in a parallel alignment and a crystalline structure with straight, stable supramolecular fibers of great tensile strength and low accessibility, which is known as cellulose microfibril. Due to the fact that cellulose possesses a substantial degree of crystallinity, it functions as a rigid, load-bearing component of the cell wall. The individual chains in these fibrils are associated in various degrees of parallelism. Regions containing highly oriented chains are called crystallites; those in which the chains are more randomly oriented are termed amorphous. In naturally occurring cellulose, the degree of crystallinity varies between 40% and 90% and the rest of the cellulose is amorphous. The amorphous regions are the target site for enzymatic hydrolysis

Valorization of Lignocellulosic Materials

7

and these regions facilitate the penetration and adsorption of enzyme. The resistance of celluloses to enzymatic breakdown is a function of their degree of crystallinity. Furthermore, the rigidity of the cellulose microfibril is strengthened within a matrix of hemicellulose lignin and pectin.

1.2.2

Hemicelluloses

Hemicellulose is the second most abundant component of lignocellulosic biomass. The dominant sugars in hemicelluloses are mannose in softwoods and xylose in hardwoods and agriculture residues. Furthermore, these heteropolymers contain galactose, glucose, arabinose, and small amounts of rhamnose, glucuronic acid, methyl glucuronic acid, and galacturonic acid [25]. The average degree of polymerization of hemicelluloses is in the range of 80–200. They are usually associated with various other cell wall components such as cellulose, cell wall proteins, lignin, and other phenolic compounds by covalent and hydrogen bonding, and by ionic and hydrophobic interactions [26]. In contrast to cellulose, which is crystalline and strong, hemicellulose have a random, amorphous, and branched structure with little resistance to hydrolysis, and they are more easily hydrolyzed by acids to their monomer components. Composition of hemicelluloses is very variable in nature and depends on the plant source.

1.2.3

Lignin

Lignin, the third main heterogeneous polymer in lignocellulosic residues, is a very complex molecule constructed of aromatic alcohols, including coniferyl alcohol, sinapyl and p-coumaryl units linked in a threedimensional structure [27]. It is present in the middle lamella and acts as cement between the plant cells. It is also located in the layers of the cell walls, forming, together with hemicelluloses, an amorphous matrix in which cellulose fibrils are embedded and protected against biodegradation. Lignin acts as a binder of the lignocellulosic constituents, giving the plant structural support, impermeability, and resistance against microbial attack and oxidative stress. Not surprisingly, lignin is the most recalcitrant component of the plant cell wall, and the higher the proportion of lignin, the higher the resistance to chemical and enzymatic degradation [28]. Generally, softwoods contain more lignin than hardwoods and most of the agriculture residues. There are chemical bonds between lignin and hemicellulose and even cellulose. Lignin is one of the drawbacks of using lignocellulosic materials in fermentation, as it makes lignocellulose resistant to chemical and biological degradation.

8 Lignocellulosic Biomass Production and Industrial Applications

1.2.4

Pectin

Pectins are polymers of d-galactopyranosyluronic acids joined by α-d(1→4) glycosidic linkages. The main chain can be modified in various ways (ramification with neutral sugars, esterification, acetylation) [29]. Pectin is an acidic cell wall polysaccharide that functions as a sol-like matrix, providing water and ion retention, support and facilitation of cell wall modifying enzymes, cell wall porosity, cell-to-cell adhesion, cell expansion, cell signaling, developmental regulation, and defense [30].

1.3

Lignocellulosic Pretreatment Techniques

The structure of cellulose imparts tightly packed arrangements that are water insoluble and resistant to depolymerization [31]. Thus, it is imperative that a pretreatment regime alter the structure of biomass to make the cellulose more accessible to hydrolysis. A glimpse of different pretreatment processes is shown in Table 1.2. An effective pretreatment must meet the following requirements: (1) increase the accessible cellulose surface area, (2) disrupt the lignin barrier as well as cellulose crystallinity to allow proper enzymatic attack, (3) limit the formation of toxic degradation products that are inhibitory towards the enzymes or fermentative microorganisms, (4) reduce the loss of sugar components (cellulose and hemicellulose) and (5) minimize the capital and operating costs. Wide spectrums of pretreatment protocols have been investigated for hydrolysis and a few of them have been developed sufficiently to be called technologies. Pretreatment approaches can be broadly classified into four categories: (1) physical; (2) chemical; (3) physicochemical and (4) biological.

1.3.1

Physical Pretreatment Techniques

Physical methods of pretreatment like milling and steam treatment will reduce particle sizes thereby increasing the available surface area for enzymatic attack. Steam explosion loosens the crystalline complex and also removes the pentose while increasing the surface area. However, the drawback of the process is that steam treatment may generate certain cellulase inhibitors that can interfere with the enzymatic hydrolysis of the cellulosic substrate.

1.3.1.1 Milling Milling can be employed to alter the inherent ultrastructure of lignocelluloses and degree of crystallinity, and consequently make it more accessible

Biological pretreatments

Hemicellulose degradation, Remove most lignin and hemicellulose, destroy the cellulose crystallinity

Removal of lignin; dissolves hemicellulose and causes cellulose decrystallization Hydrolyze lignin and hemicellulose

Acid: Sulfuric acid, Hydrochloric acid, Phosphoric acid/acetone

Oxidizing agents: Hydrogen peroxide, Wet oxidation, Ozone

Ionic Liquids

Delignification; reduction in degree of polymerization of cellulose; partial hydrolysis of hemicellulose

Remove most of lignin and hemicellulose; swell the cellulose fibers, disrupt the connections between hemicelluloses, cellulose, and lignin; break down the fiber bundles into small and loose particles

Alkali: Sodium hydroxide, Ammonia, NaOH/urea

Fungi and actinomycetes

Remove hemicellulose; swell the plant material

Decrease in degrees of polymerization

Others: Hydrothermal, High pressure Steaming, Expansion, Extrusion, Pyrolysis

Explosion: Steam explosion, Ammonia fiber explosion (AFEX), CO2 explosion, SO2 explosion

decrease the degree of polymerization of cellulose

Irradiation: Gamma-ray irradiation, Electronbeam irradiation, Microwave irradiation

Chemical and physicochemical pretreatments

Increase in accessible surface area and pore size

Milling: Ball milling, Two-roll milling, Hammer milling, Colloid milling

Physical pretreatments

Possible changes in biomass

Processes

Pretreatment method

Table 1.2 Glimpse of different physical and chemical methods for lignocellulose pretreatment.

[79]

[78]

[77]

[76]

[75]

[74]

[73]

[72]

[33]

Ref.

Valorization of Lignocellulosic Materials

9

10 Lignocellulosic Biomass Production and Industrial Applications to enzymatic degradation. Milling and particle size reduction have been applied prior to enzymatic hydrolysis, or even other pretreatment processes with dilute acid, steam or ammonia, on several lignocellulosic waste materials [31,  32]. Among the milling processes, colloid mill, fibrillator and dissolver are suitable only for wet materials, while the extruder, roller mill, cryogenic mill and hammer mill are usually used for dry materials. The ball mill can be used for both dry and wet materials. Grinding with hammer milling of waste paper is a favorable method [33]. Milling can improve susceptibility to enzymatic hydrolysis by reducing the particle size and degree of crystallinity of lignocelluloses, which improves enzymatic degradation of these materials.

1.3.1.2 Irradiation Irradiation by gamma rays, electron beam and microwaves can improve enzymatic hydrolysis of lignocelluloses. The combination of the preradiation and other methods, such as acid treatment, can further accelerate degradation of cellulose into glucose. The cellulose component of the lignocellulose materials can be degraded by irradiation to fragile fibers and low molecular weight oligosaccharides and even cellobiose, that could be due to preferential dissociation of the glucoside bonds of the cellulose chains by irradiation in the presence of lignin. But a very high irradiation can lead to the decomposition of oligosaccharides and the glucose ring structure [34].

1.3.2

Chemical Pretreatment

In general, chemical pretreatment processes selectivity degrades the biomass component, but they involve relatively harsh reaction conditions, which may not be ideal in a biosaccharification scheme due to adverse effects on downstream biological processing. Different chemical pretreatments that are generally practiced include acid, alkaline, ozonolysis, oxidative H2O2 delignification, organosolv, etc. [31, 35]. Besides these, their combinational effects have also been found suitable. However, utilization of various chemicals in the pretreatment procedures is a major drawback and affects the total economy of the bioconversion of the lignocellulosic biomass.

1.3.2.1 Acid Hydrolysis Pretreatment Both concentrated and diluted acids such as H2SO4, HCl and perchloric acids have been used to treat lignocellulosic materials. Pretreatment with acid

Valorization of Lignocellulosic Materials

11

hydrolysis can result in improvement of enzymatic hydrolysis of lignocellulosic biomasses to release fermentable sugars. Although they are powerful agents for cellulose hydrolysis, concentrated acids are toxic, corrosive, hazardous, and thus require corrosion resistant reactors, which makes the pretreatment process very expensive. In addition, the concentrated acid must be recovered after hydrolysis to make the process economically feasible [36].

1.3.2.2 Alkaline Hydrolysis The effect of alkaline pretreatment depends on the lignin content of the lignocellulosic materials. Alkali pretreatment processes can be effective at lower temperatures and pressures than many other pretreatment technologies, but it requires longer times on the order of hours or days. Compared with acid, alkaline pretreatments cause less sugar degradation, and many of the caustic salts can be recovered and/or regenerated [37]. Sodium, calcium, potassium, and ammonium hydroxides are widely used alkaline pretreatment agents. Out of these, sodium hydroxide has been studied the most. However, calcium hydroxide (slake lime) also has been shown to be an effective pretreatment agent and is the least expensive.

1.3.2.3 Oxidative Delignification by Peroxide Lignin biodegradation has been reported to be catalyzed in the presence of H2O2. The pretreatment of cane bagasse with hydrogen peroxide greatly enhanced its susceptibility to enzymatic hydrolysis [38]. About 50% of the lignin and most of the hemicellulose were solubilized by 2% H2O2 at 30 °C within 8 h, and 95% efficiency of glucose production from cellulose was achieved in the subsequent saccharification by cellulase at 45 °C for 24 h. Wet oxidation combined with base addition readily oxidizes lignin from wheat straw, thus making the polysaccharides more susceptible to enzymatic hydrolysis. Furfural and hydroxymethylfurfural, known inhibitors of microbial growth when other pretreatment systems are applied, were not observed following the wet oxidation treatment.

1.3.2.4 Organosolv Process The organosolvation method is a promising pretreatment strategy, and it has attracted much attention and has proven potential for utilization in lignocellulosic pretreatment. In this process, an organic or aqueous organic solvent mixture with inorganic acid catalysts (HCl or H2SO4) is used to break the internal lignin and hemicellulose bonds [39]. The commonly used solvents in the process are methanol, ethanol, acetone, ethylene

12 Lignocellulosic Biomass Production and Industrial Applications glycol, triethylene glycol, and tetrahydrofurfuryl alcohol. Other organic acids like oxalic, acetylsalicylic, and salicylic acids can also be used as catalysts in the organosolvation process. Treatment of lignocellulosic materials with these organosolvs at temperatures ranging from 140 to 220 °C causes lignin breakdown into fragments which are quite soluble in the solvent system [40]. This technique yields three separate fractions: dry lignin, an aqueous hemicellulose stream, and a relatively pure cellulose fraction.

1.3.2.5 Ozonolysis Pretreatment Ozone pretreatment is one way of reducing the lignin content of lignocellulosic wastes which results in an increase of the in-vitro enzymatic digestibility of the treated material, and unlike other chemical treatments, it does not yield toxic products [41]. Although ozone can be used to degrade lignin and hemicellulose in many lignocellulosic materials, such as wheat straw, rice straw, bagasse, peanut, pine, cotton straw, sawdust, etc., the degradation is mainly limited to lignin. In this process hemicellulose portions are slightly affected, but cellulose is not. Ozonolysis pretreatment has an advantage in that the reactions are carried out at room temperature and under normal pressure. Furthermore, after pretreatment ozone can be easily decomposed by using a catalytic bed or increasing the temperature to minimize environmental pollution. A drawback of ozone pretreatment is that a large amount of ozone is required, which can make the process expensive [42].

1.3.2.66 Ionic Liquids Pretreatment Another technology for lignocellulose fractionation is using ionic liquids. Ionic liquids (ILs) are organic salts which exist as liquids at low temperatures; often well below 100 °C. They have negligible (or very low) vapor pressures, generally good thermal stability and there is a variety of combinations of anions and cations that can be used to synthesize ILs [43]. Recent studies have showed that cellulose and lignin both can be dissolved in a variety of ILs and can be easily regenerated from these solutions by means of addition of a non-solvent [44]. Dadi et al. [45] used 1-n-butyl-3methylimidazolium chloride to dissolve cellulose. The regenerated cellulose had an amorphous structure allowing a greater number of sites for enzyme adsorption and improving the enzymatic hydrolysis rate by 50-fold.

1.3.3

Physico-Chemical Pretreatment

Physico-chemical pretreatment is a combination of different processes for chemical and physical treatments. In these procedures, milder chemical

Valorization of Lignocellulosic Materials

13

conditions are often used, but under more extreme operational conditions like biorefinery, relatively harsh techniques are used. Different physicochemical pretreatment techniques include mainly liquid hot water (hydrothermolysis, aqueous or steam/aqueous, uncatalyzed solvolysis aquasolv), steam explosion (autohydrolysis with and without chemical addition), ammonia fiber explosion (AFEX), and CO2 explosion.

1.3.3.1 Liquid Hot-Water Pretreatment Beginning several decades ago, treatment in liquid hot water has been one of the pretreatment methods applied for lignocellulosic materials. Pressurized water can penetrate into the biomass, hydrate cellulose, and remove hemicellulose and part of the lignin. The major advantages of this process are that no addition of chemicals and no corrosion-resistant materials are required for hydrolysis. In addition, the process has a much lower need for chemicals for neutralization of the produced hydrolyzate, and produces lower amounts of inhibitory products compared to acid or alkali pretreatment [46]. Hemicelluloses are dissolved as soluble oligosaccharides and can be separated from insoluble cellulose and lignin fractions. Due to enlargement of the accessible surface area of the cellulose, hydrolytic enzymes become more accessible for saccharification [47].

1.3.3.2 Steam Explosion Steaming with or without explosion has received substantial attention in the pretreatment of lignocellulosic materials. The pretreatment removes most of the hemicellulose, thus improving the enzymatic digestion. In this method, lignocellulosic biomass is treated with high-pressure saturated steam, and then the pressure is suddenly reduced, which makes the materials undergo an explosive decompression. High pressure and temperature (between 160 and 260 °C) for a few seconds (e.g., 30 s) to several minutes (e.g., 20 min), are used in the steam explosion process [48], which cause hemicellulose degradation and lignin transformation due to high temperature and increase the potential of cellulose hydrolysis. Its energy cost is relatively moderate, and it satisfies all the requirements of the pretreatment process. For this reason, this process is being utilized in pilot processes by several research groups and companies [49].

1.3.3.3 Ammonia Fiber Explosion (AFEX) In the AFEX process, the lignocellulosic biomass is treated with liquid ammonia at moderate temperatures (e.g., 90–100  °C) and high pressure

14 Lignocellulosic Biomass Production and Industrial Applications for a period of time (e.g., 30 min), followed by the rapid release of pressure. As a result, cellulose crystallinity is decreased, the fiber structure is expanded, and the accessible surface area to enzymes is increased. It also depolymerizes or alters lignin structure via ammonia reactions with lignin macromolecules. This pretreatment yields optimal hydrolysis rates at low enzyme loadings and is particularly suited for herbaceous and agricultural residues [31]. The major advantage of AFEX pretreatment is minimization the formation of sugar degradation inhibitory by-products, yet, part of phenolic fragments of lignin degraded products may remain on the cellulosic surface. Therefore, washing with water is necessary to remove these inhibitory components. However, AFEX is more effective on the biomass that contains less lignin, and it does not significantly solubilize hemicellulose. Besides, ammonia must be recycled after the pretreatment to reduce the cost and protect the environment [50].

1.3.4

Bological Pretreatment

Biological pretreatment includes microorganisms for the treatment of lignocellulosic biomass and to enhance enzymatic hydrolysis. Microorganisms usually secrete extracellular enzymes to degrade lignin and hemicellulose. Cellulose is degraded to a lesser extent since it is more recalcitrant to the biological attack. Several brown-, white-, and soft-rot fungi are used for this purpose. White-rot fungi are among the most effective microorganisms for biological pretreatment of lignocelluloses. The biological delignifcation of lignocellulosic materials has been attemped by different strains like Aspergillus, Streptomyces, Phelebia, and Pleurotus [51,  52]. Lignin degradation by fungi occurs through the action of lignin-degrading enzymes such as peroxidases and laccase [53]. Although the biological pretreatment have several advantages such as low-capital cost, energy input and high yields without generating inhibitory by-products, the hydrolysis rate of most biological pretreatment processes is very low [20]. Long treatment time and degradation of the residual carbohydrates are also some of the drawbacks of such processes.

1.4

Hydrolysis of Lingocellulose

The hydrolysis of cellulose is usually performed by acids, alkali or by enzymes. Acid hydrolysis of hemicelluloses and cellulose is performed by concentrated or diluted acids. Acid catalyzes the breakdown of long carbohydrate chains to shorter chain oligomers and then to monomeric sugars.

Valorization of Lignocellulosic Materials

15

Due to their amorphous nature, hemicelluloses require less severe conditions for their hydrolysis in comparison with crystalline cellulose. When concentrated acids, such as H2SO4 or HCl (10–30%), are used during the pretreatment, cellulose is degraded concomitantly. In this case, pretreatment and hydrolysis are carried out in one step. The advantages of acid hydrolysis are that the acid can penetrate lignin without pretreatment and the rate of acid hydrolysis is faster than enzyme hydrolysis; but it causes corrosion problems in the equipment, which is one of the major disadvantages of this process. When a dilute acid hydrolysis is chosen (2–5%), high temperatures are needed to achieve good rates of cellulose conversion. In this case, the high temperature increases the rates of lignocellulose biomass-derived sugars decomposition, thus causing the formation of toxic compounds which further decreases the yields of fermentable sugars. Acetic acid is released from the acetyl groups of hemicellulose, while furfural and 5-hydroxymethylfurfural (HMF) are formed from the degradation of sugars (xylose and glucose, respectively). On the other hand, subsequent degradation of these aldehydes leads to the formation of formic acid and levulinic acid [54]. The degradation of lignin produces phenolic compounds, such as vanillic acid, vanillin, syringic acid, or syringaldehyde, depending on the type of the lignin present in the biomass [50]. These are very toxic compounds to the microorganisms which will be using the hydrolysates in a subsequent step. Several detoxifying steps are required to remove these inhibitors [55]. Examples of detoxification methods are evaporation, overliming, activated charcoal treatment, membrane filtration, ion exchange resins, and biological treatments [55–57]. Beside acids, alkaline hydrolysis is used for pretreatment of lignocellulosic materials. The mechanism of alkaline hydrolysis is believed to be saponification of intermolecular ester bonds crosslinking xylan hemicelluloses and other components. With the removal of the crosslinks, the porosity of the lignocellulosic materials increases. Generally, alkaline hydrolysis enhances digestibility of the lignocellulose and reduces inhibitors formation [58, 59]. Enzymatic hydrolysis of cellulose is superior to the inorganic catalysts, because enzymes are highly specific towards its substrates and can work at mild process conditions. The cellulase enzyme system is a mixture of endoβ-1,4-glucanglucanhydrolases, exo-β-1,4-glucancellobiohydrolases, and β-glucosidase, which effectively breaks down cellulose to cellobiose and subsequently to glucose. Besides cellulases, a number of other enzymes, such as glucuronide, acetylesterase, xylanase, β-xylosidase, galactomannase, and glucomannase, are present, which can effectively degrade hemicellulose. These enzymes work synergistically to break down both cellulose

16 Lignocellulosic Biomass Production and Industrial Applications and hemicellulose in the lignocellulosic materials [60]. In spite of several advantages, the use of enzymes in industrial processes is still limited due to their relative instability at high temperatures and high costs of their purified forms. Currently, extensive research is being carried out with improved thermostability, since high temperatures could speed up the hydrolysis reaction time [59].

1.5

Lignocellulose Biomass as Substrate for PHA Production

Among various types of lignocelluloses, forest biomass represents an enormous reservoir of renewable carbon-rich material. Globally, approximately 80 billion tons of woody biomass is generated per annum, with the production of total plant matter estimated at roughly 180 billion tons annually. There is abundant availability of such agricultural wastes and they are rich sources of carbohydrates. A process diagram of PHA production from lignocellulosic biomass is shown in Figure 1.2. These wastes are mostly used

Lignocellulose (cellulose + hemicellulose + lignin) Acid pretreatment

Alkali pretreatment

Degradation of lignin and hemicellulose

Degradation of hemicellulose

5C sugar: Xylose, arabinose 6C sugar: Glucose, galactose, mannose Others: furfural, acitic acid etc

Cellulose fiber Enzymatic hydrolysis by cellulases

Removal of toxic materials 5C and 6C sugars

6C sugar: Glucose

Microbial fermentation

Microbial fermentation PHA

Figure 1.2 A process diagram of PHA production from lignocellulosic biomass.

Valorization of Lignocellulosic Materials

17

as cattle feed since they have little economic value. Several bacterial species have the innate ability to utilize such diverse and cheap carbon wastes as they possess hydrolytic enzymes capable of metabolizing these complex residues. An overview of different microbial strains utilizing lignocellulosic materials is presented in Table 1.3. Wood hydrolysate is a potentially inexpensive and renewable feedstock that can be produced through enzymatic or dilute acid hydrolysis of cellulose or hemicellulose to fermentable sugars, such as glucose, galactose, xylose, and mannose. Considering this, Pan et al. [55] utilized Sugar maple hemicellulosic hydrolysate containing 71.9 g/l of xylose as an inexpensive feedstock to produce polyhydroxyalkanoates (PHAs) by Burkholderia cepacia ATCC 17759. The inhibitory effects of selected inhibitors from wood hydrolysate were evaluated for effects on cell growth, PHA production, and physical-chemical properties of PHAs. Subsequently, membrane-purified wood hydrolysate, detoxifed to remove phenolics, was used to produce PHAs by fermentation. Wood biomass was again utilized by Bowers et al. [61] for PHAs production. Table 1.3 Microbial utilization of different lignocellulosic materials for polyhydroxyalkanoates production. Microorganisms

Carbon source

PHA production gL 1 Ref.

Azotobacter beijerinickii Coir pitch

5.0

[80]

Cupriavidus necator

Bagasse hydrolysate

6.3

[81]

Bacillus firmus NII 0830

Rice straw hydrolysate

1.7

[82]

Burkholderia sacchari

Wheat straw hydrolysate

105.0

[83]

Burkholderia cepacia ATCC 17759

Sugar maple hemicellulosic hydrolysate

8.7

[55]

Ralstonia eutropha

Bagasse hydrolysate

6.3

[84]

Brevundimona svesicularis

Acid hydrolyzed sawdust

0.3

[85]

Cupriavidus necator MTCC-1472

Water hyacinth hydrolysates

7.0

[86]

Bacillus megaterium

Oil palm empty fruit bunch

12.5

[87]

0.23

[85]

Sphingopyxis Hydrolyzed pine saw dust macrogoltabida LMG 17324

18 Lignocellulosic Biomass Production and Industrial Applications In their study, Pinus radiata wood chips were subjected to high-temperature mechanical pretreatment or steam explosion in the presence of sulphur dioxide before being enzymatically treated to produce corresponding hydrolysates. Two potent bacteria, Novosphingobium nitrogenifigens and Sphingobium scionense, were grown on these hydrolysates and the highest PHB yields of 0.4 g L–1 were observed in Sphingobium scionense. Apart from wood biomass, agriculture and food industry waste streams are another promising source of lignocellulose-based materials which can be used as a substrate for PHA production. These materials are generated in enormous amounts during processing of agricultural plants. Hence, there are numerous papers dealing with the conversion of these waste materials into PHAs. To confirm the feasibility of using agrowastes to replace glucose in the production of PHA, Gowda and Shivakumar [15] successfully utilized different carbon sources (4%, w/v), like bagasse, jowar, ragi husk, straw, rice husk, wheat bran, mango peel, jack fruit seed powder and potato peel residue, for PHA accumulation by B. thuringiensis IAM 12077 in shake flask cultures. The strain showed PHB production on all the substrates tested and the maximum PHA yield was observed with mango peel (4.03 g/L; 51.3%), followed in decreasing order by bagasse (1.26 g/L; 46.15%); rice husk (1.56 g/L; 32.7%); jackfruit seed powder (3.93 g/L; 29.32%); ragi husk (0.96 g/L; 23.2 %), etc. Bacillus species were further explored for their potential to produce poly hydroxy butyrate (PHB) using different low-cost agro-industrial materials. Ghate et al. [62] utilized diff ferent agro-industrial materials like Jawar stem, Neera, Cashew apple pulp, Sugar cane bagasse, Coconut pulp and Grapes pulp. Highest cellular PHB content was obtained from Bacillus subtilis with Neera as source of carbon, which was found to be 0.284 g/L. Chaleomrum et al. [63] attempted to investigate the potential of cassava starch wastewater for producing polyhydroxyalkanoate (PHA) from sequencing batch reactor (SBR) treatment system seeded with Bacillus tequilensis MSU 112, a PHA-producing bacterial strain. Under the optimized condition, 3,346 mg/L of PHA was produced. Another very promising waste substrate for PHA production is spent coffee grounds (SCG). This can be considered as a very promising substrate for PHA production. SCG contain approximately 15% of oil, which can be simply extracted and converted into PHB by Cupriavidus necator [64,  65]. The residual solids after oil extraction contain a significant portion of hemicelluloses and cellulose. Therefore, they were hydrolyzed and converted into PHAs employing Burkholderia cepacia. Hexoses (predominantly mannose and galactose) were substantially dominating sugars of the hydrolysate, which may be an important factor positively influencing the production of

Valorization of Lignocellulosic Materials

19

PHAs. Moreover, hydrolysate contained levulinic acid, which served as a precursor of 3-hydroxyvalerate, resulting in accumulation of P (3HB-co3HV) copolymer.

1.6

Conclusion

Biocomposites usefulness is no longer in question and more and more reports are focused on applicative aspects in the environment, packaging, agriculture devices, biomedical fields, etc. Lignocellulose materials seem to be very promising substrates for various industrial and biotechnological processes, because utilization of these resources might decrease our dependence on petroleum and reduce the impact of its gradual depletion and increasing price. The economic feasibility of using lignocellulosic hydrolysates as carbon sources to biologically produce biopolyesters strongly depends on the capacity of microorganisms to consume both the hexoses and the pentoses released from the lignocellulosic biomass and to convert these sugars into products at high conversion yields. Hence there is a necessity of utilizing potent microorganisms capable of fermenting different types of sugar for better production of PHA. This might help these valuable environmentally friendly polymers to compete with petrochemical-based plastics and, therefore, partially replace them in appropriate applications.

References 1. Madison, L.L., and Huisman, G.W., Metabolic engineering of poly (3-hydroxyalkanoates): From DNA to plastic. Microbiol. Mol. Biol. Rev. 63, 21, 1999. 2. Oliveira, F.C., Freire, D.M.G., and Castilho, L.R., Production of poly (3-hydroxybutyrate) by solid-state fermentation with Ralstonia eutropha. Biotechnol. Lett. 26, 1851, 2004. 3. Khanna, S., and Srivastava, A., Recent advances in microbial polyhydroxyalkanoates. Process Biochem. 40, 607, 2005. 4. Choi, J., and Lee, S.Y., Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl. Microbiol. Biotechnol. 51, 12, 1999. 5. Martinez, A., Rodriguez, M.E., York, S.W., Preston, J.F., and Ingram, L.O., Effects of Ca(OH)(2) treatments (“overliming”) on the composition and toxicity of bagasse hemicellulose hydrolysates. Biotechnol. Bioeng. 69, 526, 2000.

20 Lignocellulosic Biomass Production and Industrial Applications 6. Canilha, L., Silva, J.B.A., and Solenzal, A.I.N., Eucalyptus hydrolysate detoxification with activated charcoal adsorption or ion-exchange resins for xylitol production. Process Biochem. 39, 1909, 2004. 7. Liu, S., Amidon, T.E., and Wood, C.D., Membrane filtration: Concentration and purification of hydrolysates from biomass. J. Biobased Mater. Bio. 2, 121, 2008. 8. Nilvebrant, N.O., Reimann, A., Larsson, S., and Jonsson, L.J., Detoxification of lignocellulose hydrolysates with ion-exchange resins. Appl. Biochem. Biotechnol. 35, 91, 2001. 9. Okuda, N., Soneura, M., Ninomiya, K., Katakura, Y., and Shioya, S., Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production. J. Biosci. Bioeng. 106, 128, 2008. 10. Wang, Y., and Liu, S., Polyhydroxyalkanoates (PHAs): Biosynthesis, production and recovery. J. Bioproc. Eng. Bioref. 2, 61, 2013. 11. Le Meur, S., Zinn, M., Egli, T., Thöny-Meyer, L., and Ren, Q., Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440. BMC Biotechnol. 12, 53, 2012. 12. Hartmann, R., Hany, R., Pletscher, E., Ritter, A., Witholt, B., and Zinn, M., Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] by Pseudomonas putida GPo1: Batch versus chemostat production. Biotechnol. Bioeng. 93, 737, 2006. 13. Slepecky, R.A., and Law, J.H., A rapid spectro photometric assay of α, β-unsaturated acids and β-hydroxy acids. Anal. Chem. 32, 1697, 1960. 14. Madison, L.L., and Huisman, G.W., Metabolic engineering of poly(3-hydroxyalkanoates): From DNA to plastic. Microbiol. Mol. Biol. Rev. 63, 21, 1999. 15. Gowda, V., and Shivakumar, S., Agrowaste-based polyhydroxyalkanoate (PHA) production using hydrolytic potential of Bacillus thuringiensis IAM 12077. Braz. Archi. Bio. Technol. 57, 55, 2014. 16. Shivakumar, S., Polyhydroxybutyrate (PHB) production using agro-industrial residue as substrate by Bacillus thuringiensis IAM 12077. Int. J. Chem. Tech. Res. 4, 1158, 2012. 17. Nath, A., Dixit, M., Bandiya, A., Chavda, S., and Desai, A.J., Enhanced PHB production and scale up studies using cheese whey in fed batch culture of Methylobacterium sp. ZP24. Bioresour. Technol. 99, 5749, 2008. 18. Watanabe, H., and Tokuda, H., Animal cellulases. Cell. Mol. Life Sci. 58, 1167, 2001. 19. Lynd, L.R., Weimer, P.J., van Zyl, W.H., and Pretorius, I.S., Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66, 506, 2002. 20. Kumar, P., Barrett, D.M., Delwiche, M.J., and Stroeve, P., Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 48, 3713, 2009.

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22 Lignocellulosic Biomass Production and Industrial Applications 36. Jordan, D.B., and Wagschal, K., Properties and applications of microbial β-Dxylosidases featuring the catalytically efficient enzyme from Selenomonas ruminantium. Appl. Microbiol. Biotechnol. 86, 1647, 2010. 37. Singh, P., Sulaiman, S., Hashim, R., Rupani, P.F., and Peng, L.C., Biopulping of lignocellulosic material using different fungal species: A review. Rev. Environ. Sci. Biotechnol. 9, 141, 2010. 38. Martin, C., Klinke, H.B., and Thomsen, A.B., Wet oxidation as a pretreatment method for enhancing the enzymatic convertibility of sugarcane bagasse. Enzyme Microb. Tech. 40, 426, 2007. 39. Sun, Y., and Cheng, J., Hydrolysis of lignocellulosic materials for ethanol production: A review. Bioresour. Technol. 83, 1, 2002. 40. Arato, C., Pye, E.K., and Gjennestad, G., The lignol approach to biorefining of woody biomass to produce ethanol and chemicals. Appl. Biochem. Biotechnol. 121/124, 871, 2005. 41. Girio, F.M., Fonseca, C., Carvalheiro, F., Duarte, L.C., Maraues, S., BoelLakasik, R., Goel, R., Tokutomi, T., and Yasui, H., Anaerobic digestion of excess activated sludge with ozone pretreatment. Water Sci. Technol. 47, 207, 2003. 42. Freire, C.S.R., Silvestre, A.J.D., Neto, C.P., and Evtuguin, D.V., Effect of oxygen, ozone and hydrogen peroxide bleaching stages on the contents and composition of extractives of Eucalyptus globulus kraft pulps. Bioresour. Technol. 97, 420, 2006. 43. Kim, S.J., Dwiatmoko, A.A., Choi, J.W., Suh, Y.W., Suh, D.J., and Oh, M., Cellulose pretreatment with 1-n-butyl-3-methylimidazolium chloride for solid acid-catalyzed hydrolysis. Bioresour. Technol. 101, 8273, 2010. 44. Stark, A., Ionic liquids in the biorefinery: A critical assessment of their potential. Energy Environ Sci. 4, 19, 2011. 45. Dadi, A.P., Varanasi, S., and Schall, C.A., Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95, 904, 2006. 46. Wang, Y., and Liu, S., Production of (R)-3-hydroxybutyric acid by Burkholderia cepacia from wood extract hydrolysates. AMB Express 4, 28, 2014. 47. Shupe, A.M., and Liu S.J., Ethanol fermentation from hydrolysed hot-water wood extracts by pentose fermenting yeasts. Biomass Bioenergy 39, 31, 2012. 48. Fitzpatrick, M., Champagne, P., Cunningham, M.F., and Whitney, R.A., A biorefinery processing perspective: Treatment of lignocellulosic materials for the production of value-added products. Bioresour. Technol. 101(23), 8915, 2010. 49. Hayes, D.J., An examination of biorefining processes, catalysts and challenges. Catalysis Todayy 145, 138, 2009. 50. Obruca, S., Benesova, P., Marsalek, L., and Marova, I., Use of lignocellulosic materials for PHA production. Chem. Biochem. Eng. Q. 29, 135, 2015. 51. Milala, M.A., Shugaba, A., Gidado, A., Ene, A.C., and Wafar, J.A., Studies on the use of agricultural wastes for cellulase enzyme production by Aspergillus niger. Res. J. Agri. Biol. Sci. 1, 325, 2005.

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52. Bonatti, M., Karnopp, P., Soares, H.M., and Furlan, S.A., Evaluation of Pleurotus ostreatus and Pleurotus sajor-caju nutritional characteristics when cultivated in different lignocellulosic wastes. Food Chem. 88, 425, 2004. 53. Dong, Y.-C., Dai, Y.-N., Xu, T.-Y., Cai, J., and Chen, Q.-H., Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens, Phlebia radiate. Bioprocess Biosyst. Eng. 37, 755, 2014. 54. Palmqvist, E., and Hahn-Hägerdal, B., Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresour. Technol. 74, 17, 2000. 55. Pan, W., Perrotta, J.A., Stipanovic, A.J., Nomura, C.T., and Nakas, J.P., Production of polyhydroxyalkanoates by Burkholderia cepacia ATCC 17759 using a detoxified sugar maple hemicellulosic hydrolysate. J. Ind. Microbiol. Biotechnol. 39, 459, 2012. 56. Ranatunga, T., Jervis, J., Helm, R., McMillan, J., and Wooley, R., The effect of overliming on the toxicity of dilute acid pretreated lignocellulosics: The role of inorganics, uronic acids and ethersoluble organics. Enzym. Microb. Technol. 27, 240, 2000. 57. Mussatto, S.I., and Roberto, I.C., Hydrolysate detoxification with activated charcoal for xylitol production by Candida guilliermondii. Biotechnol. Lett. 23(20), 1681, 2001. 58. Lenihan, P., Orozco, A., O’Neill, E., Ahmad, M.N.M., Rooney, D.W., and Walker, G.M., Dilute acid hydrolysis of lignocellulosic biomas. Chem. Eng. J. 156, 395, 2010. 59. Verardi, A., De Bari, I., Ricca, E., and Calabro, V., Hydrolysis of lignocellulosic biomass: Current status of processes and technologies and future perspectives, in: Bioethanol, Pinheiro Lima, M.L. (Ed.), p. 95, InTech, 2012. 60. Amore, A., and Faraco, V., Potential of fungi as category I Consolidated BioProcessing organisms for cellulosic ethanol production. Renew. Sustainable Energy Rev. 16, 3286, 2012. 61. Bowers, T., Vaidya, A., Smith, D.A., and Lloyd-Jones, G.J., Softwood hydrolysate as a carbon source for polyhydroxyalkanoate production. Chem. Technol. Biotechnol. 89, 1030, 2014. 62. Ghate, B., Pandit, P., Kulkarni, C., Deepti, D., Mungi, and Patel, T.S., PHB production using novel agroindustrial sources from different Bacillus sp. Int. J. Pharm. Bio. Sci. 2, 242, 2011. 63. Chaleomrum, N., Chookietwattana, K., and Dararat, S., Production of PHA from cassava starch wastewater in sequencing batch reactor treatment system. APCBEE Procedia 8, 167, 2014. 64. Obruca, S., Petrik, S., Benesova, P., Svoboda, Z., Eremka, L., and Marova, I., Utilization of oil extracted from spent coffee grounds for sustainable production of polyhydroxyalkanoates. Appl. Microbiol. Biotechnol. 98, 5883, 2014. 65. Cruz, M.V., Paiva, A., Lisboa, P., Freitas, F., Alves, V.D., Simoes, P., Barreiros, S., and Reis, M.A.M., Production of polyhydroxyalkanoates from spent coffee

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66.

67. 68. 69.

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79. 80.

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2 Biological Gaseous Energy Recovery from Lignocellulosic Biomass Shantonu Roy Department of Biotechnology, National Institute of Technology, Arunachal Pradesh, India

Abstract Lignocellulosic biomass in nature is by far the most abundant raw material from hardwood, softwood, grasses, and agricultural residues. Annual yields of lignocellulosic biomass residues were estimated to exceed 220 billion tons worldwide, equivalent to 60–80 billion tons of crude oil, making it a promising feedstock for biofuels. Hydrogen (H2) as biofuel is considered the green fuel of the future. It offers tremendous potential as a clean, renewable energy currency. It has the highest gravimetric energy density of any known fuel and is compatible with electrochemical and combustion processes for energy conversion without producing carbon-based emissions that contribute to environmental pollution and climate change. Dark fermentation of biomass or carbohydrate-based substrates presents a promising route for biohydrogen (bioH2) production. Only 20 to 30% of total energy can be recovered through H2 production. Integration of the bioH2 production process with biomethanation could lead to 50–60% gaseous energy recovery. Keywords: Lignocellulosic biomass, dark fermentation, biohydrogen

2.1

Introduction

Biohythane production could be envisioned as a renewable source of energy because it is produced most from organic biomass. Any organic compound which is rich in carbohydrates, fats and proteins could be considered as possible substrate for biohythane production. A review of the literature Corresponding author: [email protected] Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial Applications, (27–46) © 2017 Scrivener Publishing LLC

27

28 Lignocellulosic Biomass Production and Industrial Applications Food processing industry wastes Diary industry wastes

Hexose Industrial wastewater

Monosaccharides

Alcohol beverage industry wastewater

Agricultural wastes

Lignocellulosic biomass

Complex organic wastes

Energy crops

Feedstock

Defined sugars

Glycerol

Algal biomass

Food crops

Maltose Disaccharides

Lactose Sucrose

Sewage

Household wastes

Pentose

Starch Municipal wastes

Organic litters

Polysaccharides

Cellulose, hemicellulose Xylan

Figure 2.1 Potential feedstock for biohydrogen production.

suggests that carbohydrates may be considered as the main source of hydrogen during fermentation. Substrates rich in complex carbohydrates are thus the most suitable feedstock for biohydrogen production. Wide ranges of feedstock are available for hydrogen production (Figure 2.1). Most of the reports suggested that 80% hydrogen production from dark fermentation was carried out using pure substrates. Cost-effective hydrogen production can be possible only through renewable feedstock [1]. Some woody plants, aquatic plants and algae show promise as feedstock. Furthermore, agricultural wastes, wastes from food processing, livestock effluents and other industrial wastes are important renewable feedstock for hydrogen production. In this chapter different types of feedstock are discussed based on their availability and the prospect of using them for fermentative hydrogen production are discussed. Different types of feedstock used for fermentative H2 production along with the rate of H2 production and yield are shown in Table 2.1.

2.2

Simple Sugars as Feedstock

Mono- and disaccharides are simple sugars which are mostly used as model substrate for optimization of fermentative H2 production process. Carbon, hydrogen, and oxygen are the backbone of a sugars molecule with

Ribose

Batch fermentation; pH 6.5; 37 °C

Batch fermentation; pH 6.5; 37 °C

Clostridium butyricum TM-9A

Clostridium butyricum TM-9A

Batch fermentation; pH 6.5; 60 °C

Thermophilic mixed culture

Batch fermentation; pH 6.5; 37 °C

Escherichia coli DJT135

Batch fermentation; pH 7; 70 °C

Batch fermentation; pH 6.5; 37 °C

Enterobacter aerogenes IAM 1183

Caldicellulosiruptor owensensis

Batch fermentation; pH 7; 70 °C

Thermoanaerobacter mathranii A3N

Arabinose

Batch fermentation; pH 6.5; 60 °C

Thermoanaerobacterium thermosaccharolyticum W16

CSTR; pH 5; 35 °C

Clostridium tyrobutyricum CSABR; pH 7; 75 °C

CSTR; pH 5; 35 °C

Ethanoligenens harbinese

Thermotoga neapolitana

Batch fermentation; pH 6.5; 37 °C

Clostridium beijerinckii Fanp 3

Xylose

Batch fermentation; pH 6.5; 37 °C

Clostridium beijerinckii

Batch fermentation; pH 6.5; 37 °C

Batch fermentation; pH 6.5; 60 °C

Thermoanaerobacterium thermosaccharolyticum

Clostridium butyricum TM-9A

Batch fermentation; pH 6.5; 60 °C

Thermoanaerobacterium thermosaccharolyticm

Fructose

Batch fermentation; pH 7; 75 °C

Thermotoga neapolitana DSM 4359

Bioprocess characteristics Batch fermentation; pH 7; 70 °C

Organism

Caldicellulosiruptor saccharolyticus

Monosaccharide

Glucose

Substrate type

Table 2.1 Feedstock used for hydrogen production.

8

8

1.2

2.7

0.68

2.64

2.5

2.62

3.6

2.4

1.18

1.93

2.52

2

2.5

2.7

3

2.8

HY*

[10]

[10]

[17]

[16]

[15]

[14]

[13]

[12]

[11]

[10]

[9]

[8]

[7]

[6]

[5]

[4]

[3]

[2]

Ref.

(Continued)

0.84

0.06

3.6

–

–

5.79

1.8

5.71

2.66

0.84

7.2

19.6

9.36

15

6.8

8.0

8.5

8.8

HPR# (L/L/d)

Biological Gaseous Energy Recovery

29

Batch; pH 6.5; 60 °C Batch fermentation; pH 6.5; 37 °C Batch fermentation; pH 6.5; 37 °C CSTR; pH 6.5; 37 °C

Thermophilic mixed culture

Clostridium beijerinckii RZF-1108

Enterobacter aerogenes and C. butyricum

Mixed culture

Clostridium butyricum EB6

Caldicellulosiruptor saccharolyticus

Clostridium butyricum CGS5

Thermophilic mixed culture

Enterobacter cloacae IITBT08

Palm oil mill effluent

Miscanthus hydrolysate

Chlorella vulgaris ESP6 hydrolysate

Chlorella sorokiniana

Chlorella sorokiniana

*Hydrogen yield (mol H2/mol hexose equivalent); Hydrogen production rate

#

C. acetobutylicum

Cassava wastewater

Chemical wastewater Mixed culture

Batch fermentation; pH 6; 55 °C

Clostridium thermocellum

Corn stalk

Batch fermentation; pH 6.5; 37 °C

Batch fermentation; pH 6.5; 37 °C

Batch fermentation; pH 6; 60 °C

Batch fermentation; pH 6.5; 37 °C

Batch fermentation; pH 7; 70 °C

Batch fermentation; pH 6.5; 37 °C

Batch fermentation; pH 6.5; 37 °C

Batch fermentation; pH 7; 70 °C

Pretreated wheat straw Caldicellulosiruptor saccharolyticus

Agricultural biomass

UASB; pH 6.5; 60 °C

Thermophilic mixed culture

Starch

Bioprocess characteristics

Organism

Polysaccharides

Substrate type

Table 2.1 Cont.

1.2

2.54

2.6

–

3.4

0.22

2.41

–

3.8

0.9

2.8

–

1.73

1.17

–

–

–

5.8

6.8

28.4

1.32

0.44

–

1.14

–

8.64

–

–

[30]

[29]

[28]

[27]

[26]

[25]

[24]

[23]

[22]

[21]

[20]

[6]

[19]

[18]

HPR# HY* (L/L/d) Ref.

30 Lignocellulosic Biomass Production and Industrial Applications

Biological Gaseous Energy Recovery

31

an empirical formula of CnH2nOn. In nature, pentose and hexose sugars are the most abundant, usually found in plant- and animal-based feedstock. Apart from fructose, fruits also contain xylose and arabinose, which in turn are the most abundant pentose sugars. Ribose is available in all plants. Sugar cane or sugar beets are the source of sucrose, which is a disaccharide. Similarly, maltose is found in germinating grain, corn syrup, etc., and lactose is mostly found in milk and milk-derived dairy products. Simple sugars are important because of their biodegradability, which helps in the uncomplicated study of bacterial kinetics, assessment of nutritional requirements and also in the optimization of process parameters [31]. Moreover, the use of simple sugars for fermentation generally takes less time as compared to complex sugars [32]. Various studies have highlighted the fact that fermentative bacteria have certain preferences when different types of sugars are used as substrate. Pure substrates, such as glucose, fructose, sucrose, lactose, etc., are a few of the commonly used sugars for biohydrogen production. However, pure carbohydrate sources are not suitable as feedstock for hydrogen production due to their high costs [33]. Different microbial species have different preferences towards substrates for hydrogen production. For example, it was observed that Clostridium saccharoperbutylacetonicum ATCC 27021 grown on disaccharides (lactose, sucrose and maltose) produces 2.81 mol H2/mol of sugar. Whereas, with monosaccharide, 1.29 mol H2/mol hexose was observed, which is a twofold lesser yield as compared to disaccharide [34]. It was also observed that hydrogen yields varied with different arrays of substrate (2.2 mol H2/mol glucose, 6 mol H2/mol sucrose and 5.4 mol H2/mol cellobiose) using Enterobacter cloacae IIT-BT08 [35]. Moreover, glucose, sucrose and xylose were used in many experiments to study hydrogen production by hyperthermophilic microorganism Caldicellulosiruptor saccharolyticus. It was observed that during fermentations of 10 g/L glucose and 10 g/L fructose, C. saccharolyticus could completely consume all substrates and showed identical rate of substrate consumption [36]. In contrast, Thermotoga neapolitana, which is also a hyperthermophilic microorganism, showed a higher substrate consumption rate for glucose and fructose, which suggests that in this instance glucose is preferred over fructose for hydrogen production. Moreover, higher substrate concentrations led to higher lactate production, thereby decreasing overall hydrogen yield [26]. To determine the preference of sugar utilization and fermentative behavior of the Thermoanaerobacterium thermosaccharolyticum W16, various experiments were performed where glucose, xylose, and a mixture of glucose, xylose, and arabinose with final concentration of 10 g/L was provided. These concentrations of sugars were on par with the sugar content found in the corn stover hydrolysate. It was

32 Lignocellulosic Biomass Production and Industrial Applications observed that this microorganism preferred glucose over the other types of sugars and sugar mixtures. The bacterium grew well on simple sugars and showed optical density and maximum hydrogen production rate on par with media containing hydrolysate (although H2 yield was slightly higher on using hydrolysate) [12].

2.3

Complex Substrates as Feedstock

There is a relatively high abundance of complex sugars (polysaccharides) in nature. But in order to provide information about their suitability for hydrogen production, comprehensive research is required targeting the pretreatment and saccharification process [37]. The bioH2 production process can be commercialized economically if cheap, renewable organic feedstock and low-cost sources, such as polysaccharides (starch, cellulose and xylan), are utilized. Polymeric sugars, such as starch and cellulose, consist of a monomeric unit of hundreds or thousands of glucose molecules. In plants, the atmospheric CO2 is fixed into energy storage molecules known as starch, whereas cellulose serves as a rigid building block to maintain the cell wall integrity. The cell walls of different parts of the plant (viz., leaves, stems, stalks and woody portions) are made up of cellulose, hemicelluloses and lignin. Moreover, plant cell walls and some algae also contain xylan. Xylan is a polymeric sugar made up of xylose subunits (which is a pentose sugar). Subunits of β-D-xylose are linked via β-1,4 linkage to form xylan molecules (structure is analogous to that of cellulose). Lignocellulosic raw materials may be projected as the main component of future feedstock due to their availability. Tightly bound cellulose, hemicelluloses and lignin form the basic structure of lignocellulic biomass. The cellulose and hemicellulose can be a potential source of sugar for hydrogen fermentation. The lignin does not get digested by the anaerobic fermentation process. Moreover, the presence of lignin hinders the accessibility of cellulose and hemicelluloses for enzymatic hydrolysis, thereby resisting the mobilization of sugar; and chemically degraded lignin can also inhibit microbial growth [20].

2.4

Biomass Feedstock

Enhancement of hydrogen production could be achieved through biomass. Properties such as renewability and consumption of atmospheric

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CO2 during growth may leave a smaller carbon footprint as compared to fossil fuels. However, lower energy content (40% of oxygen content of biomass) and lower hydrogen content of 6% hampers the utility of biomass as a feedstock for thermal gasification. Rather, the biomass could be converted to H2 via biological route by implementing different saccharification technologies that help in converting complex sugar to simple sugar. These techniques include thermochemical processes such as acid-heat treatment, high-pressure steam explosion, etc., and biological processes such as enzymatic hydrolysis, microbial hydrolysis, etc. Biological saccharification processes are slow but are environmentally friendly as compared to thermochemical processes.

2.4.1

Energy Crop

Feedstocks used for energy generation are regarded as energy crops (e.g., straws from rice, wheat, barley, corn stalk, miscanthus, cassava, etc.). They are commonly referred to as second generation cellulosic biomass [38]. The possibility of utilizing whole crops for energy production was first shown in the early 1980s [39]. “Energy crops” are those crops which are grown solely for their further exploitation as feedstock for energy production. The entire biomass or a part of it might be used as feedstock. Such feedstock either gets directly exploited for its energy content via combustion or it is biotransformed into biofuels via fermentation. Sustainable energy production from energy crops can be achieved by incorporating the following points [40]: a. Minimum nutrient and water requirements, thus cost effective; b. Immunity to environmental stress conditions; c. Higher biomass yields. A high sugar/carbohydrate content and low lignin content are the principle criteria for these plants to be called suitable for hydrogen production via dark fermentation. Energy crops can be categorized into three broad domains: a) fermentable simple sugar-rich crops such as sweet sorghum, sugar beet and sugar cane, etc.; b) starch-rich crops such as corn, wheat, millets and rice; and c) lignocellulosic biomass, which includes a few herbaceous plants, such as switch grass, sugar cane bagasse and fodder grass, and a few woody plants such as Miscanthus, poplar, conifers, etc.

34 Lignocellulosic Biomass Production and Industrial Applications

2.4.1.1 Miscanthus sp. Miscanthus belongs to the perennial C4 grass family. It is a rapidly growing plant capable of producing 8–15 t/ha dry weight with low inputs of nutrients and is found in Western European regions. This plant has been extensively studied as a potential feedstock for future energy generation. It needs different types of pretreatment (grinding, ball milling, steam explosion, alkali treatment, etc.) before it can be used as feedstock. The highest hydrogen yield of 3.2 mol/mol glucose was reported using Miscanthus hydrolysate fermented by an extreme thermophilic microorganism, Thermotoga elifii [41].

2.4.1.2 Sweet Sorghum Extract Another perennial C4 plant is sweet sorghum, which is also known to have high photosynthetic efficiency. The biomass of sweet sorghum is rich in carbohydrates and is also a high-yielding crop. Its stalks contain sucrose and glucose which contribute to 55% and 3.2% w/w of dry matter [42]. They also contain cellulose (12.4% w/w) and hemicellulose (10.2%). Its high fermentable sugar content makes it an enterprising prospect for fermentative hydrogen production. Overall, out of the many “new crops” that are currently being investigated as potential raw materials for energy and industry, sweet sorghum seems to be the most promising one. Using sweet sorghum lysate, the highest methane yield of 78.0 L CH4/kg was observed along with a hydrogen yield of 0.86 mol/mol hexose biomass [43].

2.4.1.3 Sugar Beet Juice In the UK, sugar beet yields around 54 t ha–1 (wet extractable sucrose content of about 170 g kg–1 wet beets). Reports suggest that the dry mass of the pulp after extraction contains mostly cellulose, hemicelluloses and pectin, with a small amount of lignin and variable nutrient content. Sugar beet can be cultivated alongside fermentation facilities to be used as an energy crop [44]. Hydrogen yields using sugar beet pulp were 1.7 ± 0.2 to 1.9 ± 0.2 mol 1 mol 1 hexose converted at a retention time of 14–15 h (16 kg total sugar m 3d 1 organic loading rate). Voices have been raised against the practice of large-scale cultivation of such crops for biofuel production. The excessive amount of water required, competition with dietary crops for fertile land, use of pesticides, decrease in fertility of soil due to less crop rotation, etc., are the pertinent issues regarding energy crop cultivation. These issues have led to the debate over “food vs. fuel,” thereby questioning the use of energy crops as feedstock for biofuels. Even

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in the case of non-food crops cultivation, the sustainability issue is under question. As a solution, second generation biofuels have entered the fray, i.e., the use of wastes and residues for biofuel production.

2.4.2

Algal Biomass

The rise of lignocellulosic biomass as a source of feedstock is due to its abundance but harsh pretreatment and saccharification processes hamper its utility to a certain extent. Moreover, limited resources for water and agriculture are the main disadvantages of using agricultural wastes as a source of bioenergy production [45]. Algal biomass has been considered a third generation feedstock for biofuel production. The cellular structure of algae is simpler as compared to higher photosynthetic plants as they do not have lignin or hemicelluloses in their cell walls. Due to their large surface-to-volume body ratio, algae can assimilate more nutrients as compared to higher plants. Moreover, algal cultivation has certain fundamental advantages [46]: Growth rate: It generally has higher growth rate as compared to other terrestrial autotrophic plants and a higher rate of biomass production (dry cell weight per unit time and volume). Carbohydrate content and quality: In the harvested biomass, the carbohydrate content varies from 20% w/w to 40% w/w. Lack of recalcitrant polymer, such as lignin, makes it an ideal feedstock. Nutrient preference and rate of substrate utilization: During growth it has minimal nutritional requirements. Ease of biomass harvesting: Filamentous algae are easier to harvest as compared to single cell algae. Cheap and largescale harvesting techniques could prove vital for usage of algal biomass as feedstock. Various pretreatment methods have been studied using algae as feedstock for hydrogen production. The algal biomass contains complex sugars that were converted into simpler forms by optimizing the different physicochemical parameters of pretreatment. Algal biomass contains complex carbohydrates which remain attached to the rigid cell wall. Therefore, to gain access to the complex sugars and to convert them into simple sugars, it is necessary to break the algal cell wall with harsh pretreatment methods. The overall cost effectiveness of the hydrogen production process largely

36 Lignocellulosic Biomass Production and Industrial Applications depends on the cost of pretreatment of biomass. Several pretreatment methods have been explored to break the algal cell wall, including physical (sonication, bead beating, grinding, milling and pyrolysis), chemical (acid, alkali, thermal, H2O2) and biological (enzymatic, microbial) methods, resulting in the release of fermentable sugars for biofuel production. All of the methods have their own merits and demerits. Due to higher saccharification efficiency, acid treatment (chemical method) is preferred over others. Energy-intensive physicochemical methods may be technologically simpler but have limited commercial potential. In the process, formation of furfurals leads to the inhibition of growth of the fermentative microorganisms. Biological methods like co-culture development may be considered as an alternative, where one organism intensifies the saccharification process, thereby helping in the fermentation process. The costly and time-consuming nature of crude enzymatic techniques is a hurdle for their utility as pretreatment methods. Use of algal biomass as feedstock for hydrogen production has gained interest recently. In one study, a two-step H2 production was used where Clostridium butyricum was fed with algal biomass hydrolysate to produce H2, and short-chain fatty acids produced in the first stage were used for H2 production in the second stage photofermentation using Rhodobacter sphaeroides KD 131 [47]. In another study, thermophilic dark fermentative hydrogen production was reported using Chlamydomonas reinhardtti biomass [48]. Anabaena variabilis biomass was hydrolyzed by using amylase and the hydrolysate was used in thermophilic dark fermentative hydrogen production [49]. In a similar study, Chlorella sorokiniana biomass was treated with acid-heat pretreatment and the hydrolysate was used to produce hydrogen by Enterobacter cloacae IITBT08 [29].

2.5

Waste as Feedstock

Agricultural wastes as a whole are a bright prospect for use as a feedstock for biohydrogen generation because of their vast repertoire and patentability. Various industrial wastewaters could serve a dual purpose in the biohydrogen production process. Firstly, they would lead to generation of clean energy in the form of hydrogen; secondly, they would also help waste management by reducing its COD and BOD content, thereby reducing the toxic effects of wastewater when discharged in receiving land or water bodies. The food manufacturing industry generally produces high strength organic wastewaters which are rich in carbohydrates, mainly simple sugars, starch and cellulose, etc., and could be used as feedstock for biohydrogen production. A variety of food industry wastes, such as

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noodle manufacturing wastes, sugar beet wastewater, rice and wheat bran processing by-product, bean curd manufacturing waste, sugar factory wastes, distillery effluents, rice winery wastewater, molasses, dehydrated brewery mixture, starch manufacturing waste, organic wastewater, and cheese whey [50]. Solid organic wastes are also being explored as feedstock for fermentative hydrogen production. Solid wastes generated from the household kitchen, food processing and municipal wastes have high COD. Apart from carbohydrates, these wastes are also rich in proteins and fats [21].

2.5.1

Municipal Solid Waste (MSW)

The organic fraction of municipal solid waste (OFMSW) can be considered an important feedstock for hydrogen production because it could lead to a cost-efficient and environmentally friendly production strategy [51]. Various value-added by-products may also be derived from conversion of organic wastes into a hydrogen-rich biogas. Municipal solid waste (MSW) poses a major threat to the environment whose production is globally increasing exponentially at a rate of 6% per year. Nearly 60% of MSW is organic fraction which consists of kitchen waste, waste paper and urban greening waste. Usage of OFMSW for biohydrogen production could be considered as a sustainable source of feedstock. Its abundant availability at zero cost could improve the economic feasibility of biohythane production. Worldwide biological wastewater treatment processes generate large amounts of sewage sludge. In China, about 4.22 billion tons of municipal wastewater were treated in 150 municipal wastewater treatment plants, which generated about 0.55–1.06 million tons of dry sludge in 2001 [52]. The waste sludge is treated by anaerobic digestion for methane production with hydrogen as an intermediate product. Many reports have shown that different wastes with a high organic content produce hydrogen by anaerobic fermentation. The polysaccharides and proteins are the major component of sewage sludge and thus could be potential substrate for fermentative hydrogen production. The raw sewage sludge generally shows inferior potential for hydrogen production, i.e., 0.16 mg of H2 (g of dried solids, DS)-1. To solubilize the nutrients present in the sewage sludge, various pretreatments such as ultrasonication, acid treatment, sterilization and freezing/thawing were explored to improve fermentative hydrogen production. Sludge pretreated with sterilization or freezing/thawing showed improved hydrogen yields in the range of 1.5–2.1 mmol of H2 (g of COD) 1 [53].

38 Lignocellulosic Biomass Production and Industrial Applications

2.5.2

Food Waste

Food waste is considered to be one of the substrates with the most portential for hydrogen production as it contains about 90% volatile suspended solids favoring microbial degradation. Landfill disposal of food waste generates foul smells and pollutes the ground water. For this reason, anaerobic digestion is the most preferred method for treating food wastes. These food wastes include canteen wastes, starchy waste material, cheese whey, etc., which are highly potential carbon sources. At the present time, researchers are focusing on thermophilic fermentation for the enhancement of production of biogas. The institutional food wastes used for thermophilic hydrogen production gave 81 ml H2/g VSS as compared to 63 ml H2/g VSS by mesophilic dark fermentation [54].

2.6 2.6.1

Industrial Wastewater Dairy Industry Wastewater

Wastewater from the dairy industry is high in organic content with high biological oxygen demand (BOD) and chemical oxygen demand (COD) [55]. Furthermore, the dairy industry is one of the largest sources of industrial effluents in Europe. Carbohydrates, fats and proteins from milk contribute to the increase in the organic load in natural habitats [56]. High concentrations of organic matter in dairy waste streams may pose serious problems by increasing the organic load on local municipal sewage treatment facilities. The majority of effluent is generated during cleaning of transport lines and equipment between production cycles, cleaning of tank trucks, washing of milk silos and equipment malfunctions or operational errors  [57]. Various physicochemical and biological treatment methods were employed for treatment of dairy wastewaters. Biological treatment methods were preferred over chemical methods because of the high operational cost of the latter [58]. Treatment in oxidation ponds, activated sludge plants and anaerobic treatment are commonly employed biological treatment processes for dairy wastewater [59]. Anaerobic treatment of high COD dairy effluents is more advisable as there are no aeration requirements, low sludge production and it can be performed in smaller areas as compared to aerobic process. In dairy waste streams, the organic fraction is contributed by the residues which are derived from milk and its derivatives. Nitrogen content of such wastewater is contributed mainly from milk proteins, urea and nucleic acids or in the form inorganic nitrogen such as NH4+, NO2− and NO3−. Along

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with it, orthophosphate (PO43−) and polyphosphate (P2O74−) are also present [60]. The yardsticks generally used for the assessment of the strength and treatability of wastewater were concentrations of suspended solids (SS) and volatile suspended solids (VSS) [57]. Suspended solids in dairy wastewaters were contributed by coagulated milk solids, cheese curd fines or flavoring ingredients. The bulk portion of the total COD in cheese-processing wastewater was contributed by lactose, lactate, protein and fat [61]. The main carbohydrate present in dairy wastewater is lactose, which could prove to be potential substrate for anaerobic bacteria. Anaerobic digestion of lactose for biogas production is a mutual biological activity from acidogens, acetogens and methanogens [62]. Metabolites, viz., ethanol, acetate, butyrate, propionate, iso- and normal valerate, caproate, lactate and formate, were produced along with hydrogen during fermentation of lactose. Other components of wastewater were casein protein in milk composition and in dairy effluents and lipids. Lipids are potentially inhibitory compounds for biohydrogen and biomethanation processes. On using dairy wastewater, the maximum hydrogen production of 0.122 mmol H2 (g COD) 1 was observed when chemically treated sludge was used as inoculum [63].

2.6.2

Distillery Wastewater

Distillery wastewater is rich in biodegradable organic material such as sugars, hemicelluloses, dextrin, resins, lignin and organic acids. For every 1 L of alcohol, 8–15 L of wastewater are produced by distillery industries which have high chemical oxygen demand (COD) (80–160 g L–1). The distilleries use technologies, such as biomethanation, followed by concentration and incineration for treatment of such wastewaters. High organic content and availability of large quantities of wastewater could prove to be a potential feedstock for biohydrogen production by anaerobic fermentation. Many reports are available on biohydrogen production using distillery wastewater in different reactor configurations. In anaerobic sequencing batch biofilm reactor, maximum hydrogen production of 6.98 mol H2 (kg CODR)–1 d–1 was observe along with 70% COD removal [64].

2.6.3

Chemical Wastewaters

These wastewaters are the effluents generated from chemicals, drugs, pharmaceuticals, pesticides and various chemical processing units. In general, these wastewaters have low COD but are rich in nitrogen and complex organic compounds. The potentiality of such wastewater for biohydrogen production showed maximum hydrogen production of 1.25 mmol (g COD

40 Lignocellulosic Biomass Production and Industrial Applications added) 1 [30]. Thus, in the future such chemical industry wastewaters could also be considered as feedstock for dark fermentation.

2.6.4

Glycerol

Biodiesel is the clean-burning diesel fuel produced from animal fats, greases and vegetable oils. Diminishing fossil fuel reserves and environmental consequences have led to the importance of biodiesel. The production of biodiesel is done both chemically and enzymatically. In both cases glycerol is the major by-product. Treating glycerol waste is a major problem as it increases the overall cost of production of biodiesel. Previously, glycerol waste was treated by mesophilic organisms for hydrogen production. Because glycerol industry effluents are generally released at very high temperatures, they need to be cooled down for the treatment. Currently researchers are moving towards the treatment of this waste by thermophilic organisms to save energy. Under mesophilic conditions, Enterobacter aerogenes HU-101 could get 80 mmol L–1 h–1 of hydrogen using glycerol waste [65].

2.6.5

Palm Oil Mill Effluent

Due to its release at high temperature, palm oil mill effluent (POME) can be a potential substrate for a thermophilic dark fermentation process. The major source of palm oil mill effluent generation in the palm oil mill is the separator sludge and the sterilizer condensate. On an average, 0.9–1.5 m3 POME is generated from 1 ton of palm oil produced. It is highly enriched in organic content with BOD of more than 20 g L 1 and nitrogen more than 0.5 g L 1. It was reported that with POME using UASB reactor under thermophilic conditions, a hydrogen production rate of 4.4 L (g POME) 1 d 1 was achieved [66].

2.7

Conclusion

The biohydrogen production processes reported in the literature is predominated by usage of substrates like pure monosaccharides (59%), disaccharides (10%), polysaccharides (11%) and sustainable feedstock (20%) [38]. At present, wheat straw, algal biomass, barely straw and different biomass hydrolysates are being explored as sustainable feedstocks. Another domain is feedstocks of high-strength industrial effluents, agro-based residues and dairy wastes. Utilization of such feedstocks would not only help in clean energy generation but also in waste management.

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Acknowledgments The author is grateful for the financial assistance from INSPIRE Faculty Scheme, Department of Science and Technology (DST), Government of India.

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44 Lignocellulosic Biomass Production and Industrial Applications 39. Helsel, Z.R., and Wedin, W., Direct combustion energy from crops and crop residues produced in Iowa. Energy Agric. 1, 317–329, 1981. 40. Hawkes, F., Sustainable fermentative hydrogen production: Challenges for process optimisation. Int. J. Hydrogen Energy 27, 1339–1347, 2002. 41. Devrije, T., Pretreatment of Miscanthus for hydrogen production by Thermotoga elfii. Int. J. Hydrogen Energyy 27, 1381–1390, 2002. 42. Antonopoulou, G., Gavala, H.N., Skiadas, I.V., Angelopoulos, K., and Lyberatos, G., Biofuels generation from sweet sorghum: Fermentative hydrogen production and anaerobic digestion of the remaining biomass. Bioresour. Technol. 99, 110–119, 2008. 43. Ntaikou, I., Gavala, H., Kornaros, M., and Lyberatos, G., Hydrogen production from sugars and sweet sorghum biomass using Ruminococcus albus. Int. J. Hydrogen Energyy 33, 1153–1163, 2008. 44. Hussy, I., Hawkes, F., Dinsdale, R., and Hawkes, D., Continuous fermentative hydrogen production from sucrose and sugarbeet. Int. J. Hydrogen Energyy 30, 471–483, 2005. 45. Li, Y., Horsman, M., Wu, N., Lan, C.Q., and Dubois-Calero, N., Biofuels from microalgae. Biotechnol. Progr. 24(4), 815–820, 2008. 46. Singh, S., Kate, B.N., and Banerjee, U.C., Bioactive compounds from cyanobacteria and microalgae: An overview. Crit. Rev. Biotechnol. 25, 73–95, 2005. 47. Kim, M., Baek, J., Yun, Y., Junsim, S., Park, S., and Kim, S., Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: Anaerobic conversion and photosynthetic fermentation. Int. J. Hydrogen Energyy 31, 812–816, 2006. 48. Nguyen, T.-A.D., Kim, K.-R., Nguyen, M.-T., Kim, M.S., Kim, D., and Sim, S.J., Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods. Int. J. Hydrogen Energyy 35, 13035–13040, 2010. 49. Nayak, B.K., Roy, S., and Das, D., Biohydrogen production from algal biomass (Anabaena sp. PCC 7120) cultivated in airlift photobioreactor. Int. J. Hydrogen Energyy 39, 7553–7560, 2014. 50. Mizuno, O., Ohara, T., Shinya, M., and Noike, T., Characteristics of hydrogen production from bean curd manufacturing waste by anaerobic microflora. Water Sci. Technol. 41, 25–32, 2000. 51. Noike, T., and Mizuno, O., Hydrogen fermentation of organic municipal wastes. Water Sci. Technol. 42, 155–162, 2000. 52. Dunn, S., Hydrogen futures: Toward a sustainable energy system. Int. J. Hydrogen Energyy 27, 235–264, 2002. 53. Wang, C.C., Chang, C., Chu, C.P., Lee, D.J., Chang, B.-V., and Liao, C.S., Producing hydrogen from wastewater sludge by Clostridium bifermentans. J. Biotechnol. 102, 83–92, 2003. 54. Chen, C.-C., Chuang, Y.-S., Lin, C.-Y., Lay, C.-H., and Sen, B., Thermophilic dark fermentation of untreated rice straw using mixed cultures for hydrogen production. Int. J. Hydrogen Energy 37, 15540–15546, 2012.

Biological Gaseous Energy Recovery

45

55. Orhon, D., Görgün, E., Germirli, F., and Artan, N., Biological treatability of dairy wastewaters. Water Res. 27, 625–633, 1993. 56. Perle, M., Kimchie, S., and Shelef, G., Some biochemical aspects of the anaerobic degradation of dairy wastewater. Water Res. 29, 1549–1554, 1995. 57. Danalewich, J.R., Papagiannis, T.G., Belyea, R.L., Tumbleson, M.E., and Raskin, L., Characterization of dairy waste streams, current treatment practices, and potential for biological nutrient removal. Water Res. 32, 3555–3568, 1998. 58. Vidal, G., Influence of the content in fats and proteins on the anaerobic biodegradability of dairy wastewaters. Bioresour. Technol. 74, 231–239, 2000. 59. Tawfik, A., Sobhey, M., and Badawy, M., Treatment of a combined dairy and domestic wastewater in an up-flow anaerobic sludge blanket (UASB) reactor followed by activated sludge (AS system). Desalination 227, 167–177, 2008. 60. Guillen-Jimenez, E., Bio-mineralization of organic matter in dairy wastewater, as affected by pH. The evolution of ammonium and phosphates. Water Res. 34, 1215–1224, 2000. 61. Hwang, S., and Hansen, C.L., Characterization of and bioproduction of shortchain organic acids from mixed dairy-processing wastewater. Trans. Am. Soc. Agric. Eng. 41, 795–802, 1998. 62. Yu, J., and Pinder, K.L., Intrinsic fermentation kinetics of lactose in acidogenic biofilms. Biotechnol. Bioeng. 41, 479–488, 1993. 63. Venkata Mohan, S., Lalit Babu, V., and Sarma, P.N., Effect of various pretreatment methods on anaerobic mixed microflora to enhance biohydrogen production utilizing dairy wastewater as substrate. Bioresour. Technol. 99, 59–67, 2008. 64. Venkata Mohan, S., Simultaneous biohydrogen production and wastewater treatment in biofilm configured anaerobic periodic discontinuous batch reactor using distillery wastewater. Int. J. Hydrogen Energyy 33, 550–558, 2008. 65. Ito, T., Nakashimada, Y., Senba, K., Matsui, T., and Nishio, N., Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process. J. Biosci. Bioeng. 100, 260–265, 2005. 66. O-Thong, S., and Mamimin, C., and Prasertsan, P., Effect of temperature and initial pH on biohydrogen production from palm oil mill effluent: Long-term evaluation and microbial community analysis. Electron. J. Biotechnol. 14, 9, 2011.

3 Alkali Treatment to Improve Physical, Mechanical and Chemical Properties of Lignocellulosic Natural Fibers for Use in Various Applications Suvendu Manna1*, Prosenjit Saha2, Sukanya Chowdhury2 and Sabu Thomas1,3 1

School of Chemical Sciences, Mahatma Gandhi University, Kerala, India Dr. MN Dastur School of Materials Science, Indian Institute of Engineering Science and Technology Shibpur, West Bengal, India 3 International and Interuniversity Center for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kerala, India 2

Abstract Due to economical and environmental friendly nature, lignocellulosic (LC) natural fibers are emerging as an alternative to petroleum and synthetic polymeric materials. These LC natural fibers contain diverse types of surface functional groups which make them suitable alternatives for various applications like preparation of technical textiles, biocomposites and filter materials. However, their high biodegradability and poor mechanical properties restrict the use of LC natural fibers. Thus suitable physical or chemical treatment is necessary to enhance the physical, mechanical and chemical properties of LC natural fibers. The treatment methods should be economical and easy to execute. After treatment, these LC natural fibers can be used for preparing biocomposites or technical textiles with improved properties. Treated LC natural fibers can also be used to remove pollutants from wastewater. In this chapter, alkali treatment is discussed for improvement of the physical, mechanical and chemical properties of lignocellulosic materials in connection with their applications. Keywords: Lignocellulose, mechanical strength improvement, surface modification, alkali treatment, water treatment, biocomposites, fibers *Corresponding author: [email protected] Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial Applications, (47–64) © 2017 Scrivener Publishing LLC

47

48 Lignocellulosic Biomass Production and Industrial Applications

3.1

Introduction

Lignocellulosic natural fibers, such as sisal, bamboo, coir and jute, are renewable, nonabrasive to process and can be incinerated at the end of their life cycle. These types of materials are also very safe during handling, processing and use. Furthermore, these types of materials have physicomechanical properties comparable to the widely used alternative engineering materials (Table 3.1). Growing sustainable issues have enhanced the use of natural materials like lignocellulosic matter as a replacement for synthetic materials for application in composites, technical textiles and water filter development. The synthetic fiber-based composites are difficult to recycle after their designed service life. However, natural fiber-based composites are environmental friendly to a large extent. Although natural fibers have several advantages, they have some disadvantages too. The strong hydrophilic character of natural fibers makes them less usable in high-end applications, such as for preparation of composites for use in car interior design. Also, natural fibers often show poor interaction with the adhesive used in composite preparation. Use of lignocellulosic materials for water treatment is an innovative and alternative technology. The efficiency of these types of materials depends on their capacity, affinity and specificity, including their physicochemical nature. Surfaces of these types of materials are known to contain various functional groups, e.g., carboxyl, amine, hydroxyl, phosphate, sulfhydryl groups, that often bind to waterborne chemical constituents nonspecifically and have potential to be used as filter materials. But drawbacks like poor recyclability, less removal efficacy and high water absorption restrict their use as water filter. Table 3.1 Properties of engineering materials [2].

Materials

Strength- Degradation to-weight Insulation ratio Thermal UV light Acid

Swelling

Lignocellulose Good

High

Yes (Fire)

Yes

Yes

Yes (Moisture)

Metal

Poor

Low

Yes (Melt) No

Yes

Yes (Temp)

Plastic

Poor to good

Fair

Yes (Fire)

Yes/ Yes (Temp) No

Glass

Poor

Low

Yes (Melt) No

No

Yes (Temp)

Concrete

Poor

Low

No

Yes

No

Yes/No

No

Lignocellulosic Natural Fibers for Use in Various Applications

49

These types of drawbacks can be nullified through a simple and easily executable pretreatment, such as alkali treatment. This treatment will expose a variety of surface functional groups by removing the wax layer, part of hemicellulose and lignin from the surface of the fibers [1]. The surface functional groups exposed during alkali treatment will improve the interactions between adhesive and lignocellulose during composite preparation. Also, the heterogenic surface functional groups improve the pollutant removal capacity of the lignocellulosic materials. In this chapter, alkali treatment of lignocellulosic materials and use of alkali treated lignocellulosic materials for composite preparation and water treatment are discussed.

3.1.1

Composition of Natural Fibers

Lignocellulosic fibers can be classified according to their origin and grouped into a) bast: flax, hemp, jute, ramie; b) fruit: coir, kapok, oil palm; c) grass: alfa, bagasse, bamboo; c) leaf: abaca, cantala, curaua, date palm, henequen, pineapple, sisal, banana; d) seed: cotton; and e) stalk: straw (cereal). The most commonly used plant fibers are cotton, jute, hemp, flax, ramie, sisal, coir, henequen and kapok. These plant fibers are constitutes of cellulose, hemicellulose, lignin and small amounts of extractives and ash (Table 3.2). The cellulose fibers consist of helical microfibrils bound together by amorphous lignin matrix. Lignin acts as a protection against biological attack and stiffens jute stem, improving its resistance against gravity forces and wind. Hemicellulose acts as a compatibilizer between cellulose and lignin [3]. Cellulose microfibrils, typically with a diameter of about 10–30 nm, provide mechanical strength to the lignocellulosic fibers. Cellulose, a linear polymer of D-glucopyranose sugar units linked in a beta configuration, has an average degree of polymerization of about 9,000 to 10,000 units. It was reported that approximately 65% of the total cellulose is highly oriented, crystalline and not accessible to water or other solvents [4]. The remaining cellulose is composed of less oriented chains partially accessible to water and other solvents due to its association with hemicellulose and lignin. The hemicelluloses are amorphous polymers of pentose, hexose and methylglucuronic acid, with a much lower degree of polymerization than cellulose and variable sugar composition depending on the source. Lignin is a highly branched amorphous polymer of nine carbon units derived from substituted cinnamyl alcohol; that is, coumaryl, coniferyl, and syringyl alcohols associated with the hemicelluloses. The other composition of lignocelluloses consists of water, organic soluble extractives, and inorganic materials. The extractives are primarily composed of cyclic hydrocarbons

50 Lignocellulosic Biomass Production and Industrial Applications Table 3.2 Composition of different lignocellulosic fibers [2]. Composition (%) Type of fiber

Cellulose Lignin Hemicelluloses

Ash

Silica

Stalk fiber Rice

28–36

12–16

23–28

15–20

9–14

Wheat

29–35

16–21

26–32

4.5–9

3–6

Barley

31–34

14–15

24–29

5–7

3–6

Oat

31–37

16–19

27–38

6–8

4–6.5

Rye

33–35

16–19

27–30

2–5

0.5–4

Sugar

32–44

19–24

27–32

1.5–5

0.7–3.5

Bamboo

26–43

21–31

15–26

1.7–5

–

33–38

17–19

27–32

6–8

–

–

22

23.9

6

–

44.75

22.8

20.0

2.9

2

47

23

25

5

–

Kenaf

31–39

15–19

22–23

2–5

–

Jute

45–55

21–26

18–21

0.5–2

–

60.8

8.8

17.3

1.1

–

43–56

7–9

21–24

0.6–101

–

80–85

–

–

0.8–1.8

–

Coniferous

40–45

26–34

7–34

bacteria > yeast > animals > plants since microbial sources contribute 80% in commercial enzyme production. Various filamentous fungi, such as Trichoderma reesei, Aspergillus niger, Rhizomucor miehei, Fusarium venenatum, Humicola

Microbial Enzymes and Lignocellulosic Fuel Production

141

Table 7.2 Sources of enzymes/proteins used in lignocellulosic biomass conversion to biofuels. Source of enzyme

Enzyme

Plant Rhus vernicifera, p9apaya leaves Malted barley

Laccase β-Glucanase

Animal Pancreas

Lipase

Fungal Pleurotus sp., Trametes sp. Rhizopus sp. Trichoderma reesei T. harzianum Trichoderma sp., Fusarium proliferatum A. usamii Penicillium janthinellum T. reesei Sporotrichum thermophile

Laccase Lipase Cellulase Endo-1,4-β-glucanases β-Glucosidases Endo-β-1,4-xylanase β-xylosidase Acetyl xylan esterase Glucuronoyl esterase

Bacterial Azospirillum lipoferum, Streptomyces sp. Bacillus sp. Caldicellulosiruptor sp., Ruminococcus flavefaciens, R. albus Cellulomonas fimi Clostridium josui, R. albus Amycolatopsis orientalis R. albus Thermomonospora fusca Bacillus licheniformis Geobacillus stearothermophilus Geobacillus sp. Lactobacillus fermentum

Laccase Xylanase Mannanase Endoglucanase Exo-β-D-glucosaminidase Cellobiohydrolase Exo-glucanases Endo-(1,4)-β-D-glucanase Endo-β-1,4-xylanase β-xylosidase Feruloyl esterase

Yeast Candida

Lipase

142 Lignocellulosic Biomass Production and Industrial Applications lanuginosa and Chrysosporium lucknowense, are currently used for largescale enzyme production. Among these fungi, A. niger and T. reesei are considered as potential sources for extracellular enzymes and are extensively used in industries. A. niger is an unique organism that is reported to produce 40 different commercial enzymes, including cellulase, lipase, pectinase, protease and amylase. The productivity of microbial enzymes is enhanced by enforcing some alterations in the genome of the microorganism through mutation, protoplast fusion or recombinant DNA technology. Most of the enzymes (approximately 60%) used on the industrial level are recombinant proteins [20]. According to Liu et al., low-energy ion irradiated Pichia pastoris produced mutants that yielded higher laccase (7.7 mg/L, 92.5% increase) with enhanced catalytic activity and higher thermal stability [21]. In order to make the enzyme production process more economical, lignocellulosic biomass obtained as agro-industrial residues may be used as carbon source instead of molasses, yeast extract, starch hydrolysate, corn steep liquor, soyabean meal, whey, pulses and cereals owing to their high holocellulosic content. Several lignocellulosics which are being used as substrate for enzyme production are tabulated in Table 7.3.

7.4

Microbial Enzymes towards Lignocellulosic Biomass Degradation

Lignocellulosic fuel production is driven by utilization of a multitude of enzymes that perform substrate-specific degradation of lignocellulosic biomass which is mainly composed of lignin, cellulose and hemicellulose. Based on the nature of polymer, industrial enzymes used in bioethanol production may be categorized into three major groups, namely group I, II and III, specific to pretreatment, saccharification and fermentation steps respectively. Group I enzymes include ligninases which are used for lignin degradation, group II involves cellulases, hemicellulases, carboxyl esterases and other auxiliary enzymes, referred to together as holocellulases, that catalyze the breakdown of holocelluloses to hexoses and pentoses. Whereas group III enzymes involve zymase and its associated enzymes that are used for fermenting sugars to ethanol. Apart from these major groups of enzymes, others such as lipases and esterases also exist that play a crucial role in transesterification of lipids for biodiesel production. This chapter emphasizes group I, II and III enzymes (Table 7.4) and their application in biobased products such as bioethanol, biomethane, etc.

Microorganism Ganoderma lucidum, P. ostreatus, Phanerochaete chrysosporium, Phlebia radiata P. dryinus, P. tuberregium, Lentinula edodes T. reesei QM 9414 Aspergillus terreus M11 and Myceliophthora sp. IMI 387099 Fusarium oxisporum A. nigerr 38 A. sydowii BTMFS 55 Aureobasidium pullulans ER-16 and Thermoscus aurantiacus CBMAI 456 Neosartorya spinosa D19 Coprinellus disseminatus Fomes sclerodemeus A. terreus A. carneus M34 A. terreus and A. niger Thermoascus aurantiacus Bacillus sp.

Biomass

Pineapple leaf, neem hull, wheat bran, sugarcane bagasse, and wheat straw Beech tree leaves Eucalyptus residue

Rice bran Corn cob, sugarcane bagasse and wheat bran Rice straw Corn cob Wheat bran and straw

Wheat bran

Wheat bran Palm waste Sugarcane bagasse, rice straw and soya bean hulls Sugarcane bagasse Rice bran Peanut shells Banana stalk

Table 7.3 Production of microbial enzymes from lignocellulosic biomass.

Xylanase

β-glucosidase

Endoglucanase

Laccase, Manganese peroxidase and Lignin peroxidase

Enzyme

(Continued)

[34–36] [37–40] [41] [42] [43] [44]

[32] [33]

[27] [28, 29] [30] [31]

[22–24] [25] [26]

Ref. Microbial Enzymes and Lignocellulosic Fuel Production

143

T. versicolor Streptomyces sp. HM29 T. viride A. niger Bacillus megatherium T. viride

Coffee husk and sugarcane bagasse

Wheat bran

Orange peel waste

P. chrysosporium Trametes versicolor and P. ostreatus

Microorganism

Corn cob Sugarcane bagasse Apple pomace

Biomass

Table 7.3 Cont.

Endoglucanase, exoglucanase, β-glucosidase

Pectinase, xylanase and α- amylase

Pectinase

Cellulase and xylanase Cellulase Cellulase

Enzyme

[47]

[49]

[48]

[45] [46] [47]

Ref.

144 Lignocellulosic Biomass Production and Industrial Applications

Microbial Enzymes and Lignocellulosic Fuel Production

145

Table 7.4 Group I, II and III enzymes used in lignocellulosic ethanol production. Group I: Lignindegrading enzymes for pretreatment

Group II: Holocellulases and auxiliary enzymes/proteins for saccharification

Group III: Enzyme complex involved in fermentation

Ligninases: Lignin peroxidase Manganese peroxidase Versatile peroxidase Phenol oxidase: (i) catechol oxidase, (ii) monophenol monooxygenase, (iii) laccase, (iv) cresolases, (v) monophenol oxidases

Cellulase: Endoglucanases, Exoglucanases/ cellobiohydrolases, β-Glucosidases, Oxidative cellulases, Cellulose phosphorylases

Zymase: Invertase, Hexogenase, Zymohexase, Isomerase, Phosphatase, Triosedehydrogenase, Mutase, Enolase, Carboxylase

Other enzymes: Aryl alcohol dehydrogenases, Glyoxal oxidase, Cellobiose dehydrogenases

Hemicellulase: Endoxylanase, Exoxylanases, β-Xylosidase, Glucuronidase, α-Galactosidase, Arabinofuranosidase, α-L-arabinanase, β-Mannanase, β-Mannosidase, α-L-fucosidase, Acetylxylan esterase, Endomannanases, α-Galactosidases, β-Mannosidases Carboxyl esterases: Acetyl xylan esterase Feruloyl esterase Auxiliary cellulose degrading enzymes: Cellobiose dehydrogenases, Copper-dependent lytic polysaccharide monooxygenases Cell wall proteins: Expansin and swollenins

146 Lignocellulosic Biomass Production and Industrial Applications

7.4.1

Ligninases

This group of enzymes are also referred to as lignin modifying enzymes as they are oxidative rather than hydrolytic by their mechanism. These include heme peroxidases, phenol oxidases, along with some other accessory enzymes. These enzymes play a crucial role in benefiting the subsequent step of the biofuels production process, i.e., saccharification, by allowing better access of carbohydratases to the holocellulosic biomass. Further, the action of ligninases upon the biomass generally leads to an increase in the cellulose cystallinity, which represents the extent to which cellulosic biomass is more exposed towards cellulase and xylanase. This fact has been studied by a number of researchers using XRD spectra of the lignocellulosic biomass before and after enzymatic treatment. In addition to the crystallinity, pore size of the biomass is another criterion which is equally crucial to investigate while studying the efficacy of delignification, since pore size of the untreated substrate limits the access of carbohydrates towards its degrading enzymes. It has also been reported that the action of ligninases upon the biomass leads to an increase in the average pore size of the biomass, resulting in better accessibility of holocelluloses towards cellulase and xylanase. Pore size and the available surface area influence the enzymatic action synergistically such that the greater the size of the pores, the greater is the surface area available for ligninases to act on the biomass, thereby resulting in efficient saccharification.

7.4.1.1 Lignin Peroxidase Lignin peroxidases are popular ligninolytic enzymes with the enzyme classification number, EC 1.11.1.14. These cause the oxidative depolymerization of the phenolic and non-phenolic lignin units present in the lignocellulosic biomass. This process is generally referred to as delignification of the biomass and is aimed at selectively removing the recalcitrant lignin molecules and loosening the holocellulosic macrofibril to enable easy access to cell wall carbohydrates. These N-glycosylated enzymes with heme core conduct multiple electron transfer reactions through hydrogen peroxide (H2O2)-dependent pathway, resulting in several redox intermediates, viz., methoxybenzaldehyde derivatives, positively charged phenoxy and veratryl alcohol radicals with higher redox potentials (1.4 V) which in turn are capable of further propagating the free radical reaction in an enzyme independent manner through polymerization, side-group removal and rearrangement reactions. It is reported that these enzymes are capable

Microbial Enzymes and Lignocellulosic Fuel Production

147

of degrading lignin without the requirement of external mediators [50]. The microbial sources for lignin peroxidases mainly include the white rot fungi P. chrysosporium, P. ostreatus, Phlebia floridensis, P. eryngii, P. radiata and Trametes versicolor; other groups of fungi include Perenniporia medulla-panis, Pycnoporus sanguineus, Panus sp., P. coccineus, etc. Besides fungi, certain bacterial sources are also known for producing this enzyme, namely Acinetobacter calcoaceticus and Streptomyces sp. [51]. These high redox oxidoreductases are extracellularly produced and are reported to be stable at a broad range of pH (1–5) and temperature (35–55 °C).

7.4.1.2 Manganese Peroxidase Manganese peroxidases are the other significant ligninases with the enzyme classification number, EC 1.11.1.13. These are the hemecontaining N-glycosylated proteins that cause the oxidation of Mn2+ to Mn3+ oxides through hydrogen peroxide-dependent pathway similar to lignin peroxidases. These oxides are exposed towards the outer surfaces of the enzyme coupled with primary redox intermediates, such as oxalic acid, malonic acid and other chelators, such that manganese oxide coupled chelator complex in turn acts as a redox mediator to depolymerize the phenolic lignin. However, for the effective breakdown of non-phenolic lignin, highly reactive free radicals, namely carbon core radicals, peroxyl, superoxide, formate radicals, etc., derived from the mediator like primary intermediates are essential. The microbial sources for manganese peroxidases include the members of the basidiomycetes class, mainly Panus tigrinus, P. chrysosporium, Lenzites betulinus, P. floridensis, Phanerochaete flavido-alba, P. radiata, Physisporinus rivulosus, Agaricus bisporus, Ceriporiopsis subvermispora, Bjerkandera sp., T. versicolor, Nematoloma frowardii, P. eryngii, P. ostreatus, etc. [51]. These metal-ion (Ca2+, Cd2+ and Mn2+)-dependent oxidoreductases are extracellularly produced on lignin-rich biomass and are reported to be stable at a broad range of pH (2.5–6.8) and temperature (30–60 °C). Yet their industrial application is limited mainly due to the low yield of the enzyme. To solve this issue several recombinant strains with enhanced yields are being considered globally [51, 52].

7.4.1.3 Hybrid Peroxidase Versatile peroxidases (VP, EC 1.11.1.16) are referred to as hybrid peroxidases as they exhibit the characteristics of both manganese and lignin peroxidases. These dual oxidases act on phenolic and non-phenolic lignin moieties without the need for mediators. They can oxidize the substrates

148 Lignocellulosic Biomass Production and Industrial Applications ranging from low to high redox potentials owing to the presence of hybrid structure with multiple substrate binding sites.

7.4.1.4 Phenol Oxidases Phenol oxidases are copper-containing proteins that catalyze the oxidation of aromatic compounds using molecular oxygen as terminal acceptor. These include three types of enzymes, catechol oxidase (EC 1.10.3.1) with o-diphenol oxidation and cresolase activity, monophenol monooxygenase (EC 1.18.14.1) with monophenol oxidation and laccase (EC 1.10.3.2) with ortho- and para-diphenol activity. Catechol oxidases contain copper (three) proteins that catalyze the oxidation of catechols to their corresponding o-quinones. Cresolases (o-hydroxylation of monophenols) are enzymes involved in secondary metabolism and act in the metabolic processes of phenolic compounds derived from the shikimate pathway; whereas monophenol oxidases are involved in the oxidation of catechols and monooxygenation of monophenols. All three enzymes differ in their substrate specificity and their relative amount produced varies depending on the source of the microbes. On the other hand, laccase (benzenediol:oxygen oxidoreductases) belongs to the family of multicopper oxidase that uses O2 as the terminal electron acceptor for oxidation of aromatic and non-aromatic compounds of lignin polymer. Based on the spectroscopic features, four copper ions of laccase are placed under three groups: type 1 (T1, blue copper), type 2 (T2, normal copper) and type 3 (T3, binuclear copper) where T2 and T3 together form a trinuclear cluster. Oxidation of the substrate occurs at T1 and electrons are then shifted to a trinuclear cluster, where reduction of oxygen and release of water molecule takes place [53]. Based on the electron paramagnetic resonance (EPR) spectroscopy, the three copper atoms are further classified as type 1, type 2 and type 3, where type 1 and 2 copper are EPR detectable, whereas type 3 copper is EPR undetectable. UV-visible spectrum of laccase shows peaks at 280 nm, 330 nm and 600 nm [54]. The absorption maxima for T1 and T3 are observed at 600 nm and 330 nm respectively in the case of blue laccases; while some other laccases exist that do not exhibit absorption maxima at 600 nm due to the absence of T1 copper and are referred to as yellow/white laccases [55]. Yellow laccase has been reported to have less than four Cu atoms whose absence is compensated for by substituting it with metal atoms such as manganese. Yellow laccases lack absorption maximum at 600 nm in the absorption spectrum but are EPR detectable. Ademakinwa and Agboola reported the presence of three copper atoms and one manganese atom in

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yellow laccase produced from Aureobasidium pullulans NAC8 through atomic spectrophotometry studies [56]. Absorption ratio at 280/610 nm was found to be 26, which was higher than that of blue laccase, which generally ranges between 15–20, thus referred to as yellow laccase. In the case of white laccase, different studies have been conducted in reference to the metal content of the enzyme. Palmieri and coworkers investigated two isoenzymes from Pleurotus ostreatus named POXA1 and POXA2, and found that POXA1 displayed different laccase characteristic not reported in blue and yellow laccase, which was referred to as white laccase. This newly identified laccase was observed to consist of one copper atom instead of four, along with two zinc atoms and one iron atom [57]. In another study, white laccase produced from Myrothecium verrucaria NF-05 showed copper and iron in the ratio of 3:1 and was found to be both UV visible and EPR undetectable [58]. Depending upon the source of laccase, redox potential may vary between 0.5 to 0.8 V, with the maximum (0.8 V) reported in whiterot fungi. Laccases with low redox potential are efficient in oxidizing only phenolic subunits where the oxidation of phenolic β-O-4 and β-1 lignin dimers occur, resulting in the formation of phenolic radicals. The dimers undergo either Cα-oxidations or cleavage through Cα-Cβ/aryl-alkyl linkage [59]. In order to oxidize the non-phenolic subunits having 1.5 V redox potential, blue laccases need a mediator which carries an electron to the enzyme, resulting in the formation of high redox laccase-mediator system that in turn oxidizes the non-phenolic subunits of lignin [60]. Mediators are organic compounds with low molecular weight and high redox potential (> 900 mV) that act as an electron shuttle. After the mediator is oxidized by laccase, generation of co-mediators occurs, which are strong oxidizing intermediates that move away from the enzymatic pockets and oxidize substrates that were unable to enter the active site. Some notable examples of mediators are 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 1-hydroxy benzotriazole (HBT), etc. [61]. On the contrary, yellow laccases do not require external mediators for the oxidation of non-phenolic subunits as it is reported that lignin-derived high redox radicals formed as intermediates during microbial fermentation tend to act as natural mediators by binding to the catalytic site of the enzyme and aid in non-phenolic lignin degradation [55].

7.4.1.5 Other Lignin-Degrading Enzymes Besides ligninases, some extracellular microbial oxidases and myceliumassociated dehydrogenases act as accessory enzymes for lignin degradation.

150 Lignocellulosic Biomass Production and Industrial Applications Oxidases are involved in generating H2O2 required for peroxidases, which include aryl-alcohol oxidase (EC 1.1.3.7) and glyoxal oxidase found in P. eryngii and P. chrysosporium respectively [62]. Mycelium-associated dehydrogenases, such as aryl-alcohol dehydrogenases or cellobiose dehydrogenases (under cellulolytic conditions), act synergistically along with quinone reductases in the presence of H2O2 for lignin degradation [63]. It is suggested that the action of cellobiose dehydrogenases on lignin is owing to the reduction of quinones that further aid ligninolytic enzymes such as manganese peroxidases in lignin degradation.

7.4.2

Carbohydratases

This group of enzymes are involved in the hydrolysis of carbohydrate polymers. The carbohydratases that specifically act on the holocelluloses of lignocellulosics include cellulase, hemicellulase, carboxyl esterases, including acetyl xylan esterases, feruloyl esterases and p-coumaroyl esterases.

7.4.2.1 Cellulase Agro-industrial residues and wastes are potent sources of cellulose and can support the growth of cellulolytic microorganisms for cellulase production. The sources of production include fungi, bacteria, protozoans, plants and animals. Cellulases hydrolyze β-1,4-linkages present in cellulose of lignocellulosic biomass. These contain non-catalytic carbohydrate-binding modules (CBMs) and cellulose-binding domains (CBDs), located either at the N- or C-terminus of a catalytic module. The classification of catalytic modules of the enzyme into various families was made based on their crystal structure and amino acid sequence [64]. Hydrolysis of cellulose is a combined effect of endoglucanases (EGs, EC 3.2.1.4), exoglucanases or cellobiohydrolases (CBHs) (EC 3.2.1.91) and β-glucosidases (BG) (EC 3.2.1.21). The role of endoglucanase is to randomly hydrolyze β-1,4-linkages of cellulose to generate new ends which are attacked by exoglucanases that act progressively on the reducing or nonreducing ends of cellulose, releasing cellodextrins/cellobiose by slowly decreasing the degree of polymerization of the substrate. Further, β-glucosidases hydrolyze these to glucose. Cellulolytic microorganisms utilize their cellulases as discrete complexed and noncomplexed cellulases [65]. In order to degrade cellulose, aerobic microorganisms secrete different cellulases, which contain CBM connected to catalytic module through a flexible linker peptide, whereas the anaerobic cellulolytic microorganisms produce cellulosomes which are

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multienzyme complexes [66]. Only a few of the enzymes in cellulosomes contain a CBM, but most of them are attached to the protein scaffold containing CBM. Apart from the above major group of cellulases, two other types, namely oxidative cellulases and cellulose phosphorylases, are also reported to be adopted for depolymerizing cellulose that differ in their mode of action. Oxidative cellulases are known to act on celluloses through generation of a series of free radicals following an oxidation-reduction mechanism; while cellulose phosphorylases are reported to attack cellulose using phosphates instead of water, unlike other cellulolytic enzymes. The preliminary product obtained upon the action of oxidative cellulases on cellulose is cellobionic acid, which is an aldonic sugar acid in nature arising from cellobiose units. Since the mode of action involves oxidative depolymerization of cellulose, the residence time may be significantly reduced and thus with the discovery of oxidative cellulases the shift from hydrolytic conventional cellulases to oxidative cellulases is reported [67]. In order to further improve the cellulase production, several strategies have been opted for: (1) directed evolution for cellulase, (2) rational design for cellulase and (3) the reconstitution of designer cellulosome or cellulase mixtures that are active on insoluble cellulosic substrates. These strategies are reported to yield higher reducing sugars with improved hydrolysis rate due to better cellulose digestibility [68]. It is reported that the overall cellulase production system is inducible and is found to be elicited in the presence of cellulose-rich substrates; but, to the contrary, it is repressed in the presence of excess glucose produced as the final hydrolysis product. Therefore, continuous reactor systems with the provision to sap the reducing sugars produced can subdue this problem. Also, a detailed study on the product inhibition kinetics of the enzymatic system may aid in maximizing the efficiency of the enzymes and thus result in higher reducing sugar yield.

7.4.2.2 Auxiliary Cellulose-Degrading Enzymes A new concept for cellulose depolymerization focuses on oxidative mechanism which involves cellobiose dehydrogenase (CDH, EC 1.1.99.18) and copper-dependent lytic polysaccharide monooxygenase (LPMO) produced by fungi. The CDH has heme-binding cytochrome (CYT) attached via a flexible linker to a flavin-dependent dehydrogenase (DH). The CDH and LPMO act synergistically for cellulose degradation, where the electrons generated through cellobiose oxidation upon the catalytic action of DH are shuttled via CYT to LPMO [69].  LPMOs are involved in the

152 Lignocellulosic Biomass Production and Industrial Applications oxidative cleavage of crystalline cellulose along with CBHs and Egs by using reducing agents like ascorbate, reduced glutathione, gallate and lignin fragments [70]. It employs several electron acceptors to catalyze the oxidation reaction of the reducing end of cellodextrins/cellobiose/other oligosaccharides to their corresponding lactones that are instantly converted to aldonic acids such as cellobionic acid. Along with LMPOs, CDHs are reported to enhance the depolymerization of crystalline cellulose, thus reducing its crystallinity [71]. LPMOs (copper oxidases) are advantageous over other cellulolytic enzymes since they are capable of attacking the highly crystalline regions in cellulose in contrast to endoglucanases, which are active on amorphous parts, while CBHs require a cellodextrin end in order to start the cleavage of crystalline cellulose [72]. Thus, with the advances in cellulolytic enzyme research an efficient cellulase system may be formulated that can synergistically act on cellulose for enhanced lignocellulosic sugar yield.

7.4.2.3 Hemicellulase Degradation of hemicellulose involves the synergistic action of endoxylanase (EC 3.2.1.8), β-xylosidase (EC 3.2.1.37), exoxylanses (exo-1,4β-xylanase, EC 3.2.1.37), glucuronidase (EC 3.2.1.139), α-galactosidase (EC 3.2.1.22), arabinofuranosidase (EC 3.2.1.55), α-L-arabinanase (EC 3.2.1.99), β-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), α-L-fucosidase (EC 3.2.1.51) and acetylxylan esterase (EC 3.1.1.72). Exoxylanses are xylan-degrading enzymes that belong to the GH8 family. Xylan consists of β-1,4-linked xylopyranosyl residues consisting of O-acetyl, α-L-arabinofuranosyl, α-1,2-linked glucuronic acid or 4-O-methylglucuronic acid units. The lateral side chains of xylan are linked with the D-galactopyranosyl, D-glucoronopyranosyl, feruloyl, L-arabinofuranosyl, acetyl and p-coumaroyl groups. The side-chain composition varies depending on the type of plant species (hard wood and soft wood) [73]. This structural diversity of xylan affects the degradation potentiality by enzymes, solubility and interactions with other polymeric cell wall materials. Therefore, for the depolymerization of xylan, enzymes which can cleave internal glycosidic linkages and break the branch point are necessary. Different types of branch point-degrading enzymes are acetylxylan esterase, α-D-glucuronidase and α-L-arabinofuranosidase. Endo-1,4β-xylanases randomly cleave the glycosidic bonds in xylan and yield xylo-oligomers. Exoxylanases hydrolyze β-1,4-glycosidic bonds of xylooligomers from the reducing and non-reducing ends, releasing xylobiose

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and xylooligosaccharides. β-Xylosidases act on these exoxylanase-derived products to produce xylose monomers. Depending upon the hydrolytic activities, substrate specificities, physicochemical characteristics and mode of action, xylanase from different sources can be categorized into different glycoside hydrolase (GH) families like GH7, GH8, GH10, GH11, GH26, GH30 and GH43. Among these, GH10 exhibits broader substrate specificity than the others. β-Xylosidases have high specificity towards unsubstituted xylose oligosaccharides and even express transxylosylation activity. Family GH30 enyzymes are single-domain enzymes which have methyl-glucuronosyl (MeGlcA) side residues and thus help to hydrolyze glucuronoxylan [74]. Another important polymer present in hemicellulose is β-mannan, which consists of glucomannan, galactoglucomannan and galactomannan. Galactomannan contains β-1,4-linked mannose residues which are branched with galactosyl residues. On the other hand, galactoglucomannan contains β-1,4-linked glucose and mannose branched with galactosyl residues. Thus, for complete degradation, different enzymes (endomannanases, α-galactosidases and β-mannosidases) are required. Endomannanases hydrolyze β-1,4-mannosidic bonds and belong to three different glycoside hydrolase (GH) groups, namely GH5, GH26 and GH76. Whereas α-galactosidases break the terminal α-1,6-linked D-galactosyl residues, β-mannosidases cleave β-1,4-linked mannose residues to release β-1,4-manno-oligomers such as mannobiose and mannotriose. The manno-oligomers are further acted upon by β-mannosidases for the liberation of mannose. Whereas the galactose substituents in the galactoglucomannans are cleaved by α-galactosidase, another enzyme, α-L-fucosidase, acts on O-glycosyl bonds present in the xyloglucans and releases L-fucose residues [75]. On the other hand, arabinofuranosyl-based hemicelluloses are hydrolyzed by the synergistic action of arabinofuranosidase (AFases) and α-L-arabinanases (ABNases). AFases specifically act on the terminal nonreducing arabinofuranosyl residues of arabinose-containing oligosaccharides/polysaccharides. AFases are further classified into three types based on their substrate specificity and mode of action. Type-A AFase acts on α-(1,5)-L-arabinooligosaccharides to degrade into arabinose, whereas type-B AFase acts on the debranching L-arabinose residues of the arabinoxylan/arabinan side chain. Both these types act effectively on synthetic substrate like p-nitrophenyl-α-L-arabinofuranoside. Whereas α-L-arabinofuranohydrolases, referred to as type-C Afase, act on arabinosidic linkages present in arabinoxylans of oat spelt, barely or wheat,

154 Lignocellulosic Biomass Production and Industrial Applications ABNases act on α-(1,5)-L-arabinofurano side linkages present within the arabinan polymer. Secretion of five arabinan-degrading enzymes, including two endoarabinanases, two AFases and one exo-arabinanase, by Pencillium chrysogenum 31B was reported by Sakamoto and Kawasaki [76]. From this study it was observed that AFases and ABNases act along with other hemicellulases for complete degradation of hemicellulosic backbone. Moreover, it is also reported that some cellulases also exhibit hemicellulolytic activity towards hemicelluloses. Endoglucanases belonging to family GH7 act on xylan, xyloglucan and arabinoxylan, while family GH5 endoglucanases act on mannans and galactomannans [77]. Xylanases from microbial sources are preferred for the hydrolysis of xylan because of their high specificity, mild reaction conditions and low substrate loss. Fungi, actinomycetes and bacteria are widely used for the production of xylan-degrading enzymes. Some examples of xylanolytic producers are Aspergillus sp., Bacillus sp., Clostridia sp., Fibrobacteres sp., Streptomyces sp., and Trichoderma sp., etc. Utilization of enzymes having broad specificity is beneficial for the biomass hydrolysis due to heterogeneity of lignocellulosics. Release of the side chains present on the main hemicellulose backbone through the synergistic action of different types of hemicellulases can ensure the complete degradation of hemicelluloses.

7.4.2.4 Expansins and Swollenins Expansins, a group of non-hydrolytic proteins, are believed to be involved in the modifications of cell wall during physiological processes, resulting in loosening and disruption of hydrogen bonds of the structural polysaccharides without solubilizing them [78]. Expansins are categorized into two major groups, namely plant expansins and bacterial expansins. Plant expansins are further divided into α-expansins that normally bind to cellulose enriched with xyloglucans [79] and β-expansins that specifically bind with arabinoxylans [80]. On the other hand, bacterial expansins bind to cellulose or whole cell walls depending upon their electrostatic behavior; however, their preferred substrate is cellulose [81]. Acidic expansin proteins are mainly encoded by Gram negative bacterial strains, whereas a majority of the basic expansin proteins are obtained from the Gram positive species [82]. The first plant expansin, referred to as Ex29/30, was isolated from cucumber and was reported to increase the production of both hexose and pentose sugars when used along with cellulases [83]. Similarly, there

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are various expansin-type proteins derived from fungi and nematodes that share similar structure and sequence as plant and bacterial expansin proteins [84]. The structure of expansin consists of two domains, D1 and D2, that are tightly bound together and are stabilized by ionic interactions and disulphide bonds [85, 82]. A shallow groove present on the protein surface consists of conserved residues that extend to D1 and D2 domains and form a binding site for polysaccharides. The D2 domain of expansin binds to the crystalline cellulose via aromatic residues [86]. There is relatively less information available regarding the interaction and mode of action of D1 domain with the substrate. Swollenins are a type of expansin-like proteins which were first isolated and characterized from T. reesei [87]. Swollenin is capable of swelling and weakening filter paper and cotton fibers without releasing any detectable amount of reducing sugars. Due to this property, attention has been shifted towards the application of swollenin in combination with cellulase for efficient hydrolysis of cellulosic biomass [88]. Further, it has been reported that swollenin does not cleave β-1,4-glycosidic linkages in cellulose, indicating its similarity with expansins in bulging the cellulosic networks of the cell wall. The role of swollenins in biorefineries is to increase the rate of enzymatic hydrolysis by improving the accessibility of the cellulases towards cellulose fibrils through dispersal of cellulose aggregations, thereby exposing it to cellulolytic enzymes [89]. The synergistic effects of plant expansins and microbial cellulases were first reported in 2001 [90]. Moreover, in a reaction mixture consisting of both expansin and cellulose, it is observed that expansin first binds to the accessible region of the cellulose and subsequently degrades it. From the inner side of the degraded cellulose, a new accessible area appears that is rapidly degraded alternatively by cellulase and expansin. Cellulose crystallinity is one of the major factors that governs the biomass digestibility. The highly ordered crystalline structure of cellulose is maintained by a number of inter- and intramolecular hydrogen bonds. According to the X-ray diffraction studies, Terinte et al. suggested the role of expansin in transformation of cellulose crystal structure and its degradation [91]. The crystal structure of cellulose transformed to amorphous form and was subsequently hydrolyzed by cellulases which in turn reduced the crystallinity of cellulose. The obtained spectra from the above study indicated that crystalline cellulose existed in the form of cellulose I. Transformation in the crystal structure of cellulose is confirmed by the change or shift in the peaks obtained during X-ray diffraction studies [92].

156 Lignocellulosic Biomass Production and Industrial Applications Kang et al. isolated swollenin from T. reesei and reported that the obtained protein was able to hydrolyze the crystalline cellulose (Avicel) into smaller molecules, significantly increasing the surface area and reducing crystallinity of the substrate and subsequently leading to higher adsorption of cellulase, which was confirmed through cellulase-binding assay. The maximum synergistic activity of swollenin in this assay was reported to be 51.5% at 48 h [93]. Thus, expansins and swollenins are promising non-hydrolytic proteins that can be used to strengthen the efficiency of cellulase for improved enzymatic hydrolysis of cellulosic biomass. In depth studies on the structure and functional relationship of expansins and swollenins may contribute to a better understanding of the underlying molecular mechanism that can gear up their application in the field of biomass hydrolysis and reduce the need for purified cellulase, making the lignocellulosic fuel production cost-effective.

7.4.2.5 Carboxyl Esterases Acetyl and feruloyl side-chain removal from hemicellulose is mainly achieved by acetyl xylan esterase (AXE, EC 3.1.1.72) and feruloyl esterase (FE, EC 3.1.1.73) respectively. These enzymes are together known as carboxyl esterases that are reported to be produced along with xylanases by fungi. They are usually found to synergistically act along with xylanases to assist them in overcoming the stearic hindrance from hemicellulose side groups. AXE specifically cleaves the acetyl groups present at 2nd or 3rd oxygen in the glucuronoxylan backbone, whereas FE selectively cleaves the ester bonds of hydroxycinnamoyl derivatives, including feruloyl esters present as side groups at 2nd or 5th oxygen of α-L-arabinose or 6th oxygen of β-galactose moieties [94]. FE plays a significant role in the breakdown of ester bonds present within hemicelluloses and also between hemicellulose and lignin by targeting the key bridging moieties, i.e., ferulic acid ester groups. This enzymatic action results in increased surface area of the lignocellulosic biomass for further interaction with endoxylanases and β-xylosidases and thus enhances the overall reducing sugar yield. These esterases are extracellularly produced on hemicellulose-rich biomass and might decrease the pH of the medium due to the release of carboxylic acids, such as acetic acid, ferulic acid and other sugar acids, namely gulonic acid, as end products [95]. The microbial sources for carboxyl esterase production include the basidiomycetes, namely Schizophyllum commune, A. niger, P. chrysosporium, A. awamori, A. tubingensis, Penicillium funiculosum, Neurospora crassa, etc.

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7.4.2.66 Zymase Zymase is a complex of enzymes that catalyze the fermentation of sugars to ethanol and carbon dioxide. This is a naturally occurring enzyme complex varying in its activity according to its presence in different yeasts. Zymase loses its strength outside the cell due to the oxidation and partly due to the tryptic enzyme present in the yeast that has destructive properties towards zymase [96]. Zymase has the ability to readily convert sugars, such as maltose and sucrose, into ethanol, while galactose is not easily fermented. The sugars that are directly fermentable by yeast are dextrose, D-galactose, D-mannose and levulose (a keto-hexose) [97]. Zymase is a complex of many enzymes, such as invertase, that converts polysaccharides to hexoses, hexogenase which has the ability to convert glucose into glucose-6-phosphate, zymohexase which acts on hexose to give two trioses, isomerase that converts glucose into fructose, phosphatase which releases phosphoric acid from organic phosphates, triosedehydrogenase that oxidizes phosphoglyceraldehyde into phosphoglyceric acid, mutase that alters the location of phosphoric radical within the phosphoglyceric molecule, enolase that imparts energy to the carbon atom linked to phosphoric radical and carboxylase that separates carbohydrates without the addition of water. Zymase is composed of two elements, namely a dialyzable and thermostable element, and the other a nondialyzable and heat-labile element [98]. The nondialyzable element, also referred to as inactive residue, does not possess sugar fermentation ability while the dialyzable liquid is known as co-ferment. The union of these two elements is essential for fermentation to take place. This enzyme complex has a tertiary structure and is inhibited in the presence of weak acids. It acts best in alkaline medium and it can be easily denatured by heating at high temperature (55 °C). It is also reported that zymase activity is enhanced in the presence of phosphates of sodium and potassium. In first generation bioethanol production, sucrose is one of the disaccharides which is fermented using commercial yeast. In this process, invertase which is present in the yeast hydrolyzes the disaccharide into glucose and fructose. These monomeric sugars are then acted upon by other components of zymase, converting them into ethanol and carbon dioxide [99]. In a similar manner, starch is also used in the first generation ethanol production process where with the help of diastase starch is converted into maltose. This sugar in turn is acted upon by maltase that converts maltose into glucose, which is subsequently attacked by zymase to convert it into ethanol and carbon dioxide [100].

158 Lignocellulosic Biomass Production and Industrial Applications In 1920, Guilliermond reported that the concentration of sugars plays a crucial role in the activity of zymase. It was observed that the rate of fermentation increased with the increase in sugar concentration (up to 25%), which in turn suppressed the antagonistic endotryptic action and enhanced zymase activity, improving the overall fermentation. Fermentation of glucose to alcohol and carbon dioxide was initially theoretically assumed to occur in two-phases such that in the primary phase zymase converted glucose into lactic acid and in the secondary phase another enzyme, lactacidase, converted lactic acid into ethanol and carbon dioxide. But this theory was later negated as this process of lactic acid to ethanol conversion mediated by lactacidase could not occur in natural yeasts due to the lack of lactacidases. Another separate theory stated that alcohol fermentation took place in two different phases where the glucose moieties are first converted to dioxyacetones which are acted upon by another enzyme referred to as dioxyacetonase to form ethanol and carbon dioxide. However, formation of dioxyacetone during fermentation process is not yet established. To the contrary, a third theory was framed for zymase mediated alcohol fermentation according to which glucose is first decomposed into two molecules of dioxyacetone, which are then converted into two molecules of phosphoric ether upon combining with two phosphate groups. These phosphoric ethers are then assumed to combine with one hexose phosphate to finally yield ethanol, carbon dioxide and phosphate. Nevertheless, this assumption was later considered invalid. With the advancement in alcohol fermentation research it was found that zymase-mediated ethanol formation involves the conversion of glucose into two pyruvic acid molecules which are in turn converted into two acetaldehydes by pyruvate decarboxylase, followed by their subsequent conversion into two ethanol molecules catalyzed by alcohol dehydrogenase. This theory was globally accepted and is still considered valid to date [101]. Zymase directly influences the fermentation efficiency. This was proved by Angelov and coworkers who conducted a study on Saccharomyces cerevisiae strains by inducing mutation with the help of ethyl methanesulfonate and checked their fermentation activities. Among the 14 mutated strains generated, two strains showed an increase in their fermentation activity by 14%. Further investigation on the maltase and zymase activities showed that strain 3 depicted high maltase activity while strain 10 showed higher zymase activity. The strain with high zymase function showed maximum carbon di-oxide accumulation and improved fermentation rate. This study substantiates the potential of this strain having higher zymase activity for enhanced ethanol production [102].

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Nevertheless, in-depth studies at the molecular level emphasizing the pathways of fermentation and the different intermediates involved in inducing or repressing zymase can help in the development of the present glycolysis pathway and alcohol formation. This cell-free extract in combination with holocellulases and cell wall modifying proteins, such as expansins and swollenins, can improve the lignocellulosic biomass depolymerization and enhance ethanol yield. Biodiversity of carbon-rich renewable sources is high. In this context, depending on the nature of the raw material and whether it is starchy or lignocellulosic feedstock, the initial steps before fermentation may vary. Lignocellulosics need pretreatment/delignification of biomass prior to saccharifcation, whereas starch-rich first generation feedstocks do not contain recalcitrant lignin and thus do not require pretreatment. However, irrespective of the source employed, ranging from pure glucose, sucrose, molasses, sugar beet, sugarcane, cellulose, holocellulose, edible/non-edible lignocellulosic residues, etc., yeast fermentation follows the same universally accepted pathway mediated by the action of zymase and associated enzymes for ethanol generation. Owing to the positive role of zymase cell-free extract in ethanol fermentation, an opportunity has been opened up for its utilization in biorefineries for fermenting sugars derived from various sources, including lignocellulosics, for improved ethanol yield.

7.5

Applications in Biofuel Production

Production of biofuels from lignocellulosic biomass containing high energy is a viable alternative to petroleum-based fuels. A range of biofuels, such as solid, liquid and gaseous biofuels, can be produced from biomass through thermochemical or biological methods. The rising cost of petroleum-based fuels is the prime motive behind the shift in focus towards biofuels such as bioethanol, biomethane, biodiesel, etc. Apart from biofuels, other biobased products, such as biomanure/biofertilizer, as value-added deliverables may be produced from the residues obtained from the bioethanol and biomethane production process, leading to complete utilization of the biomass. This chapter focuses on the role of different enzymes in the generation of various biobased products from lignocellulosic biomass.

7.5.1

Bioethanol

Lignocellulosic biomass is an inexpensive, abundantly available and sustainable source for large-scale ethanol production in developing countries.

160 Lignocellulosic Biomass Production and Industrial Applications The high octane number, flame speed and heat of vaporization of ethanol enables high blend (85%) with gasoline. Lignocellulosic bioethanol production includes three major steps, viz., pretreatment/delignification, saccharification and fermentation. The pretreatment is accompanied by the breakdown of lignin barrier for the recovery of holocelluloses which are the target molecules for bioethanol production. Various pretreatment methods, such as physical, chemical, physicochemical and biological methods, are implemented for lignin removal/degradation. Laccase-mediated pretreatment is advantageous owing to its substrate specificity, meagre inhibitor formation, less requirement for water, efficiency in working under mild conditions and eco-friendliness. Apart from laccases, several other ligninolytic enzymes, such as Mn-dependent peroxidase (MnP) and lignin peroxidase (LiP), find their application in detoxification of chemically pretreated biomass. Carbohydrate polymers of the lignocellulosic biomass, i.e., cellulose and hemicellulose, are transformed into monomers of glucose and xylose by the effective action of cellulase and xylanase through the saccharification process. The recovery of fermentable sugars can be enhanced by employing an enzyme mixture consisting of key carbohydratases along with some accessory enzymes, thereby facilitating the complete utilization of lignocellulosic biomass. The reducing sugar obtained from the saccharification process is converted into bioethanol in the presence of yeast/bacteria during fermentation. Apart from separate hydrolysis and fermentation (SHF), various other fermentation strategies, such as simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF) and consolidated bioprocessing (CBP), are implemented to achieve maximum ethanol yield in a short incubation time. Microbial Biotechnology and Downstream Processing Laboratory, IIT Kharagpur, optimized an enzymatic method for bioethanol production from various non-edible lignocellulosic biomasses. Laccase produced from P. djamor and Lentinus squarrosulus MR13, cellulase and xylanases produced from T. reesei RUT C30 were used for pretreatment and saccharification of the feedstocks respectively. Kuila et al. reported 88.79% delignification of laccase-treated Lantana camara, with subsequent release of 713.33 mg/g (dry substrate) reducing sugar and 9.63 g/L ethanol after saccharification and fermentation processes respectively [103]. Yanase et al. reported a genetically engineered Kluyveromyces marxianus with endoglucanase (from T. reesei) and β-glucosidase (from A. aculeatus) activity on its surface that converted cellulosic β-glucan into ethanol at 48 °C with 92.2% of theoretical ethanol yield [104].

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161

Biomethane and Biomanure

Anaerobic digestion is one of the cost-effective bioconversion technologies commonly implemented for the commercial production of biogas from the organic material of the substrate. Biomethanation process includes three stages, namely hydrolysis, acetogenesis and methanogenesis. Hydrolysis of various components of biomass, such as lignin, cellulose and hemicellulose, has a profound influence on the yield of biogas/biomethane and requires the intervention of enzymes, such as laccase, cellulases and hemicellulases, for the degradation of lignin and conversion of complex carbohydrate polymer into monomers (hexose and pentose). The monomer units are acted upon by the acidogens and acetogens for the production of fatty acids and alcohols, which are further converted into methane by methanogens. Zieminski et al. reported the effect of enzymatic pretreatment (with mixture of celustar XL and agropect pomace [3:1, v/v], endoglucanase, xylanase and pectinase) prior to methane fermentation for improved biogas production from sugar beet pulp and spent hops [105]. The reducing sugar concentration in hydrolysates of spent hops and sugar beet pulp was reported to be 59.4% and 88.9% higher than the undigested substrates with biogas yield of 121.47 mL/d and 183.39 mL/d from spent hops and sugar beet pulp hydrolysates respectively. Kumar et al. reported biomethane production (yield 48.8–72.45%) from pineapple waste using a newly isolated anaerobic consortia substituting cow dung, where lignin present in the substrate was degraded by laccase-mediated delignification process [106]. The solid residue obtained after ethanol and biomethane production contains several nutrients which upon further enrichment can be effectively utilized as biomanure/biofertilizer. The enriched residue acts as an excellent source for essential nutrients vital for plant growth and humus to develop the soil structure [107]. There are various organisms and techniques that effectively carry out the enrichment process, among which the well-known vermicomposting approach is the one that is mostly followed. Kale reported an extensive usage of an exotic and epigeic earthworm, Eudrilus eugeniae, for vermicomposting of organic wastes in India. During this process, earthworms feed upon organic wastes and utilize them partially for their growth and metabolism; but the major portion of the ingested material is excreted out in half-digested form. This is further treated with digestive enzymes, such as cellulase, amylase, lipase, protease, urease, chitinase, etc., as well as by cellulase and mannase containing microorganisms present within the intestines of earthworms to transform them into vermicompost within 4–8 weeks. Vermicompost consists of a

162 Lignocellulosic Biomass Production and Industrial Applications plethora of microorganisms, namely Actinomycetes sp., Azotobacter sp., Nitrobacterr sp., Rhizobium sp., phosphate solubilizing bacteria (PSB), etc., in the range of 102–106 per gram of vermicompost [108]. Some mushroom fungi, such as Lentinus conatus and Pleurotus sojarcaju, were found to enrich the nutrient content of organic residue owing to their ability to degrade the residue using cellulases and laccases secreted by them. For instance, L. conatus was found to be effective in degrading coir residues, paddy straw, sugarcane bagasse, etc., which led to the decrease in the C/N ratio and increase in macro- and micronutrients of these lignocellulosics within 30 days. The soil application of L. conatus enriched coir residue as biomanure to the rice planted fields to the tune of 15.5 tonnes/ hectare supplemented with 60 kg nitrogen/hectare in the form of urea was reported to reduce the intensity of sheath blight disease and increase the rice yield [109]. Cyanobacterial mediated enrichment of the waste residue is another approach for biomanure production. This enriched residue may serve as an eco-friendly alternative to the chemical fertilizers to improve productivity. Chintagunta et al. studied the nitrogen, potassium and phosphorous (NPK) enrichment of potato wastes using various cyanobacterial strains, namely Nostoc muscorum, Fischerella muscicola, Anabaena variabilis, Aulosira fertilissima, Cylindrospermum muscicola and their concoction. It was observed that maximum enrichment was achieved using A. variabilis [110]. The enrichment of nitrogen content of the residue by cyanobacterial strains may be due to the presence of heterocyst, which is known to play a crucial role in nitrogen fixation. On the other hand, conversion of immobilized and insoluble potassium and phosphorous into their soluble forms by the action of organic acids, such as oxalic, succinic, oxaloacetic, tartaric and lactic acid, released by the action of cyanobacteria may be responsible for potassium and phosphorous enrichment. This strategy of residual biomass enrichment for the production of biomanure/biofertilizer as a value-added product may be considered as an ultimate step in the biorefineries to utilize the substrate completely. This effective mode of waste management renders the overall lignocellulosic ethanol production process as a zero waste generating and environmentally safe process.

7.6

Conclusion

Bioenergy generated from biofuels is capable of sustaining the rising demand for energy with a check on greenhouse gas emissions, resolving

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the issues pertaining to the import of fossil fuels and their escalating prices due to their depletion. The vital factor to be considered when developing bioenergy as an alternative form of vehicular fuel is the availability of sufficient raw material to cater to the demand. In this aspect, lignocellulosic biomass has a major role to play due to its surplus availability. Safer and greener production of fuels using lignocellulosics involves the use of a group of industrial enzymes. Increasing awareness and other environmental issues have triggered the world to move towards biological and ecofriendly products and technologies and thus enzyme-based processes are currently in the limelight in the biofuel sector. In order to meet the demand for industrial enzymes, microbes are the most reliable and viable sources. Microbial enzyme production coupled with lignocellulosic biomass as raw materials can reduce the overall cost of production and strengthen the whole essence of replacing chemical- or physical-based technologies in biorefineries.

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Kazlauskas, R.J. (Ed.), pp. 361–389, John Wiley & Sons, Inc.: Hoboken, New Jersey, 2011. Moreno, A.D., Ibarra, D., Mialon, A., and Ballesteros, M., A bacterial laccase for enhancing saccharification and ethanol fermentation of steam-pretreated biomass. Fermentation 2, 1–15, 2016. Bowden, A.C., New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge, Cornish-Bowden, A. (Ed.), Universitat De Valencia, 1997. Simmonds, C., Alcohol: Its Production, Properties, Chemistry, and Industrial Applications, Macmillan and Co. Ltd: London, 1919. Ellis, W.C., and Conde, J.C., Fermentation of sugar to ethyl alcohol in the presence of proteolytic enzymes, US Patent 3093548 A, assigned to Central Anejadora Guatemalteca, 1963. Baeyens, J., Kang, Q., Appels, L., Dewil, R., Lv, Y., and Tan, T., Challenges and opportunities in improving the production of bio-ethanol. Prog. Energy. Combust. Sci. 47, 60–88, 2015. Palanna, O.G., Engineering Chemistry, Tata McGraw Hill Education Pvt. Ltd: New Delhi, 2009. Guilliermond, A., Yeasts: Culture, Identification, and Microbiology, pp. 91–95, Watchmaker Publishing, 2003. Angelov, A.I., Karadjo, G.I., and Roshkova, Z.G., Strains selection of baker’s yeast with improved technological properties. Food Res. Int. 29, 235–239, 1996. Kuila, A., Mukhopadhyay, M., Tuli, D.K., and Banerjee, R., Production of ethanol from lignocellulosics: An enzymatic venture. EXCLI. J. 10, 85–96, 2011. Yanase, S., Hasunuma, T., Yamada, R., Tanaka, T., Ogino, C., Fukuda, H., and Kondo, A., Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes. Appl. Microbiol. Biotechnol. 88, 381–388, 2010. Zieminski, K., Romanowska, I., and Kowalska, M., Enzymatic pretreatment of lignocellulosic wastes to improve biogas production. Waste. Manage. 32, 1131–1137, 2012. Kumar, M., Jacob, S.B., Lakshmishri, U., and Banerjee, R., Biomethanation of pineapple wastes using potent anaerobic consortia substituting cow manure. Environ. Eng. Manag. J. 2014. Bhattacharyya, B.C., and Banerjee, R., Environmental Biotechnology, Oxford University Press: USA, 2007. Kale, R.D., in: Earthworm Cinderella of Organic Farming, g Prism Books Pvt. Ltd: Bangalore, 48, 1998. The Hindu, 2003. www.thehindu.com/seta/2003/12/11/stories/20031211 00321700.htm (accessed on 13th September 2016). Chintagunta, A.D., Jacob, S.B., and Banerjee, R., Integrated bioethanol and biomanure production from potato waste. Waste Manage. 49, 320–325, 2016.

8 Sugarcane: A Potential Agricultural Crop for Bioeconomy through Biorefinery Knawang Chhunji Sherpa1, Rajiv Chandra Rajak1 and Rintu Banerjee1,2* 1

Advanced Technology Development Center, Indian Institute of Technology, Kharagpur, West Bengal, India 2 Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur, West Bengal, India

Abstract Sustainability remains one of the critical issues highlighted in the topic of availability and utilization of biomass. There is an urge to explore alternatives to improve the quality of crops and utilize every part of the plant for food, fossilbased fuels and chemicals. Among the different alternative energy sources, biomass is a potential candidate for such a dualistic application. Comprehensive yet careful biomass exploitation is therefore essential. In this perspective, sugarcane (Saccharum officinarum), an efficient energy crop of the grass family grown in tropical and subtropical regions of the world, is considered as an energy cane. Since every part of the sugarcane is useful, it creates an opportunity for the people of sugarcane producing countries in terms of their development and economy. This chapter highlights the growth, production and integrated scheme of using both products and by-products of sugarcane that can further improve the economics of the process. Keywords: Biomass, Saccharum officinarum, biofuels, biochemical

8.1

Introduction

Sugarcane is an efficient crop that can be used for biorefinery feedstock. It is a major C4 crop grown in the tropical and subtropical areas of the

*Corresponding author: [email protected] Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial Applications, (171–196) © 2017 Scrivener Publishing LLC

171

172 Lignocellulosic Biomass Production and Industrial Applications world. Brazil is the largest producer of sugarcane with India taking second place in the world. In 2013–14, sugarcane accounted for 6% of the overall value of agriculture output and covered 2.5% of India’s gross cropped area. Sugarcane production in 2013–14 increased from the previous year to 348.4 million tonnes [1]. Sugarcane is mainly grown for its sucrose and consumes about 92% of sugarcane for sugar production [2]. The sugar industry in India is the second largest agro industry, second only to the textile industry. The sugar industry plays a vital role in developing the socioeconomic status in rural areas by making use of its resources and generating employment. Having a well-established agricultural production system and infrastructure of processing; it is one of the most advanced feedstocks that can be used for sugar production as well as for bioenergy. Besides being in demand for sugar, sugarcane also has various other purposes such as in the paper and pulp industry, as animal fodder and for biofuels. In comparison to the petroleum-based fuels, sugarcane releases less carbon dioxide and has an overall net energy balance [3]. Huge amounts of lignocellulosic residue, such as bagasse and trash, are generated. During the harvest, sugarcane stalks are taken to the sugar mills for juice extraction, generating bagasse as the residue and the trash is generally left in the field as waste during harvest. The trash of sugarcane usually consists of green leaves, dry leaves and tops [4]. In a sugar mill, 100 tonnes of sugarcane on average is able to produce 10 tonnes of sugar, subsequently producing 4 tonnes of molasses that is used for ethanol production. Along with molasses, 3 tonnes of press mud is produced that is used as biofertilizer and 30 tonnes of bagasse is produced which is utilized for electricity generation of 1500 KW and also used in the paper and pulp industry. During the harvesting of 100 tonnes of sugarcane, approximately 30 tonnes of sugarcane tops and leaves are left in the field that can also be utilized for various purposes, adding to the economy [5]. In India, there are about nine states where sugarcane is grown on a large scale. Among these, Uttar Pradesh occupies the highest total area of 2.25 m ha with production of 134 MT in terms of productivity. There are a number of sugarcane varieties that are grown according to the soil properties and climatic conditions. This chapter thoroughly discusses the different practices for the growth of sugarcane, area of production, and its productivity. It also sheds light on the potential of sugarcane as an energy crop to not only produce sugar but also other by-products such as biofuels and other biobased chemicals. This chapter addresses the current performance and adaptation of other

Sugarcane: A Potential Agricultural Crop for Bioeconomy

173

technologies through techno-economic analysis that can increase the performance on a longer term, benefitting the economy and the environment. It gives an insight into how well we can completely utilize sugarcane without wasting a single part and making the most of it.

8.2

Present Status of Sugarcane Production and its Availability

Brazil has remarkable historical development in sugarcane production. The sugarcane production in Brazil was not controlled by colonial powers but significantly forced by domestic policies to make self-reliance on ethanol production and to reduce the import of petroleum products. In 2013, the Food and Agriculture Organization (FAO) of the United Nations disclosed that the global production of sugarcane was 448 MT during the 1960s [6]. After five decades, the sugarcane production for the year 2011 indicates a 1.75 times enhanced output. The global production of sugarcane hit the level of 1800 MT with average productivity of 71 tonnes per hectare. In a nutshell, from 1961–2012, the global production of sugarcane was increased by nearly four times. The average global production of sugarcane during this period was also enhanced from 50 to 71 tonnes per hectare. The global sugarcane production in the year 2014/2015 reached 175.1 MMT [7]. Sugarcane has been cultivated widely in more than 100 countries around the world. According to the FAO database there are 12 major countries contributing the maximum towards the production of sugarcane. It is apparent that the ranks for global sugarcane production vary from decade to decade. Prior to the 1980s, India ranked first in terms of sugarcane production, and after the 1980s Brazil became the leading producer of sugarcane followed by India. From the perspective of availability, 40% of global sugarcane production is contributed by Brazil alone, followed by 20% by India and 6% by China. Among the 12 main countries for sugarcane production, Indonesia, Philippines, and Cuba showed a decreasing trend in sugarcane production. During 1961–2011, the average productivity of sugarcane increased from 47 to 71 tonnes/hectare by the leading sugarcane producing countries. The improved productivity was due to advancements in the research and development sector and application of high yielding sugarcane varieties. Technological advancements and governments added suitable policy changes that affected the socio-economic status of sugarcane farmers in diff ferent countries. Moreover, the production of biofuels, such as bioethanol,

174 Lignocellulosic Biomass Production and Industrial Applications has played a major role in the sugarcane production in Brazil. The Brazilian supremacy in global sugar, ethanol and energy production was responsible for the huge expansion of sugarcane-based agro-industrialization during the first decade of the 21st century. In India, there has been a rise in sugarcane cultivation over the area of 5.06 million hectares (MH) in 2015–16 compared to 4.9 MH in 2013–14. Sugarcane production in the year 2014–15 was reported to be 347.00 MMT by the Ministry of Agriculture, which is estimated to increase to around 350 MMT for the year 2015–16 [8].

8.3

Morphology of Sugarcane

The morphology of sugarcane includes its stem, root and leaf parts. Sugarcane is a tropical grass having multiple stems consisting of nodes divided by internodes. Upon germination, the vegetative bud present at the terminal position of each shoot set has a number of nodes. The node consists of a dormant bud, growth ring, and root primordial [9]. The sugarcane stem is similar to the stems of sorghum and maize in that it is not hollow as observed in grasses [10]. The leaf appears on the node as the stem develops and forms two alternate divisions on each side of the stem. Apical meristem is present on the top of the stem. The mature stem contains a number of young and senescent leaves and the number increases with the age of the plant. New leaves appear and expand between two to three weeks. The length of the internode reaches over 30 cm based on the growth conditions and a two to three meter increase in stem length is observed during a normal season [11]. The leaves of the sugarcane possess blades containing hairs on the underside of the leaf and without hairs on the upper side of the leaf. Similar to other grasses, the C4 pathway of photosynthesis was observed in sugarcane and, consequently, the leaf anatomy reveals this underlying C4 physiology. The lower portion of the leaf attaches to the node of the stem and wraps around the stem to form a sheath that loosely surrounds the internode from which the node extends [9, 10]. The node contains intercalary meristem, the root band contains root primordia and a bud on top of the leaf scar to which the leaf sheath attaches. The root system of sugarcane is fibrous and shallow, similar to grasses. However, sugarcane develops buttress roots to attach the plant beneath the soil and during water stress, deep penetrating roots develop downwards about 5–7 meters to absorb water [9].

Sugarcane: A Potential Agricultural Crop for Bioeconomy

8.4 8.4.1

175

Factors Involved in Sugarcane Production Climatic Conditions

8.4.1.1 Temperature Sugarcane thrives best in tropical and subtropical regions with a long, warm growing season and adequate moisture. A moderately dry and sunny season without frost is required for ripening and harvesting. Temperature required for germination of stem cuttings is 32–38 °C, beyond which the rate of photosynthesis decreases and respiration increases. High temperature also leads to low sugar content due to reversion of sucrose into fructose and glucose. During ripening, a temperature range of 12–14 °C is desirable since it helps in increasing the sucrose content. At low temperature, bud sprouting is inhibited and cane growth is seized. Young leaves and lateral buds get frozen when temperature falls below 0°C. Temperature also plays an important role in the spread of sugarcane disease, such as smut, that spreads at favorable temperatures of 25–30 °C [12].

8.4.1.2 Rainfall and Relative Humidity Rainfall in the range of 110–150 cm is adequate during its vegetative growth and humidity of 80–85% supports cane elongation during its growth. For obtaining maximum yield, ample moisture should be available throughout the growing period since cane growth is directly proportional to transpiration. Water deficit leads to reduction in its yield by slowing down germination and rate of stalk elongation. During the ripening period, water deprivation of sugarcane leads to sugar content loss in comparison to the sugar being formed.

8.4.1.3 Sunlight Sugarcane being a C4 plant, it is an efficient photosynthesizer that is able to convert up to 2% of incident solar energy into biomass. Regions that receive solar energy from 18–36 MJ/m2 are favorable for the growth of sugarcane since it can adapt to high photosynthesis with high saturation range with respect to light. Growth of sugarcane stalk depends on the duration and intensity of light the plant is exposed to. Plant exposed to daylight in the range of 10–14 hours is favorable for the growth of stalk. Long days of sunlight give thick and short sugarcane with high sucrose content while short daylight produces thin and long sugarcane.

176 Lignocellulosic Biomass Production and Industrial Applications

8.4.2

Soil Quality

Sugarcane can grow on a variety of soils ranging from loamy soils to heavy clays and even laterites. Maintenance of soil conditions is necessary for providing proper nutrients, water and anchorage to the plants so as to have a good yield and quality of sugarcane. For ideal sugarcane cultivation, a well-drained deep loamy soil of bulk density 1.1–1.2 g/cm3 with adequate balance between pores of the soil and 15% of water holding capacity is necessary. Sugarcane can grow in the pH range of 5–8.5 with an optimum pH being 6.5. With such a wide range of pH, the plant is able to grow in soils that are acidic or basic in nature. Testing of soil is necessary before cultivation for determining the quality of soil. Improving soil quality can be done by applying lime or gypsum in case the pH of the soil is too acidic or too basic. Testing of soil before cultivation of sugarcane is vital to determine the ideal amount of micro- and macronutrients that is to be applied in the field for high productivity.

8.4.3

Varieties of Sugarcane

Sugarcane variety is an important factor for considering the yield of sugarcane, which encompasses high sucrose content, age of the plant, plant suitability to grow under different conditions, resistance to pest and disease and ratooning capability. Since sugarcane grows under a wide range of soil and climatic conditions, different varieties of sugarcane can be used according to their suitability. Variety is an important factor for good productivity; therefore, sugarcane varieties are continuously being developed and improved upon in sugarcane breeding institutes and R&D centers so as to elicit the maximum expression of yield potential influenced by these varieties. After sugarcane attains 16% sucrose and 85% cane juice purity it is considered to be ready for harvest. The different varieties developed are broadly classified as early, midlate and late maturing types, depending on the rate of their maturity that corresponds to 12, 14 and 16 months respectively. Classification based on maturity of varieties is done to enable sugarcane variety harvesting at an appropriate time so as to get maximum yield. Yield per hectare, sugar recovery, increased tillering capacity, stalk dimensions, and ratooning ability are some of the factors to be considered while developing a new variety. Sometimes during variety development defects occur such as vulnerability towards disease, affinity for lodging, cavity formation, heavy pith formation or growth of spines on leaf sheath. Sugarcane varieties are given names according to the countries where they were developed such as India – Co;

Sugarcane: A Potential Agricultural Crop for Bioeconomy

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Brazil – CB, IAC, PB, RB, SP; Argentina – NA; Egypt – E, etc. Some of the different varieties used in the world are SP-77–5181, RB-85–5453 (Brazil), CoS.687, Co.7527 (India), 91–2-29, K92–181 (Thailand), etc. There are a number of varieties in India that have been developed according to the suitability of the region. Some of the varieties that are recommended for commercial use are given as in Table 8.1. The different states of India are divided into four different zones with respect to sugarcane cultivation: North Central Zone (NCZ), North West Zone (NWZ), East Coast Zone (ECZ) and Peninsular Zone (PZ). Sugarcane varieties have special characteristics which make them favorable to grow in a particular zone. Table 8.2 shows some of the specific characteristics of the varieties which are favorable to their growth in a specific zone.

8.4.4

Land Requirement

Preparation of land is an important factor for the production of sugarcane. A proper area for land utilization is necessary for easy planting, improved transportation, pest and weed control and reduction in steps during harvesting that can help reduce the cost and pollutants [14]. A well-planned method is to be followed that takes into consideration the shape of the land with a design of the tracks and quality of the soil of that particular area. Land preparation is done so as to keep good soil bed for early proliff eration of plants and to assist in proper soil chemical and microbial activity. Rectangle-shaped plots are recommended for sugarcane cultivation since these plots evade sharp turns and lessen maneuvering, thus saving time and fuel. When preparing the land for cultivation any previous residue should be removed from the field, followed by tillage using tractors for a quick and easy operation. Tillage is conducted to keep the soil free from any leftovers of previous plants or weeds, followed by deep ploughing of the field for facilitating proper aeration and penetration of water into the soil. While ploughing, it should be kept in mind that the land should be leveled. Furrows are prepared with a depth of 25 m and 75 cm apart for early maturing variety and 90 cm apart for medium maturing variety.

8.4.5

Propagation

In subtropical states sugarcane takes a year to mature and is called Eksali in the local language, while the sugarcane that takes 18 months to mature  in tropical states is called Adsali. Generally, the planting season for sugarcane in subtropical areas is September-October and in the

State

Uttar Pradesh

Maharashtra

Tamil Nadu

Bihar

West Bengal

Orrisa

Punjab

Haryana

Rajasthan

Karnataka

S. no.

1

2

3

4

5

6

7

8

9

10

Co 6415, Co 7704, CoC 671, Co 85002

Co 29, Co 997, Co 527, Co 6617

Co 89003, Co 7717, CoJ 64

CoS 8436, Co 89003

Co 7508, Co 7704, Co 62175, Co 740

BO 90

BO 90, BO 99, BO 102, BO 120

Coc 671, CoC 8001, Coc 85061, Co 7704, Co 8208, CoC 92061, CoC 90063

Co 419, Co 775, Co 7219, CoC 671

CoS 687, CoS 8436, CoS 88230, CoS 95435, CoSe 91232

Early maturing type varieties

Table 8.1 Different varieties of sugarcane grown in different states of India [13].

Co 62175, Co 740, Co 8014, Co 8021, Co 8011, Co 8371, Co 7804, Co 86032

Co 1253, Co 419, Co 1007, CoJ 111, Co 449, Co 527

Co 1148, CoS 767

CoJ 67, CoJ 83, CoJ 84

Co 7219, CoJ 8201, Co 975, Co 7706, Co 8402, Co 62175

Co 1148, Co 7224

BO 104, CoS 767, BO 109, BO 89, BO 91, BO 106, BO 108, Co 1148

Co 6304, CoSi 776, CoSi 86071, Co 8021, Co 85019, Co 86032, Co 86010, Co 86249, CoSi 95071, CoSi 96071, CoSi 98071, CoG 93076

Co 740, Co 7219, CoM 7125, Co 7527, Co 86032

Co 1148, CoS 767, BO 91

Mid-late and late maturing type varieties

178 Lignocellulosic Biomass Production and Industrial Applications

Variety name

Co 85004 (Prabha)

Co 86032 (Nayana)

Co 86249 (Bhavani)

BO 128 (Pramod)

CoSe 95422 (Rasbhari)

CoH 2201 (Haryana-92)

CoS 1230

S. no.

1

2

3

4

5

6

7

NWZ

NWZ

NCZ

NCZ

ECZ

PZ

PZ

Favorable area of growth

Feb-March

Feb–March

Feb–March

Feb–March

Nov–Jan

Nov–Jan

Nov–Jan

Season for planting

Table 8.2 Sugarcane varieties and their salient features.

18.8

18.2

17.7

17.6

18.7

20.1

19.5

Sucrose content (% in juice)

68.2

70

67.8

69.2

104.2

102

90.5

Yield of sugarcane (t/ha)

Mid-late, moderate resistance to red rot, top borer resistant, shoot borer and stalk borer resistant

Early maturing variety, moderate resistance to red rot

Early maturing variety, moderate resistance to red rot

Mid-late maturing variety, temperately resistant to red rot and smut, water logging tolerant and saline-sodic soil

Mid-late maturing variety, smut and red rot resistant, good ratooning capability

Mid-late maturing variety, resistant towards smut and red rot, drought tolerant, good ratooning capability

Early variety with good ratooning capability, resistant to smut and slightly vulnerable to red rot

Salient features

Sugarcane: A Potential Agricultural Crop for Bioeconomy

179

180 Lignocellulosic Biomass Production and Industrial Applications spring it is February-March. In tropical areas, sugarcane is planted during June-August for Adsali and January-February and October–November for Eksali. Vegetative propagation of sugarcane is done by stem cutting, in which the cane setts that include two or three nodes are taken from a healthy plant and sowed during the spring season, which is considered the best time of the year with an average temperature of 25 °C. Cane setts are the cuttings of immature canes with the presence of 2–3 lateral buds that is taken from the 8–12 months old plant from the upper third of the stem and incase of ratoon from 6–8 months. The setts, which are usually 30–45 cm in length, are sowed in either trenches or furrows of depth 15–30 cm. These stem cuttings are sorted carefully before planting, taking all the good ones and subjecting them to treatment in moist hot air at 52 °C for 20–30 minutes and adding fungicide to control disease. The setts are treated after cutting so as to avoid decrease in yield since they tend to be susceptible to diseases and pests. The most common diseases that affect the sugarcane yield are smut, grassy shoot disease and ratoon stunting. In order to control such diseases, the setts are subjected to 0.1% Carbendazim solution treatment for 15 minutes and for insect and pest control Malathion 50 EC or Dimethoate 30 EC are used for 15 minutes. The improved methods of irrigation employed for sugarcane plantation, such as flat planting, ridges and furrows, ring-pit method, trench method and drip irrigation [15], are described below. a. Flat planting: This method is followed in areas containing high soil moisture and is popular in North India. The sets are placed in shallow furrows of depth 75 cm apart. One viable bud of a sett is placed per 10 cm length in each furrow [12]. b. Ridges and furrows: This type of irrigation method is better than flat planting with respect to germination, water using capacity, aeration and yield. The ridges and furrows are usually prepared manually with the help of tractors or bullock carts. The spaces between the rows are in the range of 60–135  cm. The spaces between rows in the range of 60–75  cm are favorable for short duration varieties, early varieties and shy tillering varieties. Even under poor soil quality and adverse climatic condition, close spacing is desirable. In good soil and high fertility conditions with good irrigation facilities, long duration and high tillering varieties can be grown in wide row spacing with a range of 100–120 cm.

Sugarcane: A Potential Agricultural Crop for Bioeconomy

c. Ring-pit method: The setts are planted in round pits with a spacing of 150 cm between the pits in a single row and a spacing of 180 cm between two rows. Tractors with power tillers are used for digging pits. The depth of the pit is usually in the range of 1.25–1.5 feet. Approximately 2700 pits can be dug per acre. Before planting, the pits are filled with top soil and manure and properly irrigated before planting. While planting the setts it should be kept in mind that only 30 of the mother shoots are allowed to develop, which subsequently give rise to thicker and heavier canes of weight 1.25–1.75 kg each. This system is suitable for drought-prone regions, saline-sodic soils and regions with light textured or undulating areas. It has several advantages such as less water requirement, enhancement of nutrient use, increased yield of cane and ratoon, increased efficiency of solar energy and no ploughing and lodging, saving labor and machining cost [16]. d. Trench method: In this method, deep trenches are dug at a depth of 30–45 cm and width of 60 cm with a spacing of 120  cm between the centers of two nearby trenches. This type of system is favorable for early drought and waterlogged areas. During planting, the setts are placed on both sides of the trench bottom and covered lightly with soil. The spaces between the trenches are used for growing other crops. Such planting method gives increased yield. The trench gradually gets filled with manure as the crop keeps growing. In the end between the two sets of paired rows a small trench is formed that serves as a channel to drain out the excess water during monsoons. e. Drip irrigation: In the present world where the water and energy crisis is an issue of debate, such technology can be the answer for solving this problem. This is an inventive technology that is used for conservation of water and energy. In this method, the water is applied on the field with the help of a point or line source emitter at a pressure of 20–200 kPa and discharge rate of 0.6–20 LPH on or below the surface of the soil that results in partial dampening of the soil surface. The most widespread type of drip irrigation followed by farmers is surface and subsurface drip irrigation. Drip irrigation accounts for higher yields, water management and enhancement of sucrose content.

181

182 Lignocellulosic Biomass Production and Industrial Applications

8.4.6

Nutrient Management

Maintenance of soil fertility is very important for improvement of physical, chemical and biological properties of soil. This can be achieved by the application of organic manures and fertilizers. Organic manures, such as cow dung, compost, press mud, etc., can be used. Sugarcane trash, which is an agricultural waste that is usually left in the field, can also be applied as mulch. In addition to the manures, fertilizers are also used, which are generally applied after soil testing. When applied at early growing stages fertilizer can help in enhancing the sucrose content of sugarcane. For every hectare of land, 300 kg nitrogen, 80 kg phosphorus, 80 kg potash and 80 kg calcium are applied. Dosage of the nutrient is based on the requirement of the crop. Soil testing should be conducted in order to find out the nutrient and organic status of the soil so that the sugarcane variety can be grown accordingly.

8.4.7

Water Management

Sugarcane requires a huge amount of water since it is a long duration crop and produces large quantities of biomass. For good growth of sugarcane, 20,000–30,000 m3 of water are required annually. The requirement for water depends on different factors such as the quality of soil, climatic conditions, cultivation practices, application of water and the duration of crop. In its different growth phases, sugarcane requires different amounts of water. a. Germination phase: In this phase, the field is casually irrigated so that the soil is just moist and neither dry or over moist. This phase is for 0–45 days and requires 300 mm of water. b. Tillering phase: This is a vital phase of growth and usually extends from 45–120 days with a 550 mm water requirement. c. Grand growth phase: This phase usually occurs during hot weather, due to which more water is required. Inadequate water supply can lead to reduction in cane length and weight. This period extends from 120–270 days with a water requirement of 1000 mm. d. Ripening phase: This phase lasts for about 270–360 days with a prerequisite of 650 mm water. A moderate amount of water is needed since excess water hinders sugar buildup. Subjecting the crop to a stress condition can lead to loss in sugar yield; therefore, an optimum amount of water should be supplied to the plant [15].

Sugarcane: A Potential Agricultural Crop for Bioeconomy

8.4.8

183

Weed Management

During its initial growing period, sugarcane usually does not make use of the field, due to which the chance of weeds growing increases. All types of weeds are grown in the field that can cause serious damage to the crop, such as the competition for nutrients, sunlight, and space, which make the crop susceptible to pests and diseases. Infestation of weeds can lead to heavy losses that can range from 12–72% due to a decrease in the quality of sugarcane. Annually, around 25 million tonnes of losses occur due to the decrease in the sugarcane yield. Some of the weeds that infest the sugarcane field are Dacryloctanum aegyptium (makra), Echinochloa crusgalli (grasses), Cyperus rotundus (motha), Sorghum halepense (banchari), Lathyrus sativa (matri), etc. In order to overcome this problem, various steps can be adopted such as mechanical method, chemical method, cropping and cultural practices and an integrated approach.

8.4.9

Biotic Factors: Pests and Pathogens

Pests and pathogens affect the productivity of sugarcane by causing diseases that can get transmitted from one plant to another or from one field to another with the help of spores or soil contamination. Some of the diseases caused by pests and pathogens are given in Table 8.3.

8.4.10

Crop Rotation

The same field is used for 2 to 3 years for sugarcane growth, due to which the probability of the plant being infected by diseases, insects and pests becomes high, making the field condition unfeasible for crops to grow in a healthy manner. In order to overcome these deficiencies, crop rotation is practiced by growing non-sugarcane crops in order to maintain the fertility of the soil. For example, in subtropical countries different strategies are followed such as: Kharif Crops-Potato-Spring Sugarcane-ratoon-Wheat, while in tropical countries Paddy-Sugarcane-Ratoon-Wheat cultivation is followed [12].

8.4.11

Ratooning

Ratooning is practiced in sugarcane cultivation because it saves cultivation costs. Well-maintained ratoon gives high yields and this is possible when the ratoon crop is from a healthy plant. After harvesting for a week, proper ratoon management should be followed, such as stubble shaving, off barring, gap filling, etc. Practicing ratooning is economically viable due to the

Organism

Chilo infuscatellus

Holotrichia sp.

Colletotrichum falcatum

Sporisorium scitamineum

Disease

Early shoot borer

White Grub

Red rot

Sugarcane smut

Ratoon crops are more prone to this disease Elongated whip-like structure is produced from the terminal bud and is black in color and contains smut spores Excessive tillers produced with stunted growth and sprouts

Yellowing and drying of the leaves, discolored legions on the rind The internal color of the stalk becomes brown and the pith becomes gray in color and emanates a sour and alcoholic smell

Grubs feed on the root system and underground stalk Entire shoot dries up and gets dislodged easily

Attacks sugarcane from germination phase till internode formation Larvae enters the growing point through holes in the shoot, causing dead heart symptoms

Symptoms

Use of resistant variety crop and avoidance of infected seed cane Removal and destruction of whip-like structure before the spore is released Treatment of setts with hot water at 50 °C for 1 hour

Cultivation of resistant varieties Crop rotation and avoidance of rationing Disinfection of soil around the diseased area

Spraying of insecticides for killing the beetles Flooding of the infested field for 2 days and deep ploughing before planting of sugarcane Can also use crop rotation with paddy

Late planting during hot climate should be avoided Removal of dead hearts and killing of larvae by piercing small iron rods or spokes in the affected shoots Light earthing and profuse irrigation should be provided

Management

Table 8.3 Few examples of disease-causing organisms, symptoms and management of sugarcane crop [17].

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money saved on preparing tillage and seed material. Ratoon crop matures faster than plant sugarcane, leading to early harvesting and enough time for planting the next crop. Generally, the ratoon crop gives better quality sugarcane and high sugar recovery but at times a low sugarcane yield occurs due to poor management practices, depletion of nutrients in the soil, low nutrient uptake and use of pest- and disease-infected ratoon. For a good ratoon yield, late maturing varieties and spring harvested crops should be used. Ratoon crop has a shallow root system, thus requiring irrigation a number of times. Mulching of the earlier crop residue is done to improve the moisture content of the soil, increase the organic matter, stop weed growth and enhance the efficiency of the crop for using fertilizer.

8.4.12

Intercropping

When planting sugarcane a wide space is left between rows which can be utilized by growing other intercrops like potato, cowpea, watermelon, etc., depending on the location of the field. The intercropping method helps to control weeds, provides extra income to the farmers, acts as mulch for providing moisture, acts as an alternate host for the pest to attack and increases the fertility of the soil [5]. The planting of leguminous crops as intercrops can help in fixing atmospheric nitrogen in the soil, thus enriching it. Intercrops can also help in improving the structure of the soil. The main disadvantage of intercropping is the decrease in yield due to tillering reduction or nutrient and water loss. The intercrops chosen for planting in the sugarcane field should have a short growth period with less competition with sugarcane. The plant selected should not be bushy in nature; neither should it be branching and non-shading.

8.5

Major Limitations of Sugarcane Production

The leading sugarcane industries have pointed out several limitations associated with sugarcane production such as low soil quality, drought stress, lack of fertile land, shortage of improved varieties, expensive inorganic fertilizers, fluctuating prices, lack of disease-resistant varieties and poor management practices. In many parts of the world, drought stress has become one of the major constraints hindering the production of sugarcane. Climate change in the world is associated with reduced rainfall, which causes a reduction in sugarcane production [18]. Monocropping drastically reduces the fertility of the soil and is considered to be an important limitation in sugarcane production. Though

186 Lignocellulosic Biomass Production and Industrial Applications biomanure and compost somewhat help to improve the soil quality, they are required in huge quantities, which restricts large-scale application [19]. Efforts to replenish compost and manure were not enough to retain the quality of the soils. The high cost of inorganic fertilizer, its unavailability and its poor distribution are some of the common problems in sugarcane production. In India, three major limitations prevail in restricting the full phase production of sugarcane, namely low yield of cane and sugar recovery, expensive cane cultivation and reduction in factor productivity; besides constraints such as soil fertility, water availability, land fragmentation, etc. [20]. These major limitations can be overcome by adopting the following strategies; I. Maximization in cane yield and sugar recovery Introduction of novel genes into the gene pool; Application of marker-assisted selection (MAS) for developing disease- and pest-resistant varieties; Increasing the yield of ratoon cane. II. Cost-effective cane cultivation Improvement in the area of rhizosphere engineering and integrated nutrient management technology to enhance the efficiency of nutrient uptake; Usage of pesticides in an eco-friendly manner through integrated pest management (IPM); Mechanistic way of sugarcane farming. III. Limiting the reduction in factor productivity Retainment of nutrients in the soil through crop residue recycling; Carbon sequestration to balance the nutrient requirement. The sugarcane industry has carried out intensive research and made many efforts to maximize cane production and recovery of sugar that will help in sustaining the future demand for sugar and energy in the everexpanding population of India.

8.6

An Overview of Biotechnological Developments for Sugarcane Improvement

Sugarcane is the source of half of the world’s sugar production. Conventional methods have already contributed a lot towards improvement of sugarcane

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but limitations like poor fertility, narrow genetic base, proneness to biotic and abiotic stresses, complex genome, and long interval time to cross with elite cultivars still exist as challenges for biotechnologists. Sugarcane therefore is an appropriate feedstock for application of genetic engineering and biotechnological tools. from this perspective, novel strategies such as in-vitro culture systems along with related technologies for sugarcane improvement have been developed. Biotechnological and conventional methods both need to be combined to overcome such types of challenges. Sustained efforts to produce genetically improved sugarcane varieties are being made that can substantially produce high-value-added compounds such as biopolymers, proteins, precursors, biopigments, enzymes, nutraceuticals, and functional foods. It will take a long time before sugarcane acts as a biofactory [21]. Molecular marker technologies, specifically DNA markers, play a major role in developing superior genetic stocks, rapid assessment of genetic diversity, and efficiency for trait selection. For instance, markers based on short DNA fragments, i.e., rapid amplification DNA (RAPD) markers, have been extensively used for amplification of the genomic sites and assessment of the superior variety of sugarcane germplasm [22, 23]. The potential of inter-simple sequence repeats (ISSR) markers was investigated for molecular profiling in 42 varieties of sugarcane of subtropical India [24]. Cytoplasmic and mitochondrial ISSR were used to analyze red rot disease in resistant as well as susceptible genotypes of sugarcane [25]. The results reflected that these types of markers might be employed as a new biotechnological tool to identify the disease-resistant varieties. Amplified fragment length polymorphism (AFLP) markers were used to study the innate diversity among sugarcane cultivars of India [26]. However, progress in modern science and technologies, such as automated DNA sequencing, single-nucleotide polymorphisms (SNPs), microarray technologies, RNA interference (RNAi) technology and data mining techniques, have a major impact on future improvement programs for sugarcane. Both macro- and microarrays are being exploited for identification of the genes linked to carbohydrate metabolism and disease resistance genes [27]. In-vitro techniques have proved to be very useful for mass propagation of plantlets of sugarcane through direct or indirect regeneration pathways and there are numerous ongoing efforts for the development of sugarcane germplasm via genetic engineering [28]. In general, propagation of sugarcane through stems of mature sugarcane is not cost-effective and is susceptible to diseases of bacterial, viral and fungal origin. Thus, in order to overcome such types of problems, synthetic seed technology is becoming a potential novel tool in crop biotechnology [29]. Synthetic seeds are considered as effective delivery systems and a substitute for the expensive

188 Lignocellulosic Biomass Production and Industrial Applications method of vegetative propagation, and are also proved to be useful for the storage of novel germplasm. Research on synthetic seeds in the case of sugarcane has been less explored over the last few years. The somatic embryos of sugarcane obtained from direct somatic embryogenesis [30] showed the highest percent of germination (73%) when encapsulated in the beads of sodium alginate. Simultaneous somatic embryo production and regeneration of plants can be helpful in sugarcane synthetic seed technology. Somaclonal variation observed during cell or plant tissue culture process is an important tool of in-vitro cell culture that helps in crop improvement. This technique has been widely adopted for improving the sugarcane quality and the somaclones for better sugar yield, recovery, tolerance to drought, resistance to disease, etc. Somaclonal variation was first reported in sugarcane species [31]. Embryogenic plants and cells tolerant to red rot were isolated through mutagenesis and selection [32,  33]. Several genes (drought and salt tolerance, pest or disease resistance, and sugar accumulation) responsible for the improvement have been transferred into different varieties of sugarcane cultivars [34, 35]. The production of transgenic sugarcane varieties relies on the method of transformation, tissue/explants type and/or system for tissue culture regeneration. Improvements in gene transfer techniques, mainly microprojectile methods of gene transfer in plants, are remarkable in sugarcane genetic engineering. However, Agrobacterium mediated gene transfer in plants has gained more attention because of its well-studied genome that led to efficient integration of transgene in the host organism. The advancement in sugarcane genetic engineering could become significant in the future in terms of improved productivity and yield that substantially raise the utility and value of this crop.

8.7

By-Products of Sugarcane Processing

Diversification and utilization of by-products from the sugar industry is a financially viable strategy that can make the industry self-sufficient and financially stable so as to help boost the economy. There are many byproducts formed, among which some of the important ones are presented below.

8.7.1

Bagasse

Bagasse is the key by-product of the sugarcane industry. It is a fibrous residue that is left behind after juice extraction that can be used as a cheap feedstock in industries. It is surplus in amount since each metric tonne

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of sugarcane is able to generate 280 kg of bagasse [36]. Conventionally, bagasse was used as fuel for boiler generating steam and also for electricity generation. But with the advent of sustainability and economic viability on the world scene, bagasse is being used for the production of value-added products as well, such as for paper making, animal feed, filter cake used as soil improver, biofuel production, xylitol production, etc.

8.7.2

Molasses

Molasses is a viscous by-product that is usually the leftover liquor after crystallization of sugar. Even though it is a waste product, it still consists of 30–35% sugar along with 15–20% reducing sugar, thereby making it an important raw material that can be used for the production of various value-added products. Several products have been produced from molasses on a commercial scale such as acids (citric acid, lactic acid, acetic acid), ethyl alcohol, acetone-butanol-alcohol, ethyl benzene, polyethylene, monosodium glutamate, baker’s yeast, etc.

8.7.3

Vinasse

Vinasse is a dark brown, acidic (pH 3.5–5) slurry with high organic content (COD: 50–150 g L–1), with an unpleasant odor. Each ton of sugarcane processed gives 700–900 liters of vinasse [37]. Due to increasing environmental concerns, instead of being disposed of in the environment vinasse is now being used for different purposes, such as ferti-irrigation, that can replace the use of chemical fertilizers, especially those containing phosphorus, and help in reducing the cost of the process. Other than this, it can also be used in boilers for energy generation and the organic load of vinasse can be used for biogas production [38].

8.8

Applications of Sugarcane for Biorefinery Concept

The concept of biorefinery facilitates the utilization of all the elements of biomass for the production of fuels, heat, power, and biobased chemical and materials that is illustrated in Figure 8.1. This concept is analogous to the petrochemical refinery. The main difference between these two refineries is that instead of fossil feedstock, renewable organic matter is used in the biorefineries. Utilization of all parts of a biomass plays a pivotal role in the economy of the process. During this process, there is production of

190 Lignocellulosic Biomass Production and Industrial Applications

Sugar Juice (sucrose)

Ethanol

Stalks

Biobutanol Bioplastics Polyethyleneglycol Diethyl ether Ethylene Others

Sugarcane Bagasse

Pretreatment & hydrolysis

Trash Lignocellulosic biomass

Pentoses

Glucose Residual solid biomass

Fuel

Chemical products Biomethane

Ethanol Xylitol, furfural

Field

Ethanol

Energy to the plant

Residual biomass

Biogas Biomanure

Figure 8.1 Biorefinery concept for sugarcane [39].

both low-value, high-volume products and high-value, low-volume products that provide scale and enhance profitability respectively. Due to global competition, the sugar industry is under a lot of pressure to diversify its value-added products. The economic diversification of sucrose other than food utilization is just now being realized. Following the biorefinery concept many value-added products can be derived from sugarcane other than sugar such as biofuels, inositol, xylitol, glycerol, succinic acid, animal feed ingredient, etc., as mentioned in Table 8.4.

8.9

Utilization of Sugarcane Residue for Bioethanol Production

Depletion of fossil fuel reserves and instability in oil-producing countries has added to the woes of already existing problems. In order to mitigate these problems, biobased products, such as biofuels from lignocelluloses, are one of the solutions that can ease our burden. Production of biofuels from lignocellulosics is the best alternative for substituting fossil-based fuels. Ethanol produced from cane sugar is called first generation bioethanol, which has given rise to the food versus fuel controversy. Unlike Brazil, which produces ethanol directly from sugarcane, other developing countries cannot spare the crop for bioethanol production as hundreds

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Table 8.4 Use of sugarcane for different biofuels and biobased products in a biorefinery concept. Products

Substrate

Ref.

Bioethanol

Sugarcane bagasse, trash and juice

[40, 41]

Biobutanol

Sugarcane bagasse

[4, 42]

Biohydrogen

Sugarcane leaves

[43]

Sugarcane bagasse

[44]

Biodiesel

Sugarcane bagasse

[45]

Bioelectricity

Sugarcane bagasse

[46, 47]

Bioplastics

Sugarcane bagasse

[48]

Xylitol

Sugarcane straw

[49]

Bio oil and activated carbon

Sugarcane bagasse and molasses

[50]

L-glutamic acid

Sugarcane bagasse

[51]

5-hydroxymethyl furfural

Sugarcane bagasse

[52]

Cellulose nanocrystals Butyric acid/Cellulase Itaconic acid/Lipase Propionic acid Lactic acid

Sugarcane bagasse fibers and pith Bagasse Molasses Molasses Molasses

[53] [54, 55] [56, 57] [58] [59]

of people are starving for food. Therefore, in order to overcome this issue sugarcane residues like bagasse, sugarcane tops and sugarcane leaves are being used for ethanol production. Dias et al. [40] conducted a study to compare three different plants where ethanol was produced from sugarcane bagasse and trash (sugarcane tops and leaves); the second plant produced ethanol from sugarcane alone and the third plant was an integration of 1G and 2G ethanol. After the development of different simulations, the results showed that an integrated process of first and second generation for ethanol production was more economically viable than stand-alone plant. Integrating the process will lead to some common operations like fermentation and distillation which will lessen the investment, and the inhibitors generated during the pretreatment process will have a minor effect since the hydrolyzed liquor from second generation will be mixed with the sugarcane juice [41].

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8.10

Conclusion

In the wake of the energy crisis and economic instability, it is necessary to come up with strategies that can benefit nations and the environment. Complete utilization of a biomass without any wastage should be the aim, as well as producing many value-added products. Adopting a biorefinery concept for sugarcane can produce an infinite number of biobased chemicals but the most important aspect to be considered is a well-defined methodology for the production of crops and value-added products according to market demand that can give maximum profit in terms of the economy and the environment. It has become imperative to explore the possibilities of biomass, such as sugarcane, so that we can free ourselves from our dependency on fossil-based products.

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9 Lignocellulosic Biomass Availability Map: A GIS-Based Approach for Assessing Production Statistics of Lignocellulosics and its Application in Biorefinery Sanjeev Kumar1, G. Lohit Kumar Srinivas1 and Rintu Banerjee1,2* 1

Advanced Technology Development Centre, Indian Institute of Technology, Kharagpur, West Bengal, India 2 Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur, West Bengal, India

Abstract Since India is a thickly populated agricultural country, most of the land utilized is under cultivation throughout the year. After the “green revolution,” the present day emphasis is on “evergreen revolution,” which not only indicates an improvement in productivity by cultivating various agricultural products but also covers overall agricultural productivity, which also includes by-product generation. As  India is a huge country with a wide range of habitats, different agricultural crops are cultivated in different parts of the country having wide ecological variations. Along with improved productivity, the by-product generation associated with it can be efficiently utilized for useful applications like bioenergy. A geographical information system (GIS) is a modern day decision support system which is being efficiently used for mapping bioresources for energy applications. Modern day energy policy reckons with the utilization of lignocellulosic biomass owing to the food, fodder vs. fuel controversy. Comprehensive statistics about the spatial distribution of different lignocelluloiscs along with the prevailing soil and climatic conditions, etc., play a crucial role in formulating efficient policy and locating the position of a biorefinery. In this chapter, a comprehensive outlook on

*Corresponding author: [email protected] Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial Applications, (197–214) © 2017 Scrivener Publishing LLC

197

198 Lignocellulosic Biomass Production and Industrial Applications the existing GIS models used to date for mapping lignocellulosic cover throughout the world is detailed along with the production statistics of biomass availability. Keywords: Geographical information system (GIS), biorefinery, lignocellulosics

9.1

Introduction

Government agencies around the world have pressed the accelerator button for funding research on bioenergy considering the non-renewable nature of the fossil fuels and alarming issues like global warming. The Government of India’s Ministry of New and Renewable Energy has set a goal for 20% blending of petro-based fuels (petrol and diesel) with their biobased counterparts by the year 2017 in order to curb the volatile geopolitical issues governing their price control while simultaneously building indigenous technology for biofuels production [1]. This type of policy has been laid down by government agencies throughout the world, which has created a drive for producing biofuels so that the projected demand for blending can be fulfilled. Moreover, bioenergy as an energy option has many advantages, viz., its carbon-neutral nature and the utilization of lignocellulosic biomass (LCB) for its production. A report by the International Finance Corporation suggests that populations belonging to the lower income category spend nearly US$37 for meeting their daily energy needs, which may be mainly due to the adoption of less efficient energy options [2]. Thus, popularizing the utilization of bioenergy, viz., biogas for conducting household activities, will not only reduce their incurred expenses for it but also improve the ambient environment so that their health is not at risk [3]. Nowadays, commercial production of biofuels has been established using edible starch-based feedstocks and its utilization invites the controversy of food vs. fuel. Thus, considering their rich biochemical potential and sustainable nature, LCBs are being utilized for bioenergy production. Utilization of LCB for biofuels production comes under the category of second generation fuel production and alleviates the controversy of food vs. fuel. This resource is a boon for agriculture-driven countries since the residues left over after the crops have been harvested can be efficiently converted to biofuels, which would otherwise have been disposed of at the expense of environmental safety. The advantages of utilizing LCB for biofuel production are: Potential of utilizing LCB is comparable to any other source for biofuels production considering the rich source of carbohydrates within the biomass;

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LCB can be grown in wastelands which cannot be utilized for commercial gains; Biofuels produced from LCB are carbon neutral; Utilization of LCB leads to the generation of jobs for the rural youth. For a biorefinery to operate completely upon LCB, one of the major preconditions is the copious availability of LCB so that the biofuel processing plant can be logically stationed. Although LCB is portrayed as one of the potential substrates for biofuel production, a major bottleneck in utilizing this source lies with the broad distribution of biomass within the geographical landscape, which makes collection and transportation to the nearby processing centers a challenging task. This bottleneck can be overcome by studying the area under investigation through the scanner of the geographic information system (GIS) by identifying the availability and the distribution of the biomass within the study area. The computational platform known as GIS facilitates the creation of maps for further critical analysis for comprehending any existing patterns, which is inconceivable through geographical maps [4]. This computational interface converts the geographical data into useful information which can be reviewed in different forms which suit the end user [5]. Traditionally GIS is used as a tool for studying the effect of environmental changes, viz., climate change on agricultural productivity, monsoon prediction, etc., and also for purposes like chalking optimized routes for municipal solid waste (MSW) carrying vehicles, etc. In this chapter, a comprehensive review of the available works on utilizing GIS as a technology for mapping and quantifying the availability of LCB has been conducted.

9.2

Geographical Information System (GIS)

The GIS is a scientific tool which envisages the study of the physical world around us through the help of scientifically developed models revealing hidden patterns lying within the geographical maps of the study area, which can be analyzed to extract the desired information of interest. The geographical aspect of the GIS represents a particular data entity and links it with a specific location on the surface of the earth. This specific entity can refer to a point, line or an area within the earth’s surface. There are five basic functions through which GIS performs the above-mentioned activities: Accessing the maps of the study area; Conversion of the input map into useful data;

200 Lignocellulosic Biomass Production and Industrial Applications Storage of the data obtained for further processing; Analysis of the stored data to derive useful information from the pool of data collected Display of the result in the form of data or updated map. Analysis of the data obtained from the input images is mainly based upon multi-criteria evaluation, which works on the principle of formulating a problem by scrutinizing similarities within spatially co-registered data points within input images [6]. Suitability of a particular land for a predetermined purpose has traditionally been achieved using linear overlay technique and using binary thresholds. Overlaying technique refers to the operation of superimposing maps representing different informations: land use, landcover, regional cartography, administrative boundary, populated areas, road network, terrain map, lithological map, climate map, industry, civil census, etc. These attributes are overlapped in order to locate potential areas from where agricultural residues and non-edible lignocellulosics can be collected and then the data collected can be analyzed for assessing the net availability of the biomass. The GIS data is organized in the form of either a vector, raster or both. A vector form of the data contains points, lines and polygons and is useful in describing physical boundaries in the surface of the earth like boundaries of countries, streets, etc., while raster form describes a surface segregated in the form of grids and is useful in showcasing objects varying continuously, viz., elevated surface, aerial photograph, satellite image, etc. Among the two, vector form is the one mostly used and is also very accurate while the raster form contains more than one value corresponding to each pixel. The maps used in GIS are generally acquired from remote sensing satellites, and since it is recorded in the form of pixels each of the vector data is represented as a polygon. Remote sensing is a technique which envisages collection and

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analysis of data without any physical contact with the object. It is generally conducted via telescopes, satellites, etc., which allows the observer to gather information about objects from far distances. The basic principle behind this technique is the measurement of energy by a sensor which is reflected from the target under consideration [7]. Every specific target has a unique reflection pattern and is often used for identifying the target under consideration. Sensors can be categorized into two types: active and passive. Active sensors contain a radar instrument which is positioned in order to measure signals transmitted by the target object upon receiving radiation incident by the sensor itself. Thus they provide their own energy for the purpose of illumination and thus are independent of the day and night cycles and can also be used for evaluating wavelengths in the microwave range, which are seldom emitted by sun. Examples of active sensor include fluorosensor, synthetic aperture radar, etc. Passive sensors are microwave instruments which receive and analyze signals radiated by objects on the surface of the earth only after receiving energy incident from the sun. The image received from remote sensing satellite is categorized into true and false images where true images represent the actual photograph of the earth’s surface captured through the satellite while a false image is represented in different shades of red, which is easy to distinguish in comparison to the varying shades of green. That is the reason behind converting red-green-blue color images in a true image into near infrared, red and green spectral bands which is the form of a false image. In the case of visualizing agricultural residues, false images are useful since they reflect more light in the near-infrared region. Apart from true and false images, two other forms of images also exist, viz., panchromatic (PAN) and multispectral (MS) images. A PAN image is generated by adopting two sensors which work in synchrony in order to generate higher resolution image while an MS image contains rich information in terms of color but the resolution is low. These two types of images are generally clumped together in order to obtain both high-resolution and color-rich images. Generally the satellites used are U.S. Landsat, French SPOT, U.S. IKONOS, India (INSAT, Aryabhatta, Bhaskara, Rohini, SROSS, IRS, RISAT), etc. These satellites are specific for their spectral resolution which denotes the range of the electromagnetic spectrum and the definite number of spectral bands in which the image will be taken. This feature is important since different spectral ranges are specific for studying specific objects on the surface of the earth; for example, 0.45–0.52 μm for studying coastal morphology and sedimentation, 0.52–0.59 μm for deciphering vegetation, rock-soil discrimination turbidity and bathymetry, 1.55–1.70 μm for

202 Lignocellulosic Biomass Production and Industrial Applications

GIS ARC/ INFO-GRID

Biomass availability index calculated by assigning numbers to the vegetation depending on their density

Climatic index map Precipitation map Soil map Topography map Ecofloristic map Vegetation map National boundary map Population map

Different layers overlapped over each other

Influence of anthropogenic activities in maintaining the resources

Final estimate of the biomass availability validated by the inventory data collected from the concerned authorities.

Figure 9.1 Flowchart for assessing the biomass estimates using GIS.

observing crop types, forest types, canopy status, etc. The flowchart generally adopted while implementing GIS for estimating biomass availability is shown in Figure 9.1.

9.3

Application of GIS in Mapping Lignocellulosic Biomass

In the last decade, several attempts have been made to gather information for mapping biomass availability throughout the world. These attempts can be broadly divided into destructive and non-destructive, where the destructive option includes harvesting the entire area under consideration or chopping trees belonging to a particular size class in order to establish a relation between the available biomass and the significant features of the plants actually present in the study area, viz., diameter/girth or height ratio [8]. However, considering the looming climatic issues, a destructive solution is not feasible and hence efforts were channeled for framing nondestructive options which would incur minimum damage to the trees. A possible solution can be attained by adopting a remote sensing led GIS approach for mapping the abundance of biomass and researchers have successfully worked out a vegetation index which relates to the vegetation amount with high accuracy [9]. Canopy reflectance is one more technique which provides useful information regarding the amount of photosynthesis and transpiration taking place within the study area and scientists have

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successfully linked this information to the biomass availability [10,  11]. This technique has also been proven to be effective in assessing the abundance of woody biomass, as was suggested by Roy [12]. Accurate assessment of the biomass envisages the inclusion of the structural conditions of the study area which has been proven to be true by ground sampling of a few homogenous vegetations since their spectral properties are unique and hence reliable [8, 13]. However, this information needs further crosschecking since most of the confirmatory works have been conducted on homogeneous conifer forests or grassland and the challenge lies with deciduous forests coming into the picture since they are heterogeneous as far as background reflectance is concerned. As far as the use of GIS models for identifying suitable lands for growing energy crops are concerned, mostly simple suitability models based on linear combinations of layers with Boolean overlays are used. Differences in the approach lies in the sequence of the inputs accessed by the model. Some models initially mapped marginal land followed by identifying the biomass cover [14–16] while a few others used land use constraints in-between the demarcating marginal lands and assessing biomass cover [17, 18]. Several researchers have also performed the assessment employing a hierarchical overlay technique by classifying marginal lands by physical and biological means [19]. Physical classification includes salient features like slope of the area, rock depth, fragments of rock, etc., while biological classification was done by considering temperature, soil conditions, water table etc., of the area under consideration. Fuzzy set theory has also been used for identifying suitable land for energy crops, for example, FAO’s Agro-Ecological Zone, which utilizes a membership function based on fuzzy logic [20]. In this attempt, soil indicators of the land were divided into five categories referred to as “bins” and assigned values such as 1.0 for best suitable range for the crops while the range selected was 0.5–1. Based on this logic they identified land suitable for growing specific crops. Another such attempt to use fuzzy theory was based on plant growth index which includes rating the soil content and thus enabling the decision support system of the software to analyze the plot qualitatively [17]. Lu et al. [21] utilized GIS overlay technique to locate land for Chinese pistache and identified 19.9 Mha of land, which accounted for 2.08% of the land area available for China. Zhuang et al. [14] used binary threshold with GIS overlay technique to map the availability of five different biomasses, viz., Jerusalem artichoke, castor, cassava, tung tree and Chinese pistache, and observed that nearly 43.75 Mha of land was available in China for growing these biomasses. There have also been plenty of efforts to estimate the abundance of biomass in the study area. One such study was conducted by Fiorese and

204 Lignocellulosic Biomass Production and Industrial Applications Guariso [22], who proposed a decision support model based on GIS in order to maximize the energy production from the biomass after considering factors like environment, climatic conditions, land usage map, and geographical morphology for studying biomass availability in the EmiliaRomanga area of Italy. Thomas et al. [23] presented a GIS-based method for assessing the biomass abundance in England. Their study was focused specifically on Miscanthus and they concluded that out of the 25,21,996 ha of land available for growing this energy crop, 19,98,435 ha were found to be suitable and located within 25 km of its processing area. Fernandes and Costa [24] used a GIS-based approach in order to assess the availability of biomass residues within the Marvão region of Portugal and observed that the annual biomass availability was in the tune of 10,600 tons, which translates to 1,06,000 GJ of energy produced in a year. They also studied the integration of this biomass with energy supplied to a hotel situated in the study area for heating purposes and found that in comparison to the conventional fossil fuel-based resources, biomass-based source was less energy intensive and economical. Jiang and coworkers [25] did a comprehensive study to estimate the crop residue available in China by adopting a GIS-based approach, considering factors like spatial and temporal distribution of the biomass in the study area, and identified that nearly 505.5 × 106 tons of crop residues were available per year, which amounts to a net processed energy of 7.4 EJ generated in a year. A slightly different approach was adopted by Yoshioka et al. [26], where GIS was used to estimate the available biomass in Japan along with demarcation of the transportation system, finally establishing a link between these factors and cost of procuring the biomass. This study pointed out that logging residues were the cheapest while residues from thin trees were the costliest. In India, a country with wide theoretical residual biomass, resource assessment was conducted by an initiative of the Ministry of New and Nonrenewable Energy [27]. It was observed that a minimum amount was available in Mizoram (0.21 MT) while the maximum amount was estimated in Uttar Pradesh (UP) (121 MT). Within UP, majority of the biomass residue was contributed due to crops, viz., sugarcane, wheat and rice, as they contribute 90% of the available biomass. Surplus availability of biomass was maximum in UP (40 MT, 33% of the total) while the minimum amount was found in Arunachal Pradesh, i.e., 21% of the total. Among the 33% of the surplus availability in UP, 10–22% of the biomass was attributed to wheat straw and rice straw was found to be burnt in the field [28]. This figure represents a dismal picture of the loss which has to be diverted towards utilization for constructive purposes, viz., bioenergy. The observations gathered through this study have been tabulated in Table 9.1.

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Table 9.1 Surplus biomass available in different states of India. Sl. No.

State

Biomass Surplus (kT/Yr)

1

Andhra Pradesh

1172.80

2

Assam

1398.40

3

Bihar

4286.20

4

Chhattisgarh

1907.80

5

Goa

6

Gujarat

7505.50

7

Haryana

9796.10

8

Himachal Pradesh

988.30

9

Jammu & Kashmir

237.70

10

Jharkhand

567.70

11

Karnataka

6400.60

12

Kerala

5702.60

13

Madhya Pradesh

8033.30

14

Maharashtra

15

Manipur

16

Meghalaya

8.40

17

Nagaland

27.20

18

Orissa

1163.40

19

Punjab

21267.00

20

Rajasthan

35531.10

21

Tamil Nadu

22

Uttar Pradesh

23

Uttaranchal

51.60

24

West Bengal

2959.70

129.90

11803.90 31.90

6658.70 11725.90

Along with the availability of the surplus biomass across the different states of India, the abundance of specific crops was also measured and is tabulated in Table 9.2 [29]. Among the crops studied, banana residues (2,40,000 MT) and rice straw (2,17,575 MT) were observed to be most abundant, while the least available were areca and coffee residues. The trend observed is quite predictable considering the fact that rice is the staple

206 Lignocellulosic Biomass Production and Industrial Applications Table 9.2 Availability of specific crops in India. Total available residue, thousand (MT)

Sl. no.

Name of the crop residue

1

Areca husks and fronds

2

Arhar (tur) stalks and husks

5,460.30

3

Bajra stalks, husks and cobs

20,224.40

4

Banana residue

5

Barley stalks

1,560.00

6

Coconut shell, husk and pith

9,843.90

7

Coffee husk, pruning and wastes

151.48

8

Coriander stalks

287.50

9

Cotton husk and stalk

10

Cumin stalks

11

Dry Chili stalks

12

Rice Straw

2,17,575.00

13

Sugarcane top, leaves and bagasse

1,04,975.00

14

Wheat stalks

1,17,000.00

15

Maize stalks and cobs

42,550.00

16

Cassava Starch from roots and solid waste

14,544.00

17

Millet stalks

14,892.00

18

Black Gram stalks and husk

264.86

2,40,000.00

3,335.00 310.00 1,200.15

975.00

food of India and thus utilization of rice straw for bioenergy can solve the issue  of ever-increasing energy demand while simultaneously mitigating the volatile geopolitical issues which control the price of petro-fuels. For the United States of America, one such study was conducted by Milbrandt [4] using the GIS approach. In this study, the following crops were selected: corn wheat soybean

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207

cotton sorghum barley oats rice rye canola beans peas peanuts potato sunflower sugarcane flaxseed In order to estimate the total biomass available, assumptions like total grain production, residue generated per crop, amount of moisture in the crop and the residue leftover in the field for protecting soil (30%), for grazing (20–25%) and other agricultural purposes (10–15%) were assumed and thus only 35% of the available biomass was proposed to be used for bioenergy applications. The estimated biomass amount has been tabulated in Table 9.3. Although GIS has been extensively used for estimating biomass availability throughout the world, attempts to map the abundance of non-edible lignocellulosics have been scarce and thus efforts have to be focused on getting an exact measure, since these biomasses negate the food vs. fuel controversy and thus can be an ideal feedstock for bioenergy applications. The National Renewable Energy Laboratory (NREL) [18] conducted a thorough study which estimated the availability of lignocellulosic biomass in APEC (Asia Pacific Economic Cooperation) countries and found that in Australia nearly 36.7 × 106 tons of biomass are available, out of which 30 × 106 tons are represented by the crop residues while the rest is attributed to wood waste, forest residues, etc. On the other hand, its neighbor New Zealand mainly depends upon wheat, barley, sugar beet, etc., as first generation feedstocks for bioethanol production and the potential of bioethanol from the residues of these biomasses is 4100 × 106 L while the second generation feedstocks amount to 5.5 × 106 tons of crops and woody residues. In Canada, about 78.27 × 106 tons of crop residue are available for bioenergy applications out of which only 43.89 × 106 tons could be recovered for bioenergy applications. Second generation biomass availability in Chile was estimated to be 5.5 × 106 m3 and the majority of it was from the

208 Lignocellulosic Biomass Production and Industrial Applications Table 9.3 Biomass residue available in different states of the USA. Biomass Surplus (dry Ton × 1000)

Sl. no.

State

1

Alabama, Arizona, Arkansas

2

California, Colarado

3

Delaware

245

4

Florida

3263

5

Georgia

997

6

Hawaii

396

7

Idaho

1788

8

Illinois, Indiana, Iowa

9

Kansas, Kentucky

10

Louisiana

4335

11

Maryland

584

12

Michigan, Minnesota, Mississippi, Missouri, Montana

13

Nebraska, New Jersey, New Mexico, New York, North Calorina, North Dakota

14

Ohio, Oklahoma, Oregon

15

Pennsylvania

16

South Carolina, South Dakota

331, 5140

17

Tennessee, Texas

1501, 6089

18

Utah

88

19

Virginia

502

20

Wyoming, Wisconsin, West Virginia, Washington

391, 351, 4796 1659, 1550

19,593, 8976, 23590 7614, 1722

3586, 14231, 2919, 6007, 1560 10931, 91, 168, 507, 1494, 6602 5001, 1641, 567 810

106, 4419, 32, 1746

processed residues of mills. Assessment of the first generation crop residues within China amounted to 112.5 × 106 tons, mainly represented by cassava and sweet potato, while second generation residues amounted to 788 × 106 tons. Indonesia is one of the richest countries in the world in

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209

terms of biomass availability as the reserve of sugarcane is quite predominant (annual production: 17 × 106 tons), while second generation feedstocks are available at the rate of 105.5 × 106 tons/year. In the case of Japan, the availability of agricultural residues for biofuel production is quite limited since there are only 30 × 106 tons/year from first generation feedstocks, while second generation woody residues amounted to only 32 × 106 tons/ year. Korea is also not far behind Japan in terms of its limited resources for bioenergy applications since only 13.1 × 106 tons of agricultural residues are available per year. In Russia, a thorough inventory was conducted on the available first generation resources and it was observed that nearly 82 × 106 tons are available while the inventory on the second generation residues amounted to 100 × 106 tons. Small countries like Papua New Guinea (15000 tons FG), Peru (No specific data), Phillipines (17.7 × 106 tons SG), Singapore (5.6 × 106 tons SG), Thailand (12 × 106 tons FG; 46 × 106 tons SG) and Vietnam (35 × 106 tons FG; 93 × 106 tons SG) were also surveyed for the biomass estimation (FG: first generation; SG: second generation).

9.4

Biofuels from Lignocellulosics

Efforts to map the lignocellulosic availability will be justified only if the biomass has potential for optimum biofuel production. A number of efforts have already been made and the overall scenario is very encouraging. One such study was conducted by Kim and Dale [30], who estimated that the ethanol production from the residues of crops, viz., corn, barley, oat, rice, wheat, sorghum and sugarcane, was 491 GL/yr. This quantity of ethanol has the potential to substitute nearly 353 GL of gasoline if it is used as E85 fuel. Asia is the world’s largest producer of ethanol from crop residues, amounting to nearly 291 GL/yr, and the majority of the residues used belong to crops like rice straw, wheat straw and corn stover. Among these three residues, rice straw is the most potent since it leads to the production of nearly 205 GL of bioethanol. Binod et al. [31] carried out a review on the potential of ethanol production from rice straw and observed that it is both abundantly available and carries the appropriate composition of cellulose and hemicelluloses for optimum conversion into fermentable sugars, which can be efficiently converted into ethanol by yeast used during fermentation process. The major challenge encountered with rice straw as a substrate is the presence of high silica and ash content, which poses major challenges during ethanol production. Apart from ethanol, conversion of biomass into fuels other than ethanol were also tried, viz., the production of biobutanol. One such study was conducted by Ranjan et al. [32],

210 Lignocellulosic Biomass Production and Industrial Applications who evaluated the potential of acid pretreated rice straw for conversion into biobutanol by utilizing Clostridium sp., which was followed by acidogenesis and then solventogenesis as the route for biobutanol production. They reported that the potential of rice straw for producing the four carbon fuel is encouraging and is worth trying. Even gaseous fuels were also formed using rice straw in a study by Mussoline et al. [33], who reported that nearly 92–280 L of methane can be obtained per kg of the volatile solids content of rice straw. The anaerobic digestion process performed was maintained for slightly alkaline pH of 6.5–8, mesophilic temperature of 35–40 °C and C/N ratio of 25–35. Along with rice straw, switchgrass—a tall prairie grass which is abundantly grown as an energy crop—also has the appropriate characteristics to be reckoned with for bioenergy applications. Its advantageous features include a high yield of production per hectare with minimum input in terms of nutrients and its adjustable nature which accomodates a range of growth conditions [34]. According to the US Department of Energy [35], about 1150 GL of ethanol can be produced from the harvest from an acre of land within a year. Other useful features of switchgrass are the fact that it grows fast and also retains useful nutrients within the soil for the next harvest. Miscanthus giganteus is also a feasible option for ethanol production considering its high yield per hectare and minimum amount of fertilizer requirement for its growth. This biomass is also industrially beneficial due to the fact that it can be stored in the form of bales for a long period of time. As per Dohleman [36], nearly 7300 L of ethanol can be produced per hectare of land. In Australia, the majority of ethanol production is attributed to the crops, viz., wheat, sugarcane, sorghum, etc. According to a study by NREL [18], in Australia about 3.11 × 106 m3 of ethanol can be produced, which represents an equivalent energy of 1.5 M tons of gasoline from first generation feedstocks and 11 × 106 m3 from second generation feedstocks. In Canada, nearly 2.18 × 106 tons of ethanol can be produced from crop residues, mainly from wheat straw and corn. In Chile nearly 0.26 and 0.98 × 106 m3 of ethanol can be produced if the available resources respectively (first and second generation) of biomass can be utilized for bioenergy applications. Bioenergy applications from first generation residues of China have been very promising since 19 × 106 m3 of ethanol can be produced from the residual biomass belonging to the first generation, while second generation residues have the potential of 236 × 106 m3 of ethanol. In Asia, apart from China, Indonesia is also a significant contributor of biomass-based ethanol since the potential of bioethanol is to the tune of 6.7 and 22 × 106 m3 from first and second generation resources respectively. Japan on the other hand is not very gifted

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in terms of biomass and hence only about 265.3 PJ of energy equivalent can be produced from the agricultural residues per year. Considering the dismal situation, the government of Japan has started taking measures to promote the utilization of abandoned arable lands for energy crop production and subsequent utilization for bioenergy applications. In this regard, a study was conducted by Yamamoto et al. [37], who estimated the amount of such abandoned lands and estimated that about 4,00,000 ha of land could be utilized for energy crop plantation. Even Korea is unfortunate in this area as its resources for bioenergy applications are quite limited owing to the fact that only 0.33 × 106 m3 of ethanol can be produced if 20% of the entire rice production is diverted towards the bioenergy sector, and the produced ethanol would only satisfy 0.1% of the imported oil. Inventory from developing countries like Papua New Guinea (0.06 × 106 m3 FG), Peru (1 × 106 tons FG), Phillipines (0.33 × 106 tons FG; 5.4 × 106 tons SG), Singapore (No specific data), Thailand (2.7 × 106 m3 FG; 14 × 106 m3 SG) and Vietnam (4.57 × 106 m3 FG; 28 × 106 m3 SG) was also conducted for ethanol production.

9.5 Conclusion Governments throughout the world are itching to formulate a sustainable policy for second generation biofuels which can see the daylight. In their quest a major factor to be considered is an estimation of the amount of biomass in their jurisdiction along with its spatial distribution. This data will allow the policy makers to predict the capacity of processing biorefineries and their location so that biofuels from these sources can be made sustainable and profitable. The GIS is a useful tool in this quest as it allows the user to visualize the resources by superimposing all the constraints possible within the actual biomass map so that a workable solution can be arrived upon. Input to the GIS analysis is mainly the images retrieved through remote sensing satellites which utilize energy incident from the target objects within the earth’s surface. A significant amount of work has already been carried out throughout the world for estimating the potential biomass resources for bioenergy application. However, most of the efforts had been concentrated on mapping the first generation resources and it is high time for second generation feedstocks to be updated in the map. This data will allow policy makers to look back upon the energy demand and compare it with the available resources and decide whether further plans for energy crop plantations are required for which locations like marginal lands can be utilized.

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16. Fahd, S., Fiorentino, G., Mellino, S., and Ulgiati, S., Cropping bioenergy and biomaterials in marginal land: The added value of the biorefinery concept. Energyy 37, 79–93, 2012. 17. Cai, X., Zhang, X., and Wang, D., Land availability for biofuel production. Environ. Sci. Technol. 45, 334–339, 2011. 18. Milbrandt, A., and Overend, R.P., Assessment of biomass resources from marginal lands in APEC economies, National Renewable Energy Laboratory Report, NREL Golden, CO, USA, 2009. 19. Kang, S., Post, W.M., Nichols, J.A., Wang, D., West, T.O., Bandaru, V., and Izaurralde, R.C., Marginal lands: Concept, assessment and management. J. Agric. Sci. 5, 2013. 20. Wu, W., Huang, J., and Deng, X., Potential land for plantation of Jatropha curcas as feedstocks for biodiesel in China. Sci. China Ser. 53, 120–127, 2010. 21. Lu, L., Jiang, D., Zhuang, D., and Huang, Y., Evaluating the marginal land resources suitable for developing pistacia chinensis-based biodiesel in China. Energies 5, 2165–2177, 2012. 22. Fiorese, G., and Guariso, G., A GIS-based approach to evaluate biomass potential from energy crops at regional scale. Environ. Modell. Softw. 25, 702–711, 2010. 23. Thomas, A., Bond, A., and Hiscock, K., A GIS based assessment of bioenergy potential in England within existing energy systems. Biomass Bioenerg. 55, 107–121, 2013. 24. Fernandes, U., and Costa, M., Potential of biomass residues for energy production and utilization in a region of Portugal. Biomass Bioenerg. 34, 661–666, 2010. 25. Jiang, D., Zhuang, D., Fu, J., Huang, Y., and Wen, K., Bioenergy potential from crop residues in China: Availability and distribution. Renew Sust. Energ. Rev. 16, 1377–1382, 2012. 26. Yoshioka, T., Sakurai, R., Aruga, K., Sakai, H., and Kobayashi, H., A GISbased analysis on the relationship between the annual available amount and the procurement cost of forest biomass in a mountainous region in Japan. Biomass Bioenerg. 35, 4530–4537, 2011. 27. Ministry of Renewable and New Energy, Biomass Resource Atlas, 2010. 28. Hiloidhari, M., Das, D., and Baruah, D.C., Bioenergy potential from crop residue biomass in India. Renew. Sust. Energ. Rev. 32, 504–512, 2014. 29. Singh, J., and Gu, S., Biomass conversion to energy in India—A critique. Renew. Sust. Energ. Rev. 14, 1367–1378, 2010. 30. Kim, S., and Dale, B.E., Global potential bioethanol production from wasted crops and crop residues. Biomass and Bioenerg. 26, 361–375, 2004. 31. Binod, P., Sindhu, R., Singhania, R.R., Vikram, S., Devi, L., Nagalakshmi, S., Kurien, N., Sukumaran, R.K., and Pandey, A., Bioethanol from rice straw: An overview. Bioresource Technol. 101, 4767–4774, 2010. 32. Ranjan, R.K., Ramanathan, A.L., Parthasarathy, P., and Kumar, A., Hydrochemical characteristics of groundwater in the plains of Phalgu River in Gaya, Bihar, India. Arab. J. Geosci. 6, 3257–3267, 2013.

214 Lignocellulosic Biomass Production and Industrial Applications 33. Mussoline, W., Esposito, G., Giordano, A., and Lens, P., The anaerobic digestion of rice straw: A review. Crit. Rev. Environ. Sci. Technol. 9, 895–915, 2013. 34. Johnson, K., Switch grass—On corn acreage or CRP?. Biofuels and Bio-Based Carbon Mitigation 2009. 35. U.S. Department of Energy.  Biofuels from switchgrass: Greener energy pastures. http://bioenergy.ornl.gov/papers/misc/switgrs.html (last accessed 21.08.2016), 2007. 36. Dohleman, F., Miscanthus bests switchgrass as biofuel source, 2007. http:// news.mongabay.com/2007/0711-miscanthus.html (accessed 21.08.2016). 37. Yamamoto, Y., Quick construction of a lawn grass type grazed pasture through Eremochloa ophiuroides seeding. Sustainable Livestock Production Human Welfare 59, 131–134, 2005.

10 Lignocellulosic Biomass Utilization for the Production of Sustainable Chemicals and Polymers Gunjan Mukherjee1,2*, Gourav Dhiman1 and Nadeem Akhtar3 1

Department of Biotechnology, Chandigarh University, Punjab, India 2 The Energy and Resources Institute, New Delhi, India 3 Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada

Abstract The ever-increasing demand for petroleum-derived chemicals and materials has a pronounced effect on energy security and stability across the globe. Utilization of lignocellulosic biomass, which is the most abundant and sustainable biomass, has shown enormous potential with an established technology to occupy the market place, limited to the production of bioethanol and lactic acid. Great strides are required to harness biobased chemicals and polymers from lignocellulosic biomass to mitigate the energy crisis and demand for polymeric materials for human welfare. The valorization of lignocellulosic complex is still a big challenge as effective fractionation of the major biomass components—cellulose, hemicelluloses and lignin—lacks economic viability. To expand the diverse use of biorenewable resources in a biorefinery, effective pretreatment technologies are gaining in importance for use in an economical eco-friendly process. Biobased commodities and specific chemicals have garnered special interest for imitating their petroleum-based counterparts, which they are expected to to be the replacement in near future. Moreover, commercialization of these technologies needs robust techno-economical analysis, evaluation of product performance and consumer acceptance for a biobased economy. Strategic amendments to government mandates and subsidies may conquer the daunting challenge of establishing a biorefinery to attain the successful global transition to attain a sustainable future. Keywords: Biobased chemicals, biorefinery, lignocellulosic biomass, sustainable polymers *Corresponding author: [email protected] Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial Applications, (215–246) © 2017 Scrivener Publishing LLC

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216 Lignocellulosic Biomass Production and Industrial Applications

10.1

Introduction

Worldwide economic sustainability and environmental concerns have raised research interest towards renewable sources of energy to substitute the petroleum-dependent fuels, chemicals and materials. The elevated demand for petroleum-based fuels has engendered the utilization of renewable energy resources to supply biobased fuels and co-products [1]. Lignocellulose-based biorenewable resources lessen the dependence from fossil-derived resources for an established economy and waste management [2]. Lignocellulosic biomasses, such as agricultural, forest and agro-industrial residues [3] along with municipal solid wastes [4], act as a potential candidate due to their abundance across the globe. It has been estimated that around 150–170 × 109 tons of lignocellulosic biomass is produced every year [5], which remains a huge source of various hydrocarbons. Therefore, it is believed that cellulosic materials are the only sustainable source of organic carbon that can replace petroleum for the production of bioenergy and other green chemicals without affecting environmental health [6, 7]. Despite their abundance, the conversion of lignocellulosic biomass to biofuels and other biobased products remains a big challenge due to the recalcitrant nature of the biomass. This inherent property of the biomass limits the hydrolysis step through biochemical conversion routes [8]. In this context, a low-cost pretreatment strategy is a prerequisite to make biomass compatible for an integrated biorefinery [9]. Although lignocellulosic biomass is abundant and available at low cost, the production of biofuels and value-added products and their selective recovery remain a bottleneck for a biorefinery due to the lack of economic viability [10]. Exhaustive research is being carried out across the globe to address this concern worldwide [11]. Technologies involved in biobased biorefineries are reinforced to produce renewable fuels and green monomers [12]. However, many companies based on biorefining technologies have come into existence for the production of fuels, chemicals and materials derived from lignocellulosic materials [3, 13].

10.2

Lignocellulosic Biomass

Lignocelluloses are composed mainly of three organic compounds in the form of polymer: cellulose, hemicellulose, and lignin, along with pectin, glycosylated proteins, extractives, minerals and some phenolic compounds. Cellulose is the most abundant molecule, which consists of a linear chain of glucose units linked by β-1,4 glycosidic bonds [14], selfassembled into microfibrils through hydrogen bonding and van der Waals

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interactions  [15]. The second most abundant polymer is hemicellulose, which consists of xylan, galactomannan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan, and exists in the form of heteropolymer [3]. Finally, among biopolymers, lignin stands third most abundant polymer in nature, composed of three phenylpropane building blocks, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol units, linked to build a complex with hemicellulose with ester bonds to encapsulate cellulose for biochemical conversion [9]. Cellulosic microfibrils are packed linearly in the crosslinked structure of hemicellulose and lignin to provide structural support to the plant [16]. A typical lignocellulosic biomass usually contains 30–55% cellulose, 20–35% hemicellulose, and 10–25% lignin. The distribution of cellulose, hemicellulose and lignin varies within the cell wall [3] and the chemical and molecular characteristics also differ among species, tissues and age of the biomass [8]. From a biorefinery point of view, the fractionation of cellulose, hemicellulose and lignin from lignocelluloses is the biggest challenge for subsequent processing to assort products of choice with optimal recovery. Accordingly, suitable pretreatment prior to biochemical conversion make the cellulose and hemicellulose accessible for enzymatic and/or chemical action [8, 10]. The common path followed for valorization of the biomass is presented in Figure 10.1 for various applications using lignocelluose-based susbtrates (Table 10.1).

Cellulose

Ethanol Fermentation Butanol Acetone Hydrogen

Pre-treatment

Lignocellulosic biomass

Pulping

Hemicellulose

Chemical modification

Lignin

Chemical modification

Fermentation

Paper or cellulose products

Furfural Hydroxymethylfurfural Levulinic acid Thickeners Vanillin Vinyl ester resins Cyanate ester resins Polyuretnabes Polyesters Citric acid Rhamnolipid Lactic acid

Figure 10.1 A generalized concept for production of value-added chemicals and polymers from lignocellulosic biomass.

Biochemical /Chemical process(es)

Alkaline oxidation

Free radical polymerization

Microbial fermentation

Condensation reaction

Polycondensation reactions

Microbial fermentation

Microbial fermentation

Microbial fermentation

Substrate

Lignin

Lignin

Glucose

Lignin

Cellulose, Hemicellulose

Glucose

Cellulosic biomass

Ethanol, Glucose

Glutamic acid

3-Hydroxy propionic acid

Di-acids

Polyurethanes, Polyamides, Polyesters and Polycarbonates

Polyester

Fumaric acid

Vinyl ester resins

Vallinin

Product(s)

Polymerizes into poly-γ- glutamic acid

Acts as precursor for various valueadded products, e.g., acrylic monomers, malonic acids, etc.

Homopolymers of di-acid are used for various biomedical applications

Formation of various biodegradable polymers

Thermostable biobased polyester

Acts as precursor for the production of aspartic acid and its polymer

Biobased substitute for styrene

Used as additive in food and pharmaceutical industry

Use(s)

Table 10.1 Value-added chemicals and polymers obtained from lignocellulose-based substrate and their applications.

[87]

[81]

[78]

[73]

[70]

[84]

[67]

[66]

Ref.

218 Lignocellulosic Biomass Production and Industrial Applications

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10.3

219

Pretreatment Strategies

To date, various approaches of pretreatment have been employed for hydrolysis of the lignocellulosic biomasses, which include physical, chemical, thermophysical, thermochemical and biological, along with their combinatorial possibilities. Different approaches have been tried for deconstructing of lignocellulosic biomasses but any novel method has yet to be established for industrial welfare. This section briefly highlights the different pretreatment techniques for efficient hydrolysis of biomasses in context of their applicability and economic feasibility at an industrial scale.

10.3.1

Physical Pretreatment

10.3.1.1 Physical Comminution and Extrusion Various modes of physical pretreatment like grinding, chipping or milling use microwave irradiations are used to enhance enzymatic hydrolysis of the biomass [17]. The mechanical disintegration (grinding, chipping or milling) is of great importance as it reduces the size of the biomass and therefore enhances the enzymatic hydrolysis [18]. There are different methods of size reduction such as hammer milling, ball milling, two-roll milling, colloid milling, wet-disk milling and vibro-disk milling, vibratory ball milling, jet milling and centrifugal milling [17]. Extrusion is a promising approach that uses extruder in which the biomass is heated, mixed and sheared at the same time for size reduction and effective enzymatic hydrolysis [14, 19].

10.3.1.2 Pyrolysis, Irradiation and Pulsed Electric Field Pyrolysis is meant for depolymerization of cellulose to H2, CO and biochar at a relatively high temperature [14]. The pretreatment strategy is employed under high temperature for breaking the lignin-hemicelluloses or lignin-cellulose bonds. Irradiation pretreatment uses γ-rays and electron beams to release sugars from cellulosic materials [14]. The heat is provided either by direct heat explosion or high pressure steam explosion. In order to prevent the formation of temperature gradient in the treated substrate usually biomass is crushed into small fragments. The temperature gradient is undesirable because hemicelluloses into furfural and reduces sugar recovery. However microwave irradiation provides an alternative to this problem by using uniform heat supply to the substrate in a very short time [20]. Microwave irradiation technique has been applied with various

220 Lignocellulosic Biomass Production and Industrial Applications mediums like high boiling solvents, i.e., glycerol, where 5 min treatment resulted in 5.4% of the lignin and 11.3% xylan degradation in sugarcane baggase biomass [21]. Similarly, ILs-assisted microwave irradiation pretreatments are a present area of research in terms of cellulose dissolution, where the 25% cellulose was dissolved with the 1-butyl-3-methylimidazolium cations and various anions containing ionic liquid were subjected to microwave irradiation for 2–5 seconds [22]. Acid pretreatment is the most widely adopted pretreatment method even though the use of microwaves for heating purposes has resulted in increased efficiency of the pretreatment. A 43.3 g of sugar per 100 g of hyacinth was found when it was treated in 1% H2SO4 with microwave irradiation at 140 °C for 15 min [23]. In addition, pyrolyisis could also be an effective pretreatment strategy where biomass is degraded at a very high temperature (>300 °C) for fermentable sugar in the residual char for the purpose of further fermentation [14, 24]. Pulsed electric field is also another approach where membrane porosity is increased for effective hydrolysis [25]. However, there is no established a single pretreatment which has a distinct industrial application. Each pretreatment give distinct advantages and disadvantages over others [14].

10.3.2

Chemical Pretreatment

10.3.2.1 Acid and Alkali Pretreatment Acid treatment is the most widely accepted chemical pretreatment method which is currently feasible for industrial use. Both concentrated and dilute acids are employed for pretreatment studies. Whereas concentrated acids are less used due to the damaging effect on the carbohydrate component of the biomass, while weak acids show a mild effect. Acids like H3PO4, H2SO4 and HCl were used for pretreatment studies [26]; however, dilute H2SO4 and HCl are known for their cost-effectiveness. Dilute H2SO4 (0.5–2.5%) pretreatment of Moringa olifera and Jatropha curcas at high temperature (100–200 °C) resulted in an 87% and 80% recovery of cellulose, respectively [27, 28]. The use of NaOH, NH4OH, KOH, Ca(OH)2, NH3, and (NH4)2SO3 for pretreatment purpose has been widely explored and extensively used [14]. Sodium hydroxide is a very strong base which acts as a catalyst that cleave the ester and ether bonds between the hemicelluloses-lignin and carbon-carbon bonds in a lignin molecule. Therefore, it is effective to facilitate cellulose degradation to release sugars as a result of enzymatic hydrolysis [29].

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10.3.2.1.1 Oxidative Delignification and Organosolv Pretreatments Delignification of the biomasses can be achieved using different oxidizing agents, such as hydrogen peroxide, ozone, oxygen or air, to act on the aromatic rings of lignin, resulting in cleavage without affecting carbohydrate decomposition [14]. Another important pretreatment is the organic solvent pretreatment that is also widely explored. The method selectively dissolves the lignin and hemicellulose with minimum effect on the cellulosic part of the lignocelluloses. The selective dissolution of the biomass also results in increased surface area available for enzymatic hydrolysis. Furthermore, the lignin recovered from the organic solvents could be further utilized for other lignin-derived commercially important products [30]. Various types of organic solvents have also been exploited for pretreatment processes where the use of low boiling point alcohols is widely employed. The condition required for the pretreatment in alcohol depends upon biochemical and molecular characteristics, generally the biomass is subjected to 35–70% (w/w) alcohol solvent at cooking temperature ranges from 180–195 °C in 2.0–3.8 pH range for 30–90 minute. The liquor is then separated for the recovery of solvent and other dissolved components, while the solid biomass is subjected to saccharification and subsequent fermentation [31]. The most commonly used solvent is ethanol, which is successful among alcohols and is best for its use in pre-treatment. The low cost, ease of recovery and low boiling point make it more user-friendly than the others. However, methanol and high boiling point alcohols have also been used for the purpose of pretreatment [30]. 10.3.2.1.2 Ionic Liquids (ILs) and Surfactants A variety of ILs has been studied for the development of an effective pretreatment strategy. Ionic liquids are very effective, nontoxic, less volatile and odorfree green chemicals which selectively dissolve the lignin and hemicellulosic component for effective enzymatic actions [32]. Despite other solvents, ILs can be used for the separation of different compounds for derivation of various value-added products in biorefineries [33]. Use of surfactants like PEG (polyethylene glycol), Tween 80, Tween 20, SDS (sodium dodecyl sulfate), dodecyltrimethylammonium bromide, Triton X-100, Triton X-114 and Neopelex F-25 are effective in pretreatment as they prevent the unwanted adsorption of enzymes on lignin and trigger hydrolysis mechanism [34].

10.3.4

Thermophysical Pretreatments

There are various thermo-physical pretreatment approaches developed so far such as hydrothermal (steam explosion, hot compressed water, sub- and

222 Lignocellulosic Biomass Production and Industrial Applications supercritical water) and ultrasound. In the hydrothermal pretreatment approach, lignocellulosic biomass is treated at varied temperatures and under high pressure, where the ultrastructure of the biomass is degraded and exposed for enzymatic hydrolysis [35]. However, thermophysical pretreatment using ultrasoncation is a better mode of pretreatment, where high-intensity sound waves loosen the cellulose structure to enhance enzymatic degradation. The pretreatment of sawdust using ultrasonic treatment (10W, 5–10 minute) yielded 61% glucose recovery [36], which provided an evidence of a vital pretreatment strategy.

10.3.5

Thermochemical Pretreatments

10.3.5.1 Oxidation Ozone, one of the strongest oxidizing agents, has been widely exploited for its potential in lignocellulosic pretreatment. When the lignocellulosic substrate is subjected to ozone-mediated pretreatment, the ozone attacks the lignin specifically, as it is electron rich, however the carbohydrate part remains largely unaffected. The ozonolysis of lignocellulosic biomass is highly advantageous as it generate less concentration of inhibitory compound which may hinder the enzymatic hydrolysis. Unfortunately, some properties of ozone such as highly reactive and inflammable and highly inflammable properties of ozone pose limitations of using this method [37]. Different studies have shown different yields of products produced by the ozone pretreatment of lignocellulosic biomass. The lignin content was reduced to 60% when wheat straw was pretreated for 3 hours with ozone and 52% of alcohol was obtained via simultaneous saccharification and fermentation (SSF) of pretreated biomass [38]. Also, there was an enhancement of 158–168% biohydrogen production from the ozone-pretreated wheat and barley straw in comparison to the non-pretreated ones [39, 40]. Similarly, oxidation at a very high temperature in water (wet oxidation) yields the dissolution of lignin and hemicelluose and therefore increases the biomass’s potency for hydrolysis [41]. 10.3.5.1.1

Ammonia Fiber Explosion (AFEX) and Ammonia Recycle Percolation (ARP) Ammonia fiber explosion pretreatment method exposes the lignocellulosic biomass to ammonia at high temperature and pressure in the presence of water. The pretreatment method involves the penetration of ammonia into the cell wall and the cleavage of various ester linkages. When pressure is released the resultant cleaved diferulate linkages cause the pore in the middle lamella of the cell wall to provide the route for penetration into different cellulases in the cell wall for the release of sugars. Usually the

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pore size as a result of AFEX pretreatment is larger than 10 nm, which facilitates the easy entry for cellulases [42]. The efficacy of AFEX treatment is better with biomass with low lignin content biomass while high-lignincontaining biomass yields low sugars [43]. Another mode of pretreatment using ammonia in a fixed-bed reactor is flow-through mode (ARP, ammonia recycle percolation), which has been found to be an effective pretreatment strategy where lignin is dissolved and removed simultaneously from the mixture in a flow through, preventing its redeposition [44]. 10.3.5.1.2 Supercritical-CO2 (SC-CO2) Pretreatment Another pretreatment approach is the usage of SC-CO2, where it acts as both a solid and liquid diffusing through the lignocellulosic biomass, resulting in an explosion and biomass degradation, thereby providing greater surface area for hydrolysis [26].

10.3.6

Biological Pretreatment

The majority of chemical pretreatment techniques, except the dilute acid and alkali method, are not very cost-effective and incur elevated cost for ligocellulose processing. The downstream processing and waste treatment are another challenge. In order to find another eco-friendly and cost-effective alternative, significant advancement has been made in the direction biological pretreatment. Various fungi, such as white-rot, brown-rot and soft-rot fungi, are being used to counteract the high processing cost and formation of treatment-based inhibitors. White-rot fungi like Phenerochaete crysosporium, Pycnoporus cinnabarinus, Ceriporia lacerate, etc., have been wellknown for pretreatment of biomasses for decades. Similarly, soft-rot fungus Daldinia concentrica and brown-rot fungi Serpula lacrymans and Coniophora puteana have also been exploited for lignocellulosic biomass pretreatment [45]. In one study, 51 isolates of white-rot fungi belonging to Punctularia sp. were used to show 50% lignin removal from bamboo culms [46]. Another white-rot fungus, Irpex lacteus, has shown 82% hydrolysis of corn stalk in biological pretreatment [47]. Although the fungal isolates have shown more promising results compared to bacteria, though co-culture strategy has shown even more enhanced saccharification [48]. Among bacteria, Bacillus spp. have also been reported for biodegradation of cellulosic biomass for saccharification studies [49, 50]. Hence we can say that the biological pretreatment provides an additional advantage over the conventional pretreatment, because they produces less inhibitors and the chances of equipment corrosion is also low. Further development in this line of research could provide a vital pretreatment alternative with improved efficiency at low cost.

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10.4

Value-Added Chemicals from Lignocellulosic Biomass

10.4.1

Lignocellulose-Derived Sugars

Glucose is the primary and most abundant sugar produced from lignocelluoses, that are rich in cellulose content. However, further degradation of the hemicellulose can give rise to other pentose sugars such as xylose and arabinose and hexoses like mannose, galactose and rhamnose [3, 14]. The produced sugars could easily be exploited for subsequent fermentation for the production of bioethanol and other value-added products. The extraction of sugars and its concentration depends upon the type of biomass used, employed pretreatment technique, physical and physiological process parameters and type of microorganism and enzyme used. Steam explosion method along with dilute NaOH at 210 °C resulted in the highest hemicellulose yield using horticultural waste [51]. The dilute acid pretreatment is thought to be the most efficient method for lignocellulosic biomass degradation and liberation of fermentable sugar, since the processing cost is reasonable. For instance, dilute phosphoric acid pretreatment at 135 °C on potato peel biomass resulted in 55.2 g sugar/100 g of biomass after 8 min [52]. The ionic liquid pretreatment resulted in effective hydrolysis of various lignocellulosic substrates and release of sugars. The oil palm biomass, when pretreated with ionic liquid, resulted in 100% glucose recovery after 15 minutes of treatment at 80 °C [53]. As discussed above, the concentration of released sugar will depend upon the nature and carbohydrate content of the feedstock and the pretreatment method. The treatment of rice husk with tetrabutylphosphonium hydroxide (TBPH) resulted in significant disruption of the cellulose mass. A study has revealed that the TBPH-treated rice husk resulted in 325 mg of glucose per gram of biomass when hydrolyzed with H2SO4 [54]. The pretreatment of corn stover by acetylation followed by dilute acid pretreatment resulted in the liberation of different sugars in varied concentrations, i.e., glucose (13.60 g/L), xylose (99.00 g/L), galactose (6.66 g/L) and arabinose (15.60 g/L) [55]. The different sugars yielded can be further processed for the production of different value-added products. The organoslov pretreatment of rice straw at 180 °C followed by enzymatic scarification yielded 15.1 g/L glucose, 5.6 g/L xylose and 0.9 g/L arabinose [56]. However, the yield of different sugars usually varies depending on the time and temperature used in the organosolv pretreatment. If we particularly look at the yield of the xylose, the acid pretreatment at 140  °C resulted in 37.3 g/L of xylose, which accounted for 93% of the recovery [57]. The aqueous ammonia soaking pretreatment of rice straw resulted

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in 87–98% glucan recovery depending upon the time and concentration of the ammonia [58]. As already discussed above, the yield of the sugars released from the lignocellulosic substrate will depend upon the pretreatment method used. Thus the two sequential pretreatments with H2SO4 resulted in the remarkable sugar release from softwood. The first pretreatment with dilute H2SO4 (0.5% w/w) at 180 °C for 10 min resulted in 91% of the glucan release and 100% of the mannan release. After the second pretreatment the amount of glucan remaining was hydrolyzed into the glucose and its concentration varied between 14–77% [59]. The salix chips, when pretreated under varying concentrations of H2SO4 and SO2 gas, can recover glucan and xylan from 96–99% and 47–74%, respectively [60]. The steam explosion pretreatment of poplar biomass yielded 68–70% of the glucose when pretreated at high temperature (230– 240 °C) and 41% of the xylose was recovered when the poplar biomass was pretreated at 210 °C for 4 min [61]. Another attempt of pretreatment of olive tree pruning with different concentrations of H2SO4 at different temperatures (190–240 °C) resulted in 38–48% glucose recovery and 0.3–9.3% xylose recovery, whereas the concentration of other sugars like galactose, mannose and arabinose varied from 0.9–1.6% after the pretreatment [62]. In a nutshell, it can be concluded that the quantity of different sugars liberated from diverse lignocellulosic substrate is directly controlled by the nature and composition of the feedstock and the pretreatment method opted for it. The vitality of the technology will depend upon the different sugar concentrations and their further conversion into value-added products through economical pretreatment routes for actualization of a circular economy.

10.4.2

Lignin-Derived Chemicals

Lignin is usually considered as the mechanical component of lignocellulosic biomass, which is known for providing rigidity to the plant. Four diff ferent processes, viz., sulfite, kraft and organosolv, have been developed industrially for isolation of the lignin. In the sulfite process the aqueous SO2 is used at different pH for lignin fractionation. Whereas, in the soda lignin process the NaOH is used for the dissolution of lignin and in the kraft process the lignocellulosic biomass is dissolved in the solution of NaOH and Na2SO3 at about 170 °C. The organosolv-based lignin isolation from lignocellulosic biomass is fractionated ethanol-water solution and resulted in production of high quality lignin [63]. The higher aromaticity of the lignin makes it a desirable candidate for production of commercially valuable products. There have been multiple

226 Lignocellulosic Biomass Production and Industrial Applications approaches for the depolymerization of lignin for harnessing its aromatic monomeric units for the modification and generation of economically viable organic products. Hydrogenolysis, oxidation and acidolysis have been used in the presence of various catalysts for the extraction and modification of monomeric aromatic units for lignin-based chemical production. The viability of the above-mentioned processes is still limited because of the cost and generation of non-aromatic monomers during the process, which is why very few lignin-based chemicals been commercialized so far [64]. Very few lignin-derived products have been commercialized, i.e., vanillin, dimethyl sulfide and dimethyl sulfoxide, which are produced by oxidation of lignin in alkaline conditions, reaction of craft lignin with molten sulfur and oxidation of isolated sulfoxide, respectively [65]. The further polymerization of vanillin has been potentially exploited for the production of various polymers of great economic importance. The generated product carries thermosetting properties for production of thermostable polymers such as thermoset plastics. The advantage of using lignin as a raw material for producing such polymers is not only to enhance renewability but also to make it biodegradable as compared to petroleum-derived polymers.

10.4.2.1 Vanillin Vanillin is the most common type of polymer synthesized from lignin with vital commercial values in current times. It is synthesized via alkaline oxidation of the lignin and also used as a flavoring agent in various food and pharmaceutical products [66]. Various chemical methods have been developed for producing vanillin and its polymers which can be further used for synthesis of other products.

10.4.2.2 Vanillin-Based Resins Vinyl ester resins are produced by polymerization of vanillin methacrylate and glycerol dimethacrylate. The resins are produced from the free radical polymerization of the vinyl ester produced. The formed resins possess high thermal stability over conventional vinyl ester resins where styrene is used. Therefore, vanillin methacrylate can act as a suitable biobased substitute for the styrene [67].

10.4.2.3 Cyanate Ester Resins Similar to the vinyl ester resins, another thermostable resin could be obtained from bisphenols produced by the reductive coupling and hydrogenation of the vanillin aldehyde and vanillin, respectively. The yielded

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vanillin-based cyanate ester resins possess very high thermostable properties comparable to that of commercial petroleum-based products [68].

10.4.2.4 Epoxide Resins The epoxide resins, also have thermosetting properties and are widely used in different coatings, electronics and for different adhesive purposes. The pure lignin serves an important precursor for production of these resins. Chemical modifications of the various epoxides from the vanillin from lignin yield the epoxide resins [66].

10.4.2.5 Benaoxazine Resins Benaoxazine resins are another important lignin-derived high temperature adhesive polymer with an excellent stiffness. The polybenzoxazines have great thermomechanical and chemical properties. They are produced using guaiacol and vanillin as monomeric units [69].

10.4.2.66 Polyester In addition to the above-described thermostable polymers, the use of lignin has expanded to the synthesis of thermostable polyester. Lignin condensation under basic conditions with the sebacoyl chloride resulted in the polyester having a transition temperature of 70 °C [70]. It has been observed that with the increase in the lignin content the thermal stability of the polymer increased significantly. Also, the variation in lignin proportions in various different processes resulted in the change of other properties like rigidity, flexibility and toughness. [64].

10.4.2.77 Polyurethanes The modification of lignin for the generation of new active sites for further chemical modification has opened another door for expanding the range of various chemicals synthesized from lignin. Polyurethanes are an example of such an approach, where demethylation of the methoxy group in the lignin and their reaction with the toluene 2,4-diisocyanide results in the generation of the same [71]. Also, the biodegradability of such polymers is enhanced when used with lignin-based polyols in comparison to the non-lignin-based ones. The physical properties of the polyurethanes from lignin are better than the conventional petroleum-based ones. The only difficulty lies in the lignin properties, as they differ from source to source and need to be characterized before utilization.

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10.5

Sustainable Polymers from Lignocellulosic Biomass

10.5.1

Sugar-Containing Polymers

As discussed earlier, lignocellulosic biomass is a rich source of carbohydrates and can provide a variety of C5 and C6 sugars and their derivatives for different polymerization reactions. Extensive research is being conducted to develop glycopolymers with therapeutic potential for widespread diseases such as acquired immune deficiency disease (AIDS) and Alzheimer’s disease [72]. There are multiple types of base polymers which are produced by different chemical methods; for instance, hyperbranched polymers, block polymers, polymer brushes, etc. In order to synthesize glycopolymers of these categories, monomeric units of sugar are subjected to various polymeric reactions like reversible addition-fragment chain transfer (RAFT) polymerization, nitroxide-mediated polymerization (NMP) and atom-transfer radical polymerization (ATRP) reaction [3]. Other than C5 and C6 sugars, other monosaccharides obtained from lignocellulosic biomass can be used for the synthesis of biopolymers which are nontoxic and biodegradeable polymer. The abundance of OH and COOH groups in sugar moieties makes them suitable for polycondensation reactions. Polymers like polyurethanes, polyamides, polyesters and polycarbonates have been reported to be synthesized by polycondensation reaction [73]. In conclusion more study is required to come up with economically feasible sugar-derived functional polymers for market entry and stability.

10.5.1.1 1,4-Diacid-Based Polymers The successful microbial production of succinic acid has now drawn the interest of scientists towards production of biobased polymers. The condensation of succinic acid with its ester derivative (succinic acid diesters) can produce various polymers such as polyamides, polyesters and polyester amides. To date, high melting point succinic acid polyesters like polyethylene succinate and polypropylene succinate have been successfully commercialized [74]. They are biodegradable and are good to process and recycle; at the same time they possess similar mechanical properties to that of polyethylene [75]. Polybutylene terephthalate (PBT) is another polymer with high thermal stability, strength and chemical resistance, which is produced from 1,4-butanediol generated from the hydrogenation of succinic acid [76]. Another polymer resulting from succinic acid hydrogenation, i.e., gamma butyrolacetone (GBL), is suitable for the production of poly-4-hydroxybutyrate via

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the process of ring-opening polymerization reaction [77]. The ring-opening process for tetrahydrofuran (THF) has resulted in the commercially more successful polymer, i.e., polytetramethylene ether glycol (PTMEG), which is used to make thermoplastic elastomers. In addition to succinic acid, other diacids like fumaric acid, when homopolymerized, have a wide range of application. The production of these diacids can be done through fermentation with organisms, which produces more C4 diacids [78].

10.5.1.2 5-(Hydroxymethyl) Furfural (HMF)- and 2,5-Furandicarboxylic Acid (FDCA)-Based Polymers Both HMF and FDCA possess huge market potential for their intermediate chemicals and their derived products. Their capacity to undergo various chemical modifications and generate economically important polymer is a matter of extensive research. The Wittig reaction can convert 5-HMF to vinyl monomers in a solid liquid-phase transfer process followed by a free radical polymerization reaction [79]. The 5-HMF can be easily converted into various furan-based derivatives which provide a distinct advantage of polymerization due to the presence of the furan ring. One of the important furan monomers is 2,5-FDCA, which is produced as a result of oxidation of the HMF [80]. The 2,5-FDCA shows comparable properties as that of terephthalic acid (TA), therefore significant efforts are being made to use 2,5FDCA in biobased polymer in spite of oil-based polyesters. A well-known organization named Avantium has successfully developed polyethylene furanoate from 2,5-FDCA derived from lignocellulosic sugar [3].

10.5.1.3 3-HPA (3-Hydroxy Propionic Acid) Platform-Based Polymers The 3-HPA platform-based polymers are an important component for the production of various high-value products such as 3-hydroxypropionic esters, acrylic monomers 1,3-propanediol, propene, β-propiolactone and malonic acids [81]. There are various chemical methods available for the production of these value-added products from 3-HPA but lack of cost effectiveness has initiated the exploration of biological means. A microbial process was used for the production of 3-HPA from cellulosic feedstock via fermentation [82]. The dehydration reaction of 3-HPA in the presence of acid, catalyst and metal powder is being used for the production of acrylic acid. The technology to dehydrate 3-HPA in to propene through decarboxylation-coupled reaction is yet to be commercialized. Therefore, the dehydration reactions of 3-HPA could easily be used for production of acrylic monomers [83]. Using 3-HPA platform for the production of

230 Lignocellulosic Biomass Production and Industrial Applications acrylic acid can provide vital biobased acrylic polymers for commercial usage, since all commercially available acrylic polymers contain acrylic acid. Therefore, the possibility of 3-HPA production by biological means may prove insightful for the significant utilization of lignocelluloses for commercial polymer production.

10.5.1.4 Aspartic Acid Platform-Based Polymers Aspartic acid-based polymers, such as polyaspartic acid (PASA) polymers, are more desirable as they have properties of polyacrylic acid and are biodegradable. The synergy between the polymeric properties of these two molecules lead to a huge market potential for aspartic acid-based polymers. The production of poly aspartic acid polymers (PASA), mainly thermal polyaspartate (TPA) production, is a more commercially viable process for the purpose of polymerization [84]. Aspartic acid is produced mainly from ammonia and fumaric acid in the presence of microorganism or immobilized enzyme aspartate. Therefore, the production of fumaric acid from lignocellulosic feedstock seems to be a viable option for commercial production of fumaric acid, which can easily be converted into aspartic acid [85].

10.5.1.5 Glutamic Acid Platform-Based Polymers Glutamic acid is mainly produced via microbial fermentation in the presence of Brevibacterium spp. and Corynebacterium spp. using glucose, ethanol and glycerol as a substrate. However, the downstream processing is still a daunting challenge for a cost-effective process. Currently, new membrane-based technologies are being developed to reduce the downstream processing cost [86]. The most successful commercial polymer of glutamic acid is poly-γ-glutamic (γ-PGA) acid, which has a wide range of applications in the pharmaceutical, cosmetics, food and water treatment industries. It is mainly produced from various carbon sources like glutamic acid, fructose, glucose, glycerol, etc., using different Bacillus spp. [87]. The production of γ-PGA from lignocellulosic biomass has been successfully optimized in a pilot-scale study [88]. Therefore, an efficient utilization of lignocellulosic biomass as a renewable source for glutamic acid and its polymer synthesis may provide greater commercial usage of these feedstocks.

10.5.1.66 Glucaric Acid-Based Polymers The synthesis of sugar-containing polymers provides a huge scope for utilization of glucaric acid-based platform for their synthesis. The oxidation

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of D-glucose using an enzymatic treatment provides D-gluconic acid comonomer for synthesis of such polymer [89]. The chemical conversion of D-gluconic acid to dilactone and its ring-opening reaction with the lactide yield an important biodegradable polyester with enhanced physical properties which can be used for various biomedical applications [90]. Another important advancement in the usage of glucaric acid lies in the development of an efficient polymer which may be used for effective gene delivery. An effective gene delivery system contains a carbohydrate moiety, secondary amine and amine bonds, where glucaric acid acts as a carbohydrate co-monomer for synthesis of the polymer. The synthesized polymers are called poly(D-glucaramidoamine)s (PGAAs), which are proven to be a great gene delivery vehicle without any toxic effects [91]. Thus, this new path can be seen for providing cellulosic raw material to create carbohydrate moieties for polymer synthesis for the market place.

10.5.1.77 Itaconic Acid (ITA) Platform-Based Polymers Itaconic acid (ITA) is a compound of great interest in polymer chemistry because of the availability of two acid groups and one vinyl group. Currently, ITA is produced from the fermentation of carbohydrate substrate using fungi. The similarity between ITA and petroleum- based acrylic acid provides the advantage of using ITA for polymeric synthesis owing to its ecofriendly and cost-effective synthesis, while the chemical synthesis of acrylic acid from petroleum is an expensive multistep chemical process. The ITA is already in use for commercial production of various binders, thickeners, fibers and adhesives [92]. A homoploymer of ITA has already been commercialized and is being produced using Aspergillus niger via fermentation process of the biomass for its wide range applications [93].

10.5.1.8 Levulinic Acid Platform-Based Polymer Levulinic acid is a monomer which has been successfully produced at industrial scale using lignocellulosic biomass via acid-catalyzed dehydration process [94]. The acid has its own importance as it acts as a precursor for various valuable compounds with novel applications in pharmaceuticals, additives and plasticizers [95]. The homopolymer of levulinic acid provides an important replacement for the petro-chemical (bisphenol A)-based polymers, as the use of the latter has adverse effects on human health. The levulinic acid-based diphenolic acid is considered more suitable for various polymer syntheses than bisphenol A (BPA). Therefore the petroleum-based BPA can easily be replaced by lignocellulosic-based diphenolic acid [3]. Hence, the use of levulinic acid for various industrial

232 Lignocellulosic Biomass Production and Industrial Applications productions is very much of a reality and its production from lignocellulosic biomass further opens the door for production of different polymers.

10.5.1.99 3-Hydroxy-Butyrolactone (3-HBL) Platform-Based Polymer The importance of 3-hydroxy-butyrolactone (3-HBL) is widely known in polymer producing biorefinery. Its application in pharmaceuticals (cholesterol-lowering drugs, antibiotics, and antihyperlipidemic drugs), polymers and solvents have made it one of the most important chemicals produced for commercial use. Most 3-HBL production is still via chemical synthesis, which is a relatively an expensive process [96]. In the past few years, metabolically engineered microbes have been used for its biological production using glucose and glycolic acid as substrate [97]. Therefore, the scope exists for enormous production of 3-HBL though biochemical routes using lignocellulosic biomass.

10.5.1.10 Sorbitol-Based Polymers Sorbitol is one of the most commonly used polyalcohols because of its wide application as artificial sweeteners, in cosmetics, excipients, and ascorbic acid. Additionally, sorbitol can also be used for the production of other lower alcohols, such as glycerol, ethanol and methanol, which can be further used for the production of value-added chemicals, materials and polymers. For wide application in various industries, sorbitol is produced via enzymatic hydrolysis at a larger scale, mainly using wheat and corn [3]. However, production of sorbitol from cellulose in the presence of Ru/ magnatite nanoparticle catalysis has also been exploited [98].

10.5.1.11 Glycerol-Based Polymers Over the decades, the microbial production of biodiesel has been well known; however, 90% of glycerol is produced as a by-product of the biodiesel industry. Glycerol has applications in the cosmetics, pharmaceutical, food and polymer industries, with a major portion being utilized for the polymer industry [99]. There are many products which can be produced from glycerol; for instance, production of 1-propanol and propane via hydrogenolysis, which are very common fuels for industry. The conversion of lignocellulosic biomass-derived glycerol for the production of bi-propanol has been studied [100], ever since hydrogenolysis is an expensive process. Similarly, 1-propanol has also been successfully produced from lignocellulosic biomass using the genetically modified bacterium Thermobifida fusca

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[101]. However, there has yet to be a techno-economical analysis of the commercialization of the biological process.

10.5.1.12 Lactic Acid-Based Platform Lactic acid-based polymers have great for application in the production of biodegradable polymers, especially in the food, cosmetics and pharmaceutical industries. A novel hyperbranched polylactic acid (PLA) can easily be produced by polycondensation of lactic acid or ring-opening polymerization of lactide in the presence of glyceric acid, which can compete with other biodegradable polyesters. The commercial production of lactic acid is common and it is being produced on a large scale across the globe via fermentation process using glucose, sucrose and starch as a substrates. The lignocellulosic biomass usage for lactic acid production provides a vital cheaper alternative. Moreover, a direct conversion of the lignocellulosic biomass into lactic acid has been achieved without the addition of any enzymes [3].

10.5.1.13 Acetone-Butanol-Ethanol-Based Polymer Bioethanol production technology using lignocellulosic biomass is wellestablished and commercialized. The efficient production and betterment of this technology is being further investigated [102]. However, significant attention is being paid to the production of butanol and acetone, where butanol provides a better alternative for gasoline than ethanol [103]. The acetone, butanol and ethanol compounds are very important precursors of many value-added chemicals and can be converted via various chemical processes. For instance, acid-based conversion of acetone to 2-propanol using Ir (0)n nanoclusters [104]. Furthermore, butanol and ethanol conversion to butyric and acetic acid via oxidation have been achieved, which could further act as polymeric precursors [105, 106]

10.5.1.14 Xylose/Furfural/Arabinitol Platform-Based Polymer The sugars of lignocellulosic biomass like xylose and arabinitol act as a potent precursor for polymer production. Their tendency to undergo various chemical reactions gives rise to various value-added chemicals. The new xylitol- and L-arabinitol-based polymers possessing polyethylene teraphthalate (PET)- and polybutylene teraphthalate (PBT)-like properties have been successfully produced [107]. Similarly, the production of furfural compounds from xylose has been employed further for the production of furans and various furfural-based polymers [3].

234 Lignocellulosic Biomass Production and Industrial Applications

10.5.1.15 Polyhydroxyalkanoate (PHA) Polyhydroxyalkanoate is a naturally occurring polymer which is completely biodegradable and has a wide range of applications in different industries. It has been observed that nearly 30% of the total bacteria are capable of producing it and they usually reside in extreme conditions [23]. The lignocellulosic substrate for the production of this polymer is very promising as it supplies hexose and pentose sugars to establish a fermentation process [108].

10.5.1.166 Rubber Polymers The monomers for rubber production from lignocelluosic biomass could substitute the raw material for synthetic rubber. The most important monomers used in the rubber industry are isoprene, isobutene and butadiene [92]. The technology involved in isoprene production from metabolically engineered organisms has been successfully achieved [3]. In a nutshell, the biomass-based rubber polymer carries huge potential to replace the synthetic one in the near future and plays a key role in actualization of a circular economy.

10.5.1.177 Other Lignocelluolse-Derived Polymers There are many varieties of polymer precursors which can easily be produced from lignocellulosic biomass; citric acid production using A. niger is one example [109]. It can be used for the production of polyester elastomers; for instance, urethane-doped polyester, polyalkene maleate citrates, poly xylitol-co-citrate and many more [110]. Similarly, production of limonene, an interesting monomer from the orange juice industry, has also shown great potential in the polymer industry [92]. Furthermore, erythritrol, 2,3-butanediol and L-lysine can also be produced from lignocellulosic biomass via various chemical and biological means [3]. Rhamnolipid is an important glycolipid in the pharmaceutical and food industry that has been produced via fermentation of lignocelluosic biomass by Pseudomonas aeruginosa NCIM 2036 [111].

10.6

Potential Challenges for a Sustainable Biorefinery

Real-time biorefinery implementation is still facing various challenges, mainly due to the lack of cost-effectiveness of the process. Cost and yield

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are not in synergy for a biorefinery, which is the bottleneck in commercialilization at large-scale as per consumer demand. Great strides have been made in the search for suitable pretreatment techniques for industrial applications, which are cost-effective and eco-friendly in nature. Production of ethanol using oxalic acids and alkali at a high temperature have been successfully implemented at industrial scale [112]. Besides hunting for an effective pretreatment, selection of the biomass which is rich in carbohydrates with minimum pretreatment-based inhibitory products remains a necessity [113]. Effective enzymatic hydrolysis and sugar liberation is another aspect after selection and pretreatment of the biomasses. The availability of biomass surface area that is accessible for enzymes and non-specific bindings, is other problem that is frequently encountered. Furthermore, formation of various inhibitory products like furfural, 4-HMF, aliphatic and other aromatic compounds directly affect either the enzyme activity or microbial growth during the fermentation process, which ultimately reduces the product yield and recovery [114]. For decades, anaerobic digestion of the biomass has been a practice mainly for waste management and manure production. The challenges and opportunities associated with anaerobic biorefinery are being critically analyzed and resolved for industrial and household applications [115].

10.7

Environmental Effects of Biorefineries

A variety of value-added products from lignocellulosic biomasses provide an additional advantage over conventional fuels and polymers in term of their eco-friendly nature. However, there are various environmental effects which have been observed in the lignocellulose-based biorefinery. The main frightening effect on the environment is deforestation and industrialization; eutrophication of water is another setback. Generation of carcinogens and particulate matters create respiratory problems, which were less in fossil-based fuel industry. Although the exact biorefinery mechanism is yet to be established in terms of the industrial implementation, the current environmental effects are evaluated for successful implementation [116]. Furthermore, gasification of lignocellulosic biomass results in tar formation, which is less useful when compared to the one produced from gasification of coal. In this context, the generated tar may trigger serious health and environmental issues, hence need to be addressed properly. Research studies are being carried out to counteract this problem using different temperatures and catalysts, all with limited success [117]. A robust environmental impact assessment needs to be carried out before establishment

236 Lignocellulosic Biomass Production and Industrial Applications of such a biorefinery, which should be continuously monitored during operation as per government mandates and policies.

10.8

Future Perspectives of Biorefineries and Their Products

To date, the major portion of lignocellulosic biomass is used in the paper and pulp industry. Despite extensive studies concerning the valorization of lignocellulosic biomass, there is still a long way to go for successful implementation of technologies for the actualization of a circular economy. So far the main focus has been directed towards biofuel production and the unutilized biomass converted for animal feed or biogas production. The development of a biorefinery on economic and environmental ends, more effective utilization of biomass needs to be investigated for the production of more value-added products [118]. Besides the effective pretreatment technology, the selective recovery of lignin and lignin-based polymer needs to take shape in the future because it is an integral part of the industrial biorefinery [119]. Amelioration of the process and techniques involved in the product formation and recovery also plays an important role in a sustainable biorefinery. A rational evaluation of other potential products will contribute to the development of a sustainable lignocellulosic biorefinery. For instance, bio-oils which form lignocellulosic feedstock via pyrolysis can give an additional fuel alternative to bioethanol [120]. The majority of polymers defined above are very low yielding, expensive and produced via chemical conversion, they need an advanced technology in order to compete with the market. Low-cost polymer production via developing some biological route, i.e., direct conversion, through microorganisms can be highly useful and eco-friendly. However, the biobased biorefinery concept is going to flourish in the coming decades. Various industrial players like Lignol, Verenium, NatureWorks, BioAmber and Mascoma are wellestablished and keenly working in the direction of biorefinery and polymer development [3].

10.9

Conclusion

Lignocellulose-based biorefineries for production of biofuels, biobased chemicals and materials are reaching their tipping point and are expected to flourish in upcoming years. Thermochemical and biochemical conversion technologies for cellulosic materials are extensively employed mainly

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for production of bio-oil, syngas, ethanol and butanol. Currently, syngas fermentation is the advanced biomass conversion technology for gas-toliquid fuels using mesophilic and thermophilic microbes. In the past few years, lignocellulose-based biorefinery has been increasing and has been successful for the production of ethanol, butanol and lactic acid at largescale. However, other co-products in the form of acids and aldehydes, such as sorbitol, xylitol, glycerol, propanediol, levulinic acid, acetic acid, itaconic acid, adipic acid, succinic acid, PBT, PLA, PHA and FDCA, are other potential products seeking consumer attention for market establishment. An integrative approach for simultaneous production of value-added coproducts with bioethanol or biodiesel may hit the “zero waste” concept to maximize the economic sustainability of biorefineries.

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242 Lignocellulosic Biomass Production and Industrial Applications 69. Wang, C., Sun, J., Liu, X., Sudo, A., and Endo, T., Synthesis and copolymerization  of fully bio-based  benzoxazines  from  guaiacol, furfurylamine  and stearylamine. Green Chem. 14, 2799, 2012. 70. Binh, N.T.T., Luong, N.D., Kim, D.O., Lee, S.H., Kim, B.J., Lee, Y.S., and Nam, J.D., Synthesis of lignin-based thermoplastic copolyester using kraft lignin as a macromonomer. Compos. Interface. 16, 923, 2009. 71. Chung, H., and Washburn, N.R., Improved lignin polyurethane properties with Lewis acid treatment. ACS Appl. Mater. Interfaces 4, 2840, 2012. 72. Becer, C.R., The glycopolymer code: Synthesis of glycopolymers and multivalent carbohydrate–lectin interactions. Macromol. Rapid. Commun. 33, 742, 2012. 73. Munoz-Guerra, S., Carbohydrate-based polyamides and polyesters: An overview illustrated with two selected examples. High Perform. Polym. 24, 9, 2012. 74. Bechthold, I., Bretz, K., Kabasci, S., and Kopitzky, R., Succinic acid: A new platform chemical for biobased polymers from renewable resources. Chem. Eng. Technol. 31, 647, 2008. 75. Kabasci, S., and Bretz, I., Succinic acid: Synthesis of biobased polymers from renewable resources, in: Renewable Polymers, Mittal, V. (Ed.), pp. 355–379, John Wiley & Sons, Inc., 2011. 76. Luque, R., Clark, J.H., Yoshida, K., and Gai, P.L., Efficient aqueous hydrogenation of biomass platform molecules using supported metal nanoparticles on Starbons®. Chem. Commun. 45, 5305, 2009. 77. Moore, T., Adhikari, R., and Gunatillake, P., Chemosynthesis of bioresorbable poly(γ butyrolactone) by ring-opening polymerisation: A review. Biomaterials 26, 3771, 2005. 78. Roa Engel, C.A., Straathof, A.J., Zijlmans, T.W., van Gulik, W.M., and van der Wielen, L.A., Fumaric acid production by fermentation. Appl. Microbiol. Biotechnol. 78, 379, 2008. 79. Yoshida, N., Kasuya, N., Haga, N., and Fukuda, K., Brand-new biomass-based vinyl polymers from 5-hydroxymethylfurfural. Polym. J. 40, 1164, 2008. 80. Gandini, A., Furan monomers and their polymers: Synthesis, properties and applications, in: Biopolymers: New Materials for Sustainable Films and Coatings, Plackett, D. (Ed.), pp. 179–209, John Wiley & Sons, Ltd: Chichester, UK, 2011. 81. Rajagopal, R., Sustainable Value Creation in the Fine and Speciality Chemicals Industry, pp. 153–197, John Wiley & Sons Ltd, 2014. 82. Jiang, X., Meng, X., and Xian, M., Biosynthetic pathways for 3-hydroxypropionic acid production. Appl. Microbiol. Biotechnol. 82, 995, 2009. 83. Corma, A., Iborra, S., and Velty, A., Chemical routes for the transformation of biomass into chemicals. Chem. Rev. 107, 2411, 2007 84. Gross, R.A., and Kalra, B., Biodegradable polymers for the environment. Science 2002, 803, 2003.

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11 Utilization of Lignocellulosic Biomass for Biobutanol Production Anand Prakash1, Vinay Sharma1, Deepak Kumar2, Arindam Kuila1* and Arun Kumar Sharma1 1

Department of Bioscience and Biotechnology, Banasthali University, Rajasthan, India Department of Plant Sciences, Central University of Jammu, Jammu, India

Abstract Nowadays, research has been focused on n-butanol production from lignocellulosic biomass. Compared to traditional biofuel, butanol has several advantages: its higher energy content (29.2 MJ/dm3) can be used in pure form or mixed in any concentration with gasoline or diesel; it can be used without modifications in automobile engines; it is non-hygroscopic so less susceptible to separation in the presence of water; it has a lower vapor pressure and can be easily added to conventional gasoline. The challenge for industrial lignocellulosic biobutanol production are reducing the cost of pretreatment, saccharification and fermentation steps. These problems can be solved with an expansion of research in the biobutanol area. This chapter deals with the pretreatment, enzymatic hydrolysis and fermentation steps involved in lignocellulosic butanol production. Keywords: Biobutanol, lignocellulosic biomass, pretreatment, enzymatic hydrolysis, fermentation

11.1

Introduction

Biobutanol is a renewable fuel source that is increasingly being considered as blending component or replacement for traditional petroleum fuels, particularly for use in internal combustion engines and heating appliances [1, 2].

*Corresponding author: [email protected] Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial Applications, (247–264) © 2017 Scrivener Publishing LLC

247

248 Lignocellulosic Biomass Production and Industrial Applications Environmental, economic and geopolitical factors have created a market for biobutanol [3–5]. It is introduced in low blend percentages for use in engines and appliances designed for traditional fuels. As an expanding market, biobutanol usage is expected to increase significantly as production capacity is brought in line and additional market incentives are made available.

11.2

Bioconversion of Lignocellulosic Biomass to Biobutanol

Biobutanol produced from lignocellulosic biomass is considered as second generation biofuels. Conversion of abundant lignocellulosic biomass to biobutanol to be used as transportation fuels presents a viable option for improving energy security and reducing greenhouse gas emissions [6]. In comparison to fossil fuels, biobutanol emits a lesser amount of CO2 into the atmosphere. It has been reported that biobutanol produced from biomass resources has the potential to cut greenhouse gas emissions by 86% [7]. Lignocellulosic materials, such as agricultural residues (e.g., wheat straw, sugarcane bagasse, corn stover), forest products (hardwood and softwood), and crops such as switchgrass and salix, are becoming a potent source for generation of bioethanol. Prior to the application of lignocellulosic feedstocks, detailed compositional analysis is mandatory for assessment of their suitability and efficient process development for biobutanol production.

11.3

Composition of Lignocellulosic Biomass

Plant biomass is mainly composed of cellulose, hemicellulose and lignin, along with smaller amounts of pectin, protein and extractives (soluble nonstructural materials such as nonstructural sugars, nitrogenous material, chlorophyll and waxes) [8–11]. The composition of these constituents can vary from one plant species to another [12–20]. For example, hardwood has greater amounts of cellulose, whereas wheat straw and leaves have more hemicellulose content. In addition, the ratio between various constituents within a single plant varies with age, stage of growth and other factors.

11.4

Structure of Lignocellulosic Biomass

Cellulose is the main constituent of lignocellulosic biomass occupying a major portion of plant cell wall, which is an organized fibrous structure.

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The linear polymer of cellulose consists of D-glucose subunits linked to each other by β-(1,4)-glycosidic bonds. Cellobiose is the repetitive unit established through this linkage which constitutes cellulose chains. The long-chain cellulose polymers along with hemicellulose and lignin are linked together by hydrogen bonds and van der Waals forces, making compact microfibrils [21]. Cellulose in biomass is present in both crystalline and amorphous forms. Crystalline cellulose comprises the major proportion of cellulose, whereas a small percentage of unorganized cellulose chains form amorphous cellulose. Fermentable D-glucose units can be produced from cellulose through the action of either acid or enzyme breaking the β-(1,4)-glycosidic linkages. Especially, the cellulose in its amorphous form is more susceptible to enzymatic degradation. The main feature that differentiates hemicellulose from cellulose is that hemicellulose has branches with short lateral chains consisting of different sugars. These monosaccharides include pentoses (xylose, rhamnose, and arabinose), hexoses (glucose, mannose, and galactose), and uronic acids (e.g., 4-O-methylglucuronic, D-glucuronic, and D-galacturonic acids) [22]. The backbone of hemicellulose is either a homopolymer or a heteropolymer with short branches linked by β-(1,4)glycosidic bonds and occasionally β-(1,3)-glycosidic bonds. Additionally, hemicellulose possesses some degree of acetylation, for example, in heteroxylan. In contrast to cellulose, the polymers present in hemicellulose are easily hydrolyzable. These polymers do not aggregate even when they co-crystallize with cellulose chains. Lignin is a large, complex molecular structure containing crosslinked polymers of phenolic monomers. It is present in the primary cell wall, imparting structural support, impermeability, and resistance against microbial attack. Three phenyl propionic alcohols exist as monomers of lignin: coniferyl alcohol (guaiacyl propanol), coumaryl alcohol (p-hydroxyphenyl propanol) and sinapyl alcohol (syringyl alcohol). Alkyl-aryl, alkyl-alkyl, and aryl-aryl ether bonds link these phenolic monomers together [23]. In general, herbaceous plants such as grasses have the lowest content of lignin, whereas softwoods have the highest lignin content.

11.5

Biobutanol Production from Lignocellulosic Biomass

Nowadays, biobutanol production from lignocellulosic biomass is gaining momentum because of its blending with gasoline, which increases the octane level, consequently diminishing the CO2 emissions by 12–15%

250 Lignocellulosic Biomass Production and Industrial Applications over gasoline. Butanol yields 25% more energy than the energy invested in its production [3]. Biobutanol production from lignocellulosic biomass includes three main steps: biomass pretreatment, hydrolysis of free cellulose and hemicellulose and fermentation of reducing sugars.

11.5.1

Pretreatment

Prior to enzymatic hydrolysis most lignocellulosic substrates need to undergo some sort of pretreatment to enhance the accessibility of the substrate for efficient hydrolysis and biofuel production. Pretreatment preferably results in removal of lignin, subsequently increasing the surface area of cellulose as well as substrate porosity [19, 20]. In particular, the removal of lignin has a large influence on the rate and extent of enzymatic hydrolysis of the substrate. Several methods have been developed for the pretreatment of lignocellulosic biomass such as mechanical, thermal, chemical, biological and combinations thereof. There are several advantages and disadvantages of several types of pretreatment methods [24–26]. Among them, enzymatic pretreatment process has several important advantages such as higher yields, minimal by-product formation, meager substrate loss, low energy requirements, involvement of mild operating conditions and low chemical disposal costs [27, 28].

11.5.2

Hydrolysis

Enzymatic hydrolysis of lignocellulosic biomass to sugars for subsequent biobutanol production has become one of the major thrust areas of research in recent years [29]. Hassan et al. [30] worked on enzymatic saccharification of liquid hot water pretreated alfalfa fiber using pectinase and xylanase. Enzymatic saccharification was optimized in terms of substrate and enzyme concentration. The main components of hydrolyzing sugar were glucose, sucrose, xylose and arabinose. A study on the enzymatic hydrolysis of cellulosic biosludges generated in a water treatment plant of a Kraft pulp mill was carried out by Aloia et al. [31]. The effect of the operational conditions (cellulase-to-solid ratio (CSR), liquid-to-solid ratio (LSR), surfactant concentration (SC) and reaction time) on hydrolysate composition was evaluated and a set of mathematical models were proposed to predict the glucose and xylose concentrations in the reaction media. Using low cellulase loading (8 FPU/g) and high liquid-to-solid ratios (28–30 g/g), a quantitative conversion of the glucan fraction could be reached in 48 h, although diluted solutions were produced [31].

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11.5.2.1 Cellulases and Xylanases Cellulase is used for enzymatic hydrolysis of plant carbohydrate polymers. It is a hydrolytic enzyme that degrades cellulose into glucose. Cellulase consists of four major components: endo-β-glucanase, exo-βglucanase (cellobiohydrolase or glucanase), exo-β-glucanase (exoglucanase or glucohydrolase) and β-glucosidase. Their specific mode of action is as follows: 1. Endo-β-glucanase, β-(1,4)-D-glucan glucanohydrolase and CMCase randomly cleave cellulose chains yielding glucose and cello-oligosaccharides. Generally, the endoglucanases randomly attack H3PO4-swollen cellulose, CM-cellulose and amorphous regions of the cellulose, subsequently releasing cello-oligosaccharides. 2. Exo-β-glucanase (cellobiohydrolase (CBH) or glucanase) releases cellobiose either from the reducing or non-reducing end. The CBHs hydrolyze H3PO4-swollen cellulose and avicel sequentially by removing the cellobiose units from the non-reducing end of the cellulose chain. 3. Exo-β-glucanase (exoglucanase or glucohydrolase) releases glucose from the non-reducing end. 4. β-glucosidase and cellobiase release glucose from cellobiose and short-chain cello-oligosaccharides. Endoglucanase and CBH act synergistically to influence the hydrolysis of crystalline cellulose, whereas β-glucosidase completes the hydrolysis by converting the resultant cellobiose into glucose. Cellulase is mainly produced by some fungi, bacteria (including the symbiotic bacteria residing within termites and ruminant herbivores) and protozoans. Fungal cellulases are produced in large amounts which include all the components of a multi-enzyme system with different specificities and modes of action, acting in synergism for complete hydrolysis of cellulose [32]. One of the most extensively studied cellulolytic microorganisms is the soft rot fungus Trichoderma reesei. The cellulases produced by the aerobic fungi Trichoderma viride, Penicillium pinophilum, Phanerochaete chrysosporium (Sporotrichum pulverulentum), Fusarium solani, Talaromyces emersonii, Trichoderma koningii and Rhizopus oryzae are also well studied [33, 34]. Several cellulolytic anaerobic bacteria secrete multienzyme cellulase complexes called cellulosomes [35]. Several cellulolytic aerobic

252 Lignocellulosic Biomass Production and Industrial Applications bacteria secrete cellulases in a very low amount and have been found to lack cellobiohydrolases [36]. Cellulase finds its application in varied industrial sectors, with major utility in textile industries where it is employed for biopolishing of fabrics. Apart from this, it is also employed in commercial food processing (e.g., fruit juices, baking, etc.), paper and pulp industries for de-inking of paper and improvement of the nutritional quality and digestibility of animal feeds. The naturally occurring lignocellulosic plant biomass consists of 20–30% hemicellulosic materials, which are heterogeneous polysaccharides found in association with cellulose. Xylan is the major constituent of hemicellulose and is the second most abundant renewable resource. Hemicellulose is degraded by hemicellulase and classified according to the substrates on which they act. They are collectively grouped as glycan hydrolases. Three different types of xylanases are involved in xylan degradation. 1. Endo-β-(1,4)-D-xylanase [β-(1,4)-D-xylan xylano hydrolase]: These enzymes act randomly on xylan to produce a large amount of xylo-oligosaccharides of various chain lengths. These are of four types and their mode of action is as follows: a. Non-arabinose liberating endoxylanases-I: These cannot act on L-arabinosyl initiated branch points at β-(1,4) linkages and produce only xylobiose and xylose as the major end products. These enzymes can break down xylo-oligosaccharides as small as xylobiose. b. Non-arabinose liberating endoxylanases-II: These cannot cleave branch points at α-(1,2) and α-(1,3) and produce mainly xylo-oligosaccharides larger than xylobiose. These endoxylanases have no action on xylotriose and xylobiose. c. Arabinose liberating endoxylanase-I: These can cleave xylan chain at the branch points and produce mainly xylobiose, xylose and arabinose. d. Arabinose liberating endoxylanases-II: These can hydrolyze branch points and produce intermediate size xylooligosaccharides and arabinose. 2. Exo-β-(1,4)-D-xylanase [β-(1,4)-D-xylan xylohydrolase]: These enzymes remove single xylose units from the nonreducing end of the xylan chain.

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3. β-xylosidase or xylobiase: These enzymes hydrolyze disaccharides like xylobiose and the higher xylo-oligosaccharides with decreasing specific affinity. 4. Thus, in a nut shell, it can be stated that xylan hydrolysis is mainly carried out by β-1,4-endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-glucuronidase, acetyl xylan esterase and phenolic acid (ferulic and p-coumaric acid) esterase. All of these enzymes act co-operatively to convert xylan into its constituent sugars. The xylanases have been reported mainly from bacteria, fungi, actinomycetes and yeast. The presence of such a multifunctional xylanolytic enzyme system is quite widespread among fungi [37, 38]. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. Xylanases from Trichoderma reesei, Trichoderma harzianum, Penicillium purpurogenum, Thermomyces lanuginosus, Fusarium oxysporium, and Cephalosporium sp. are also well characterized [39, 40]. Major industrial applications of xylanases are in the pulp and paper industries for biobleaching. Xylanases are also used in bioethanol industries along with cellulase for efficient enzymatic hydrolysis.

11.5.2.2 The Cellulase of Trichoderma reesei RUT-C30 Cellulase from T. reesei Rut-C30 has long been available for cereal foods, brewing, and fruit and vegetable processing industries [41]. It has also been widely evaluated and applied in relation to bioethanol production processes. T. reesei secretes high amounts of enzymes, up to 40 g/L, that consist of endoglucanase, cellobiohydrolase, β-glucosidase and different hemicellulolytic enzymes which catalyze the degradation of cellulose and hemicellulose of plant cell walls [42]. The growth and enzyme production by Trichoderma reesei Rut-C30 using different lignocellulosic materials (wheat bran and rice husk) as carbon source were also investigated by Olsson et al. [43]. Juhász et al. [44] used pretreated spruce, willow, corn stover and Solka-Floc for cellulase production using Trichoderma reesei Rut-C30. They found maximum cellulase production using pretreated corn stover as substrate. Table 11.1 depicts the reducing sugar yield from different lignocellulosic biomass by using cellulase enzyme [45–59].

Mango residue

Seaweed waste

Kinnow mandarin waste

Rice straw

Corncob

Switch grass

Rice straw

Kikuyu Grass

Lantana camara

3

4

5

6

7

8

9

10

11

Commercial cellulase

Hybrid grass

Corncob

Commercial cellulase

Water hyacinth

2

Commercial cellulase

Sorghum biomass

Commercial cellulase

Commercial cellulase

Commercial T. viride cellulase

Commercial cellulase

Commercial cellulase

Cellulase from T. viride and xylanase from B. pumilus

Commercial cellulase

Commercial cellulase

α-amylase from B. licheniformis

Commercial cellulase

Commercial cellulase

Cellulase from T. reesei NCIM 1186

Wheat straw

Yellow poplar

Types of enzyme

Lignocellulosic substrate

1

Sl. no.

Table 11.1 Reducing sugar yield from several types of lignocellulosic biomass.

28

6

96

72

48

72

24

48

4

24

24

144

36

48

24

Incubation time (h)

777.13

75.00

693.00

400.00

170.00

331.00

688.00

277.50

780.00

210.00

233.00

380

465

408

371.44

Maximum reducing sugar yield (mg/g dry substrate)

[59]

[58]

[57]

[56]

[55]

[54]

[53]

[52]

[51]

[50]

[49]

[48]

[47]

[46]

[45]

Ref.

254 Lignocellulosic Biomass Production and Industrial Applications

Utilization of Lignocellulosic Biomass for Biobutanol Production

11.5.3

255

Fermentation

Fermentation is the last step of lignocellulosic biobutanol production. Clostridium sp. [60] is used widely for industrial butanol production because of its ability to produce high concentrations of butanol as well as its high tolerance to butanol and other inhibitory compounds. Table 11.2 shows the bioethanol yield from several lignocellulosic substrates. Solventogenic clostridia are rod-shaped, typically anaerobic, sporeforming, Gram-positive bacteria, which can utilize a wide range of substrates from monosaccharides (pentoses and hexoses) to polysaccharides. Clostridial fermentative solvent production is typically biphasic. The first phase is the acidogenic phase and the second phase is the solventogenic phase. During the acidogenic phase, acetate, butyrate, hydrogen and carbon dioxide forming pathways are activated, which are reassimilated and used in production of acetone, butanol and ethanol during the solventogenic phase [68]. Lignocellulosic biobutanol fermentation can be carried out in different ways such as batch, fed-batch and continuous fermentation process. The batch process is a simple, traditional and commonly studied fermentation method for butanol production. Usual ABE product ratio up to 3:6:1 (15–20 g/L) is achieved within 2–6 days of fermentation [69]. There are various challenges associated with this mode of process related to low butanol yield (~20% w/w), product inhibition, fermentable sugar or substrate limitation (less than 60 g/L), product stream is much diluted (productivity higher than 0.5–0.6 g/L h rarely achieved), the utility cost in a butanol Table 11.2 Biobutanol production from several types of lignocellulosic biomass. Incubation Butanol time (h) (g/L) Ref.

Microorganisms

Substrate

C. acetobutylicum ATCC 824

Hardwood kraft pulp

72

7.3

[61]

Clostridium beijerinckii B592

Pinus banksiana

60

11.9

[62]

Clostridium beijerinckii CC101 Wood pulp

72

13.46

[63]

C. beijerinckii NCIMB 8052

Corncob

48

8.2

[64]

C. beijerinckii ATCC 55025

Wheat bran

72

8.8

[65]

Clostridium beijerinckii BA101 Corn fiber

88

7.2

[66]

Clostridium beijerinckii P260

42

12.0

[67]

Wheat straw

256 Lignocellulosic Biomass Production and Industrial Applications plant being approximately three times higher than that of an ethanol plant with the same scale of production and other important economical drawbacks related to steam-consuming operations, including mash sterilization, downstream recovery by distillation and wastewater treatment [70]. To overcome butanol toxicity, new mutant strains have been developed for the continuous removal of butanol from the fermenter. Such modifications reduced the effect of product inhibition, improved productivity and minimized wastewater generation [69]. Other authors reported maximum butanol production of 26.64 g/L and 21.42 g/L from barley straw and wheat straw, respectively [71, 72]. Fed-batch process begins with low substrate concentration; more substrate is added when fermentation culture starts consuming the substrate to maintain the fermentation process, but the substrate is not allowed to exceed the detrimental level. Fed-batch is applied to a process where high product concentration is inhibitory to the culture [73]. Biobutanol production by feeding butyric acid in a pH-stat fed-batch fermentation process has a reported butanol concentration of 16 g/L using C. saccharoperbutylacetonicum [74]. Butanol production of 16.59 g/L has been reported from C. beijerinckii P260 using wheat straw hydrolysate as substrate [75]. Using cassava bagasse hydrolysate with additional glucose as the substrate in fed-batch operation has a reported butanol production of ~10g/L after 30 h of incubation [76]. High butanol concentration is toxic to strains like C. acetobutylicum or C. beijerinckii. Therefore, for greater productivity C. beijerinckii with 500 g/L glucose was used with simultaneous product recovery by gas stripping or pervaporation [77]. Maximum productivity with gas stripping and pervaporation product removal techniques was reported to be 1.16 g/L per h and 0.98 g/L/h, respectively, with fedbatch fermentation process in comparison to batch fermentation where maximum productivity was 0.60 g/L/h [77]. In continuous fermentation process, one time inoculation is sufficient for long time fermentation, drastically reducing sterilization time as well as inoculation time, which leads to better productivity. The continuous culture technique was also used to study the physiology of culture in exponential phase, while fermentation is not allowed to enter the stationary phase because of butanol toxicity. Maximum butanol productivity of 1.74 g/L/h has been reported by C. beijerinckii BA101 [77, 78]. There are some serious problems linked to continuous culture such as the instability of solvent production for long periods and simultaneous increase in acid concentration. High productivity might be obtained at the cost of low product concentration compared to the batch process in a single stage continuous fermentation system. Therefore, due to this complexity, the

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use of single-stage continuous reactor is not suitable at an industrial scale and two or more multistage continuous systems have been investigated. Two-stage system using C. acetobutylicum DSM 1731 reported a butanol concentration up to 18.2 g/L, which was comparable to the solvent concentration in a batch process [77]. In a two-stage continuous study, acidogenesis was carried out using Butyribacterium methylotrophicum and solventogenesis was achieved from acids using C. acetobutylicum. It was observed that the final concentration of products also depends on pH [74].

11.5.3.1 Development of New Fermentation Technologies Modification in conventional butanol fermentation technology includes free cell, immobilized cell and cell recycle options in continuous bioreactors; along with this, development of integrated fermentation systems with product recovery increased fermentation yield and kept butanol concentration below the toxic level during the process [79]. Final butanol concentration was kept lower than 6–10 g/L with respect to batch process (achieved 10–13 g/L). Two-stage or multistage continuous fermentation systems have been developed where acidogenic and solventogenic phases were performed separately to overcome the problems associated with single-stage continuous system [79, 80]. Furthermore, to reduce production cost and achieve high productivity, immobilized cell reactors or membrane cell recycle technique was employed. Immobilized cell reactors sustain cell survival for a longer time in the solventogenic phase due to the absence of mechanical agitation; overcome the problem of frequent cell regeneration; are easy to operate; can be applied to various substrates such as corn, lactose/yeast extract, deliberated-sweet-potato-slurry (DSPS), etc.; and can be applied in a fibrous bed bioreactor, fluidized bed bioreactor, hydrophobic packed bed reactor, etc. [74, 78]. However, a more economical process can be developed with the integration of new recovery techniques such as gas stripping, liquid-liquid extraction system and advanced membrane separation with supercritical extraction. In-situ removal of solvents in the conventional system can reduce the butanol toxicity of culture and facilitate high production of butanol (25.32 g/L) with maximum utilization of glucose [76, 78]. This may give the flexibility to maintain high dilution rates of the feed and can be more economical at an industrial scale. The economical process also depends on the microbiological butanol synthesis which can also be accompanied by co-culture fermentation processes with efficient technology, which may be a future innovation.

258 Lignocellulosic Biomass Production and Industrial Applications

11.6

Conclusion

In this chapter, different studies and findings concerning the different steps (pretreatment, enzymatic hydrolysis and fermentation) involved in lignocellulosic butanol production were described. Nowadays, industrial production of butanol is lagging behind the production of ethanol and biodiesel. To make butanol production commercially feasible, we need to use cost-effective substrate and develop efficient processes and strains (by genetic engineering or metabolic engineering approach).

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262 Lignocellulosic Biomass Production and Industrial Applications

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

degraded by Trichoderma viride and Bacillus pumilus. Afr. J. Microbiol. Res. 5, 5757–5764, 2011. Hsu, C.L., Chang, K.S., Lai, M.Z., Chang, T.C., Chang, Y.H., and Jang, H.D., Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulaseproducing Streptomyces for bioethanol production. Biomass Bioenerg. 35, 1878–1884, 2011. Xu, J., and Cheng, J.J., Pretreatment of switchgrass for sugar production with the combination of sodium hydroxide and lime. Bioresour. Technol. 102, 3861–3868, 2011. Cheng, J., Su, H., Zhou, J., Song, W., and Cen, K., Microwave-assisted alkali pretreatment of  rice  straw  to promote enzymatic hydrolysis and hydrogen production in dark- and photo-fermentation. Int. J. Hydrogen Energ. 35, 2093–2101, 2011. Vásquez, A.F.L., Rey, G.A.O., and Rodríguez, F.A.R., Obtaining of reducing sugars from kikuyu grass (Pennisetum Clandestinum). Avances: Investigación en Ingeniería 13, 98–101, 2010. Kuhad, R.C., Gupta, R., Khasa, Y.P., and Singh, A., Bioethanol production from Lantana camara (red sage): Pretreatment, saccharification and fermentation. Bioresour. Technol. 101, 8348–8354, 2010. Lee, J., Jang, Y.S., Choi, S.J., Im, J.A., Song, H., Cho, J.H., Seung, D.Y., Papoutsakis, E.T., Bennett, G.N., and Lee, S.Y., Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation. Appl. Environ. Microbiol. 78, 1416–1423, 2012. Kudahettige-Nilsson, R.L., Helmerius, J., Nilsson, R.T., Sjöblom, M., Hodge, D.B., and Rova, U., Biobutanol production by Clostridium acetobutylicum using xylose recovered from birch Kraft black liquor. Bioresour. Technol. 176, 71–79, 2015. Nanda, S., Dalai, A.K., and Kozinski, J.A., Butanol and ethanol production from lignocellulosic feedstock: Biomass pretreatment and bioconversion. Energ. Sci. Eng. 2, 138–148, 2014. Lu, C., Dong, J., and Yang, S.T., Butanol production from wood pulping hydrolysate in an integrated fermentation–gas stripping process. Bioresour. Technol. 143, 467–475, 2013. Zhang, W.L., Liu, Z.Y., Liu, Z., and Li, F.L., Butanol production from corncob residue using Clostridium beijerinckii NCIMB 8052. Lett. Appl. Microbiol. 55, 240–246, 2012. Liu, Z., Ying, Y., Li, F., Ma, C., and Xu, P., Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran. J. Ind. Microbiol. Biotechnol. 37, 495–501, 2010. Ezeji, T., Qureshi, N., and Blaschek, H.P., Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnol. Bioeng. 97, 1460–1469, 2007.

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67. Qureshi, N., Saha, B.C., and Cotta, M.A., Butanol production from wheat straw hydrolysate using Clostridium beijerinckii. Bioprocess Biosyst. Eng. 30, 419–427, 2007. 68. Lee, S.Y., Park, J.H., Jang, S.H., Nielsen, L.K., Kim, J., and Jung, K.S., Fermentative Butanol Production by Clostridia. Biotechnol. Bioeng. 101, 209– 228, 2008. 69. Mariano, A.P., and Filho, R.M., Improvements in biobutanol fermentation and their impacts on distillation energy consumption and wastewater generation. Bioenerg. Res. 5, 504–514, 2012. 70. Visioli, L.J., Enzweiler, H., Kuhn, R.C., Schwaab, M., and Mazutti, M.A., Recent advances on biobutanol production. Sustain. Chem. Process. 2, 1–9, 2014. 71. Qureshi, N., Saha, B.C., Hector, R.E., Hughes, S.R., and Cotta, M.A., Butanol production from wheat straw by simultaneous saccharification and fermentation using Clostridium beijerinckii: Part I – Batch fermentation. Biomass Bioenerg. 32, 168–175, 2008. 72. Qureshi, N., Saha, B.C., Dien, B., Hector, R.E., and Cotta, M.A., Production of butanol (a biofuel) from agricultural residues: Part I – Use of barley straw hydrolysate. Biomass Bioenerg. 34, 559–565, 2010. 73. Li, S.Y., Srivastava, R., Suib, S.L., Li, Y., and Parnas, R.S., Performance of batch, fed-batch, and continuous A–B–E fermentation with pH-control. Bioresour. Technol. 102, 4241–4250, 2014. 74. Kumar, M., and Gayen, K., Developments in biobutanol production: New insights. Appl. Energ. 88, 1999–2012, 2011. 75. Qureshi, N., Saha, B.C., and Cotta, M.A., Butanol production from wheat straw by simultaneous saccharification and fermentation using Clostridium beijerinckii: Part II – Fed-batch fermentation. Biomass Bioenerg. 32, 176–183, 2008. 76. Lu, C., Zhao, J., Yang, S.T., and Wei, D., Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour. Technol. 104, 380–387, 2012. 77. Ezeji, T.C., Qureshi, N., and Blaschek, H.P., Butanol fermentation research: Upstream and downstream manipulations. Chem. Rec. 4, 305–314, 2004. 78. Bankar, S.B., Survase, S.A., Singhal, R.S., and Granström, T., Continuous two stage acetone–butanol–ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313. Bioresour. Technol. 106, 110–116, 2012. 79. Mariano, A.P., and Filho, R.M., Improvements in biobutanol fermentation and their impacts on distillation energy consumption and wastewater generation. Bioenerg. Res. 5, 504–514, 2012. 80. Ni, Y., and Sun, Z., Recent progress on industrial fermentative production of acetone–butanol–ethanol by Clostridium acetobutylicum in China. Appl. Microbiol. Biotechnol. 83, 415–423, 2009.

12 Application of Lignocellulosic Biomass in the Paper Industry Mainak Mukhopadhyay1* and Debalina Bhattacharya2 1

Department of Biotechnology, JIS University, Kolkata, West Bengal, India Department of Biochemistry, University of Calcutta, Kolkata, West Bengal, India

2

Abstract One of the main approaches of green chemistry is the use of lignocellulosic biomass, which is becoming a key tool for transforming an existing process or developing new processes with a sustainable approach. It basically expresses the ultimate principles of sustainable chemistry with basic traits that minimize the environmental impact of a given process. The pulp and paper industry is known to be one of the most polluting industries, requiring a large amount of water and chemicals and producing large volumes of effluents. As these effluents are produced from lignocellulosic materials, they have the potential of being converted into many other economically important compounds. This chapter not only focuses on the feasibility of using lignocellulosic biomass for the production of paper and pulp, but also on the use of potentially important waste matter for the production of biologically important compounds using a green chemistry approach. Keywords: Cellulose, lignin, lignocellulosics, biomass, effluents, wastewater

12.1

Introduction

The pulp and paper industry is one of the biggest industrial segments with a massive influence on worldwide environment. This sector, which contains goods such as glossy paper, office and catalog paper, tissue and paper-based packaging, uses over 40 percent of all industrial wood traded globally. Paper is only the obvious product of this industry; however, *Corresponding author: [email protected] Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial Applications, (265–278) © 2017 Scrivener Publishing LLC

265

266 Lignocellulosic Biomass Production and Industrial Applications there are also various chemicals produced as a by-product of the pulp and paper industry. Pulp and paper production is usually an energy demanding and polluting zone [1]. This industry primarily utilizes conservative technologies which have highly accurate prerequisites for utilization of raw material, chemicals, energy and water; thus producing elevated amounts of diff ferentiate effluents characterized by chemical oxygen demand (COD), biochemical oxygen demand (BOD), toxicity, suspended solids (SS) and the presence of lignin-introduced dark brown color to the effluent when unprocessed crude effluents are released into receiving water bodies [2, 3]. This industry is a large consumer of lignocellulosic biomass. Lignocellulosic residues in pulp and paper mills are a major contribution of wood-based energy [4]. The lignocellulosic materials have a composite structure of cellulose, hemicellulose and lignin. Cellulose is a homopolymer with successive units of D-glucose along with hemicellulose covered by a layer of lignin [5]. The ratios between various elements within a single plant vary with age, stage of growth, and other conditions. These polymers are allied with each other in a heterogeneous manner to diverse degrees and variable opus depending on the type, species, and source of the biomass. The comparatively large quantity of cellulose, hemicellulose and lignin are key aspects in determining the best energy conversion method for every kind of lignocellulosic [6, 7]. Internationally, this industry is supposed to have a diminutive pace of innovation, with only some current progress like the upgrading bleaching technology and improved treatment of recycled paper. Certainly the improvements in this industry are also mostly determined by environmental considerations. Wide ranging usage of lignocellulosic materials and diverse technologies, are equally compelled by restrictions and upgradation of the processes. A boost in output all the way through the implementation of other competents, and cleaner/green technologies in the industrialized process will enhance the fiscal, green and collective development objectives of the pulp and paper sector.

12.2

Major Raw Materials Used in the Paper Industry

12.2.1

Agricultural Residues

12.2.1.1 Sugarcane Bagasse Several agricultural residue fibers are currently being applied for pulp production, among them sugarcane bagasse is the most assuring. Sugarcane bagasse is easily obtained in several countries, irrespective of their economic

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conditions. After subsequent cane processing to remove sugar juice, the tough bagasse remaining is generally blistered. In various countries, this bagasse has superior economic value if it is used for pulping. In fact, sugarcane bagasse can satisfactorily supply a booming papermaking industry with fiber superior to that of any other crop. It can be blended in various proportions, and used to create printing and writing paper, bristol board, tissue paper, glassine and greaseproof paper [8].

12.2.1.2 Corn Stalks Corn is produced as a chief crop in numerous countries, and corn stalks have measured up to be a high-quality fiber source meant for squat quality of paper. Corn stalk fiber qualities are comparable to the fiber quality of sugarcane in structural features, with an average fiber length of 1.5 mm and average fiber width of 0.018 mm [5].

12.2.1.3 Rice Straw, Wheat Straw and Cereal Straw Rice straw is one of the most readily available biomasses for the papermaking industry in Asian countries like India, China and Sri Lanka. On the other hand, rice straw is expensive to accumulate and stockpile, and also has a relatively elevated silica content [5]. Wheat straw is also a chief resource of fiber for the paper making industry, mainly in Europe and North America. Presently, wheat straw is used in areas where wood is relatively in short supply, like Central and South America, Europe, Asia and Africa [5]. Quite a few types of cereal straw are woven together with those from rye, oat and barley. Of these, rye is the most appropriate for pulp production due to its accessibility and the superior yield and potency of its pulp [5].

12.2.1.4 Bamboo Bamboo generally grows well in a temperate humid environment. Bamboos include some of the fastest-growing plants of several areas of the world. Generally it is observed that bamboo can grow under diverse environmental circumstances, ranging from sea level to the high hills. In blends it is used in different fractions, which are utilized to make writing and printing paper, linerboard, wrapping and bag paper, newsprint substitute, etc. [5].

12.2.1.5 Sabai Grass Sabai grass is an undomesticated species that grows in the lower Himalayas and central India. It is a tufted grass having stems 60–90 cm in height and long leaves which are mostly at the bottom. Sabai grass has been an

268 Lignocellulosic Biomass Production and Industrial Applications important fiber resource for the paper making industry in India and Nepal. The species has a tacky, fibrous, raw substance and is typified by its toughness, potency, and rigidity [9]. The quality of the pulp is similar to that of esparto and is therefore used for high-class books and printing paper [9].

12.2.1.66 Jute Jute is characterized by its high cellulose content and long fibers. Its biochemical and morphological properties are favored and come into play in the manufacture of pulp [10]. Jute is chiefly grown in Bangladesh, India, China and Thailand and the plant develops to a height of 2.5–3.5 meters. The bark comprises about 40% of the stem by mass and is mostly used for low value-added goods such as rope, cordage and gunnysacks [11].

12.2.1.77 Ramie Ramie, also known as China grass, is native to China and neighboring countries. It is a permanent plant growing to a height of 1.5–2.5 m. It is commercially grown in a moderate climate in many countries, including China, Japan, Russia, Egypt, Libya, and North and South America. Ramie fibers consist of pure cellulose, and are remarkable for their length and width. They are the strongest and most robust of the vegetable fibers [5].

12.2.1.8 Leaf Fibers A lot of plants and shrubs have leaves together with fibers that are easily appropriated to papermaking. In fact, some of these are frequently used as brilliant papermaking fibers [9].

12.2.1.99 Cotton Fibers Cotton fibers are derived from the seedpod segments of cotton plants. Standard cotton fibers are extremely long as well as costly for usual papermaking processes. These materials are thus used only in the production of specialty papers. Most of the cotton fibers produced goes to the textile industry. The average fiber length of cotton fibers is 25 mm and the average fiber diameter is 0.02 mm [12]. In blends of various proportions, it is used to make high-grade bond ledger books and writing paper.

12.2.1.10 Cotton Rags Cotton rags are the finest fibers available for the paper production process. Due to the motorized exploitation these fibers go through they can be used as cloth. These materials do not need several steps of refining preceding the

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papermaking process. The fiber length of cotton rag fibers is comparable to that of cotton fibers [12].

12.3

Pulp and Papermaking Process

12.3.1

Pulping Process

Pulp production begins with the preparation of raw lignocellulosic substances [13, 14]. This consists of debarking, chipping, and other procedures such as depithing. Cellulosic pulp is produced from lignocellulosic materials by chemical and mechanical methods. The construction of pulp for paper utilizes mechanical and chemical processes.

12.3.1.1 Mechanical Pulping The mechanical pulp production process begins with the pulverization of wood against a water lubricated revolving stone. The heat produced by crushing softens the lignin binding fibers and along with the mechanical forces split the fibers to form ground wood. In the late 20th century, new mechanical techniques were developed using “refiners.” There woodchips and lignocellulosic biomasses are introduced into rigorous shearing forces sandwiched between a revolving steel disc and an unchanging plate [15]. In a subsequent alteration to this method, the biomasses are presoftened by heat to make the fibrillation more efficient and this process is called thermomechanical pulping (TMP). The pulp produced by means of this process is light in color and has longer fibers. An additional enhancement of thermomechanical pulping is chemothermo mechanical pulping (CTMP), in which the wood chips and lignocellulosic biomasses are saturated with the chemical sodium sulphite prior to the milling process. The result of this process is a smooth and much lighter colored pulp along with remarkable improved strength [11, 15]. Following milling, the pulp is arranged by testing for appropriate status. After that it can be bleached with peroxide for utilization in superior value-added products. Mechanical pulps are made up of a blend of whole fibers and fiber fragments of diverse sizes. Paper having a high quantity of mechanical pulp and low quantity of chemical pulp is termed “woodcontaining paper.” Mechanical pulp gives the paper a yellowish/grey tone with high murkiness and an extremely even and smooth surface [16]. Mechanical pulping provides a good yield from the pulpwood because it uses the complete biomass apart from the bark. However, the energy required for refining is elevated and can merely be partially compensated

270 Lignocellulosic Biomass Production and Industrial Applications for by using the bark lignocellulosic directly as fuel or converting it into lignocellulosic-derived fuel.

12.3.1.2 Chemical Pulping For the chemical pulping process, biomasses are first sliced into small chips which are then cooked with chemicals using high pressure. This chemical cooking process eliminates lignin and breaks up the lignocellulosic biomass into cellulose fibers. The consequential slurry holds wobbly but undamaged fibers which preserve their strength. During the process, about half of the biomass liquefies into so-called black liquor. The chemically cooked pulp is subsequently cleansed and screened to attain a more consistent feature. The black liquor is separated from the pulp prior to the bleaching procedure [17]. The majority of the chemical pulp is prepared by the sulphate or Kraft process, in which biomasses are cooked with caustic soda and sodium sulphate. In the unbleached phase, a dark brown but extremely strong pulp results, which can be bleached to an elevated brightness if required. The sulphite-based chemical pulping process is a substitute technique most excellently matched for specialty pulp, which can be easily bleached with hydrogen peroxide (H2O2). These pulps fulfill the requirement for ““chlorine-free” goods in the sanitation and cleanliness paper division and also in the area of printing and writing papers [18]. The yield in both the sulphate- or Kraft-based chemical procedures is lesser in comparison than the production of mechanical pulp, because the lignin portion of the biomass is entirely dissolved and alienated from the fibers. Though, the lignin portion of the biomass extracted from the sulphate and some sulphite methods can be burnt as a fuel oil substitute or, in a recent development, it can be utilized for the formation of jet fuel. In contemporary plants, recovery boiler operations and the controlled smoldering of bark converts the chemical pulp plant into a net energy manufacturer which can frequently provide power to the grid, or steam to neighboring household heating plants. A chemically produced pulp or paper is called wood-free, even though in practice a little proportion of mechanical fiber is generally accepted [18, 19]. The differences between the mechanical pulping and chemical pulping process with respect to different parameters are given in Table 12.1.

12.3.2

Bleaching Process

The pulp bleaching process is an important step to achieve clarity and brightness of the pulp. The type of pulp produced by chemical or

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Table 12.1 Differences between mechanical and chemical pulping. Parameters

Mechanical pulping

Chemical pulping

Energy consumption

1000 KW/tonne of pulp

Self-sufficient

Yield (from wood material)

95%

45%

Fiber length

Fiber fragments of different sizes

Mainly longer fibers

Paper strength

Low

High

Production cost

Lower

Higher

mechanical pulping process and its intended utilization are imperative features in the bleaching procedure [20]. A maximum percentage of bleached chemical pulp is created by the kraft method. An exceptionally small percentage of mechanical pulp was bleached in earlier periods; yet, this trend is presently shifting. Worldwide mechanical pulp bleaching is increasing at twice the rate of chemical pulp bleaching. Several bleaching agents used for these purposes are sodium bisulphite, hydrogen peroxide (H2O2), oxygen, ozone, hypochlorite, chlorine (Cl2), chlorine dioxide (ClO2) and caustic soda [20, 21].

12.3.2.1 Chlorine Bleaching The residual lignin part of the pulp imparts the color to the end product. Therefore, it is necessary to employ a process to eliminate the leftover lignin, which ranges from 5–10%. This process is carried out by numerous phases of treatment with hypochlorite or chlorine dioxide to brighten the pulp [21].

12.3.2.2 Elemental Chlorine Free Bleaching (ECF Bleaching) The ECF bleaching process has been adopted in numerous big mills as it employs oxygen delignification (ODL), followed by ClO2 and other chemical compounds to achieve brightness. Oxygen delignification is a key pulp bleaching processes. It is cost effective and is an accepted way to increase yield in the bleached kraft pulping process [13].

12.3.2.3 Total Chlorine Free Bleaching (TCF Bleaching) Combining ODL with ozone/peroxide-based lightening of pulp is known as TCF bleaching. The effectiveness of the bleaching procedure can be improved by using biological molecules like enzymes, and also a

272 Lignocellulosic Biomass Production and Industrial Applications supplementary “chelating” mediator, to combine the metal ions enclosed in the pulp and prevent them from being decayed by hydrogen peroxide [21].

12.3.2.4 Hydrogen Peroxide (H H2O2) Brightening Hydrogen peroxide is also a useful agent for bleaching pulp produced by mechanical pulping method with high lignin content. The H2O2 modifies the chemical configuration of lignin by oxidation and residue of the pulp. Though H2O2 is environmentally friendly, it is pricey [20].

12.4

Waste Generation

The most important forms of solid waste and pollutants produced in the pulp and paper industry are described below.

12.4.1

Wastewater

Based on the kind of pulping methods, the amount of water utilization varies because all of these procedures require an intensive quantity of water. The future value of wastewater produced from pulping and bleaching processes is as extensive and unique as the type of process and chemical used [22, 23]. Roughly 200 m3 of water is needed per ton of pulp for the production. Most of the wastewaters produced in this process are extremely contaminated, particularly those produced from chemical pulping [24]. Wastewaters produced from the pulping process typically consist of wood wreckage, soluble wood resources, and some chemicals originated from chemical pulping. Wastewater produced from bleaching method has diverse feature. These wastes do not have the superior potency of pulping wastewater, but they may have several toxic components [24].

12.4.2

Rejects

The castoff from pulp consists of bark, wood residues and sand from wood treatment, as these are detrimental products for the papermaking process. Rejects usually have comparatively little moisture content, considerable heating values, are commonly scorched in the mill’s boiler for energy recuperation.

12.4.3

Green Liquor Sludge, Dregs and Lime Mud

Green liquor sludge, dregs and lime mud are mainly categorized into inorganic sludges separated from the chemical treated pulp manufacture

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procedure. These sludges are mainly used as land fill in several sectors once they are dehydrated and dried out [25].

12.4.4

Wastewater Treatment Sludge

Wastewater treatment sludge is generated mainly in the form of two resources called primary sludge and biological sludge. These two sludges are normally mixed together collectively, a biological polymer is added and then it is dried out to form a 25–40% dry solid [26].

12.4.5

Primary Sludge

This type of sludge is generated in the clearing up of processed water by dissolved air flotation. The sludge is typically composed of fines and fillers on the recovered paper being processed and is comparatively simple to dehydrate. This sludge has the prospect of being reentered into the method used for board industry, but for high-grade products it can be burned, dumped or, otherwise, mixed with deinking or secondary sludge [27].

12.4.6

Secondary or Biological Sludge

This sludge is generated in the clarifier of the biological units of the wastewater treatment, and it is recycled to the product or condensed, dewatered and then burned or disposed of in land. Primary sludge volumes are always higher than the secondary sludge volumes, since nearly all of the weighty, stringy or inorganic solids are removed in the primary clarifier [27].

12.4.7

Organic Pollutants and Suspended Solids

Impermanent fibers, starch, cellulose, hemicellulose, lignins and organic acids are the main reason for organic pollution in effluents. Emission of these compounds into the environment causes consequences in COD emancipation. The outcome of elevated BOD/COD concentration is reduction of oxygen accessible to microorganisms of the water bodies downstream of the effluent discharge. A lot of pollutants, such as resin, fatty acids and heavy metals, present in the mill effluents are wrapped up by the organic solids. This can have long-term effects as a consequence of bioaccumulation and moving all the way through the food chain [28].

12.4.8

Organochlorine Compounds

During paper production, a large number of organochlorine compounds, such as chlorinated derivatives of phenols, acids, dibenzo-p-dioxins/furans

274 Lignocellulosic Biomass Production and Industrial Applications and other neutral compounds, are generated, which is a cause for environmental concern. Bleaching process effluents may contain chloroform and carbon tetrachloride, which are classified as carcinogens. The hypochlorite stage is the major producer of chloroform. Various micropollutants like chlorinated benzenes, phenols, epoxy stearic acid and dichloromethane present in the effluents are also classified as suspected carcinogens [28].

12.4.9

Inorganic Chemicals

Several toxic chemicals, such as chloroform, chloroacetones, aldehydes and acetic acid, are created throughout the bleaching process, although in lower concentrations than chlorophenolics. Usually these compounds are non-persistent and do not accumulate in biological organisms, but some of them are moderately toxic, mutagenic and suspected carcinogens [29].

12.4.10

Chlorophenolics

The chlorophenolics are also toxic, importunate and accumulative in biological organisms, and have the ability to convert into new composites such as trichlorophenol or pentachlorophenol. Application of elemental chlorine in the bleaching step drastically amplifies chlorophenol production [28, 29].

12.4.11

Dioxins and Furans

Dioxins, i.e., polychlorinated dibenzo-dioxins, are very poisonous, unrelenting and cancer-causing chemicals. Furans, namely polychlorinated dibenzofurans, are chemically alike, however, of a lesser magnitude. Dioxins and furans are both activated in the wastewater treatment of sludge, which is a reason for enormous alarm [29].

12.5

Waste to Value-Added Products

12.5.1

Biogas

Biogas production is based on anaerobic digestion of high moisturecontaining biomass, such as effluent, coming from the pulp and paper processing wastes. This type of digestion results in the formation of methane gas which can be used directly for cooking purposes or its energetic value can be converted into the production of electrical energy [30].

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12.5.2

275

C5 and C6 Sugar Fermentation

Six and five carbon components can be derived from the cellulose, hemicelluloses and starch-based components of wastewater effluents. These can be converted into several types of fermentable sugars and value-added products. Six and five carbon sugar compounds can undergo dehydration, hydrogenation and oxidation reactions to deliver several value-added products like ethanol, lactic acid, furfural, hydroxymethyl furfural, sorbitol, levulinic acid, etc. [30, 31].

12.5.3

Value-Added Compounds from Lignins

Lignin has considerable prospects for enhancing the cost-benefit operation of the lignocellulosic industry. It is the second most abundant biopolymer on earth, contributing as much as 30% of the weight of lignocellulosic materials and 40% of the energy content of lignocellulosic biomass. Lignin can play an important role for the production of new chemicals, mostly supramolecular and aromatic chemicals [32].

12.5.4

Organic Solutions

A green biorefinery needs fresh wet biomass for its processing. The first step of processing involves dewatering of wastewater to acquire nutrientrich fluid and fiber-rich lignocellulosic cake. The organic part of the solution contains several industrially precious compounds like carbohydrates, proteins, free amino acids, etc. The fluid part has been established as feedstock for the generation of biochemicals and fuels such as lactic acid, bioethanol, methane, etc. [30, 32].

12.6

Conclusion

The need for different types of paper is increasing every day as a consequence of urbanized population and industrialization. Along with utilization of water and energy, the generation of different types of waste is predominantly becoming a source of significant apprehension globally. The most important objective is to reduce harm to the environment by waste minimization, reuse and recycling. Use of the best available techniques and a search for further innovative methods to convert waste materials into several value-added products is becoming a much more important matter. Consequently, end-of-pipe management is key to moving towards

276 Lignocellulosic Biomass Production and Industrial Applications risk minimization. In order to assess noxious wastes and convert them into a wealth of products a holistic approach is needed.

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Index

ABE fermentation, 255 Acid treatment, 122 Acidogenesis, 210 Adsali, 177, 180 Agro-industrialization, 174 Algal biomass, 35–36 Alkaline hydrolysis, 11 Alkaline treatment, 123 Ammonia fiber expansion, 121 Ammonia fiber explosion, 13 Amplified fragment length polymorphism (AFLP), 187 Animal fats, 65, 66, 80, 83 Arabinofyranosyl, 152–154 Arabinose, 75–76 Arabinoxylan, 96 Aryl alcohol oxidase, 103 Auxiliary cellulose-degrading enzyme, 151–152 Bagasse, 172, 188–191 Ball milling, 219 Bamboo, 267 Basidiomycetes, 98–99, 101, 105 Biobutanol, 247–248, 249, 255–259 Biocomposites bionanocomposites, 56 green biocomposites, 55–56 Biodiesel, 40 Bioenergy, 172 Bioethanol, 159, 173, 190, 191 Bioethanol production, 127 Biofertilizer, 172 Biofuels, 171–173, 190–191

Biogas early shoot borer, 184 red rot, 179, 184, 187–188 smut, 175, 179–180, 184 white grub, 184 Biohydrogen, 36–37, 39–40 Biohythane, 27, 37 Biological oxygen demand, 38 Biological pretreatment, 124 Biomanure, 161–162 Biomethanation, 161 Biopolymers, 217, 228 Biopulping, 98–100 Biorefineries, 136–138, 155, 159, 162 Biorefinery, 171–172, 189–192 Biotic factors, 183 Bleaching process, 270 Blue laccase, 148–149 Bological pretreatment, 14 Buttress, 174 By-products bagasse, 172, 188–191 molasses, 172, 189, 191 vinasse, 189 Cane setts, 180 Canopy reflectance, 202 Carbohydratases, 150–156 Carbon sequestration, 186 Carboxyl esterases, 142, 145, 150, 156 Catabolite repression, 73 Catechol oxidases, 145, 148 Cell wall polymers, 138–139 Cellobiohydrolase, 103

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280 Index Cellulase, 103, 136, 141–142, 144–145, 150–151, 251–254 Cellulose, 5, 95–97, 138–140, 249 Cellulose phosphorylase, 145, 151 Centrifugal milling, 219 Cetane index, 66 Chemical oxygen demand, 38–39 Chemical pulping, 270 Chlamydomonas reinhardtti, 36 Chlorophenolics, 274 Climatic conditions rainfall and relative humidity, 175 sunlight, 175, 183 temperature, 175, 180 Clostridium butyricum, 29–30, 36 Colloid milling, 219 Coniferyl, 139 Corn stalks, 267 Cotton fibers, 268 Cotton rags, 268 Cresolases, 145, 148 Crop rotation, 183–184 Cyanate ester resins, 217, 227 Delignification, 136–137, 146 Dioxins, 274 Disease causing organisms early shoot borer, 184 red rot, 179, 184, 187–188 smut, 175, 179–180, 184 white grub, 184 Drip irrigation, 180–181 Dyes, 58 Eksali, 177, 180 Endo-1,4-β-xylanases, 152–153 Endoglucanase, 141, 143–145, 150 Endo-β-glucanase, 251–252 Enterobacter aerogenes, 29–30, 40 Enzymatic transesterification, 81 Exoglucanases, 145, 150 Exoxylanases, 152–153 Exo-β-glucanase, 251–252 Expansin, 145, 154

Extractives, 95, 97 Extrusion, 219 Fatty acid methyl ester (FAMEs), 66, 69, 80, 83 Fed-batch process, 256 Fermentation, 125–126, 158 Flat planting, 180 Fluoride, 57 Food waste, 38 Fungus, 251, 253 Furans, 274 Furrows, 177, 180 Fuzzy set theory, 203 Geographic information system (GIS), 198–200, 202, 203, 207, 211 Germination, 174–175, 180, 182, 184, 188 Glucomannan, 96 Glucuronoxylan, 96 Glycerol, 40 Glyoxal oxidase, 103 Grand growth phase, 182 Green fuels, 136 Hammer milling, 219 Heavy metals, 59 Hemicellulase, 142, 145, 150, 152–154 Hemicelluloses, 7, 95–97, 138–139, 249 Hybrid peroxidase, 147 Hydrolysis, 124–125, 250 Hydrothermal pretreatment, 221, 222 In-situ transesterification, 82 Integrated biorefinery, 216 Integrated pest management (IPM), 186 Intercropping, 185 Internode, 174, 184 Inter-simple sequence repeats (ISSR), 187

Index Ionic liquids, 123–124 Ionic liquids pretreatment, 12 Irradiation, 219 Jet milling, 219 Jute, 268 Kharif crops, 183 Laccase, 102–104 Lactacidase, 158 Land requirement, 177 Leaf fibers, 268 Lignin, 7, 95–97, 138–140, 142, 249 Lignin peroxidase, 102–104, 143–146 Ligninases, 142, 145–149 Lignocellulose, 5 Lignocellulose pretreatment, 8 Lignocellulosic biomass, 32–33, 95–97, 198, 200, 207, 209, 248–249 Lignocellulosics, 65, 66–67, 69, 77, 84, 135, 138–139, 142, 150, 154, 159, 162–163, 172, 190 Linoleic acid, 79, 83 Linolenic acid, 79, 83 Lipases, 81–82 Liquid hot-water pretreatment, 13 Manganese peroxidase, 102–104, 143, 145, 147 Marker assisted selection (MAS), 186 Mechanical processes, 116–117 Mechanical pulping, 269 Mediators, 149 Metabolic engineering, 73–74 Microalgae, 70, 78–80 Microbial lipids, 83 Microwave irradiation, 117–118 Miscanthus sp., 34 Monocropping, 185

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Morphology, 174 Multicriteria evaluation, 200 Multispectral image, 201 Municipal solid waste, 37 Nanoclusters, 233 Natural fibers abaca, 49 alfa, 49 bagasse, 49 bamboo, 48, 49 banana, 49 cantala, 49 coir, 48, 49 cotton, 49 flax, 49 hemp, 49 henequen, 49 jute, 49 kapok, 49 oil palm, 49 pineapple, 49 ramie, 49 sisal, 48 straw, 49 Nitroxide-mediated polymerization (NMP), 228 Node, 174, 180 Nutrient management, 182 Octane number, 66 Oleaginous microbes, 84 Oleaginous yeast, 72, 73, 75 Oleic acid, 78, 83 Organic manure, 182 Organic pollutants and suspended solids, 273 Organochlorine compounds, 273 Organosolv, 11 Organosolv process, 123 Overlaying technique, 200, 203 Oxidative delignification, 11 Ozonolysis, 121, 222 Ozonolysis pretreatment, 12

282 Index Palm oil mill effluent, 40 Palmitic acid, 78, 83 Panchromatic image, 201 Pectin, 8 Pentose phosphate pathway, 76 Pentose sugar unit, 139 PHA production, 16 Phenol oxidases, 145–146, 148 Phenyl propionic monomeric alcohols, 139 Photosynthesis, 174–175 Poly(D-glucaramidoamine)s (PGAAs), 231 Polyaspartic acid (PASA), 230 Polybenzoxazines, 226, 227 Polybutylene teraphthalate (PBT), 233 Polybutylene terephthalate (PBT), 228 Polyethylene furanoate, 229 Polyethylene teraphthalate (PET), 233 Polyhydroxyalkanoates, 3 Polylactic acid (PLA), 233 Polytetramethylene ether glycol (PTMEG), 229 Pretreatment, 250 Primary sludge, 273 Production, 171–174, 177, 185–186, 188–192 Propagation, 177, 180, 187–188 Pulping process, 269 Pulsed electric field, 219 Pyrolysis, 118, 219 Ramie, 268 Rapid amplification DNA (RAPD), 187 Raster, 200 Ratooning, 176, 179, 183 Residual biomass, 112–113 Reversible addition-fragment chain transfer (RAFT), 228 Rhamnolipid, 234 Rhizosphere, 186

Rhodobacter sphaeroides, 36 Rice and Processing, 113–115 Ridges, 180 Ring-pit method, 180–181 Ripening, 175, 182 RNA interference (RNAi) technology, 187 Sabai grass, 267 Secondary or biological sludge, 273 Sensor, 201 Setts, 180–181, 184 Shikimate pathway, 148 Sinapyl, 139 Single-nucleotide polymorphisms (SNPs), 187 Soil quality, 176, 180, 185–186 Solid state fermentation, 79 Somaclonal variation, 188 Spatial distribution, 204 Steam Explosion, 119 Stearic acid, 79, 83 Submerged fermentation, 79 Subtropical, 171, 175, 177, 183, 187 Sucrose, 172, 175–176, 179–180, 182, 190 Sugar Beet, 34 Sugarcane, 171–192 Sugarcane bagasse, 266 Supercritical CO2 explosion, 120–121 Sweet Sorghum, 34 Swollenin, 155 Techno-economic, 173 Temporal distribution, 204 Terephthalic acid (TA), 229 Tetrahydrofuran (THF), 229 Thermal polyaspartate (TPA), 230 Thermostable polymers, 227 Tillering, 176, 180, 182, 185 Transesterification, 84 Trash, 172, 182, 190–191

Index Treatment alkali, 52–55 steam, 54 steam-alkali, 54–55 Tree based oils, 69 Trench method, 180–181 Triacyl glycerol (TAGs), 66, 82 Trinuclear cluster, 148 Tropical, 171, 174–175, 177, 180, 183 Two-roll milling, 219 Ultrasoncation, 222 Ultrasonic pretreatment, 120 Ultrasound, 222 Valorization, 215, 218, 236 Vanillin, 226, 227 Varieties of sugarcane, 176 Vector, 200 Vegetable oils, 65, 68, 70, 72, 83 Vegetation index, 202 Vibratory ball milling, 219 Vibro-disk milling, 219 Vinyl ester resins, 217, 226

Waste generation, 272 Wastewater treatment sludge, 273 Water management, 182 Weed management, 183 Wet oxidation, 119–120 Wet-disk milling, 219 White laccase, 148–149 Wood biomass, 100–101 Xylan, 96, 152–154 Xylanases, 103, 145, 152, 154, 251–254 Xyloglucan, Xylose, 75–76 Yellow laccase, 148–149 Zymase, 142, 145, 157–159 α-Galactosidases, 145, 153 β-glucosidase, 141, 143–145, 150, 251–252 β-mannan, 153 β-Xylosidases, 153

283