Lignocellulosic Ethanol Production from a Biorefinery Perspective: Sustainable Valorization of Waste [1st ed.] 9789811545726, 9789811545733

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Deepansh Sharma Anita Saini

Lignocellulosic Ethanol Production from a Biorefinery Perspective Sustainable Valorization of Waste

Lignocellulosic Ethanol Production from a Biorefinery Perspective

Deepansh Sharma • Anita Saini

Lignocellulosic Ethanol Production from a Biorefinery Perspective Sustainable Valorization of Waste

Deepansh Sharma Amity Institute of Microbial Technology Amity University Jaipur, Rajasthan, India

Anita Saini Department of Microbiology Shoolini Institute of Life Sciences and Business Management Solan, Himachal Pradesh, India

ISBN 978-981-15-4572-6 ISBN 978-981-15-4573-3 https://doi.org/10.1007/978-981-15-4573-3

(eBook)

# Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Contents

1

Introduction to Lignocellulosic Ethanol . . . . . . . . . . . . . . . . . . . . . . . Need for Renewable Fuels/General Background . . . . . . . . . . . . . . . . . . Bioethanol as a Fuel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of Bioethanol Fuel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . First-Generation Bioethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Second-Generation Bioethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . Third-Generation Bioethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Waste Valorization Through Cellulosic Ethanol Production . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 4 7 7 10 14 15 16

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Cellulosic Ethanol Feedstock: Diversity and Potential . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lignocellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Wall Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Feedstock for Cellulosic Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agricultural Wastes as Bioethanol Feedstock . . . . . . . . . . . . . . . . . . . Rice Feedstock Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wheat Straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sugarcane Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Industrial Waste as Bioethanol Feedstock . . . . . . . . . . . . . . . . . . . . . . Forest Waste as Bioethanol Feedstock . . . . . . . . . . . . . . . . . . . . . . . . Municipal Solid Waste as Bioethanol Feedstock . . . . . . . . . . . . . . . . . Dedicated Energy Crops as Bioethanol Feedstock . . . . . . . . . . . . . . . . Switchgrass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miscanthus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Napier Grass (Pennisetum Purpureum) . . . . . . . . . . . . . . . . . . . . . . . . Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 24 24 25 28 31 32 33 33 34 35 38 42 42 44 45 46 46 47 49 49

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Contents

Weed Biomass as Bioethanol Feedstock . . . . . . . . . . . . . . . . . . . . . . . . Water Hyacinth (Eichhornia Crassipes) . . . . . . . . . . . . . . . . . . . . . . . . Parthenium Hysterophorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50 50 52 52 53

3

Pretreatment Technologies for Biomass Deconstruction . . . . . . . . . Need for Biomass Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods for Biomass Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . Physical Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanical Comminution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extrusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microwave Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ultrasonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electron Beam Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physicochemical Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alkali Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acid Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organosolv Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wet Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Steam Explosion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ammonia Fiber Explosion Method (AFEX) . . . . . . . . . . . . . . . . . . . . Supercritical CO2 (or CO2 Explosion) . . . . . . . . . . . . . . . . . . . . . . . . SO2 Explosion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alkaline Peroxide Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ionic Liquids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 65 . 65 . 68 . 68 . 71 . 72 . 74 . 75 . 76 . 77 . 77 . 79 . 84 . 88 . 89 . 91 . 92 . 93 . 95 . 96 . 97 . 101

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Saccharification Fermentation and Process Integration . . . . . . . . . . Biomass Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acid Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cost of Cellulases: An Impediment in Bioethanol Production . . . . . . . . Strategies for Improving Cellulases . . . . . . . . . . . . . . . . . . . . . . . . . . Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbes for Ethanol Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . Modes of Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Process Integration for Second-Generation Ethanol . . . . . . . . . . . . . . . Separate Hydrolysis and Fermentation (SHF) . . . . . . . . . . . . . . . . . . . Separate Hydrolysis and Co-Fermentation (SHCF) . . . . . . . . . . . . . . . Simultaneous Saccharification and Fermentation (SSF) . . . . . . . . . . . . Simultaneous Saccharification and Co-Fermentation (SSCF) . . . . . . . . Consolidated Bioprocessing (CBP) . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

111 112 112 115 122 123 126 126 129 131 132 134 136 138 143 145

Contents

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Ethanol Production from High Biomass Loading . . . . . . . . . . . . . . . . . . 147 On-Site Cellulase Production for Enzyme Cost Reduction . . . . . . . . . . . 150 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 5

Microbial and Plant Genetic Engineering for Efficient Conversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Engineered Yeast to Produce Cellulosic Biofuels . . . . . . . . . . . . . . . . . Engineering of Cell for Efficient Conversions . . . . . . . . . . . . . . . . . . . . Engineering of Substrate Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . Tolerance Against Inhibitors, Temperature, and Solvents . . . . . . . . . . . . Genetic Modification of Plants for Bioethanol Production . . . . . . . . . . . Reduction and Modification of Lignin . . . . . . . . . . . . . . . . . . . . . . . . . Increase in Cellulose Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Increase in Biomass Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis of Cell Wall Hydrolytic Enzymes by Transgenic Plants . . . . . Decrease in Cellulose Crystallinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

159 159 161 161 163 166 167 168 169 170 170 171 172

6

Bioethanol: Product Separation Methods . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane-Based Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pervaporation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reverse Osmosis Based Separations . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid–Liquid Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vapor Permeation Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adsorption Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extraction Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extractive Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pressure-Swing Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Azeotropic Distillation Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . Adsorption–Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . .

177 177 178 178 179 180 181 181 182 183 183 184 185 185 186

7

Lignocellulosic Waste Valorization and Biorefineries Concept . . . . Background and Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lignocellulosic Biomass Valorization . . . . . . . . . . . . . . . . . . . . . . . . . Biorefinery Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Utility of a Lignocellulosic Substrates . . . . . . . . . . . . . . . . . . . . . . . . Biorefinery of Lignocellulosic Biomass . . . . . . . . . . . . . . . . . . . . . . . Sustainability Assessment of Biorefineries . . . . . . . . . . . . . . . . . . . . . Biorefinery Life Cycle Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . Technology and Socioeconomic Analysis of Biorefinery (TEA) . . . . . . Regulations for Forthcoming Biorefineries . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

195 196 197 199 200 201 203 204 206 207

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Contents

Case Studies: Lignocellulosic Valorization Through Biorefinery . . . . . . 210 Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 8

Fermentation Economics and Future Prospects . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting Fermentation Economics . . . . . . . . . . . . . . . . . . . . . . Market Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fermentation and Product Recovery Costs . . . . . . . . . . . . . . . . . . . . Process Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strategies to Improve Fermentation Economics of Bioethanol . . . . . . . . Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

217 217 219 219 220 223 224 226 226 227

About the Authors

Deepansh Sharma is working as an Assistant Professor (Microbiology) at Amity Institute of Microbial Technology, Amity University, Rajasthan. He has started his academic career as an Assistant Professor (Microbiology) at the School of Biotechnology and Bio-engineering, Lovely Professional University, Punjab, India. He has extensive teaching and research experience in the field of Fermentation Technology, Food Microbiology, Industrial Microbiology, and Microbial Technology. Previously, he has been selected for the Short-term scholarship (DAAD, Germany2012) to work as an international visiting researcher at Technical Biology Branch II, Karlsruhe Institute of Technology, Germany. Furthermore, he is an active member of many scientific societies and organizations, including the Association of Microbiologists of India, American Society of Microbiology, European Federation of Biotechnology, and International Scientific Association for Prebiotics and Probiotics. Till now, he has published more than 35 peer-reviewed research articles, 5 books on microbial biosurfactants and applied microbiology and authored/coauthored chapters in various edited books. Anita Saini is working as an Assistant Professor of Microbiology at Shoolini Institute of Life Sciences and Business Management, Solan, Himachal Pradesh, India. Her research expertise involves microbial bioprospecting, production of lignocellulolytic and esterases enzymes, biomass pretreatment, and secondgeneration ethanol production. Till now, she has published more than 10 research and review articles in various peer-reviewed national/international journals and has authored/co-authored chapters in various edited books.

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Introduction to Lignocellulosic Ethanol

Abstract

The current human population growth worldwide, tied with industrial advancement and urbanization, has led to the grave concern about energy security to future generations. Approximately 86% of the primary energy demands of the world are being met by fossil fuels. The rise in population and the increasing economic growth of nations will cause an exponential rise in fossil fuels’ demands in near future. The atmospheric pollution can lead to more serious problems such as acid rain, which is more prevalent in industrial areas with high consumption rate of fossil fuels. The use of biofuels is encouraged because of their potential for contributing in reduced dependence on fossil fuels and mitigating atmospheric pollution by lowering the level of CO2 emissions. One promising waste valorization strategy is energy recovery from lignocellulosic biomass. The estimates show that the biomass can contribute to 20–90% of the energy demands of the world. Several countries have already started taking initiatives for commercializing cellulosic ethanol production and studying their feasibility for future developments. In 2016, 38 million liters of the total 58 billion liters of bioethanol in the USA, and six million liters of total 25 billion liters of bioethanol in Brazil, were cellulosic ethanol. The technological advancements to reduce the production cost can enhance the production levels significantly, which suggests that this sector can be tapped for meeting future energy demands of the world. Keywords

Biofuel · Fermentation · Lignocellulosic waste · Waste Valorization · Yeast

# Springer Nature Singapore Pte Ltd. 2020 D. Sharma, A. Saini, Lignocellulosic Ethanol Production from a Biorefinery Perspective, https://doi.org/10.1007/978-981-15-4573-3_1

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Introduction to Lignocellulosic Ethanol

Need for Renewable Fuels/General Background The global human population growth, coupled with industrial advancement and urbanization, has led to the grave concern about energy security to future generations. According to the World population prospects (2017), the population of the world is likely to reach 9.8 billion in 2050, which can rise further to 11.2 billion in 2100. Therefore, the energy demands of the world are projected to grow at an unexpected rate. Presently, fossil fuels are the dominant source of energy across the globe. Approximately 86% of the primary energy demands of the world are being met by fossil fuels (Abas et al. 2015). The rise in population and the increasing economic growth of nations will cause an exponential rise in fossil fuels’ demands in near future (Stephens et al. 2010). Several analysts have foreshadowed the unsustainability of fossil fuels. The reserves of the fossil fuels, based on the probability of their recovery, have been divided into different categories, i.e., 1P (proven reserves: discovered, high probability of recovery), 2P (proved and probable: discovered, moderate chance of recovery), 3P (proved, probable, and possible: discovered, less chances of recovery), and contingent (1C, 2C, 3C with decreasing certainty, sub-commercial) reserves (Abas et al. 2015; Speirs et al. 2015). Additionally, several undiscovered (prospective) resources are also considered important. The recoverability takes both technical and economic aspects into account. It has been reckoned that fossil fuel reserves on earth are available in finite quantities and they are undergoing rapid exhaustion. Fossil fuels are formed from organic matter over the course of millions of years. The long duration involved in their production cannot ensure their adequate supply in the long run. Thus, they are regarded unviable in warranting energy security after few decades. Recent studies have shed light on this issue. It has been estimated that the proven fossil fuel reserves are likely to be obtainable until 2068–2088. If population stabilizes near 9 billion after 2050 and economies grow annually at a rate of 1.5–3%, the estimated reserves can supply energy no longer beyond 2084–2112 (Stephens et al. 2010). This necessitates the search for unconventional and renewable sources of energy. A more serious issue is the global environmental protection, which propounds the restrained use of fossil fuels. The combustion of fossil fuels generates greenhouse gases (GHG) such as carbon dioxide. The emission of other pollutants such as CO, NOx, and SO2 also is harmful for humans and other living organisms. The atmospheric pollution can lead to more serious problems such as acid rain, which is more prevalent in industrial areas with high consumption rate of fossil fuels (Yusoff et al. 2015). The magnitude of greenhouse gases is rising exceedingly due to massive consumption of fossil fuels at a global scale. Fossil fuels contribute to 73% of the global CO2 production (Kumar et al. 2015; Lokhorst and Wildenborg 2005). The world’s carbon dioxide emissions are predicted to rise from 35.6 billion metric tons in 2020 to 43.2 billion metric tons in 2040 (IEO 2016). Only a small proportion of the emitted CO2 is absorbed through natural processes, while most of the other part enters the atmosphere. The resulted imbalance between the release of CO2 and its withdrawal from the atmosphere is a serious issue. It is a well-known fact that the net increase in atmospheric greenhouse gases due to fossil fuels’ use is a major

Need for Renewable Fuels/General Background

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contributor to the global climate change. The studies have already revealed a direct correlation between the concentration of atmospheric greenhouse gases and temperature changes of the planet (Tandon and Mallik 2018). Thus, mitigation of CO2 release is essential to combat the problem of global warming. It has been proposed that in order to restrict the global temperature change to below 2  C, mitigation efforts cannot be delayed, and this necessitates a shift to the renewables by 2030 (Stephens et al. 2010; IPCC 2014). Also, a sizeable lot of the global fossil fuel reserves need to be held unused (Heede and Oreskes 2016). Hence, to address the twin problems of energy supply and climate change, the need for sustainable and eco-friendly sources of energy has become imperative in recent years (Dias De Oliveira et al. 2005; Muktham et al. 2016). Consequently, research studies have focused their attention on the production of fuels from renewable sources (Ciubota-Rosie et al. 2008; Singh et al. 2016). Renewable energy can be obtained from various sources such as solar power, wind power, hydropower, geothermic heat, tides, waves, and biomass (Demirbas 2005; Abolhosseini et al. 2013; Owusu and Asumadu-Sarkodie 2016). The energy from biomass, bioenergy, is one of the potent alternatives to the fossil fuels. The biomass refers to the renewable material having the biological origin. The plant matter is a valuable biomass in the bioenergy sector. The renewability of plant biomass confers to the remarkable property of photosynthesis in green plants, which has offered necessary evolution to the bioenergy industry towards more sustainable technology. The plants are equipped with special photosynthetic systems, which convert the solar energy efficiently into the biochemical energy. The latter is locked in the plant matter. Thus, the inexhaustible reservoir of sunlight and the capture and conversion efficiency of the plants, together promise the continuous supply of energy represented by the biomass. The estimates have revealed that the carbon content of the world’s total terrestrial biomass is nearly 100 times the global energy consumed in a year (Klass 2004). The plant biomass, primarily the terrestrial biomass, accounts for 450 gigatons of total carbon on the earth and this corresponds to 80% of the total biomass composition of the biosphere (Bar-On et al. 2018). This highlights the significance of plant biomass as a source of bioenergy. Bioenergy refers to the energy and energy related products obtained from the biomass (Schuck 2006). The energy can be generated from the biomass as heat, power, or biofuels. The biofuels are available as solid, liquid, or gaseous fuels (Cossu et al. 2018). The biomass can be to biofuels through thermochemical or biochemical processes. Unlike fossil fuels, they are not based on geologic deposits of organic matter but are produced from fresh biological materials. The unprocessed biomass had been used as primary biofuel for a long time for cooking and heating. But, later on, owing to the problems of pollution and inefficiency associated with primary biofuels, the technological advancements led to the development of secondary biofuels. In fact, the biofuels nowadays allude to secondary biofuels primarily. Biofuels are known as the prominent representative among renewable and cleaner fuels (Demirbas 2008; Putro et al. 2016). They are a kind of solar fuels, which are produced from solar energy in an indirect process (Styring 2012).

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Introduction to Lignocellulosic Ethanol

The use of biofuels is encouraged because of their potential for contributing to reduced dependence on fossil fuels and mitigating atmospheric pollution by lowering the level of CO2 emissions (Cheng et al. 2014). The potential of biofuels in minimizing the CO2 release is attributed to the closed carbon cycle, wherein the rate of CO2 released by burning biofuels equates the CO2 fixed by the plant during photosynthesis (Bessou et al. 2011; Gross 2014). Thus, biofuels help in alleviating the problem of global warming contributed by fuel combustion (Nanda et al. 2014). The current world, therefore, is advancing towards production of fuels from renewable biomass resources. In addition to offering energy security and cleaner environment, the bioenergy sector offers many other socio-economic benefits. The utilization of local resources for biofuel production can strengthen the economy of a nation in terms of reduced import of petroleum oils. Besides, it holds huge potential to offer income and employment opportunities, thus providing a way for developing rural economy (Gheewala et al. 2013). It also aids in efficient utilization of organic waste and can thus prove beneficial in eliminating the problem of their disposal (Cossu et al. 2018; Skaggs et al. 2018). Consequently, the national policies across the globe have started advocating the investments in the production of biofuels. The bioenergy domain is growing vastly along with the technological advancements.

Bioethanol as a Fuel Among biofuels, bioethanol has gained popularity across the globe, and this sector is soon expected to grow expeditiously (Shah et al. 2011). Estimates have reckoned that world ethanol production has escalated from 50 billion liters to over 100 billion liters from the year 2007 till 2012 (Kang et al. 2014). In fact, most of the biofuel has been produced in the form of ethanol (Dutta et al. 2014). Bioethanol, C2H5OH (ethyl alcohol or EtOH), is mostly attractive as a transportation fuel (Sarkar et al. 2012). Presently, fossil fuels are the dominant source of energy in the transportation sector. The consumption of petroleum and other liquid transportation fuels is estimated to rise from 85.7 million barrels/day in 2008 to 112.2 million barrels/day by 2035 (IEO 2011). The estimates have projected that energy consumption by the transport sector alone can grow by 1.1% per year from 2012 to 2040 (IEO 2016). This raises the concern of atmospheric pollution as the transportation sector alone has been known to account for 14% of the global CO2 emissions (IPCC 2014). Bioethanol is an attractive alternative to this problem. It is used widely in transportation sector. The characteristics of the ethanol make it a good fuel to be used in spark ignition engines (Balat 2009; Sharma 2016). Ethanol contains around 35% oxygen (Balat et al. 2008). The presence of oxygen ensures cleaner combustion of ethanol and lesser emission of particulates, hydrocarbons, and nitrogen oxides (Balat 2009; Thangavelu et al. 2016). The blending of ethanol in gasoline greatly reduces GHG emissions from the transportation sector (Sharma and Dhanjal 2016; Robak and Balcerek 2018). Bioethanol also has higher octane number (108), which is a measure of gasoline quality or an engine performance. A fuel with lower octane value often gives the problem of engine

Bioethanol as a Fuel

5

knocking. The higher octane number allows the fuel to withstand compression and guards against detonation (premature ignition). Thus, ethanol eliminates the requirement of costly octane boosters. Additionally, the bioethanol has lower cetane number, broader flammability limits, higher flame speeds, and higher heats of vaporization than gasoline (Balat 2009; Thangavelu et al. 2016). The internal combustion engines run efficiently on bioethanol compared to gasoline showing reasonable antiknock value, higher compression ratio, and reduced burn time (Balat 2009; Thangavelu et al. 2016). Ethanol can either replace transport fuel completely or it can be blended with petrol or diesel. E10 (10% ethanol in gasoline) is the most commonly used gasohol. The ethanol blending in lower ratios usually does not require modifications of the conventional combustion engines, though very old engines are not compatible for blending in any ratio. The higher ethanol blends, however, require flexible fuel vehicles (FFVs, dual fuel vehicles) (Balat 2009; Yusoff et al. 2015). Owing to the advantages of ethanol, various ethanol-gasoline blending programs have been implemented by Government in several countries such as E20 in Brazil (E25 for FFV), E10 in USA, Canada (E85 for FFV), Australia, Thailand, Columbia and Peru, E7 in Paraguay, and E5 in India (Balat 2009; Balat 2011). E85, a blend of 15% gasoline with 85% ethanol, when used to run medium sized passenger vehicles, can replace 32% of world’s gasoline consumption (Kim and Dale 2004). The use of ethanol as a fuel has a long record in history. Fermentation of sugars to the ethanol is a process known from ancient times, and widely used in the brewing industry. The burning of ethanol is an exothermic reaction, which reflects its potential as a fuel. The reaction produces CO2 and water as the end products. Before the discovery of petroleum oil, ethanol was the leading fuel being used in illuminants as a blend with 20 to 50 percent turpentine (mixture was called as camphene or burning fluid) (Kovarik et al. 1998). In 1826, a prototype of internal combustion engine was constructed by Samuel Morey, which could be run using ethanol as a fuel (Kovarik et al. 1998, Abebe 2008). His work, however, failed to get recognition due to the lack of funding, and thus remained unpopular. In 1830s, the use of alcohol blends for oil lamps was escalated as an economical replacement for whale oil; and ethanol accounted for 90 million gallons production in the USA in 1860 until kerosene from petroleum started becoming available as a relatively cheaper and efficient lamp fuel in 1860s (Kovarik et al. 1998; Tomes et al. 2010). Later on, between 1862 and 1864, tax was imposed on U.S. Distilled Spirits, including beverage as well as the fuel or industrial alcohol, to generate revenue to assist in financing the Civil War. It resulted in a rapid decline in the distilleries’ market of ethanol as a burning fluid in lamps. In 1860, the period when camphene was a popular fuel for illuminants, a German inventor, Nicholas August Otto, again developed an internal combustion engine driven on ethyl alcohol (Tomes et al. 2010). He worked in association with the owner of sugar refining industry, Eugen Langen, but later produced engine run primarily on gasoline, however, adaptable to ethanol. The automobiles for a long time were widely run on gasoline only. In late nineteenth century, German, French, and British Government were concerned about the reserves as well the supply of petroleum oils. France and Germany extended their

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Introduction to Lignocellulosic Ethanol

research efforts to produce fuel from domestic products and started encouraging alcohol’s use as a fuel. In the early twentieth century, the French Ministry of Agriculture encouraged ethanol use by offering prizes for the best alcohol-fueled engines. In 1902, the automobiles, agricultural engines, and household appliances that run on alcohol were exhibited in Paris alcohol fuel exposition. In France, the alcohol production increased from 2.7 million gallons to 8.3 million gallons from 1900 to 1905 (Kovarik et al. 1998). The German government also started emphasizing on replacing steam and gasoline engines with the relatively cleaner and safer engines that run on ethanol and this resulted in the proliferation of distilleries in Germany. At the same time U.S. government also started taking interest in ethanol. In 1906, Free Alcohol bill was passed, considering alcohol as the best substitute for gasoline as gasoline was appearing as a scarcer and costly fuel. In 1908, Ford motor company, USA first launched model “T” car, which ran on alcohol-gasoline fuel (Tomes et al. 2010; Niphadkar et al. 2018). In 1909, U.S. Geological Survey reports showed that the use of alcohol as an alternative fuel for gasoline and kerosene had many advantages such as high engine compression ratio (absence of knocking) and absence of smoke and strong odor. This was followed by many reports about the efficacy of ethanol fuel as well as its environment friendliness compared to the gasoline. In 1923, Ethyl Corporation, USA, insisted on ethanol blends also as a replacement for leaded gasoline, which was responsible for lead poisoning. All these factors together lead an increase in ethanol production. In the USA, the price of molasses based alcohol became less than 20 cents/gallon compared to 28 cents/ gallon of gasoline price. In 1923, many countries like Brazil, France, and Germany encouraged the blending of ethanol in gasoline and ethanol started competing with gasoline till the 1930s. Thus, the demand of ethanol-gasoline blends started increasing in 1920s and 1930s, which peaked during World War II due to paucity of fuels (Abebe 2008; Tomes et al. 2010). Various feedstock was being explored for commercial ethanol production in different countries. In 1933, Coryell Gasoline Company, USA, started marketing corn-alcohol gasoline blend, which became popular with the name of “gasohol” implying 10% ethanol with 90% gasoline (Kovarik et al. 1998; Abebe 2008; Niphadkar et al. 2018). However, the discovery of many cheap oil reserves in the 1940s resulted in a decline in the ethanol market, which was lifted again after many years in 1974, when Arab countries imposed an oil embargo. This oil crisis stimulated various countries including the USA for energy conservation and development of alternative fuels indigenously (Balat 2009; Tomes et al. 2010). Brazil initiated National Alcohol Fuel Program (ProAlcool) in 1975 with a goal of bioethanol production (Balat 2009). In 1978, in the USA, Energy Tax Act was created and use of ethanol-blended fuel was promoted by exempting a part of the excise tax on motor fuels. Subsequently, various policies and acts for energy security were passed at different times. After that ethanol industry did not look back but kept on progressing all over the world. In 1998, Brazil was the largest contributor to world ethanol production, accounting for 16.1 billion liters of total 33.3 billion liters production (Niphadkar et al., 2018). After the oil crisis in 1970, the ethanol

Classification of Bioethanol Fuel

7

production increased at the global level. It is evident from the data showing a rise from 30%) at ambient temperature (90% for both cellulose and hemicellulose) (Chen et al. 2017). The effectiveness of this method is primarily based on the susceptibility of the glycosidic bonds between sugar units in the carbohydrate polymers to the acids. The hydronium ions produced from the acids cleave the polymer chains of cellulose and hemicellulose. The acid catalysts also disorder the van der Waals forces and hydrogen bonds between lignocellulosic components, thus, facilitating their solubilization (Amin et al. 2017). The effectiveness of the method depends on the concentration of acid, acid-biomass ratio, temperature, and retention time (Bensah and Mensah 2013). On the other hand, dilute acid pretreatment is relatively milder and therefore, is mostly preferred over concentrated acid pretreatment. In this method, acid catalyst is used at lower concentrations (0.1–10%) (Chen et al. 2017; Baruah et al. 2018). The pretreatment is carried out at relatively high temperature and pressure. The reaction time may vary from minutes to hours depending on biomass composition as well as conditions used during treatment. The dilute acid treatment solubilizes the hemicellulose (amorphous) component of lignocellulose quickly by hydrolyzing the xylosidic bonds

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and cleaving the acetyl ester groups in the polymer. The lignin undergoes modification and little deconstruction through substitution reactions. However, it is not solubilized because of condensation reactions other than the hydrolytic reactions (Bensah and Mensah 2013). The lignin alteration and hemicellulose removal results in increased accessibility of the residual cellulose. This exposure of the cellulose to hydrolytic enzymes during saccharification increases the yield of fermentable sugars. The cellulose itself may also undergo degradation but in amorphous regions, thereby, increasing the crystallinity index of the pretreated material. The acid pretreatment method has been found effective in the pretreatment of a wide variety of lignocellulosic biomass (Table 3.3). However, there are several disadvantages associated with this method. The acids are toxic and highly corrosive in nature. The treatment with high concentrations of acid necessitates the use of specialized corrosion resistant equipment or bioreactors, which increases the cost of pretreatment method. Also, the acid consumption in high concentration requires their recovery and recycling to make pretreatment process economically viable (Sun and Cheng 2002; Kumar et al. 2009). However, acid recovery in itself is energy intensive and complete recovery of acid is difficult. All these factors influence the operational and maintenance costs during the process (Chen et al. 2017). After the pretreatment, the biomass needs extensive washing or neutralization for the acid removal and this generates a waste stream (Bensah and Mensah 2013). Additionally, the acid hydrolysis generates several inhibitory compounds such as furfurals and hydroxymethylfurfural (HMF) (Brodeur et al. 2011; Ahmad et al. 2018; Amin et al. 2017). These compounds, in high concentrations, show strong negative effect on cellulolytic enzymes and the growth of microorganisms during fermentation, thus are inhibitory for the subsequent steps of the saccharification and ethanol fermentation. The removal of these inhibitors is done using an additional step of detoxification, which again increases the cost of pretreatment method (Brodeur et al. 2011). However, all these disadvantages have been minimized in various research studies by focusing on dilute acid pretreatment of various feedstock at milder conditions.

Organosolv Pretreatment The organosolv pretreatment is based on lignin extraction from the biomass by treating it with organic solvents or their aqueous solutions (Kumar et al. 2009; Nitsos et al. 2017). The method is like organosolv pulping, but with a lower level of delignification (Zhao et al. 2009). Commonly used organic solvents (or their mixture with water) include ethanol, methanol, acetone, ethylene glycol, and 4-hydrogenation furfuryl alcohol (Chen et al. 2017; Kumar and Sharma 2017). The process may be combined with a catalyst, which can be acid (organic or inorganic) (HCl, H2SO4, formic, oxalic, and salicylic acid), base (NaOH), or salts (MgCl2, Fe2(SO4)3) (Bensah and Mensah 2013). The catalyst addition either lowers the temperature or improves the delignification rate (Baruah et al. 2018). The organic solvents and their solutions, under specific conditions of temperature and pressure,

Physicochemical Pretreatment

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Table 3.3 Acid pretreatment of various lignocellulosic biomass Acid pretreatment conditions 0.75% (v/v) H2SO4

Downstream process(es) Enzymatic saccharification using cellulase, xylanase, β-glucosidase, and esterase, fermentation using recombinant E. coli FBR5

Rice straw

0.5% H2SO4, for 10 min at 15 bar pressure, in 101 reactor

SSF with Mucor indicus, Rhizopus oryzae, and S. cerevisiae, under anaerobic and anaerobic conditions

Silver grass

H2SO4 pretreatment (at combined severity factor of 2.3) 1.5% H2SO4, autoclaving at 121
C for 60 min (for St6 3E germplasm)

Xylose fermentation using Candida shehatae

Feedstock Wheat straw

Switchgrass germplasms

Sugarcane tops

3% w/w H2SO4, using 15% w/w biomass loading, at 121
C, for 60 min, followed by neutralization with NaOH

Rice straw

0.5% H2SO4, 10% biomass loading, autoclaving at 121
C, for 60 min

Enzymatic hydrolysis using 30 FPU cellulase per gram along with xylanase, ethanol production using S. cerevisiae Enzymatic saccharification using commercial cellulase (50 FPU/ g), and 0.2% w/w Tween-80 as surfactant, for 60 min, using 11.25% w/v biomass loading Enzymatic saccharification using 40 FPU enzymes load per gram biomass, 17.5% biomass load, 50
C temperature, and 72 h time

Outcome 19 g/L ethanol production (0.24 g/g yield) by separate hydrolysis and fermentation (SHF); 17 g/L ethanol production by simultaneous hydrolysis and fermentation from detoxified biomass 74% ethanol yields with R. oryzae, 68% ethanol yields with M. indicus, and 61% with S. cerevisiae under anaerobic conditions 24.2 g/L xylose after pretreatment and 64.3% ethanol after 48 h

Reference Saha et al. (2005)

91.8% glucan conversion, 0.082 g ethanol/g ethanol yield (¼53.5% of theoretical yield)

Yang et al. (2009)

Increase in cellulose content from 29.9% to 46.5%; decrease in hemicellulose content from 18.9% to 6.16%

Sindhu et al. (2011)

0.359 g/g sugar yield

Kshirsagar et al. (2015)

Karimi et al. (2006)

Guo et al. (2008)

(continued)

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Table 3.3 (continued) Feedstock Sugarcane bagasse pith

Acid pretreatment conditions 1–2% v/v H2SO4, at 121
C, for 90 min

Downstream process(es) Enzymatic hydrolysis using commercial cellulase; SHF and SSF for ethanol production using Pichia stipitis JCM 10742 as ethanologen

Jerusalem artichoke stalks

5% w/v nitric acid (HNO3), at 121
C, for 60 min

Oil palm trunk

Oxalic acid (15%, at 100
C, 60 min), citric acid (13.92%, at 131.92
C, 58.92 min), and acetic acid (8.23%, 107.3
C, 30 min)

Enzymatic hydrolysis using Cellic CTec2 (10 FPU/g) and 5% w/v solid load for 72 h; ethanol fermentation using S. cerevisiae Enzymatic hydrolysis by commercial cellulase (20 FPU/ g) and cellobiase (100 CBU) and 5% w/v solid load for 72 h; fermentation using S. cerevisiae TISTR 5606

Outcome Total sugar yield of 53.7 g per100 g biomass, (67% of total sugars); 3.7 g/ L ethanol production in 24 h (0.15 g/L/ h productivity) during SSF and 2.58 g/L ethanol production (0.09 g/L/ h productivity) in SHF in 30 h 38.5 g/L glucose production (89% of theoretical yield), 9.5 g/L ethanol production (46% of the theoretical yield)

Reference Sritrakul et al. (2017)

Highest 16.27 g/L ethanol production with citric acid pretreatment

Rattanaporn et al. (2018)

DziekońskaKubczak et al. (2018)

hydrolyze the internal bonds in hemicellulose-lignin complex, and glycosidic bonds within carbohydrate polymers (more in hemicellulose and to a lesser extent in cellulose) (Bensah and Mensah 2013). The choice of temperature during pretreatment depends on the type of biomass and type of catalyst (Baruah et al. 2018) and may vary from low temperature to as high as 250
C (Zhao et al. 2009). The lignin can be recovered conveniently by precipitation. Thus, the process may generate a cellulose-enriched biomass, a hemicellulose rich liquid stream, and a solid precipitate containing lignin (Zhao et al. 2009; Nitsos et al. 2017) (Fig. 3.5). The structural characteristics of pretreated biomass such as length of biomass fiber, cellulose DP, and crystallinity may get altered depending on process conditions. The hemicellulose is solubilized, which increases pore size. The enrichment of

Physicochemical Pretreatment

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Fig. 3.5 Organosolv pretreatment of lignocellulosic biomass (Modified from Nitsos et al. 2017)

biomass with cellulose and removal of lignin (which avoids non-selective adsorption of cellulases) enhances the digestibility of biomass. The outcome of pretreatment is influenced by the process temperature, retention time, solvent level, and presence and amount of catalyst (Bensah and Mensah 2013). However, treatment at very high temperature and acid levels for longer retention times may yield fermentation inhibitors. Organosolv pretreatment is beneficial in many ways. The recovery of organic solvents is easier through distillation, which can be recycled in the process. The lignin recovered (with high purity) as solid precipitate and the hemicellulosic liquid fraction can be used as potential substrates to produce value-added products in biorefinery processes (Zhao et al. 2009). The pretreatment generates lesser amounts of inhibitors, which has a positive impact on conversion process (da Silva et al. 2018). However, the washing of pretreated biomass is essential to prevent precipitation of dissolved lignin. High amounts of solvents may themselves be inhibitory for downstream processes (Sun and Cheng 2002). The inorganic acids, when used as catalysts, are corrosive (Bensah and Mensah 2013). Diaz et al. (2011) performed organosolv (aqueous EtOH) pretreatment of olive tree pruning biomass. The delignification was enhanced up to 64% at high pretreatment severity conditions of temperature and ethanol content, at 66% w/w aqueous ethanol, 210
C, and 60 min retention time. But, maximum xylan hydrolysis (up to 92%) was achieved at a lower ethanol level. The enzymatic hydrolysis of pretreated biomass was most effective (90% yield from glucan) when the pretreatment was done using 43% w/w aqueous ethanol, at 210
C, for 15 min. The effectiveness of various solvents may vary depending on many factors, primarily the type of feedstock. Salapa et al. (2017) evaluated the effect of organosolv pretreatment with different solvents (ethanol, methanol, butanol, acetone, and diethylene glycol) on wheat straw biomass in the presence of 23 mol/cm3 H2SO4 catalyst. The results indicated that highest cellulose conversion (89%) and ethanol

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yield (67%) (using S. cerevisiae as ethanologen) were achieved when biomass was pretreated at 180
C for 40 min using ethanol as solvent. The pretreatment with diethylene glycol at 160
C for 40 min was also promising in obtaining high ethanol yield of 65% along with a pulp yield of 51%. Raghavendran et al. (2018) compared the results of enzymatic hydrolysis of two difference biomass, i.e., spruce and birch (at 25 w/v loading), after their pretreatment with the organosolv method (50–60% ethanol +1% sulfuric acid, 10% solid load). The pretreated birch could be hydrolyzed almost completely using 12 FPU/g solids enzyme loads, while only 70% saccharification yield was possible from pretreated spruce even at high enzyme loadings of 60 FPU/g solids. When compared to the steam exploded counterpart, the organosolv pretreated biomass was having higher cellulose content resulting in higher glucose yields. Additionally, the liquid hydrolysate was free from the phenolic inhibitors.

Wet Oxidation Wet oxidation, a simple thermal oxidation method, involves oxidation of biomass with air or pure oxygen along with water as an oxidizing agent (Chen et al. 2017; Kumar and Sharma 2017). The biomass is soaked in an aqueous (acidic, alkaline, or neutral) solution and treated at a temperature higher than 120
C (125–370
C) and 0.5–2 MPa pressure for less than 30 min (Martín and Thomsen 2007; Den et al. 2018; Kumari and Singh 2018). The wet oxidation method was employed to pretreat lignocellulosic wood as in place of steam explosion pretreatment in the early 1980s (Varga et al. 2003). Earlier, the method was used mostly for the treatment of wastes and polluted soil (Palonen et al. 2004; Kumar and Sharma 2017). Depending on the conditions during oxidation, the organic matter in the wastes is oxidized to carboxylic acids, acetaldehydes, and alcohols, or CO2 and H2O as final products (Palonen et al. 2004). The method is effective in opening the structure of cellulose (Varga et al. 2003). The hemicellulose is hydrolyzed to sugar monomers and organic acids, cellulose undergoes partial degradation, and lignin is cleaved and oxidized like other contents of the organic matter (Den et al. 2018). Thus, all three components of lignocellulose are affected. The fractionation starts from hemicelluloses followed by lignin oxidation, and thus produces a cellulose-rich solid residue under controlled conditions (Brodeur et al. 2011). The hydroxyl radicals and organic acids generated in the reaction are responsible for biomass alterations (Arvaniti et al. 2012). The water at a high temperature (>170
C) behaves as an acid and catalyzes the hydrolytic reactions (Kumar and Sharma 2017). The method is most suitable for the pretreatment of lignin rich biomass (Kumar and Sharma 2017). One primary advantage of the wet oxidation method is that inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are produced in lower amounts (Varga et al. 2003; Palonen et al. 2004). The presence of very reactive aliphatic aldehydes, saturated bonds, and carbon bonds reduces the formation of inhibitors. However, it is a costly method requiring high temperature and pressure (Bensah and Mensah

Steam Explosion

89

2013; Kumari and Singh 2018). Also, the use of pure oxygen increases the cost of the method (Den et al. 2018). The efficiency of the method is determined by process temperature, oxygen pressure, and retention time (Kumar and Sharma 2017). The reaction rates can be enhanced by using higher oxygen levels or pure oxygen (Den et al. 2018). In a study by Palonen et al. (2004), the conditions for wet oxidation of softwood, Picea abies, were optimized by varying temperature, pH, and reaction time. The maximum hydrolysis yield (79%) was attained by pretreating biomass at 200
C for 10 min at a neutral pH with the addition of 12 bars of oxygen. Arvaniti et al. (2012) used the wet oxidation method for the pretreatment of biomass for bioethanol production from rape straw by simultaneous saccharification and fermentation approach. The maximum ethanol yield of 67% was achieved through SSF using biomass pretreated by wet oxidation at 205
C for 3 min at 12 bars of oxygen pressure. The biomass was presoaked in water before its pretreatment. Under these conditions, they could achieve recovery of 100% cellulose and hemicellulose, along with 85% lignin recovery. Also, the recycling of filtrate was found to be ineffective because of its inhibitory effect on the fermenting yeast. Additionally, the catalysts may be used in the reaction to make the pretreatment process less energy intensive. Varga et al. (2003) have pretreated the corn stover by wet explosion method, using 2 g/L Na2CO3 at 195
C and 12 bar O2, by treating 16 g/L biomass under these conditions for 15 min. The pretreatment solubilized 60% hemicellulose and 30% lignin, thus, enriching the biomass with 90% cellulose. Subsequently, the enzymatic hydrolysis of biomass using 25 FPU/g of the enzyme was effective in converting 85% cellulose to glucose. Morone et al. (2018) varied the amount of sodium carbonate (Na2CO3) during pretreatment of rice straw by wet air oxidation, aiming at enhanced hemicellulose and lignin solubilization, to achieve high cellulose recovery while generating minimum amounts of inhibitors with reduced chemical input. The highest cellulose recovery of 82.7% was observed from the biomass pretreated at 169
C, 4 bar, for 18 min using 6.5 g/L Na2CO3 loading. Under these conditions, the hemicellulose solubilization was 85.43%, lignin removal 65.42%, and 0.36 g/L total phenolic content in the liquid fraction.

Steam Explosion Steam explosion is one of the most successful and extensively studied physicochemical pretreatment methods for biomass deconstruction to enhance its digestibility. The method was first developed in 1924 by W. H. Mason as a process to produce chipboard panels (Jacquet et al. 2015). In the 1980s, the technology was patented for aspen woodchips pretreatment to produce sugars (Duque et al. 2016). The method is based on a combination of thermal, chemical, and mechanical treatments (Chen et al. 2017; Baruah et al. 2018). The steam pretreatment is a hydrothermal method involving treatment of biomass with high-pressure saturated steam at 160–260
C temperature and 0.5–4.8 MPa pressure (Baruah et al. 2018; Bensah and Mensah 2018). Such conditions facilitate the penetration of water molecules in the biomass.

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After the retention time (of several seconds to minutes), the pressure is released swiftly to allow the escape of water molecules with an explosive effect. In addition to the mechanical fractionation, the retention for several minutes promotes hemicellulose hydrolysis, cleavage of glycosidic bonds in hemicellulose and cellulose, and the bonds between hemicellulose and lignin. The release of acetic acid from the acetyl groups in hemicellulose catalyzes the carbohydrate hydrolysis (Mosier et al. 2005). The effect of acetic acid may aid in decreasing time and treatment temperature (Kumar et al. 2009). Water also acts as an acid at high temperature (Mosier et al. 2005). After the pretreatment, the solid biomass contains cellulose and transformed lignin, while most of the hemicellulose is solubilized in the liquid fraction (Kumar et al. 2009; Bensah and Mensah 2018). The efficiency of the method is governed by the temperature, residence time, size of biomass, biomass mixing, and the moisture content (Baruah et al. 2018; Bensah and Mensah 2018). The increase in residence time and temperature decreases the cellulose DP and plays a critical role in the complete hydrolysis of hemicellulose. Increasing these parameters beyond a level, however, may cause an increase in the production of inhibitory products such as furfural, hydroxymethylfurfural, levulinic and formic acid, and reduced accessibility of the biomass due to re-condensation of pseudo-lignin (lignin-like materials) over the cellulose (Jacquet et al. 2015; Bensah and Mensah 2018). The steam pretreatment offers many advantages such as low environmental damage, low capital investment, limited use of hazardous chemicals, high energy efficiency, no recycling requirement, high sugar yields, and feasibility at the industrial levels (Duque et al. 2016; Chen et al. 2017; Baruah et al. 2018). Pielhop et al. (2016) have suggested that the enzymatic digestibility of even highly recalcitrant biomass can be enhanced if the steam explosion effect is exploited thoroughly. They observed that an increase in a pressure difference of explosion causes more defibration and reduces the particle size. The high-pressure difference and high severity lead to enhanced digestibility. The method has been successfully used for pretreatment of various biomass. Fang et al. (2011) have demonstrated continuous steam explosion of wheat straw. They studied the impact of pretreatment conditions on total sugar recovery as well as the biomass composition and observed that longer retention time (198
C for 6 min) enhanced enzymatic hydrolyzability of biomass (93.3% hydrolysis yield compared to 88.7% in 4 min) whereas with a reduced yield of glucose (85.8% compared to 88.4% in 4 min). Ferro et al. (2015) have produced bioethanol from steam pretreated and alkali extracted Cistus ladanifer (rockrose). The steam explosion disrupted the fibers and caused partial lignin degradation, which enhanced the accessibility of biomass carbohydrate polymers. Alkali extraction after the steam explosion caused partial removal of lignin, hemicelluloses, and other degradation products, and increases the glucose yield to 75%. In the end, 16.1 g/L ethanol production (22.1 g ethanol/100 g pretreated biomass) was achieved by simultaneous saccharification and fermentation of the biomass. Another work by Liu et al. (2017) carried out steam explosion pretreatment of sweet potato vine. The work involved studying the effect of moisture content on instant catapult steam explosion (ICSE) pretreatment of biomass. The ICSE can exploit the intracellular water of lignocellulosic materials for the explosive effect during steam explosion pretreatment. The authors observed

Ammonia Fiber Explosion Method (AFEX)

91

2.5-folds improvement in the sugar yield (reaching to 88.05%) of ICSE-pretreated fresh biomass, which was equivalent to the soaking of biomass for 60 h. The scanning electron micrograph revealed a uniform reduction in particle size as a result of the steam explosion. Auxenfans et al. (2017) worked on evaluating the structural and chemical alteration in Miscanthus  giganteus, poplar, and wheat straw biomass before and after steam explosion (acid-catalyzed) pretreatment. They observed a correlation of enhanced digestibility with hemicellulose removal, reduced crosslinking in phenolic acids, and lignin redistribution.

Ammonia Fiber Explosion Method (AFEX) AFEX pretreatment is a combination of steam explosion with alkali treatment involving the use of liquid anhydrous ammonia (Chen et al. 2017; Kumari and Singh 2018). The biomass is mixed with liquid ammonia (in 1:1 ratio) and exposed to ambient temperature (60–100
C) and high pressure (1–5.2 MPa) for 5–60 min followed by the sudden release of pressure (Chen et al. 2017; Kumar and Sharma 2017; Baruah et al. 2018). The large part of ammonia (over 97%) can be recovered for its reuse because it evaporates readily (Harun et al. 2013). The recycle and reuse of ammonia increases the cost-effectiveness of the method and makes it environment-friendly. During the reaction, the combined effect of explosion and temperature causes swelling of biomass, disruption of lignocellulose ultra- and macro-structure, cleavage of ester linkages in lignin-carbohydrate complex, partial removal of lignin, hemicellulose hydrolysis, deacetylation of acetyl groups, and cellulose decrystallization, consequently enhancing the digestibility of biomass (Harun et al. 2013; Chen et al. 2017; Bensah and Mesah 2018; Baruah et al. 2018; Mokomele et al. 2018). The biomass subjected to AFEX pretreatment requires lesser enzyme loadings compared to that pretreated using other methods (Baruah et al. 2018). The optimization of the AFEX process involves variation in factors such as ammonia loading, reaction temperature, water loading, blowdown pressure, and residence time (Baruah et al. 2018; Kumari and Singh 2018). Murnen et al. (2007) optimized various parameters, including temperature, moisture, ammonia loading, residence time, and enzyme loadings for the AFEX pretreatment of Miscanthus  giganteus. They evaluated the efficacy of pretreatment by hydrolyzing the pretreated biomass with cellulase and β-glucosidase enzyme (supplemented by additives, xylanase and/or pectinase and Tween-80). The highest glucan (96%) and xylan (81%) conversions were attained at 160
C, using 2:1 (w/w) ammonia to biomass loading and 233% moisture (dry weight basis), by allowing the reaction to last for 5 min. The biomass was subjected to pre-AFEX soaking, which resulted in up to 10% increase in glucan conversion. The particle size of the biomass may also have an influence on the outcome of AFEX (Bensah and Mensah 2018). Harun et al. (2013) studied the effect of two AFEX pretreatment conditions varying in their severity on rice straw of different particle sizes (milled, 2–5 mm, and cut, 2–5 cm). The maximum sugar conversion was achieved when larger cut rice straw particles (5 cm) were subjected to high severity AFEX conditions. The method is

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advantageous in that nearly all of the ammonia used for the pretreatment can be recovered and reused, does not require waste-water recovery, and solid-liquid separations (Mokomele et al. 2018). Very fewer inhibitors are formed during the process (Taherzadeh and Karimi 2008). The method, however, is not effective for biomass having high lignin content such as hardwood. AFEX pretreatment method has been employed for a variety of biomass. Lau and Dale (2009) produced bioethanol from corn stover using AFEX as the pretreatment method. Separate hydrolysis and fermentation of the pretreated biomass resulted in the production of 40 g/L ethanol (191.5 g/kg biomass) and did not require pretreated biomass washing, detoxification, or addition of nutrient supplements. They demonstrated the advantage of degradation products generated from AFEX pretreatment of biomass in increasing the metabolic yield during fermentation. Krishnan et al. (2010) demonstrated the viability of AFEX pretreatment during cellulosic ethanol production from sugarcane bagasse and cane leaf residue. They achieved production of 34–36 g/L ethanol (92% theoretical yield) by co-fermentation of glucose and xylose, produced from high solid loading hydrolysis of AFEX-treated biomass using a recombinant strain of S. cerevisiae 424A LNH-ST. Mokomele et al. (2018) compared the effectiveness of AFEX and steam explosion method for bioethanol production from sugarcane bagasse and under-utilized cane leaf matter. They found that AFEX pretreatment was more effective compared to the steam explosion in reaching higher recovery of fermentable sugars and fermentability of enzymatic hydrolysate by S. cerevisiae 424A (LNH-ST) using high biomass loadings.

Supercritical CO2 (or CO2 Explosion) A supercritical fluid formed above its critical temperature (Tc) and pressure (pc) but below the pressure needed for its condensation has properties of both liquid and gas. At the critical point, they possess indistinguishable liquid and gas phases (Gu 2013; Morais et al. 2014). CO2 is a green solvent with a 31.1
C critical temperature (compared to 374.2
C for water) and critical pressure of 1071 psi or 73.84 bar (compared to 3208 psi for water) (Gu et al. 2013). Recently, supercritical carbon dioxide is being used for biomass processing as a pretreatment method (Morais et al. 2014; Yin et al. 2014). The supercritical CO2 was first used for avicel pretreatment by Zheng et al. (1995). The method is a combination of supercritical CO2 and steam explosion pretreatment. The gas like mass transfer and liquid like solvation properties of supercritical CO2 facilitate diffusion of CO2 in the biomass interior and dissolution of its components (Baruah et al. 2018). The pressure makes the CO2 molecules to penetrate into biomass, and the rate of penetration is increased at high pressure, resulting in higher yields of sugars. The release of pressure deconstructs the biomass lignocellulosic matrix resulting in an increase in surface area and consequent enzymatic hydrolysis. The CO2 forms carbonic acid with water, which catalyzes the hydrolysis of hemicellulose (Chen et al. 2017; Baruah et al. 2018). The high moisture content of the biomass increases the hydrolytic yield of the

SO2 Explosion

93

process (Kumar and Sharma 2017). Alinia et al. (2010), in their study, pretreated wheat straw with supercritical CO2 followed by its enzymatic hydrolysis to obtain fermentable sugars. 149.1 g sugars were produced per kg of biomass when dry wheat straw was pretreated by supercritical CO2 at 190
C for 30 min. The sugar yield was increased to 208.4 g/Kg when wheat straw was impregnated with water before its pretreatment at 185
C for 30 min. Further, 234.6 g/kg sugar yield was obtained by combining supercritical CO2 pretreatment with the steam explosion (at 12 Mpa and 190
C for 30 min). The method shows many advantages such as low cost of solvent, no degradation of sugars, non-toxicity, non-flammability, low pretreatment temperatures, effective for high biomass loadings, easy recovery after extraction, and environmental acceptability (Chen et al. 2017; Huisheng et al. 2013; Baruah et al. 2018). The transport of CO2 is easy and even if released in the environment, it does not cause pollution (Gu 2013). The pretreatment method, however, is too expensive for industrial applications because of the high cost of bioreactor which can withstand highpressure conditions (Kumar and Sharma 2017; Baruah et al. 2018). The pretreatment efficacy is influenced by several operational parameters, including reaction temperature, pressure, residence time, biomass moisture content, and CO2 to biomass ratio (Gu 2013). Liu et al. (2014) used supercritical CO2 for the pretreatment of corncob, cornstalk, and rice straw, and studied the effect of temperature, pressure, moisture content of biomass, and treatment time on reducing sugar yield. At optimized conditions of 100
C, 15 MPa, 50% moisture content, and retention time of 30 min, the reducing sugar yield from both the cellulose and hemicellulose hydrolysis was 39.6%, 27.4%, and 36.6% for corncob, cornstalk, and rice straw, respectively (compared to 26.2%, 22.5%, and 30.9% in native substrates). Several workers have combined the supercritical CO2 method with other methods in order to enhance the digestibility of lignocellulosic biomass. Huisheng et al. (2013) pretreated corn stover using supercritical CO2 with water-ethanol as a co-solvent (to remove lignin). They obtained the highest 77.8℅ sugar yield when the pretreatment was done at 15 MPa and 180
C, for 1 h. The cellulose content was increased, while hemicellulose and lignin contents were reduced in the pretreated biomass. The opening of biomass structure was evident from the results of scanning electron microscopy. Yin et al. (2014) combined supercritical CO2 with an ultrasound method for the pretreatment of corn stalk and corn cob. The enhancement in enzymatic hydrolysis was recorded to be 75% and 13.4% more (compared to native biomass) for corn cob and corn stalk, respectively, using a combined method for the pretreatment. However, 50% and 29.8% rise in enzymatic hydrolysis was observed for corn cob and corn stalk, respectively, when supercritical CO2 alone was used for biomass pretreatment.

SO2 Explosion SO2 explosion is like CO2 explosion (Chen et al. 2017). It involves steam pretreatment of the biomass in the presence of SO2 (1–4%) (Taherzadeh and Karim 2008). The impregnation of sulfur dioxide during steam explosion lowers

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the temperature required for the steam pretreatment process, and reduces the reaction time, consequently, decreasing the formation of inhibitors formed as a result of sugar degradation (Bura et al., 2002). The method is being used widely for the pretreatment of softwoods. At the end of the pretreatment, the biomass is enriched with cellulose due to solubilization of hemicellulose. The hemicellulose is removed almost completely from the biomass (Kumar et al. 2009). SO2 is distributed uniformly into the biomass, and unabsorbed SO2 can be recycled, which can make the process cost-effective, and reduce the need for catalysts. Some of the SO2 is converted to H2SO3 after reaction with water (or steam), which can be oxidized to H2SO4 under high temperature and pressure conditions (Tooyserkani et al. 2013). The acid may also be added as a catalyst to enhance the efficiency of the steam pretreatment or SO2 explosion process. The method has the limitation that the process is costly, requiring special equipment and involves the formation of many degradation products when acids are used (Chen et al. 2017). Different feedstock has been subjected to SO2 explosion as a pretreatment method to enhance the digestibility of the biomass. Bura et al. (2002) have used SO explosion pretreatment for bioethanol production from corn fiber. The pretreatment at 190
C for 5 min with 6% SO2 followed by enzymatic saccharification caused 81% hydrolysis of corn polysaccharides to sugars. The fermentation process using by S. cerevisiae yielded 90–96% of theoretical conversion. De Bari et al. (2007) carried out fractionation of Aspen chips by SO2 explosion for bioethanol production and studied the effect of the addition of an acid catalyst. The pretreatment was done at 205
C for two treatment times, i.e., 3 and 10 min. The addition of 0.9% w/w acid catalyst reduced the degree of polymerization of cellulose by 50%. The xylose yield was highest (80% of total extracted dry biomass) when steam pretreatment for 3 min was followed by water extraction. The glucose yield in 3 min was 67%, and the treatment for 10 min enhanced the recovery of glucose by 70%, but with lower xylose yield. Ninety-six percent of the glucose released from biomass pretreated for 3 min could be fermented to ethanol. Kang et al. (2013) have also demonstrated the effect of SO2 catalyzed steam explosion pretreatment on the digestibility, accessibility, and crystallinity of the Loblolly pine biomass. They observed enhanced accessibility of the biomass with an increase in severity, however, with non-selective degradation of carbohydrates. The crystallinity index of biomass was lowered with a severity beyond a point due to structure disruption and removal of amorphous regions. A correlation was found between initial hydrolysis rates and biomass crystallinity index. Wang et al. (2018) compared the effects of sulfuric acid and SO2 in continuous steam pretreatment of wood of Norway spruce. The SO2 pretreatment at high temperature caused the formation of pseudo-lignin and more inhibitors formation but exhibited enhanced digestibility than sulfuric acid. The authors proposed this increase in digestibility might happen as a result of the increase in particle size due to partial degradation of lignin and cellulose.

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95

Alkaline Peroxide Pretreatment The hydrogen peroxide is among the most commonly used oxidizing agents, other than peracetic acid, used for biomass delignification. The applicability of hydrogen peroxide for enhancing biomass digestibility was studied by Gould in 1984. Compared to alkali pretreatment, hydrogen peroxide pretreatment method is more effective in improving biomass digestibility. Under alkaline conditions, hydrogen peroxide dissociates and produces superoxide and hydroxyl radical, which act as powerful oxidizing radicals (Dutra et al. 2018). The radicals play a part in lignin degradation (Michalska and Ledakowicz 2016). Lignin is the most susceptible site for the attack by radicals (Dutra et al. 2018). The oxidative delignification occurs through the attack of oxidizing agents on the aromatic nuclei and alkyl aryl ether linkages of lignin (Bensah and Mensah 2013; Kumari and Singh 2018). Also, partial dissolution of hemicellulose may occur (Michalska and Ledakowicz 2016). Alkaline peroxide method has been used successfully to pretreat to a wide variety of biomass, including straws (wheat, barley, rice), husk (rice), bagasse (sugarcane, sweet sorghum}, bamboo, softwood, corn stover, and other plants (Banerjee et al. 2011; Dutra et al. 2018). The pretreatment can be performed under mild conditions of pressure and temperature, involving low-energy consumption, reduced cost of special reactors, and use of lower concentrations of oxidant (Bensah and Mensah 2013; Michalska and Ledakowicz 2016; Dutra et al. 2018). The decomposition of H2O2 into water and oxygen leaves no residual oxidant in the biomass (Michalska and Ledakowicz 2016). No hydroxymethylfurfural and furfural are generated (Dutra et al. 2018). However, the process is associated with the release of phenolic compounds from lignin degradation. The hemicellulose oxidation may release volatile fatty acids in high quantities in the liquid stream. These compounds act as inhibitors for the microorganisms in downstream processes (Bensah and Mensah 2013; Michalska and Ledakowicz 2016; Kumari and Singh 2018). The high cost of oxidants and loss of some sugars through non-selective oxidation are other limitations of the method (Bensah and Mensah 2013). Additionally, post-treatment neutralization of alkaline slurry by acids increases the cost of the process (Michalska and Ledakowicz 2016). The efficiency of the pretreatment depends on several parameters such as the pH, temperature, hydrogen peroxide concentration, biomass loading, and reaction time, which need to be optimized for various biomass (Michalska and Ledakowicz 2016; Dutra et al. 2018). Banerjee et al. (2011) optimized conditions for alkaline peroxide pretreatment of corn stover, including biomass loading, hydrogen peroxide loading, residence time, and pH. A glucose yield of 77% was achieved using 15% biomass loading, 0.125 g H2O2/g biomass, in 48 h, with the pH adjustment for the pretreatment at 23
C, and atmospheric pressure, followed by the enzymatic hydrolysis using a cocktail of enzymes with 5 mg protein/g glucan loading. Ayeni et al. (2016) pretreated Shea tree (Vitellaria paradoxa) sawdust by alkaline peroxide method followed by its enzymatic hydrolysis (for 96 h, using 40 g/L biomass loading, and 25 FPU/g enzyme load) and fermentation by S. cerevisiae. They could produce 12.73 g/L ethanol from 2% cellulose loading in 96 h. In another

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study, seaweed Ulva prolifera was pretreated by alkaline peroxide method using 0.2% H2O2, at 50
C, pH 4.0, for 12 h resulting in the formation of 0.42 g/g sugars from enzymatic hydrolysis and 61.7% ethanol yield after fermentation (Li et al. 2016b). Ayeni et al. (2018) compared the effect of NaOH-H2O2 and Ca(OH)2-H2O2based oxidative pretreatment of Siam weed (Chromolaena odorata) for their enzymatic conversion to reducing sugars. The group found that NaOH–H2O2, under optimized conditions of 70
C, for 3 h, resulted in enrichment of biomass with 44.29% cellulose (compared to 40.1% in native biomass) due to its delignification. The enzymatic hydrolysis produced 424.35 mg glucose/g biomass. The Ca(OH)2H2O2 pretreatment at 70
C, for 3 h increased the cellulose content to 47.18%, resulting in production of 335.81 mg glucose/g biomass. The alkaline peroxide method (with 6.25% H2O2, at 160
C, for 60 min) has also been used successfully for the pretreatment of sugarcane bagasse, giving a glucose yield of 89.1% in enzymatic hydrolysis (Zhang et al. 2019).

Ionic Liquids Pretreatment with ionic liquids is a relatively new approach. Ionic liquids are also referred to as the green solvents because they do not generate toxic gases and are not explosive in nature (Chen et al. 2017; Wang et al. 2017). The ionic liquids have a low melting temperature, below 100
C, and exist in a liquid state (Baruah et al. 2018). These solvents are typically composed of a large cation and a small anion (Brodeur et al. 2011; Chen et al. 2017). The cations are mostly organic viz. imidazolium, pyridinium, aliphatic ammonium, alkylated phosphonium, and sulfonium ions, with the imidazolium being most common among cation (Bensah and Mensah 2013; Baruah et al. 2018; Kumar and Sharma 2018). The anions include chloride, bromide, acetate, sulfate, nitrate, methanoate, and triflate (Bensah and Mensah 2013). Both the cations and anions take part in the biomass processing by cellulose and lignin solubilization. The ionic liquids can dissolve both cellulose and lignin. They interfere in the intra- and intermolecular hydrogen bonding in the lignocellulosic matrix by forming hydrogen bonds with the sugar hydroxyl groups (Chen et al. 2017; Kumar and Sharma 2017; Kumari and Singh 2018). The alkyl chains of the cationic parts consist of electron-withdrawing groups, which increase the cellulose dissolution (Baruah et al. 2018). The breaking of non-covalent interactions minimizes the generation of degradation products (Chen et al. 2017). Ionic liquids have many beneficial characteristics such as very low vapor pressure, non-volatility, temperature stability over a wide range (up to 300
C), chemical stability, and high polarities (Chen et al. 2017; Kumar and Sharma 2017; Wang et al. 2017). The low vapor pressure of these solvents allows their easy recovery for recycling, which in turn reduces the cost of the process (Brodeur et al., 2011; Elgharbawy et al. 2016). Ionic liquids form a two-phase system with most of the solvents, which allows the adjustment of acidity conveniently (Chen et al. 2017). These solvents can be used at mild conditions. The adjustability of their cations and anions, governing the solubility of polymers, makes them interesting solvents for

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biomass processing (Elgharbawy et al. 2016; Chen et al. 2017). The method has been tested on the diverse type of biomass (Agbor et al. 2011; Brodeur et al. 2011). The pretreatment is influenced by many factors such as time, temperature, the composition of ionic liquid, biomass loading, and retention time (Elgharbawy et al. 2016; Kumari and Singh 2018). Weerachanchai and Lee (2014), in their study on ionic liquid pretreatment of rice straw, recorded that the lignin and water content in a pretreatment solvent, and the number of batches during ionic liquid reuse have a great influence on the pretreatment process. EMIM-AC (1-ethyl-3methylimidazolium acetate)/ethanolamine (60/40 vol%) was more effective compared to EMIM-AC. The presence of lignin and water (5–10 wt%) decreased the effectiveness of both EMIM-AC and EMIM-AC/ethanolamine considerably. The reduction in sugar conversions and lignin extraction were observed after the fifth and seventh batch of recycling. Wang et al. (2017) evaluated the efficiency of various ionic liquids, prepared by one-step synthesis process, in pretreating poplar and bamboo. The pretreatment of poplar with 1-hexylpyridinium chloride ([Hpy]Cl) and 1-H-3-methylimidazolium chloride ([Hmim]Cl) yielded the highest 61% and 60.4% amounts of lignin, respectively. The pretreatment of bamboo with ([Hpy]Cl) and N-methyl-2-pyrrolidonium chloride ([Hnmp]Cl) yielded 51.7% and 50.3% % of lignin, respectively. The enzymatic hydrolysis of poplar yielded maximum amount of cellulose (67.5%) when biomass was pretreated with [Hpy]Cl. In the case of bamboo, maximum glucose (65.5%) was obtained in [Hmim]Cl pretreated biomass. The method is, however, under exploration and studies need to be elaborated for the adoption of the method at industrial levels. The cost of the ionic liquids, their viscosity interfering in the mass transfer, the toxicity of ionic liquids to microorganisms and enzymes, and energy-intensive recycling process are among major challenges, which need to be addressed (Buarah et al. 2018; Kumari and Singh 2018).

Biological Pretreatment Biological pretreatment of biomass involves utilization of lignocellulolytic microorganisms for biomass deconstruction (Chen et al. 2010; Isroi et al. 2011; Gao et al. 2012). A wide array of microbes from different taxonomic groups has the natural ability to degrade lignin in the natural environment, which plays an important role in the biogeochemical cycling of carbon on earth. The potential of ligninolytic enzymes can be exploited to selectively hydrolyze the complex lignin present in plant biomass to unlock the cell wall structure. The lignin is depolymerized by the hydrolytic action of ligninolytic enzymes secreted extracellularly (Chen et al. 2010). As a result, cell wall carbohydrate polymers become accessible for subsequent hydrolysis. The depolymerization can be performed by either employing microorganisms or using their enzymes for the pretreatment (Ummalyma et al. 2019). The lignin hydrolysis involves the action of laccases and peroxidases, which hydrolyze the lignin backbone or low-molecular-weight organic compounds,

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which attack lignin after diffusing through the cell wall pores (Vasco-Correa et al. 2016). Commonly bacteria and wood-rotting fungi are employed for biological pretreatment. Fungi are the most promising microorganisms and include brown, white and soft rot fungi, differing in their method of biomass degradation (Chen et al. 2010; Chen et al. 2017). Among these, white rot fungi, a diverse group of woodrotting fungi, have been studied extensively because of their proficiency in lignin degradation. They are known for their capacity to mineralize lignin to water and carbon dioxide (Isroi et al. 2011; Vasco-Correa et al. 2016). Different white rot fungi either carry out selective delignification or simultaneous degradation of holocellulose and lignin. The selective degradative microorganisms are beneficial for biomass pretreatment (Isroi et al. 2011). Many research studies have been carried out employing different microbes for different feedstock, which have revealed the potential of biological pretreatment method in biomass delignification, and, thus, increasing the yield of sugars or ethanol in subsequent steps (Table 3.4). The biomass pretreatment using microbial system is an environment-friendly approach (Mohan et al. 2012). It requires lesser input of energy as microbial processes are carried out at relatively milder conditions. Thus, it is considered an advantageous alternative over traditional chemical and physical methods. The elimination of the need for chemicals and harsher operational conditions makes it an economically feasible method (Waghmare et al. 2018). Also, biological pretreatment does not generate inhibitors, which eliminates the requirement of detoxification steps performed during chemical pretreatment methods (Waghmare et al. 2018). Other advantages associated with this method include greater specificity for substrates and reactions, no chemical recycle requirement, no generation of waste water or pollutants, more co-products production potential, and higher product yields (Chen et al. 2010; Gao et al. 2012). However, there are several limitations of the method such as industrial inapplicability due to slower rate and larger retention time (Salvachúa et al. 2013; Ramarajan and Manohar 2017). It also requires a larger space compared to conventional methods (Isroi et al. 2011). The pretreatment allows limited control over the operational conditions. Additionally, there can be a significant loss of carbohydrates due to hemicellulolytic and cellulolytic activities of enzymes synthesized by microbes employed for biological delignification. This is because lignocellulolytic enzymes are synthesized in synergism in natural systems. This demands the selection of microbes for selective lignin degradation. The method, however, is gaining researchers’ attention owing to its eco-friendliness and economic viability (Ummalyma et al. 2019). The efforts are underway to reduce the time of pretreatment by various means. Some studies have suggested combining biological pretreatment with mild physical or chemical methods (Salvachúa et al. 2013; Vasco-Correa et al. 2016). Also, enzymatic pretreatment may give faster results as it does not depend on the growth of microbes on biomass directly (Vasco-Correa et al. 2016). The limitation of the method can be overcome by manipulating various culture conditions and physiological and genetic factors (Isroi et al. 2011).

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Table 3.4 Biological pretreatment of various lignocellulosic substrates Microbe Phanerochaete chrysosporium (white rot fungus)

Feedstock Rice straw

Treatment time 40 days

Gloeophyllum trabeum (brown rot fungus)

Corn stover

30 days

Irpex lacteus and Pleurotus eryngii (white rot fungi)

Wheat straw

21 days

Trametes hirsuta

Parthenium hysterophorus weed

7 days

Irpex lacteus (white rot fungus)

Wheat straw + Mn (II) supplementation

21 days

Inonotus tropicalis, Cerrena unicolor, Chaetomium brasiliense, and Chaetomium globosum

Rice straw (RS) and sugarcane bagasse (SCB)

14 days

Outcome Enhanced production of cellulase and xylanase using pretreated biomass 43% decrease in xylan content, sevenfolds increase in initial cellulose adsorption capacity and 2.5folds increase in biomass specific surface area Maximum increase in cellulose (16–100%) and hemicellulose (12–87%) digestibility using Irpex lacteus, 84% glucose yield and 74% theoretical ethanol yield in 14 days 1.92-fold higher lignin recovery, 52.65% holocellulose availability, 485.64 mg/gds sugar yields after saccharification Increased digestibility (91% higher glucose recovery), significant lignin loss Enhanced sugar yields from RS (2.64 mg/mL) and SCB (4.39 mg/ mL), physical changes in biomass evident in scanning electron

Reference Mohan et al. (2012)

Gao et al. (2012)

LópezAbelairas et al. (2013)

Rana et al. (2013)

Salvachúa et al. (2013)

Ramarajan and Manohar (2017)

(continued)

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Table 3.4 (continued) Microbe

Feedstock

Treatment time

(Microbial consortium)

Phlebia brevispora (white rot fungus)

Corn stover

42 days

Phanerochaete chrysosporium

Rapeseed straw

15 days

Phanerochaete chrysosporium

Sorghum husk

8 days

Piptoporus betulinus (brown rot fungus)

Rice straw

24 days

Outcome microgram, reduced crystallinity index (from 56.6–42.52% in RS and 49.02–42.92% in SCB) 442 mg/g sugar yield, 3maximum 8 g/L ethanol production by fed-batch SSF using xylose utilizing recombinant S. cerevisiae YRH400 strain 6.18% and 7.27% loss of lignin and hemicellulose, respectively, increase in CI from 33. 17% to 39.47%, structural changes evident in SEM and FTIR Release of 103 mg/ g reducing sugars in enzymatic hydrolysis of pretreated biomass compared to 20.07 mg/g from untreated biomass, changes in crystallinity, functional groups, and surface topology Coupled pretreatment, cellulase production, and enzymatic hydrolysis resulted in 24.79% hemicellulose loss, and 392.96 mg/g reducing sugar yield

Reference

Saha et al. (2017)

Ghasemzadeh et al. (2018)

Waghmare et al. (2018)

Li et al. (2019)

References

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Sun, Y., & Cheng, J. (2002). Hydrolysis of lignocellulosic materials for ethanol production: A review. Bioresource Technology, 83(1), 1–11. Tabil, L., Adapa, P., & Kashaninejad, M. (2011). Biomass feedstock pre-processing–part 1: Pre-treatment. In Biofuel’s Engineering Process Technology. New York, NY: InTech. Taherzadeh, M. J., & Karimi, K. (2008). Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review. International Journal of Molecular Sciences, 9(9), 1621–1651. Tooyserkani, Z., Kumar, L., Sokhansanj, S., Saddler, J., Bi, X. T., Lim, C. J., . . . Melin, S. (2013). SO2-catalyzed steam pretreatment enhances the strength and stability of softwood pellets. Bioresource Technology, 130, 59–68. Trevorah, R. M., & Othman, M. Z. (2015). Alkali pretreatment and enzymatic hydrolysis of Australian timber mill sawdust for biofuel production. Journal of Renewable Energy, 2015, 1. Truong, N., & Kim, T. (2018). Effective saccharification of corn Stover using low-liquid aqueous ammonia pretreatment and enzymatic hydrolysis. Molecules, 23(5), 1050. Ummalyma, S. B., Supriya, R. D., Sindhu, R., Binod, P., Nair, R. B., Pandey, A., & Gnansounou, E. (2019). Biological pretreatment of lignocellulosic biomass- current trends and future perspectives. In Second and Third Generation of Feedstocks (pp. 197–212). Amsterdam, The Netherlands: Elsevier. Varga, E., Schmidt, A. S., Réczey, K., & Thomsen, A. B. (2003). Pretreatment of corn Stover using wet oxidation to enhance enzymatic digestibility. Applied Biochemistry and Biotechnology, 104 (1), 37–50. Vasco-Correa, J., Ge, X., & Li, Y. (2016). Biological pretreatment of lignocellulosic biomass. In Biomass fractionation technologies for a lignocellulosic feedstock based biorefinery (pp. 561–585). Amsterdam, The Netherlands: Elsevier. Waghmare, P. R., Khandare, R. V., Jeon, B., & Govindwar, S. P. (2018). Enzymatic hydrolysis of biologically pretreated sorghum husk for bioethanol production. Biofuel Research Journal, 5(3), 846–853. Wang, F. L., Li, S., Sun, Y. X., Han, H. Y., Zhang, B. X., Hu, B. Z., . . . Hu, X. M. (2017). Ionic liquids as efficient pretreatment solvents for lignocellulosic biomass. RSC Advances, 7(76), 47990–47998. Wang, Y., & Liu, S. (2012). Pretreatment technologies for biological and chemical conversion of woody biomass. Tappi Journal, 11(1), 9–16. Wang, Z., Wu, G., & Jönsson, L. J. (2018). Effects of impregnation of softwood with sulfuric acid and sulfur dioxide on chemical and physical characteristics, enzymatic digestibility, and fermentability. Bioresource Technology, 247, 200–208. Weerachanchai, P., & Lee, J. M. (2014). Recyclability of an ionic liquid for biomass pretreatment. Bioresource Technology, 169, 336–343. Yan, Z., Li, J., Li, S., Cui, T., Yu, M., Cong, G., & Zhao, G. (2014). Effect of lignin on recalcitrance of lignocellulose. Transactions of the Chinese Society of Agricultural Engineering, 30(19), 265–272. Yang, B., & Wyman, C. E. (2008). Pretreatment: The key to unlocking low-cost cellulosic ethanol. Biofuels, Bioproducts and Biorefining: Innovation for a sustainable economy, 2(1), 26–40. Yang, Y., Sharma-Shivappa, R., Burns, J. C., & Cheng, J. J. (2009). Dilute acid pretreatment of oven-dried switchgrass germplasms for bioethanol production. Energy & Fuels, 23(7), 3759–3766. Yin, J., Hao, L., Yu, W., Wang, E., Zhao, M., Xu, Q., & Liu, Y. (2014). Enzymatic hydrolysis enhancement of corn lignocellulose by supercritical CO2 combined with ultrasound pretreatment. Chinese Journal of Catalysis, 35(5), 763–769. Zhang, H., Huang, S., Wei, W., Zhang, J., & Xie, J. (2019). Investigation of alkaline hydrogen peroxide pretreatment and tween 80 to enhance enzymatic hydrolysis of sugarcane bagasse. Biotechnology for Biofuels, 12(1), 107. Zhao, X., Cheng, K., & Liu, D. (2009). Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Applied Microbiology and Biotechnology, 82(5), 815.

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4

Saccharification Fermentation and Process Integration

Abstract

The biomass pretreatment is followed by the next step, i.e., saccharification or hydrolysis, meant for the breakdown of carbohydrate polymers into fermentable sugars. Enzymatic hydrolysis of cellulose is a non-chemical method based on microbial lignocellulolytic enzymes. The hydrolysis of cellulose and hemicellulose can be carried out by highly specific carbohydrate-active enzymes, known as glycosyl hydrolases enzymes. The enzymes of cellulase complex vary in their substrate specificity, site of action, and mechanism of cellulose depolymerization. The cellulases are generated certainly by a wide array of microorganisms from both eukaryotic (fungi) and prokaryotic (eubacteria and actinomycetes) domains. The rate of enzymatic cellulolysis is governed by several parameters such as substrate loading, enzyme loading, the efficacy of preceding pretreatment in altering substrate characteristics, efficiency and activity of cellulases, hydrolysis conditions including mixing, pH temperature, and reaction time. The rate of enzymatic hydrolysis of cellulose is also affected by the performance of enzymes which are inhibited by their end products such as glucose and cellobiose accumulated in the reaction mixture with the progress of the process. The technology for cellulosic ethanol has not been commercialized yet. Various strategies are known which have the potential to improve the cellulases for bioethanol production. The simplest strategy involves tapping relatively unexplored habitats for the search of novel cellulolytic microbes. Thus, significant improvements can be made to enhance the in-house production of cellulases as an effort to reduce the cost of enzymes in biomass saccharification. Technoeconomic evaluation in future can reveal the price reductions attainable through on-site production of enzymes. Keywords

Saccharification · Yeast fermentation · Ethanol · Yeast metabolism · Yield improvement # Springer Nature Singapore Pte Ltd. 2020 D. Sharma, A. Saini, Lignocellulosic Ethanol Production from a Biorefinery Perspective, https://doi.org/10.1007/978-981-15-4573-3_4

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Biomass Hydrolysis The biomass pretreatment is followed by the next step, i.e., saccharification or hydrolysis, meant for the breakdown of carbohydrate polymers into fermentable sugars. The sugar-rich polymeric components of the biomass, the hemicellulose and cellulose, can be hydrolyzed into their respective monomeric sugar constituents either chemically or enzymatically. Each method has its advantages as well as disadvantages.

Acid Hydrolysis The chemical method is based on acid hydrolysis. Sometimes, the pretreatment itself involves hydrolysis of one of the carbohydrate polymers (Kumar et al. 2015b; Kennes et al. 2016), which is mostly hemicellulose because it is relatively easily hydrolysable. Such biomass pretreatment generates two fractions: the solid fraction containing cellulolignin (cellulose and lignin), and a liquid fraction containing hydrolysis products of hemicellulose, i.e., either pentose sugars such as xylose, arabinose, mannose, and galactose, or oligosaccharides from the incomplete hydrolysis of hemicellulose (Zabed et al. 2016). In this case, cellulose hydrolysis can be achieved further using either an enzymatic method or a thermochemical method. The chemical method of biomass hydrolysis involves the use of an acid, either diluted or concentrated (Kennes et al. 2016). The acids can penetrate lignin without requiring much pre-processing of biomass (Verardi et al. 2012). The most commonly used acid is sulfuric acid (H2SO4). However, many other inorganic acids such as hydrochloric acid (HCl), nitric acid (HNO3), phosphoric acid (H3PO4), and formic acid have also been employed in various studies (Verardi et al. 2012; Zabed et al. 2016). The acid is effective in breaking down the cellulose and hemicellulose polymers by hydrolyzing the covalent bonds, hydrogen bonds, van der Waals forces, and various intermolecular bridges between constituent sugars, which is catalyzed by H+ ions of the acid (Wijaya et al. 2014; Ghaffar et al. 2017). The rate of acid hydrolysis is dependent on several factors such as the concentration of H3O+ ions as well as the temperature. The rate is higher with higher acid concentration and temperature (Wijaya et al. 2014). Several variations of this method are known which are being employed at different scales. The dilute acid hydrolysis can be employed for both hemicelluloses as well as cellulose hydrolysis but at different temperatures. Low-temperature dilute acid hydrolysis is used for the hydrolysis of hemicellulose fraction, while high-temperature dilute acid hydrolysis is carried out for cellulose depolymerization into glucose (Zabed et al. 2016). The higher temperature conditions, however, increase the rate of sugar degradation (acidcatalyzed dehydration) leading to the production of products such as furfural and 5-hydroxymethyl-furfural or HMF (Moe et al. 2012; Verardi et al. 2012; Zabed et al. 2016). These compounds are known as potent inhibitors for fermentation because they inhibit yeast cells, thus causing a lower ethanol yield in the process (Verardi et al. 2012). Also, as a result of degradation, the yield of sugars is decreased in the

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Fig. 4.1 Dual-stage dilute acid hydrolysis of the feedstock for bioethanol generation

saccharification process. However, furfural production can be minimized by using mild operation conditions. The effectiveness of dilute acid hydrolysis has been studied extensively on a wide variety of biomass. The method is advantageous in that it requires low acid concentration, involves simple waste process, is environment-friendly, and does not require acid recycle (Chen 2015; Chang et al. 2018a). The technology for dilute acid hydrolysis is successful at the pilot scale (Moe et al. 2012). The dilute acid hydrolysis method is generally accomplished in two stages. A multistage process can reduce the release of inhibitors at high temperatures during acid hydrolysis (Kennes et al. 2016). The first stage involves acid hydrolysis at low temperature, which is primarily aimed at hemicellulose hydrolysis. This is mostly a part of biomass pretreatment and enhances the cellulose accessibility to chemicals or enzymes in subsequent steps through disruption of the cell wall structure (Sharma 2016; Sharma and Dhanjal 2016; Zabed et al. 2016). The second stage involves the conversion of cellulose to glucan at a higher temperature (230
C and 240
C). The liquid material from the first- and second-stage hydrolysis (comprising pentoses and hexose sugars) can be fermented to ethanol (Fig. 4.1). The industrial application of concentrated acid hydrolysis for cellulose hydrolysis can be traced back to the nineteenth and twentieth centuries (Chen 2015; Chang et al. 2018a). Compared to other acids, the technology for concentrated sulfuric acid hydrolysis is more developed at a commercial scale (Chen 2015). The success of the concentrated acid method can be attributed to the advantages of the process, i.e., faster rate, higher efficiency of sugar recovery (over 90% of theoretical yield for glucose as well as xylose), and milder conditions of temperature and pressure during

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Fig. 4.2 Arkenol model for concentrated acid hydrolysis of lignocellulose: a simplified overview (Modified original flow chart from http://www.arkenol.com/Arkenol%20Inc/tech01.html)

the process. Milder conditions in turn account for lower inhibitors formation. However, the process requires a high concentration of acid (30–70%), dilution, and heating of the contents (Chin and H’ng 2013; Kennes et al. 2016). The method is based on the findings that 72% sulfuric acid or 42% HCl or 77–83% H3PO4, at a lower temperature, is capable of hydrolyzing the crystalline cellulose into a homogeneous mixture of several oligosaccharides of glucose, which may be hydrolyzed to glucose by further dilution with water followed by heating for a specific time (Chen 2015). There are also several limitations of the method, such as the requirement of high amounts of acid as well as expensive corrosion resistant alloy for making bioreactors (Chin and H’ng 2013; Wijaya et al. 2014). The method was considered economically infeasible for some time, especially after oil embargo imposition by Arab countries and World War II. Major hurdles encountered in the commercialization of the process were lowering the energy consumption and demand for the development of an efficient technology for the recovery and recycling of the acid. Acid recovery is an energy-intensive process. In the mid-1970s, the efforts of Purdue University and Tennessee Valley Authority (TVA) improved existing processes and introduced a new process, the TVA process, which involved the recycling of dilute acid. Such advancements renewed the interest in acid hydrolysis. In 1989, Arkenol, Inc., United States, claimed the economic feasibility of the process at commercial levels by developing and demonstrating a technology to separate acid and sugar components from the hydrolysate solution (Wijaya et al. 2014; Chang et al. 2018a). The Arkenol model (Fig. 4.2) was adopted for concentrated acid hydrolysis by

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companies like Izumi Biorefinery and BlueFire Ethanol in California (Shahbazi and Zhang 2010; Chang et al. 2018a).

Enzymatic Hydrolysis Enzymatic hydrolysis method is known since the 1950s (Chen 2015). But it gained popularity in cellulosic ethanol production in the 1980s (Yang et al. 2011). It was after the oil crisis in the 1970s that the U.S. Department of Energy (DOE) took initiative to work on commercialization of ethanol production from renewable lignocellulosic materials. Initially, the enzymatic hydrolysis was considered uneconomical for commercial production of the ethanol fuel from the biomass. The chemical method exploiting sulfuric method was relatively inexpensive. However, several factors such as disposal cost of acid, cost and technical complications of sugar recovery as well as acid recycle, corrosion problem of bioreactors, the sugar loss due to degradation and inhibitory products formation, together discouraged the thermochemical process significantly. The enzymatic method for cellulose hydrolysis is generally performed under mild conditions of temperature (40–50
C) and pH (4.5–5.0) (Taherzadeh and Karimi 2007; Chen 2015), without involving the addition of corrosive acids (Kennes et al. 2016). It has many advantages including higher yields (>95%), higher selectivity, lower energy requirements, operation at milder conditions, and exclusion of corrosion problem of the equipment (Chen 2015; Kennes et al. 2016). It does not generate any sugar degradation products. The enzymatic hydrolysis, however, is slower than the chemical method (Sánchez and Montoya 2013). But it is preferred over acid hydrolysis because of its eco-friendliness and other advantages (Moe et al. 2012; Kennes et al. 2016). Recently, the advancements in biotechnology have been successful to an extent in making considerable progress towards enzymatic hydrolysis of biomass for bioethanol production. The ongoing R&D efforts seem to be promising in making cellulosic ethanol production competitive in the near future. Enzymatic hydrolysis of cellulose is a non-chemical method based on microbial lignocellulolytic enzymes. The hydrolysis of cellulose and hemicellulose can be carried out by highly specific carbohydrate-active enzymes (CAZymes), known as glycosyl hydrolases enzymes, including at least 15 protein families with several subfamilies (Taherzadeh and Karimi 2007; Alvarez et al. 2016). The enzymatic hydrolysis of cellulose is catalyzed by cellulases, which is a complex of glycosyl hydrolases, together referred to as cellulases enzymes system (Lynd et al. 2002). The cellulase enzyme system is composed of enzymes such as endoglucanases (EC 3.2.1.4), exoglucanases, or cellobiohydrolases (EC 3.2.1.74, 1,4-β-D-glucanglucanohydrolase and EC 3.2.1.91, 1,4-β-D-glucan cellobiohydrolase), and β-glucosidases (EC 3.2.1.21) (Quiroz-Castañeda and Folch-Mallol 2013; El-Naggar et al. 2014). The cellulases enzymes in their native three-dimensional state share some common structural features consisting of discrete domains: a catalytic domain at the N-terminus and a carbohydrate-binding domain, CBD (carbohydrate-binding module, CBM) at the C-terminal. The two domains are linked

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Fig. 4.3 General structure of a cellulase enzyme

together through a short linking region (Quiroz-Castañeda and Folch-Mallol 2013) (Fig. 4.3). The binding of cellulase enzyme on the cellulose molecule occurs through its CBD or CBM part. This part plays an important role in the initiation and processivity of enzymes (Lynd et al. 2002). Once attached, it brings the catalytic domain of the enzyme at correct position in close proximity to the substrate. The shape of the catalytic site also varies among different cellulases. The active site of exo-acting cellulases (CBHI and CBHII), whereas an open cleft shaped catalytic site is found in endoglucanases (Quiroz-Castañeda and Folch-Mallol 2013). The enzymes of cellulase complex vary in their substrate specificity, site of action, and mechanism of cellulose depolymerization. The cellulases enzymes are adsorbed and desorbed cyclically during the hydrolysis process (Taherzadeh and Karimi 2007; Quiroz-Castañeda and Folch-Mallol 2013). The endoglucanases act inside cellulose chains at random sites and release oligosaccharides (or dextrans) of variable lengths creating new chain ends (El-Naggar et al. 2014). These enzymes prefer amorphous (loosely packed) regions of the cellulose (Taherzadeh and Karimi 2007). The exoglucanases act at reducing and non-reducing ends of cellulose molecules and release cellobiose or glucose units as the main hydrolysis product (Quiroz-Castañeda and Folch-Mallol 2013; El-Naggar et al. 2014). CBHI exoglucanases and CBHII cellobiohydrolases cleave the glycosidic bonds at reducing and non-reducing ends, respectively (Quiroz-Castañeda and FolchMallol 2013; Volynets et al. 2017). The exoglucanases show a high degree of processivity, prefer attacking the crystalline regions of the cellulose, and make up to 40–70% of the total cellulases (El-Naggar et al. 2014). β-glucosidases enzymes are active at the last stage of hydrolysis catalyzing the cleavage of cellobiose units to release glucose monomers. The comprehensive hydrolysis of cellulose requires synergistic action of complete enzymes of the complex (Fig. 4.4). Thus, enzymes exhibit synergy between endoglucanases and exoglucanases (endo–exo synergy), the synergy between exoglucanases acting on the reducing and non-reducing ends (exo-exo synergy), the synergy between cellobiohydrolases and β-glucosidases role. Also, the interactions amid catalytic sites and carbohydrate-binding domains of cellulase enzymes play a crucial role (Lynd et al. 2002; Taherzadeh and Karimi 2007; Quiroz-Castañeda and Folch-Mallol 2013). The cellulolysis is regulated thoroughly by negative regulation of endocellulases and exocellulases by end-product inhibition. Therefore, it is generally advantageous to remove the end products to facilitate complete hydrolysis of cellulose and increase the sugar yield. The cellulases are generated certainly by a wide array of microorganisms from both eukaryotic (fungi) and prokaryotic (eubacteria and actinomycetes) domains

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Fig. 4.4 Scheme of complete cellulose hydrolysis by cellulases (Modified, adapted from Lynd et al. 2002)

(Table 4.1). These microorganisms can be grouped among different categories based on their oxygen and temperature preferences. The aerobic cellulase producers secrete soluble extracellular cellulases for cellulose degradation known as free or non-complexed cellulase systems. Trichoderma reesei, (teleomorph Hypocrea

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Table 4.1 Various cellulase-producing microorganisms reported Cellulase-producing microorganisms Fungi Bacteria Aspergillus niger; A. flavus; Bacillus subtilis; B. pumilus; A. oryzae; A. nidulans; B. amyloliquefaciens; A. terreus; A. fumigatus; B. licheniformis; B. circulan; Fusarium solani; B. flexus; B. thuringiensis; F. oxysporum; Penicillium B. cereus; Cellulomonas brasilianum; P. decumbans; biazotea; Cellvibrio gilvus; P funiculosum; P fumigosum; Eubacterium cellulosolvens; P. janthinellum; P. occitanis; Geobacillus sp.; Humicola insolens; H. grisea; Paenibacillus Melanocarpus albomyces; curdlanolyticus; Trichoderma reesei; Paenibacillus polymyxa; T. longibrachiatum; T Pseudomonas cellulosa; harzianum; T. atroviride; Salinivibrio sp.; Chaetomium cellulyticum; Rhodothermus marinus; C. thermophilum; Cytophaga hutchinsonii; Neurospora crassa; Cellvibrio japonicus; Thermoascus aurantiacus; Microbacterium sp.; Bosea Mucor circinelloides; sp.; Anoxybacillus Paecilomyces inflatus; flavithermus; Fibrobacter P. echinulatum; Coniophora succinogenes; Ruminococcus puteana; Poria placenta; albus; Caldicellulosiruptor Tyromyces palustris; saccharolyticus; Fomitopsis sp., Caldicellulosiruptor Phanerochaete obsidiansis; Rhodothermus chrysosporium; Sporotrichum marinus; Pseudomonas thermophile; Trametes fluorescens; Pseudomonas versicolor; Agaricus putida; Bacteroides sp.; arvensis; Pleurotus ostreatus; Clostridium cellulolyticum; Phlebia gigantea; C. acetobutylicum; Talaromyces emersonii; C. papyrosolvens; Anaeromyces mucronatus; C. thermocellum; Caecomyces communis; C. straminisolvens; Cyllamyces aberensis; C. stercorarium Orpinomyces sp.; Sclerotium sp.; Piromyces sp.; Neocallimastix frontalis; I. lacteus; Talaromyces sp.

Actinomycetes Cellulomonas fimi; C. bioazotea; C. uda; C. cartae; C. cellusea; C. flavigena; C. cellulans; Streptomyces sp., S. lividans; S. drozdowiczii; S. flavogrisus; S. nitrosporus; S. nitrosporeus; S. albaduncus; S. reticuli; S. albogriseolus; S. cellulolyticus; S. glomeratus; S. malachitofuscus; S. actuosus; S. stramineus; S. viridobrunneus; S. matensis; S. longispororuber; Thermomonospora sp. T. fusca; T. curvata; Microbispora sp.; M. bispora; Thermobifida fusca; Rhodococcus sp., Saccharomonospora sp., Nocardia sp.; Thermoactinomyces sp.

(Source: Saini et al. 2018)

jecorina), a saprophytic fungus, has been used as a model organism for studying non-complexed cellulase system in microorganisms (Verardi et al. 2012). The discovery of the cellulolytic potential of this fungus during World War II made a great contribution in reducing cellulosic ethanol production costs in the 1980s (Yang et al. 2011). The cellulase system of T. reesei consists of two cellobiohydrolases, at least seven endoglucanases, and several β-glucosidases (Verardi et al. 2012). The relative abundance of the enzymes in a cellulase complex, however, varies among different microbes, which accounts for variation in their cellulose hydrolytic

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Fig. 4.5 Schematic representation of a microbial cellulosome (Modified from Doi et al. 2003) (HLD hydrophilic domain, CBD cellulose-binding domain, EG exoglucanase, ED endoglucanase, BG β-glucosidase)

potential. For example, T. reesei cellulase system has fewer units of β-glucosidases, as a result of which cellobiose is produced as the major end product. The accumulation of cellobiose causes end-product inhibition of cellulases, thus reducing the efficiency of hydrolysis. The supplementation with additional β-glucosidases and a co-culture technique can prove beneficial in such situations to take cellulolysis process to completion. Contrarily, Aspergillus is an efficient producer of β-glucosidases (Taherzadeh and Karimi 2007). Most of the commercial cellulase production is achieved exploiting cellulase production potential of T. reesei and Aspergillus niger (Taherzadeh and Karimi 2007). Figure 4.4 shows the complete mechanism of cellulose hydrolysis by non-complexed cellulases. On the other hand, anaerobic cellulolytic microorganisms possess complexed systems of cellulases, referred to as cellulosomes (Verardi et al. 2012) (Fig. 4.5). The cellulosome is a complex made up of multi-functional scaffoldin proteins attached with their cohesion domains to the dockerin protein domains of different glycosyl hydrolases enzymes (endoglucanases, cellobiohydrolases, xylanases, and other CAZymes) (Doi et al. 2003; Verardi et al. 2012; Quiroz-Castañeda and FolchMallol 2013). A cellulose-binding module (CBM or CBD) making part of the scaffoldin plays a role in binding the cellulosome to the cellulosic substrate, preferably more strongly in the crystalline regions compared to the amorphous regions of the cellulose. Furthermore, the special hydrophilic domains (HLDs) in a number of scaffoldins keep cellulose bound to the host cell envelope, hence the name complexed (Doi et al. 2003). All cellulases in the cellulosome function in a synergetic manner, same as free cellulases, in order to execute complete hydrolysis of the cellulose polymer. Additionally, thermophilic cellulases from thermophilic microbes are also considered promising enzymes for bioethanol production. The hydrolytic enzymes

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produced by thermophilic bacteria and fungi show the remarkable property of thermoactivity and thermostability, and tolerance to organic solvents, detergents, and alcohols, which make them attractive candidates for their exploitation in industrial bioprocesses that are operated at harsher conditions. The enzymatic hydrolysis of cellulose is carried out at 40–50
as maximum activity of cellulases from mesophilic microbes is exhibited at 50  5
C (Verardi et al. 2012). The rate of hydrolysis is slow at this temperature and may end up in incomplete hydrolysis. The cellulases from mesophiles lose nearly 60% of their activity from 50–60
C, which is lost almost completely when the temperature rises to 80
C (Verardi et al. 2012). On the contrary, the cellulases from thermophiles enzymes show many advantages. When hydrolysis is carried out at a higher temperature, the swelling of cellulose increases the rate of reaction (Patel et al. 2019). The utilization of thermostable cellulases can reduce the hydrolysis time, facilitate recovery of volatile product likes ethanol, and cut down the cost of cooling required after thermal pretreatment (Patel et al. 2019). Also, the risk of contamination is reduced at high temperature (Verardi et al. 2012; Patel et al. 2019). However, the production of thermostable enzymes at large scale needs technological developments because of the cost of expensive media required for the cultivation of thermophiles and slow growth rates of thermophiles (Verardi et al. 2012). The rate of enzymatic cellulolysis is governed by several parameters such as substrate loading, enzyme loading, the efficacy of preceding pretreatment in altering substrate characteristics, efficiency and activity of cellulases, hydrolysis conditions including mixing, pH, temperature, and reaction time (Taherzadeh and Karimi 2007; Sánchez and Montoya 2013). The optimum temperatures and pH range for most of the mesophilic cellulases falls between 40 and 50
C and pH 4–5, respectively (Taherzadeh and Karimi 2007). The reaction time may vary depending on feedstock and other factors affecting the enzyme activity. The physicochemical characteristics of the biomass altered in a pretreatment method include lignin, acetyl, and hemicellulose content of the biomass, degree of polymerization, cellulose crystallinity, and available surface area of biomass (El-Naggar et al. 2014; Kennes et al. 2016). The lignin content of the biomass influences the process of cellulose hydrolysis by imposing physical hindrance in the accessibility to enzymes and adsorbing the cellulases non-specifically (Volynets et al. 2017). The extent of biomass delignification achieved during a pretreatment method has a direct correlation with the success of saccharification (Kennes et al. 2016). The unproductive adsorption of cellulases can also be avoided by supplementation of the hydrolysis mixture with surfactants, which can substantially lower the enzyme loading required for the hydrolysis (Taherzadeh and Karimi 2007; El-Naggar et al. 2014; Volynets et al. 2017). Additionally, hemicelluloses and acetyl groups in them pose a steric hindrance to the hydrolytic enzymes. The acetyl groups also interfere in enzyme recognition (El-Naggar et al. 2014). Thus, the removal of these components influences the saccharification positively. The cellulose crystallinity has been suggested by several reports as a significant factor in determining the rate of saccharification as amorphous cellulose is hydrolyzed faster and has higher cellulase adsorption capacity than the crystalline cellulose. The cellulose crystallinity has also

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been reported to affect the synergism among different cellulases and their processivity (Yang et al. 2011). However, cellulose crystallinity, which is likely to increase during hydrolysis due to removal of amorphous and paracrystalline cellulose and affect the further rate of the process, was found to be changed insignificantly in many studies. Thus, whether crystallinity should be considered a critical factor among others is yet to be elucidated (Yang et al. 2011; El-Naggar et al. 2014). Similarly, understanding the impact of cellulose chain length (degree of polymerization) on hydrolysis requires experimental validation with more studies (El-Naggar et al. 2014). The increased surface area of the biomass is another feature considered important by several studies for enhancing the rate of cellulolysis. Again if pore size affects the digestibility of cellulose is not understood clearly because of the contradictory observations recorded in different studies (Yang et al. 2011). The rate of enzymatic hydrolysis of cellulose is also affected by the performance of enzymes which are inhibited by their end products such as glucose and cellobiose accumulated in the reaction mixture with the progress of the process. A cocktail of enzymes containing balanced amounts of all cellulase components (acting synergistically for complete cellulolysis) as well as the strategies such as sugar removal by filtration or simultaneous saccharification and fermentation can be effective in such situations (El-Naggar et al. 2014). The enzymatic hydrolysis may also get retarded by loss of enzyme activity at higher temperatures or due to shearing forces during mixing (Volynets et al. 2017). The substrate concentration is another very important parameter that affects the rate of cellulose hydrolysis. High substrate loadings decrease the rate by substrate inhibition, increase viscosity, and cause problems in mixing and mass transfer (Taherzadeh and Karimi 2007; Alvarez et al. 2016; Zabed et al. 2016). However, enzymatic hydrolysis of biomass at industrial levels requires operation at high biomass loadings, which is essential to obtain high sugar and subsequent ethanol yields so as to make the process economically feasible by improving downstream processing of ethanol recovery by distillation (Alvarez et al. 2016; Zabed et al. 2016). A balance in enzyme to substrate ratio is one factor that can resolve this problem. The increase in cellulase loadings increases the rate of saccharification but is permissible within a limited range as increasing enzyme concentrations account for the high cost of the entire process. The studies have indicated the addition of 5–35 FPU of cellulase loading per gram of substrate beyond which the hydrolysis process would become economically uncompetitive. High biomass loadings also increase the problem of end-product inhibition (Volynets et al. 2017). The composition of the enzyme cocktail of cellulase components can minimize such limitations. Additionally, non-ionic surfactant addition may also lower the enzyme loading by increasing enzyme productivity through adsorption of surfactants to the lignin. This can be concluded that the enzymatic hydrolysis of lignocellulosic biomass will depend on multiple factors, to be taken into consideration together, including the nature of feedstock, pretreatment method, and the amount and composition of enzymes as well as the conditions during hydrolysis. The enzymatic hydrolysis of hemicellulose is easier than celluloses because of smaller chain length and amorphous nature of this polymer (El-Naggar et al. 2014). However, chemically hemicellulose is complex (made up of many sugars) and

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branched. The hydrolysis of hemicellulose requires more enzymes than cellulose (Zabed et al. 2016). The complete hydrolysis of hemicellulose involves a system of xylan-degrading enzymes such as endoxylanase (endo-1,4-β-xylanase, E.C.3.2.1.8), β-D-xylosidases (EC 3.2.1.37), α-L-arabinofuranosidase (E.C.3.2.1.55), α-glucuronidase (E.C.3.2.1.139), acetylxylan esterase (E.C.3.1.1.72), ferulic/ coumaric acid esterases (EC 3.1.1.73), and mannan degrading enzymes such as mannanases (EC 3.2.1.78), β-mannosidases (EC 3.2.1.25), and α-galactosidases (EC 3.2.1.22) (Saini et al. 2015; Alvarez et al. 2016; Zabed et al. 2016; Volynets et al. 2017). Endoxylanases attack the main xylan backbone producing xylooligomers; β-xylosidases hydrolyze xylooligosaccharides into xylose (Volynets et al. 2017). The α-arabinofuranosidases and α-glucuronidases catalyze the removal of arabinose and 4-o-methyl glucuronic acid from xylan chain, respectively. The acetyl esterases remove acetyl groups from xylose. The feruloyl esterases attack the ester bonds between arabinose substitutions and ferulic acid and facilitate the release of hemicellulose from lignin (El-Naggar et al. 2014; Zabed et al. 2016). The mannanases carry out the hydrolysis of β-1,4-glycosidic bonds in mannan and produce mannan oligomers. β-mannosidase attacks β-1,4 linked mannose units in the mannooligomers at non-reducing ends and mannobiose to produce mannose (Volynets et al. 2017). The α-galactosidases attack α 1,6 linked D-galactosyl residues at the terminal positions (Saini et al. 2015).

Cost of Cellulases: An Impediment in Bioethanol Production The technology for cellulosic ethanol has not been commercialized yet (Yang et al. 2011). This can be attributed partly to the cost of cellulases, which is one of the major impediments encountered in the hydrolysis of lignocelluloses. The higher enzyme loadings required to hydrolyze high substrate levels increase the cost of the bioconversion process. The enzyme cost goal of $0.10/gallon ethanol set by DOE Biomass Program requires that the cost of cellulases should be $2/kg cellulase protein (Yang et al. 2011). The cellulases account for 36–50% of the total cost of cellulosic ethanol production (Sánchez and Montoya 2013; El-Naggar et al. 2014). The studies suggest that second-generation ethanol production using enzymatic hydrolysis can be made competitive to existing first-generation ethanol production technology if capital costs are reduced by 30% and cost of cellulases is reduced by tenfolds the current cost (Sánchez and Montoya 2013). Thus, improved cellulases systems (enzyme cocktails) need to be created which have higher specific activity, better thermal stability, high synergy between its components, and a high binding affinity for celluloses with minimum non-specific binding to the lignin (Sánchez and Montoya 2013; Volynets et al. 2017).

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Strategies for Improving Cellulases Various strategies are known which have the potential to improve the cellulases for bioethanol production (Fig. 4.6). The simplest strategy involves tapping relatively unexplored habitats for the search of novel cellulolytic microbes. Li et al. (2019) have recently reported a cold-active cellulase from Microbacterium kitamiense. Such cold-active cellulases can be employed for higher ethanol production along with S. cerevisiae in simultaneous saccharification and fermentation at low temperature. Contrary to this, thermostable cellulases from microbes from extreme environments have the potential to reduce the required enzyme loadings during separate hydrolysis and fermentation due to their higher specificity. The thermostability also allows retention of activity for longer hydrolysis times (Volynets et al. 2017). It offers additional advantages such as lesser hydrolysis times, lowered risk of contamination, fast recovery of volatile ethanol, and reduced cost for cooling after thermal pretreatment (Patel et al. 2019). Touijer et al. (2019) have recently reported production of thermostable cellulase from the yeast of Trichosporon genus, with the ability to hydrolyze soluble and insoluble substrates at elevated temperatures and a

Fig. 4.6 Strategies for improving microbial cellulases

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wider range of pH. Another approach involves metagenome exploration for cellulolytic genes with desirable characteristics. The genes may be cloned to obtain new and robust cellulases followed by testing their potential to hydrolyze lignocellulosic biomass (Saini et al. 2015; Medina et al. 2017). de Fátima Alves et al. (2018) have identified a novel β-glucosidase (named Lfa2) from Brazilian Secondary Atlantic Forest soil metagenome. They observed that Lfa2 showed many desirable features such as considerable glucose tolerance, increased activity in the presence of 10% (v/v) ethanol, and at high concentrations of 5-hydroxymethyl furfural. The cost of the cellulases production can also be decreased by utilizing inexpensive renewable lignocellulosic biomass as a substrate for enzyme production (El-Naggar et al. 2014; Ellilä et al. 2017). The use of solid-state fermentation method for enzyme production can also enhance the enzyme yields at lower costs. Mendes et al. (2017b) have demonstrated that the solid-state fermentation process is economically beneficial. However, some technical challenges need to be addressed for cost-competitive enzyme production (Zhuang et al. 2007; El-Naggar et al. 2014; Saini et al. 2018), which include improved enzyme activity (over 12 FPU/gds) and scale-up of the process (Mendes et al. 2017b). The cellulases production can be improved significantly by the production of cellulase cocktails from microbial co-cultures exhibiting high activity, specificity, stability, and processivity (El-Naggar et al. 2014; Hernández et al. 2018; Kalbarczyk et al. 2018). Genetic modification for the advance of recombinant strains is a widespread modern age strategy for improved cellulases synthesis by microorganisms. T. reesei is one of the most popular fungi for cellulase production at industrial levels. Of the total proteins of T. reesei, the cellulase system constitutes 50–60% CBHI, 10–15% CBHI, and 6–20% endoglucanases (EGI and EGII) (Zhang et al. 2018). They secret relatively lower levels of β-glucosidases (BGL1), and this limits their efficiency in the complete hydrolysis of cellulose. Hence, genetic engineering techniques are employed to generate various strains with improved features. Qian et al. (2016) have developed a hypercellulolytic T. reesei variant SPB2 with improved β-glucosidase activity by overexpression of BGL1-encoding bgl1 gene under the control of a modified promoter, cbh1. The utilization of the strain for hydrolysis of the pretreated corncob residues resulted in high saccharification of the biomass. Zhang et al. (2018) designed novel minimal transcriptional activators, DBDace2-VP16 and DBDcre1-VP16, and replaced natural transcription factors in T. reesei RUT C30 with them. The modified strains TMTA66 and TMTA139 showed 1.3-folds and 26.5-folds higher FPase production, respectively, and proved effective in hydrolyzing the pretreated corn stover. The microbial cellulolytic systems can also be remodeled for enhancing enzyme characteristics using protein engineering techniques employing either rational design or directed evolution. Zhang et al. (2015) used a rational design for the modification of a hyperthermostable β-1,4-endoglucase Cel12B from Thermotoga maritima. They obtained two recombinant enzymes with the altered active site, by amino acid substitution, having a compact structure and changed polarity. The active site consisted of a type of fold considered to be critical for distinct thermostability feature. The variants showed 77% and 87% higher enzyme activity, respectively,

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compared to the parental enzyme. Zheng et al. (2018) produced an acidic and mesophilic GH5 cellulase (GtCel5) from Gloeophyllum trabeum CBS900.73 in P. pastoris GS115. By developing the saturation mutants of GtCel5 at position 233 by site-directed mutagenesis and verifying results by reverse mutation, they concluded that the amino acid residue at position 233 on loop 6 has a critical role in substrate binding and high catalytic performance of the enzyme. The variants N233A and N233G had decreased temperature optima (10
C) but higher activities (27 and 70%) and catalytic efficiencies (kcat/Km; 45 and 52%), respectively. T. reesei RUT-C30, a well-known mutant of T. reesei utilized for commercial applications, has been developed from 30 cycles of random mutagenesis (Singh et al. 2017a). The strain QM9414 was also developed from T. reesei QM6a through mutagenesis (Lichius et al. 2015). Abdullah et al. (2015) also developed a mutant Bacillus N3 by chemical mutagenesis using nitrous acid. The strain exhibited 1.7 folds higher enzyme production compared to native strain. Kumar et al. (2015a) improved the strain of A. terreus D34 by UV mutagenesis followed by chemical mutagenesis using ethyl methyl sulfonate. The EMS1 mutant obtained showed 4.9 folds higher β-glucosidase activity that could hydrolyze mild alkali pretreated rice straw with 95% saccharification efficiency, and achieving this level of saccharification required lesser (threefolds) enzyme loads compared to that required by wild type. The enzyme recycling or immobilization technique is also effective in reducing the cost of cellulases. Cellulase recycling is a simpler and practical approach that is based on reuse of enzymes, recovered from the enzymatic hydrolysate, for the saccharification of fresh biomass. The enzyme recycling may involve recovery of enzyme by ultrafiltration or re-adsorption of free enzymes into fresh solid. However, several technical constraints need to be overcome for recycling of cellulases for efficient biomass hydrolysis, especially when biomass loadings are higher. Kim et al. (2019) in their study on enzymatic hydrolysis of pretreated empty fruit bunches (at 20% w/v loadings) through cellulase recycling observed that the effect of recycling was hindered by several factors including end-product inhibition by glucose, activity loss of enzymes, and binding of enzymes to insoluble solid biomass. However, removal of glucose from the reused enzyme fraction and supplementation with polyethylene glycol (known to reduce no-productive binding of cellulases on solid biomass) were effective in enzyme reuse. The reuse of solids (bound with enzymes) from the first-round hydrolysis resulted in 3.5 times higher glucose yield (68%) at the second round compared to control. The immobilization technique is based on the fixation of microbial enzymes on to a solid support. The immobilization may involve enzyme(s) entrapment in a polymer matrix, adsorption or covalent-linking onto a solid support, affinity interactions, and crosslinking of enzyme aggregates (Zabed et al. 2016). Ingle et al. (2017) have reported immobilization of cellulases on iron oxide nanoparticles (magnetic nanoparticles, MNPs). They achieved 72%, 68%, and 52% conversion of cellulose to glucose after the first, second, and third round of hydrolysis, respectively. The authors concluded that the immobilization of cellulase on MNPs could facilitate easy recovery and reuse of

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enzymes for more than one cycle of hydrolysis, which can prove effective in making the process economically viable.

Fermentation Ethanol fermentation is a biological process in which sugars are fermented by microorganisms to produce ethanol and other by-products. The hydrolysis of the lignocellulosic biomass generates two types of sugar, i.e., pentoses (C5) and hexoses (C6) produced from hemicellulose and cellulose, respectively, mostly in separate streams in the traditional method and are therefore fermented separately. The fermentation of 1 mol glucose (hexose) should yield 2 mol ethanol, as shown in the equation (Kennes et al. 2016): 1C6 H12 O6 ! 2C2 H5 OH þ 2CO2 Thus, 1 kg glucose should yield a maximum of 511 g of ethanol. Theoretically, fermentation of xylose (pentose) may also result in the same amount of ethanol (511 g/kg xylose), according to the following equation (Kennes et al. 2016): 1C5 H10 O5 ! 1:67 C2 H5 OH þ 1:67 CO2 The actual yield, however, is lower because the substrate is partly utilized for the growth or biomass of microorganism and several other co-metabolites may also be formed.

Microbes for Ethanol Fermentation The sugars can be fermented to ethanol using different microorganisms, belonging to bacterial (prokarya domain) and fungal (eukarya domain) groups. These microorganisms vary in their preferences for the sugars to ferment. Generally, wild strains of microbes with the ability to ferment C6 and C5 sugars are different. An ideal microorganism for efficient fermentation process in the bioethanol industry is considered having some desirable characteristics such as (1) high ethanol yields (above 90% of theoretical yield), (2) ability to utilize a wide spectrum of sugars, (3) high tolerance to ethanol (above 40 g/L), (4) resistance to acidic pH and tolerance to higher temperatures (prevent microbial contamination in fermentation broth), (5) osmotic stress tolerance, (6) low requirements for growth in the medium, (7) high resistance to fermentation inhibitors in the hydrolysate, (8) ethanol productivity higher than 1 g/L/h (El-Naggar et al. 2014; Vohra et al. 2014; Robak and Balcerek 2018; Branco et al. 2019). However, wild microorganisms mostly have very few of these features in the same strain. The researchers, therefore, are not only focused on the search for wild ethanologens with desired features but also adopt

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various approaches to improve microbes in order to make them robust for industrial applications. Saccharomyces cerevisiae is the most commonly employed microorganism in ethanol production at industrial levels. It can convert glucose, fructose, galactose, maltose, and sucrose into ethanol (Ishizaki and Hasumi 2013; Branco et al. 2019). S. cerevisiae is GRAS (generally regarded as safe) for human consumption. The yeast is a facultative anaerobe, which produces ethanol by first converting glucose into pyruvate through glycolysis or Embden–Meyerhof pathway and then converting pyruvate anaerobically to ethanol and CO2. It grows optimally within a temperature range of 22–29
C, but cannot survive beyond 35
C. The yeast has many features which make it suitable for industrial processes. It can tolerate a wide range of pH and has optimum in the acidic range, which makes the fermentation less susceptible to contamination (Tesfaw and Assefa 2014). Other advantageous features include fast growth rates, efficient utilization of glucose, high ethanol production, and tolerance to high ethanol concentration, low oxygen levels, and other inhibitory compounds generated during pretreatment or acid hydrolysis (Balat et al. 2008; Chang et al. 2018b). However, it can ferment a sugar solution with a concentration lower than 12%. A sugar level higher than 15% is inhibitory to the process because it causes osmotic stress to yeast cells (Marin et al. 2017). The native strains are incapable of fermenting pentoses, lactose, and cellobiose (Ishizaki and Hasumi 2013; Branco et al. 2019). Therefore, genetic engineering techniques are used to construct a potent strain with the capability to ferment both hexoses and pentoses. Kluyveromyces marxianus, a thermotolerant yeast, is also widely utilized to produce bioethanol. It can metabolize various sugars including both hexoses and pentoses. K. marxianus can grow over 40
C, which makes it an ideal candidate for simultaneous saccharification and fermentation as well as consolidated bioprocessing configurations of the fermentation (Limayem and Ricke 2012; Mehmood et al. 2018). It shows faster growth rate and higher tolerance to inhibitory compounds (Amaya-Delgado et al. 2018; Mehmood et al. 2018). Owing to its thermotolerance, this ethanologen has been modified by genetic engineering for cellulosic ethanol production from various feedstocks at relatively high temperatures (Ishizaki and Hasumi 2013). Zymomonas mobilis, a facultative anaerobic gram-negative bacterium, is another commonly used ethanologen (Pinilla et al. 2011). It can ferment certain sugars such as glucose, sucrose, and fructose. It employs the Entner–Doudoroff (ED) pathway for the degradation of sugars to pyruvate, which is then converted to ethanol and CO2 (Ishizaki and Hasumi 2013). Compared to the EM pathway, the ED pathway yields 50% lower amount of ATP, which is responsible for lower biomass yields. As a result, Z. mobilis shows a higher yield per unit cell mass compared to S. cerevisiae. The bacterium shows a faster rate of sugar uptake, higher ethanol yield, and ethanol specific productivity than S. cerevisiae (3–5 times) (Balat et al. 2008; Pinilla et al. 2011; El-Naggar et al. 2014; Marin et al. 2017; Branco et al. 2019). Z. mobilis requires 30–39
C temperature and a neutral pH for its growth. It shows high tolerance to ethanol (more than 16% v/v). However, it can ferment a narrow range of sugars and shows lower tolerance to inhibitors (Pinilla et al. 2011; Branco et al.

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2019). Therefore, various genetic modifications have been done to improve the characteristics of this microorganism for utilization in bioethanol production. Escherichia coli can produce ethanol from lignocellulose-derived sugars. It uses heterofermentative pathway under anaerobic conditions in which 1 mol of glucose produces 2 mol of formate, 2 mol of acetate, and 1 mol of ethanol. As a result, lower ethanol levels are obtained. The estimates show the production of 0.26 g ethanol/g of glucose, compared to the highest possible theoretical yield of 0.51 g ethanol/g of glucose (Koppolu and Vasigala 2016). Hence, genetically engineered strains are developed with high ethanol productivity and tolerance to inhibitors. E. coli is known among most commonly used microorganisms for genetic modification for bioethanol production (Ishizaki and Hasumi 2013; El-Naggar et al. 2014). The bacterium, however, has limited tolerance to ethanol, narrow range of growth temperature and pH (6.0–8.0), and low tolerance to inhibitors (Limayem and Ricke 2012; Vohra et al. 2014). Klebsiella oxytoca, an enteric bacterium, is a well-known ethanologenic bacterium capable of growing at pH as low as 5.0 and the mesophilic temperature of 35
C (Balat et al. 2008; Tran et al. 2011). It can utilize a broad range of sugars including hexoses, pentoses as well as cellobiose, xylobiose, cellotriose, and arabinosides. The substrate range for E. coli and K. oxytoca is wider compared to Z. mobilis. Several yeasts such as Pichia stipitis (Scheffersomyces stipitis), Candida shehatae (Schefersomyces shehatae), and Pachysolen tannophilus, and bacteria such as Aerobacter sp., Aeromonas hydrophila, Bacillus macerans, B. polymyxa, Clostridium acetobutylicum, Erwinia sp., Klebsiella pneumoniae, Leuconostoc sp., and Lactobacillus sp., have been reported to possess the natural ability to ferment C5 sugars into ethanol (Chandel et al. 2011; Agbogbo and Coward-Kelly 2008; Senatham et al. 2016; Branco et al. 2019). Pichia stipitis is the industrially most promising pentose-fermenting microorganism and has been widely used for various research studies on cellulosic ethanol production. It can ferment glucose, xylose, mannose, galactose, and cellobiose. P. stipitis is known to possess low-affinity and high-affinity proton symport systems, which are used based on the concentration of sugars in the media. When sugars levels are high, the glucose is transported preferably through low-affinity transport system because glucose inhibits xylose transport by non-competitive inhibition. On the other hand, high-affinity systems operate at low sugar concentrations. In a medium containing glucose and xylose, the rate of xylose uptake is lower than the glucose consumption. Therefore, pentose fermentation occurs at a fast rate when glucose levels are low. Generally, all natural pentose-fermenters assimilate xylose slower than glucose (Sánchez and Montoya 2013). Unlike most fungi and yeast, P. stipitis produces the least amount of xylitol as a co-product during xylose metabolism under anaerobic conditions (Agbogbo and Coward-Kelly 2008). P. stipitis carries out fermentation optimally at 25–33
C and at a pH 4.5–5.5. Nowadays, researchers are focused on improving sugar transport and ethanol tolerance in P. stipitis strains. C. shehatae is efficient in mixed-sugar fermentation. However, C. shehatae, similar to P. stipitis and Pachysolen tannophilus, shows low tolerance to ethanol, low pH, and inhibitors (Limayem and Ricke 2012; Senatham et al. 2016). While utilizing microaerophilic

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ethanologenic yeasts, maintenance of microaerophilic conditions is difficult, and ethanol yields are low. The pentose-fermenting bacteria show low yields as they produce by-products like acetic acid, lactic acid, 2,3-butanediol, and CO2. Again genetic manipulations through various mutation and genetic engineering techniques are done to develop strains with desired characteristics including high xylose fermentation. Several filamentous fungal species such as Mucor indicus, Fusarium oxysporum, Rhizopus sp., Monilia sp., Neurospora intermedia, Neurospora crassa, Peniophora cinerea, Trametes suaveolens, Phanerochaete sordida, and Paecilomyces sp. have also been found having ability to ferment glucose and/or xylose (Chandel et al. 2011; Sánchez and Montoya 2013; Wang et al. 2016; Branco et al. 2019; Mori et al. 2019). Some of these fungi also possess lignocellulolytic potential in addition to sugar fermentation, thereby making them an ideal candidate for consolidated bioprocessing (Branco et al. 2019; Mori et al. 2019). However, there are several limitations associated with the use of filamentous fungi for ethanol fermentation, which include longer generation and fermentation time, co-products (organic acids) formation, low ethanol yields, low productivity, lower tolerance to sugars and ethanol (Chandel et al. 2011; Branco et al. 2019). In addition to mesophilic microorganisms, many thermophilic such as Clostridium thermocellum, C. thermohydrsulfurium, C. thermosaccharolyticum, C. thermosulfurogenes Thermoanaerobacter ethanolicus, Thermoanaerobacterium saccharolyticum, Bacillus sp., Geobacillus sp., Paenibacillus sp., and Caloramator sp. also exhibit sugar fermentation potential (Chandel et al. 2011; Limayem and Ricke 2012; Scully and Orlygsson 2015). Many of them are ideal for consolidated bioprocessing process. Use of thermophilic microbes is beneficial in many ways: (1) no requirement of mixing, cooling or heating during their cultivation, (2) the broader range of carbohydrate degradation, (3) ethanol recovery by in-situ vacuum distillation, (4) tolerance to extreme pH and salt levels, (5) low nutritional requirements, (6) reduced viscosity at elevated temperatures at high solid loads, (7) higher mass transfer rates, (8) lower risk of contamination (Scully and Orlygsson 2015). Despite many advantages, thermophilic microbes need significant improvements to be utilized in industrial applications because they show low tolerance to ethanol, inhibitors, high substrate levels, and partial pressure of hydrogen (Limayem and Ricke 2012; Scully and Orlygsson 2015).

Modes of Fermentation The fermentation can be performed in a batch, fed-batch, or continuous mode (Balat et al. 2008). Several variations of these processes are also known such as repeatedbatch culture and continuous fermentation with cell recycling, etc. (Cheng et al. 2009; Azhar et al. 2017). Batch fermentation is the oldest and most common mode of fermentation. It is the simplest mode of fermentation in which the process is carried out in a closed-loop system, and ethanologen is added to the hydrolysate containing high initial sugar levels. The end product is recovered at the end of the whole batch.

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The process can be monitored and regulated conveniently. The batch mode offers several other benefits also such as low cost, easy sterilization, less complicated process, manageable by the unskilled workforce, and flexible to various product specifications (Zabed et al. 2014; Azhar et al. 2017). However, the productivity is low due to high levels of inhibitors in the hydrolysate, which necessitates the removal of inhibitors by appropriate detoxification method. Also, high initial glucose levels in the fermentative media may result in substrate inhibition, which has a considerable impact on lowering the fermentation efficiency and increases the fermentation time (Cheng et al. 2009; Wang et al. 2013; Chang et al. 2018b). Several variations of batch mode can be employed to improve the process. Cell recycle batch fermentation (CRBF) is one of such methods. The cells recycling can reduce the time and cost involved in inoculum preparation significantly (Matano et al. 2013; Azhar et al. 2017). However, the cells need to be separated from the solid biomass when integrated methods of simultaneous saccharification and fermentation or consolidated bioprocessing are used for bioethanol production. Matano et al. (2013) have used cell recycle batch fermentation for ethanol production by consolidated bioprocessing of rice straw at high solid loads. The authors observed an average of 34.5 g/L ethanol production when five batches of fermentation were performed for 200 g/L of hydrothermally pretreated rice straw. The ethanol titer was further raised to 42.2 g/ L using a recombinant cellulase-displaying yeast, leaving only 3.3 g/L sugar in the fermentation medium at the end of the final batch. The repeated-batch process allows easy collection of cells, stable operation, and long-term productivity. However, immobilized cells can prove more efficient compared to free cells in the repeatedbatch fermentation (Azhar et al. 2017). Fed-batch mode involves intermittent addition of glucose (or hydrolysate) without the removal of fermentation broth. This is known as a common method for the production of ethanol industrially, which combines the features of both batch and continuous processes (Cheng et al. 2009; Chang et al. 2012; Zabed et al. 2014). The process consists of stages of batch-feeding-batch (Balat et al. 2008). Its operation needs optimization of feed rate time. The fed-batch process is beneficial over batch mode in that it serves as an alternative to the problem of substrate inhibition. The effect of other inhibitory components is also reduced, which are otherwise a constraint at high levels. The fed-batch, therefore, is a promising process during fermentation of hydrolysates obtained from dilute acid hydrolysis. The fed-batch mode maintains the viability of cells and extends their life (Balat et al. 2008; Zabed et al. 2014). Other advantages include higher levels of dissolved oxygen, higher productivity, reduced fermentation time, and regulation of critical process parameters such as pH, temperature, and dissolved oxygen content through the feedback activities (Cheng et al. 2009; Chang et al. 2012; Zabed et al. 2014). However, the end-product inhibition by ethanol may limit the process efficiency (Wang et al. 2013). Also, ethanol productivity may be limited by feed rate and cell biomass levels (Azhar et al. 2017). Various studies have evaluated the performance of the fed-batch process and compared the results with the batch mode of fermentation. Cheng et al. (2009) compared the production of ethanol from glucose under

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batch and fed-batch modes. They observed that the fed-batch process was more efficient producing 14 g/L ethanol, compared to the batch process, at an optimized feeding rate of 2 g/L/h for the glucose. Similarly, when Chang et al. (2012) produced ethanol from corncob hydrolysate under different modes of fermentation, the higher ethanol yield was obtained in fed-batch mode than that of the batch fermentation. They concluded that the effect of substrate inhibition was lesser in the fed-batch fermentation process. Chang et al. (2018b) demonstrated that a glucose level higher than 200 g/L showed substrate inhibition in the batch mode of fermentation, whereas higher ethanol titer and yield were obtained in fed-batch process with glucose levels up to 260 g/L. Continuous mode of fermentation involves a continuous input of ingredients (substrate, medium, and inoculum) with simultaneous removal of end product from the vessel at a specified rate (Zabed et al. 2014; Azhar et al. 2017). It can minimize the inhibitory effects and improve yield and productivity significantly. The dilution rate plays an important role during the continuous process. Maintaining constant culture volume is critical in continuous fermentation (Azhar et al. 2017). The ethanol productivity shows an increase with the dilution rate. The lower dilution rate reduces the conversion rate of the inhibitors, which has an adverse effect on the growth rate of microbes. On the other hand, the dilution rate cannot be exceeded the specific growth rate of the ethanol-producing microorganism employed in the process. The high dilution rate will lead to biomass wash-out. High dilution rate may increase ethanol productivity, but ethanol yield is declined as a result of incomplete substrate utilization by the ethanologen (Azhar et al. 2017). The retention of the microorganisms in the fermenter by immobilization, encapsulation, and filtration, cell recycling, or using flocculent microbes is an alternative for maintaining high dilution rate to obtain high ethanol productivity (Wang et al. 2013). One limitation of the method is that the risk of contamination is higher during the continuous fermentation process, and also long cultivation times in continuous process may reduce the ability of yeasts to produce ethanol (Azhar et al. 2017). Wang et al. (2013) have investigated the outcome of continuous fermentation of ethanol from sucrose using a cell-recycling two-tank system. They achieved higher ethanol productivity (6.9–7.5 g/L/h) in the continuous fermentation compared to the batch process (3.85–4.48 g/L/h), leaving only 2% of the unconverted sugar at the end of the process. Thus, the choice of fermentation mode during cellulosic ethanol production will depend on the type of lignocellulosic biomass, type of microorganisms, and other parameters governing the efficiency and economics of the overall process.

Factors Affecting Fermentation The efficiency of the ethanol fermentation is determined by several factors such as initial sugar levels, amount of inoculum, pH, temperature, and agitation rate, etc. (Zabed et al. 2014; Azhar et al. 2017). A very high concentration of sugars may lead to osmotic stress to the cells. They also require a longer incubation time for their

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fermentation (Lin et al. 2012). Contrarily, the low levels yield lower ethanol concentrations. Also, the accumulation of ethanol beyond the tolerance level of fermentation microbe shows an inhibitory effect on microbial cells. The concentration of microbial cells governs the rate of ethanol production, which is slow initially but increases with the increase in the number of cells. A higher inoculum level, on the other hand, often leads to competition for nutrients, which has a negative effect on the rate of fermentation. Additionally, the incubation temperature during fermentation has a marked impact on the growth and enzymatic activity of the ethanolproducing microbes. The optimum temperature varies depending on the type of ethanologen, being 30–35
C for S. cerevisiae and above 40
C for thermotolerant yeasts. The high temperature may cause a change in transport activity and lead to a saturation level of toxins, including ethanol, in the cells. In contrast, the microbial cells’ tolerance to ethanol may be lowered at low temperature, which might affect their growth rate (Lin et al. 2012). The temperature above optima also affects membrane fluidity and denatures enzymes, whereas that below optima decreases specific growth rates by slowing down metabolic activities. Similarly, very high or low pH beyond the optimum for the microbe selected for fermentation exerts a negative effect on the microbe’s growth as well as many other metabolic activities such as permeability of some essential nutrients into the cells. The variation in pH beyond a narrow range also changes the main fermentation pathway employed by microorganisms (Lin et al. 2012). Consequently, the yield of ethanol is reduced considerably due to the production of co-products. The low pH during fermentation is, otherwise, favorable for minimizing the risk of bacterial contamination. The fermentation time influences the growth of ethanologen. The shorter time is inadequate for desired growth level, whereas longer times lead to a toxic effect of accumulated ethanol, especially during the batch mode of fermentation.

Process Integration for Second-Generation Ethanol Despite many research efforts and successful improvements in various steps (pretreatment, cellulase production, detoxification, saccharification, and fermentation), the bioconversion of lignocellulose to ethanol is still at pilot scale (Zabed et al. 2016). Among different strategies for making cellulosic ethanol production an economically competitive process, various approaches for integration of saccharification and ethanol fermentation steps have also been proposed. The process integration represents an improved method for enhancing the energy efficiency of the industrial process (Sánchez and Montoya 2013). It offers various advantages in the cellulosic ethanol production process by reducing the number of process units. Reducing the number of steps has a direct impact on capital and operation cost, energy consumption and time for the overall process (Zabed et al. 2016). The integration also aids in improving the performance of the overall process as well as the downstream processes (Sánchez and Montoya 2013). These approaches include separate hydrolysis and fermentation (SHF), separate hydrolysis and co-fermentation (SHCF), simultaneous saccharification and fermentation (SSF),

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simultaneous saccharification and co-fermentation (SSCF), and consolidated bioprocessing (CBP) (Table 4.2). All these fermentation strategies are preceded by a pretreatment step, which is essential for the deconstruction of the complex cell wall structure. Table 4.2 Process integration: major fermentation configurations Process SHF (Separate Hydrolysis and Fermentation)

Process features Biomass hydrolysis and fermentation done separately at 40–50
C and 30
C, respectively

Advantages Optimal conditions can be maintained during hydrolysis and fermentation; continuous mode of fermentation possible with cell recycling

SHCF (Separate Hydrolysis and Co-fermentation)

Biomass hydrolysis and fermentation done separately at their respective temperature optima, resultant C6 and C5 sugars are fermented simultaneously Biomass hydrolysis and fermentation (mostly of hexoses) are done simultaneously

Co-fermentation increases the ethanol titers

SSCF (Simultaneous Saccharification and Co-fermentation)

Integration of steps of biomass hydrolysis and fermentation of C5 and C6 sugars

CBP (Consolidated Bioprocessing)

Integration of cellulase production, biomass hydrolysis and (co-) fermentation

Faster rate, high ethanol titers due to co-fermentation, end-product inhibition by sugars is avoided; reduced cost; can process high solid loads Minimum number of bioreactors; simplified process; costeffectiveness; energy efficiency; cost of exogenous enzyme and substrate for its production is reduced

SSF (Simultaneous Saccharification and Fermentation)

Lower cost than SHF; reduced end-product inhibition of cellulases; lower enzyme requirements; reduced risk of contamination due to presence of ethanol; faster rate, higher ethanol titers

Limitations End-product inhibition of enzymes by sugars; high sugar levels increase the risk of contamination and osmotic stress of yeast; cost-ineffective due to multiple units End-product inhibition by sugars during hydrolysis; limitations of co-fermenting microbes

Optimal conditions for hydrolysis and fermentation are compromised; inhibition by higher ethanol levels; high viscosity; low mixing and heat transfer; cell recycling not possible due to mixing with solid biomass Optimal conditions for hydrolysis and fermentation are compromised; limitations of co-fermenting microbes Lack of suitable CBP enabled microbes with all required characteristics; lower ethanol yields; formation of co-products (acetic acid, lactic acid)

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4 Saccharification Fermentation and Process Integration

Separate Hydrolysis and Fermentation (SHF) The SHF process is the oldest method for producing bioethanol from lignocellulosic biomass. The concept of SHF is based on the execution of hydrolysis (or saccharification) and fermentation steps of the cellulosic ethanol production process in separate bioreactors (Fig. 4.7) (Sánchez and Montoya 2013; El-Naggar et al. 2014; Kennes et al. 2016). The hydrolysis is preceded by appropriate pretreatment of biomass to enhance its digestibility. Following pretreatment, the slurry is subjected to solid–liquid separation, which produces a hydrolysate containing pentoses (released from hemicellulose hydrolysis during pretreatment) and a solid fraction. The solid biomass, containing cellulose and lignin, is fed into the hydrolysis unit along with externally produced cellulases (the cocktail of enzymes showing synergy), which degrade cellulose to monomeric sugars. The solid fraction can also be washed prior to enzymatic hydrolysis, which aids in the removal of toxic products generated during the pretreatment process. The hydrolysate is fed into the next unit, where sugars are fermented to ethanol using appropriate ethanologenic strain. The lignin residue obtained at the end of the process can be used as a source of energy or an attractive substrate for producing various valueadded products as a part of biorefinery. The SHF process has the primary advantage that the hydrolysis and fermentation processes, differing in their temperature optima, can be performed at their respective optimal conditions (Kennes et al. 2016). The hydrolysis is most effective at a temperature range of 45–50
C, whereas most commonly employed fermentation microbes have temperature optima near 30
C (Vohra et al. 2014; Sebayang et al. 2016). Thus, individual processes can be monitored and regulated separately for maximizing their outcome. Also, separation of residual biomass from hydrolysate

Fig. 4.7 Simplified overview of separate hydrolysis and fermentation process (SHF) for cellulosic ethanol production

Separate Hydrolysis and Fermentation (SHF)

135

after saccharification can allow continuous fermentation mode and microbial cells recycling in the subsequent step. This is possible because lignin removal before fermentation allows separation of fermenting microbial cells from biomass. The same is problematic if lignin or biomass is mixed with ethanologen. The lignin separation also allows its utilization in the biorefinery (Sánchez and Montoya 2013). However, a long-standing limitation of the method is low saccharification yield due to inhibitory effect of glucose and cellobiose, the major end products of cellulose hydrolysis, on cellulases (Kennes et al. 2016; Robak and Balcerek 2018). Often glucose, beyond a level, causes inhibition of β-glucosidase resulting in accumulation of cellobiose, which inhibits other cellulases. Also, a longer residence time during hydrolysis, lasting from 1 to 4 days, increases the risk of microbial contamination of nutrient-rich hydrolysate (El-Naggar et al. 2014). Additionally, the installation and operational cost of separate bioreactors make this strategy relatively cost-ineffective. Belal (2013) utilized rice straw as a possible substrate for bioethanol production. They recorded 11 g/L ethanol production from rice straw pretreated using ultrasound-assisted acid pretreatment method followed by SHF of the biomass. Another research group (Nguyen et al. 2018) worked on cellulosic ethanol production from soybean residue using SHF approach. The biomass was pretreated using thermal acid hydrolysis method followed by enzymatic hydrolysis using a mixture of commercial cellulases and xylanases. Twenty percent (w/v) soybean residue hydrolysate when subjected to fermentation by S. cerevisiae KCCM 1129 (adapted to high concentrations of galactose) in a 5 L bioreactor, yielded 31.6 g/L ethanol. Sherpa et al. (2019) worked on the bioconversion of sugarcane tops to bioethanol using SHF technique. Prior to saccharification, the biomass was subject to enzymatic pretreatment using laccase. The enzymatic hydrolysis involved use of cellulase– xylanase cocktail obtained from Trichoderma reesei Rut C30 and fermentation was performed using S. cerevisiae. The process yielded 27.2 g/L ethanol from the sugarcane tops. Different studies have employed batch or fed-batch mode for the fermentation during SHF process for bioethanol production from various biomass. Kim (2018) studied bioethanol production from alkali-thermal pretreated empty palm fruit bunch fiber in both batch and fed-batch modes of fermentation. The author observed higher ethanol production, i.e., 33.8 g/L (with 1.57 g/L/h productivity) during fed-batch fermentation in a short operation time (in 20 h) compared to 21 g/L ethanol production in 28 h in the batch fermentation. Kassim et al. (2019) demonstrated that fed-batch fermentation was more effective than batch fermentation while producing ethanol from microalgal biomass. The alkali pretreated biomass was hydrolyzed with a mixture of cellulases from T. longibrachiatum and reducing sugars obtained in the process were fermented to ethanol using S. cerevisiae. The fed-batch fermentation resulted in the production of 1008.48 mg/L ethanol compared to 921.38 mg/L ethanol produced in batch mode of fermentation.

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4 Saccharification Fermentation and Process Integration

Separate Hydrolysis and Co-Fermentation (SHCF) The first attempt to integrate different steps was to perform fermentation of C6 and C5 sugars in a single step of co-fermentation, i.e., in the same vessel. This approach is referred to as separate hydrolysis and co-fermentation (Fig. 4.8) (El-Naggar et al. 2014; Sebayang et al. 2016; Zabed et al. 2016). The pentoses and hexoses are obtained separately from the saccharification of hemicellulose and cellulose, respectively. Thereafter, the C5 and C6 sugars are co-fermented to produce ethanol. Ideally, the SHCF process yields higher ethanol titer due to the utilization of both pentoses and hexoses in the co-fermentation process. The removal of an additional vessel reduces the cost of an extra fermentation unit, thus improving the economics of the process (Sánchez and Montoya 2013; Kennes et al. 2016; Sebayang et al. 2016). Performing hydrolysis in a separate unit is also advantageous because it allows its execution at optimal conditions and eliminates the problems of high viscosity during fermentation, which is especially evident when high solid loads are used. The SSCF also has lower enzyme requirement and faster production rate (Sebayang et al. 2016). But co-fermentation is a challenging process as no wild ethanologenic microorganism is known to have the capability of fermenting both hexoses and pentoses sugars efficiently. Therefore, co-fermentation is achieved by adopting different strategies (Vohra et al. 2014). The genetic modification of fermentation microbes is a common approach involving the introduction and expression of genes for xylose uptake and fermentation in glucose fermenting microorganisms. Extensive work has been done by various groups to generate different strains of S. cerevisiae, (most common robust and industrially relevant ethanologenic yeast) capable of co-fermenting both hexose and pentose sugars. However, such microbes exhibit a faster rate of glucose

Fig. 4.8 Simplified overview of separate hydrolysis and co-fermentation process (SHCF) for cellulosic ethanol production

Separate Hydrolysis and Co-Fermentation (SHCF)

137

fermentation compared to that of xylose (Kennes et al. 2016). Additionally, utilizing a microbial co-culture for mixed-sugar fermentation is also an attractive approach (Vohra et al. 2014; Kennes et al. 2016). Chen et al. (2018) have developed engineered co-culture consortium of cellobiose-consuming S. cerevisiae strain EJ2 and xylose-consuming S. cerevisiae strain SR. They believed that a co-culture system could distribute the metabolic burden on each specialist strain, while the introduction of multiple heterologous pathways into a single microorganism could lead to metabolic stress on the strain, especially when sugar concentrations are higher. The co-culture technique, however, comes with a limitation that optimum conditions for fermentation by different microorganisms might vary. The careful selection of ethanologens with similar physiological requirements may solve this problem to an extent. Furthermore, another limitation of co-fermentation is the co-consumption of sugars as xylose consumption is repressed in the presence of glucose. The fed-batch mode of SHCF is considered an improvement in such situations as it allows the efficient uptake of xylose during fermentation. Furthermore, a pre-fermentation strategy is also adopted, aimed to decrease the glucose-toxylose ratios, which kinetically favors uptake of xylose during the co-fermentation process (Nielsen et al. 2016). Compared to other configurations, the SHCF process has been relatively less explored (Kennes et al. 2016) and need experimental optimization for various feedstocks in order to make this process commercially feasible. Erdei et al. (2012) have demonstrated separate hydrolysis and co-fermentation in a process integrating ethanol production from wheat straw and wheat meal. The steam pretreated wheat straw was hydrolyzed enzymatically using commercial preparations of Cellic CTec cellulase and Cellic HTec xylanase. The xylose and glucose in the hydrolysate were subjected to co-fermentation by a genetically modified S. cerevisiae strain TMB3400 when wheat-starch hydrolysate (firstgeneration glucose source) was used as a feed. Higher ethanol yields were obtained in the co-fermentation process. Also, the integration of first- and second-generation processes increased ethanol yield, which is desirable for reducing the distillation cost in the subsequent step. Another work by Novy et al. (2015) involved the study of SHCF of wheat straw pretreated by steam explosion method. The enzymatic hydrolysis employed the (hemi-) cellulolytic enzyme cocktail produced from T. reesei SVG17 strain under batch fermentation. Maximum sugars were recovered in the saccharification process when 30 FPU/g dry substrate enzyme loadings were used for 15% substrate loading. The co-fermentation was performed, utilizing S. cerevisiae strain IBB10B05 (modified for mixed glucose–xylose fermentation), under the batch mode of fermentation using 15% hydrolysate. In the process, 71.2 g of ethanol production was achieved per kg of the raw material. Nielsen et al. (2016) also performed separate hydrolysis and co-fermentation of wheat straw. The wheat straw was pretreated by dilute acid-catalyzed steam explosion method. The pentose-rich liquid hydrolysate was separated from the hexose-rich solids. The solid fraction was hydrolyzed enzymatically (using Cellic CTec2) to obtain a glucose-rich hydrolysate. The co-fermentation of pentoses and hexoses was done using a recombinant strain of

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4 Saccharification Fermentation and Process Integration

S. cerevisiae KE6-12. However, because high glucose concentrations repress the xylose uptake, therefore, the fermentation process was accomplished sequentially in two stages. First, the xylose-rich hydrolysate liquor was subjected to fed-batch pre-fermentation, and then the enzymatic hydrolysate was subjected to co-fermentation using a single addition of pre-hydrolyzed solids. The process yielded 0.423 g/g ethanol, accompanied with 0.036 g/g xylitol production.

Simultaneous Saccharification and Fermentation (SSF) One of the most important integration strategies for cellulosic ethanol production is simultaneous saccharification and fermentation (SSF) (Fig. 4.9). In this approach, the enzymatic hydrolysis of the cellulose and the fermentation of resulting sugars are carried out simultaneously in a single bioreactor. The concept of SSF was introduced in 1976 by Gauss et al. in a patent (Gauss et al. 1976) as an improvement to the traditional method of separate hydrolysis and fermentation. They demonstrated the production of higher ethanol yield and proposed that it resulted from the release of end-product inhibition. The appropriately pretreated biomass enriched in cellulose is mixed with cellulases (the cocktail of all components, i.e., endoglucanases, cellobiohydrolases, and β-glucosidase) and an inoculum of glucose fermenting ethanologen in a vessel. The cellulases attack the cellulose releasing cellodextrins, and cellobiose, which are ultimately hydrolyzed to glucose monomers. Because fermentation takes place in the same unit, the glucose and cellobiose are not accumulated (Vohra et al. 2014; Robak and Balcerek 2018); and cellulose hydrolysis, therefore, operates at a faster rate. This is possible because cellulases do not encounter end-product inhibition. As a consequence, the yield of ethanol is increased. Studies have also demonstrated a higher (Sánchez and Montoya 2013) (Table 4.3) and faster rate of ethanol production in SSF compared to SHF (Vohra

Fig. 4.9 Simplified overview of simultaneous saccharification and fermentation (SSF) process for cellulosic ethanol production

Feedstock Cassava pulp (starch + cellulosic fiber)

Empty fruit bunch

Oil palm frond

Rice straw, wheat straw, and sugarcane bagasse

S. No. 1

2

3

4

Acid pretreatment (4% H2SO4, 121
C, 30 min) followed by delignification using 0.5% NaOH (121
C, 30 min)

Alkali pretreatment (10% NaOH, 150
C, 30 min, solid to liquid ratio 1:15) Alkali (10% NaOH, 150
C, 4–7 kg/cm2 pressure, 30 min) pretreatment

Pretreatment None

SSF conditions Enzymatic hydrolysis employed enzyme cocktail of commercial cellulase, β-glucosidase, α-amylase, and glucoamylase; fermentation by S. cerevisiae SHY08–3; SSF at 37
C, 5.0 pH, using 6% inoculum and 20% pulp load Enzymatic hydrolysis by Cellic® CTec2 and Cellic® HTec2; fermentation by S. cerevisiae; SSF at 15% substrate loading, 0 FPU/g enzyme loading, 1% yeast, 32
C, 150 rpm, 72 h Enzymatic hydrolysis by commercial cellulases, Cellic® Ctec2 and Cellic Htec2, fermentation by S. cerevisiae,15% substrate loading, 30 FPU/g enzyme loading, 32
C, 150 rpm, 96 h Hydrolysis employed in-house cellulase produced from A. terreus; fermentation by in-house yeast strain Kluyveromyces sp.; SSF at 42
C, using 10% solid loading and 9 FPU/g enzyme loading, 60 h Lower ethanol levels in SHF, i.e., highest 4.74% in 24 h Lower ethanol production in SHF (maximum 51.74 g/L with 83.86% yield, in 72 h) Lower ethanol yields in SHF (using S. cerevisiae, in 36 h), i.e., 14.0, 13.9, 12.9 g/L from rice straw, wheat straw, and sugarcane bagasse, respectively

6.05% ethanol concentration

59.20 g/L ethanol (95.95% yield)

23.23, 18.29, and 17.91 g/L ethanol from rice straw, wheat straw, and sugarcane bagasse, respectively

Comparison with SHF Lower ethanol yield in SHF, i.e., 23.51 g/L in batch process and 29.39 g/L in fed-batch mode

Outcome/ethanol yield 34.67 g/L ethanol production in batch mode and 43.25 g/L ethanol yield during fed-batch process

Table 4.3 Simultaneous saccharification and fermentation (SSF) of various lignocellulosic substrates

(continued)

Narra et al. (2016)

Triwahyuni et al. (2015)

Dahnum et al. (2015)

Reference Zhu et al. (2012)

Simultaneous Saccharification and Fermentation (SSF) 139

Feedstock Forest (Douglas-fir) residue

Water hyacinth

Eucalyptus grandis (bark, branches and leaves)

Sugarcane bagasse pith

S. No. 5

6

7

8

Table 4.3 (continued)

Acid pretreatment (1–2% v/v H2SO4, 121
C,1.5 bar, 90 min

Acid pretreatment (1% H2SO4, 100
C, 30 min, solid–liquid ratio 1:30) Acid-catalyzed steam explosion pretreatment (2.4 wt% H2SO4, 180
C, 15 min)

Pretreatment Sulfitepretreatment (SO2 concentration of 80 g/L 140
C, liquor to wood ratio 4:1, 60 min)

SSF conditions Enzymatic hydrolysis of the whole slurry solid using commercial complex cellulase enzyme, Cellic® CTec3; fermentation by S. cerevisiae YRH400; SSF at 382
C, 150 rpm, using 15% substrate loading, 20 FPU/g enzyme loading, 96 h Enzymatic hydrolysis by commercial cellulase extracted from T. viride; fermentation by S. cerevisiae; SSF at 38.87
C, 81.87 h, 6.11 mL yeast, 120 rpm Enzymatic hydrolysis used CTec2, fermentation used S. cerevisiae; SSF at 36
C, for 96 h, using 20% substrate loading, 40 FPU/g cellulase loading, supplementation of 30 mg PEG per g of biomass Biomass hydrolysis by commercial enzyme (Celluclast® 1.5 L); fermentation by Pichia stipitis JCM 10742; SSF at 30
C, 150 rpm, at 5% solid loading, using 15 FPU/g cellulase loading and 7.50 IU/g β-glucosidase loading, for 72 h Lower ethanol levels in SHF (2.58 g/L with 0.09 g/L/ h productivity) in 30 h

Sritrakul et al. (2017)

McIntosh et al. (2017)

–

60 g/L ethanol production with 85.8% yields

3.70 g/L ethanol production with 0.15 g/L/ h productivity achieved in 24 h

Zhang et al. (2016)

Reference Yang et al. (2016)

–

Comparison with SHF Lower ethanol yields in SHF (38.6 g/L)

1.289 g/L

Outcome/ethanol yield 43.2 g/L ethanol production (75.1% theoretical yield)

140 4 Saccharification Fermentation and Process Integration

Rice straw

Mixture of Ricinus communis (RC), Lantana camara (LC), Saccharum officinarum tops (SCT), Saccharum spontaneum (KG), Ananas comosus leaf wastes (PA), and Bambusa bambos (BB)

9

10

Delignification by laccase produced by Pleurotus djamor (30% w/v substrate, level, 1666.5 U/g enzyme loading) at 35
C for 6 h

Alkali pretreatment (1 M NaOH, overnight at room temperature, followed by autoclaving at 121
C for 1 h Hydrolysis by cocktail of Cellulase Onozuka 3S, Cellulase T Amano 4, Pectinase G Amano enzymes (selected among 15 food processing cellulases using a multivariate analysis); fermentation by Mucor circinelloides fungus; SSF at 28
C, 120 rpm, for 36 h, using 100 g/L pretreated rice straw suspension and 2 g-protein/L cellulase cocktail Enzymatic hydrolysis involved use of cellulase and xylanase produced from T. reesei RUT C30; fermentation by S. cerevisiae; SSF optimized by RSM Ethanol productivity 1.396 g/L/ h (41.89 g/L ethanol) at 25% substrate loading, 8% v/v inoculum level, 44.84 inoculum age, at 38.18
C, in 30 h, using 80 U/g cellulase loading

30.5 g/L ethanol production

1.64 folds lower ethanol level in SHF (25.40 g/L) with 0.929 g/L/ h productivity, at higher cellulase loading (132.9 U/ g)

–

Althuri and Banerjee (2019)

Takano and Hoshino (2018)

Simultaneous Saccharification and Fermentation (SSF) 141

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4 Saccharification Fermentation and Process Integration

et al. 2014). Also, the process does not require the separation of hydrolysate from the lignin fraction, and this prevents the loss of significant amounts of sugars. Another advantage is the cost-effectiveness of the process. The decrease in the number of vessels due to the integration of hydrolysis and saccharification reduces the capital and operational cost of additional bioreactors (Robak and Balcerek 2018). The estimates have proposed over 20% reduction in the capital investment compared to a traditional approach. Furthermore, the presence of ethanol in the medium reduces the risk of microbial contamination (Vohra et al. 2014; Robak and Balcerek 2018). This approach may find its applications at the laboratory as well as industrial scales. However, no commercial plant has yet been set up based on this technology. There is one prominent limitation of SSF. The optimum temperature for hydrolysis is higher than that for the fermentation processes. During SSF a compromise is made between the two. The SSF is carried out at a temperature between 30
C and 50
C. However, thermotolerant microorganisms involved in fermentation, such as Candida glabrata, Kluyveromyces marxianus, thermotolerant S. cerevisiae strains, Thermoanaerobacter ethanolicus, Thermoanaerobacterium saccharolyticum, and Clostridium thermotherumare, a potent solution to this problem (Sánchez and Montoya 2013; Vohra et al. 2014; Choudhary et al. 2016; Robak and Balcerek 2018). Furthermore, the microbial cells used for the fermentation cannot be recycled as their separation from the solid biomass is difficult (Olofsson et al. 2008). Table 4.2 shows different studies on simultaneous saccharification and fermentation (SSF) of various lignocellulosic substrates. The SSF can be performed either in batch or fed-batch mode. Several studies have advocated advantages of fed-batch SSF. Mendes et al. (2017a) have performed the comparison of outcome of batch and fed-batch SSF of primary sludge from pulp and paper mills, containing 60% of carbohydrates, without any pretreatment prior to SSF process. The hydrolysis was carried out using Cellic®, CTec2 cellulases complex, and the fermentation employed S. cerevisiae and a thermotolerant Kluyveromyces marxianus NCYC 1426. The results indicated that S. cerevisiae showed better SSF efficiency. 40.7 g/L ethanol was produced in fed-batch SSF using 75% lesser enzyme loadings, at higher solid content (total carbohydrate concentration of 200 g/L) than the batch SSF producing 22.7 g/L ethanol, using total carbohydrate concentration of 50 g/L. The yield and productivity, however, were lower in the fed-batch mode. Another work by Gao et al. (2018) on fed-batch SSF of sugarcane bagasse revealed that fed-batch process was more efficient for high biomass loadings during cellulosic ethanol production. The authors achieved production of 75.57 g/L ethanol (with 0.63 g/L/h productivity and 66.17% yield) from alkali pretreated sugarcane bagasse in 120 h, when SSF was performed using S. cerevisiae Y-2034 at 33% solid loadings. Several workers such as Dimos et al. (2019), while working on cellulosic ethanol production from cotton stalks (pretreated sequentially by organosolv and hydrothermal pretreatment), proposed a variation of the SSF process referred as pre-hydrolysis and simultaneous saccharification and fermentation (PSSF). The researchers demonstrated that the extent of pre-hydrolysis and solid loading had a significant impact on ethanol production and process productivity. They achieved the highest 47 g/L ethanol

Simultaneous Saccharification and Co-Fermentation (SSCF)

143

levels at 20% (w/v) solid loads when 14 h of pre-hydrolysis was performed prior to the SSF process. Pre-hydrolysis can possibly reduce the viscosity of solid–liquid mixture before the addition of ethanologen.

Simultaneous Saccharification and Co-Fermentation (SSCF) SSCF represents a higher degree of integration and an improvement over the SSF process (Sánchez and Montoya 2013). It combines the features of both SHCF and SSF processes. It integrates the steps of enzymatic hydrolysis of biomass with co-fermentation of C6 and C5 sugars, in the single unit (Sánchez and Montoya 2013; Qin et al. 2018) (Fig. 4.10). The number of vessels is reduced further, thus reducing the cost of the process. Like SSF, SSCF eliminates the problem of feedback inhibition (Robak and Balcerek 2018). However, ethanol production is higher compared to SSF because of co-fermentation of both hexose and pentoses obtained from the hydrolysis of cellulose and hemicellulose, respectively. The co-fermentation may involve either the use of recombinant strains of ethanolproducing microbes, capable of co-consumption of xylose and glucose or use of co-cultures of C5 and C6 fermenting microorganisms. The SSCF by a co-culture requires optimization of co-culture and other conditions to maximize the ethanol production from the various feedstocks. Suriyachai et al. (2013) have performed SSCF of alkali (NaOH) pretreated rice straw using a co-culture of S. cerevisiae and Scheffersomyces stipitis. Under optimized conditions for co-culture (S. cerevisiae: S. stipitis cell ratio of 0.3, 116 rpm, and 33.1
C temperature), the SSCF yielded 99% of theoretical ethanol yield. The co-culture SSCF could produce 28.6 g/L ethanol at 10% solid loads. Paschos et al. (2015) achieved production of 58 g/L ethanol by SSCF of hydrothermally pretreated wheat straw using a co-culture of S. cerevisiae and Fusarium oxysporum. Liu et al. (2019) optimized conditions for co-culture mediated SSCF of H2O2-pretreated corn stover. They achieved 10.924 g/100 mL ethanol production at 25% substrate loads, at 32
C, using 12% S. cerevisiae-C. tropicalis ratio, at an initial pH of 5.0 in 144 h. Nevertheless, the SSCF method has

Fig. 4.10 Simplified overview of simultaneous saccharification and co-fermentation (SSCF) process for cellulosic ethanol production

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4 Saccharification Fermentation and Process Integration

limitation of SSF, i.e., a compromise in optimal temperature of hydrolysis and fermentation is made, while using mesophilic ethanologens. Other limitations, similar to co-fermentation, are slow xylose utilization by co-fermenting microbes and higher glucose to ethanol conversions than xylose due to faster growth rates of glucose fermenting microbes compared to xylose-fermenting microbes in a co-culture (Vohra et al. 2014; Sebayang et al. 2016). The SSCF can also be performed under batch and fed-batch modes, with or without a pre-hydrolysis step. Several other strategies are also employed to increase the xylose uptake and obtain higher ethanol yields at higher solid loadings. In a study by Olofsson et al. (2010) on SSCF of spruce, it was found that the controlled enzyme feeding during fed-batch SSCF operation could reduce the competitive inhibition of sugar transport resulting in enhanced total xylose uptake from 40% to nearly 80%. Jin et al. (2012b) studied SSCF of AFEX-pretreated corn stover using commercial enzyme for hydrolysis and xylose-fermenting S. cerevisiae 424A(LNH-ST) for fermentation. They carried out SSCF in a two-step process consisting of a pre-hydrolysis step using 25% enzymes followed by second-stage SSCF in which remaining 75% enzymes were added 48 h after yeast inoculation. They concluded that the glucose concentration obtained after pre-hydrolysis was closely co-related to the viability of microbial cells and, therefore, the consumption of hexose during SSCF. Another work by Erdei et al. (2013) demonstrated SSCF of steam pretreated wheat straw (lignocellulosic) and investigated the effect of supplementation of the liquefied wheat meal (starch-rich, first-generation sugar source). Enzymatic hydrolysis of wheat straw involved use of commercial cellulases, and while liquefaction of wheat meal involved use of commercial α-amylase and amyloglucosidase. The fermentation was achieved using pentose-fermenting S. cerevisiae TMB3400. Maximum 43.7 g/L ethanol production was achieved in batch operation using 7.5% wheat straw added with 1% liquefied wheat meal. The higher solid loading (10%) increased ethanol level to 53.0 g/L, but with a lower yield. The fed-batch fermentation operation, however, resulted in higher ethanol concentration and yield without requiring supplementation with the liquefied wheat meal. Koppram et al. (2013) while working on bioconversion of corn cobs to ethanol, showed that fed-batch mode of SSCF resulted in efficient xylose fermentation to ethanol (with glucose co-consumption) because of controlled release of glucose from enzymatic hydrolysis and maintenance of low levels of glucose concentration during SSCF. The authors demonstrated the reproduction of lab results at process development unit (PDU) (30 L) and further to the demo scale (10 m3). Another study by Yasuda et al. (2014) observed SSCF of low-moisture anhydrous ammonia-pretreated napier grass using recombinant Escherichia coli KO11, S. cerevisiae, along with cellulase and xylanase enzymes. They recorded higher ethanol yields (74%) during SSCF compared to 69% achieved in a previous study using SSF approach under similar conditions. Similarly, Liu and Chen (2016) working on SSCF of steam exploded corn stover found SSCF as more efficient method compared to SHF, SHCF, and SSF methods. They observed that SHF, SSCF, and SSF processes could increase the ethanol concentration and productivity, but not the ethanol yields at higher solid loadings. SSCF, however, resulted in increased concentration (60.8 g/L), productivity (0.63 g/L/h) as

Consolidated Bioprocessing (CBP)

145

well as yield (75.3%) of ethanol at 20% solid loadings. Furthermore, the efficiency of SSCF was improved by following feeding strategy within 24 h of SSCF. Nielsen et al. (2017) proposed a strategy of sequential SSCF to enhance the xylose conversion in the process. They used wheat straw as feedstock, subjected it to acidcatalyzed steam pretreatment, separated the xylose-rich hydrolysate liquor followed by its pre-fermentation and then fed-batch SSCF of all hydrolysates. The sequential process resulted in higher ethanol yield (92% of theoretical yield) when the slurry contained lower inhibitor concentrations. The authors proposed that xylose utilization was improved because sequential targeting of xylose and glucose conversion could sustain the xylose fermentation, which could result in higher ethanol yields. Qin et al. (2018) have used recycling SSCF approach for bioethanol ethylenediamine-pretreated corn stover. The authors demonstrated that similar ethanol concentration could be achieved using relatively lower enzyme loadings (nearly 40%) compared to conventional SSCF, when solid (enzyme)-recycled SSCF was employed.

Consolidated Bioprocessing (CBP) CBP represents the highest level of integration process during cellulosic ethanol production, which consolidates the steps of enzyme production, biomass hydrolysis, and fermentation in a single step (Sánchez and Montoya 2013; El-Naggar et al. 2014; Kennes et al. 2016) (Fig. 4.11). The pretreated biomass is directly converted into ethanol in a single bioreactor leading to the maximum reduction of bioreactors during bioconversion process. This configuration of bioethanol production from lignocellulose is also referred to as “Direct Microbial Conversion, DMC” (Sebayang et al. 2016). This is because the process does not require addition of (hemi-) cellulolytic enzymes produced exogenously. However, mostly it is the cellulose polysaccharide which is hydrolyzed enzymatically involving cellulases during CBP

Fig. 4.11 Simplified overview of consolidated bioprocessing (CBP) process for cellulosic ethanol production

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4 Saccharification Fermentation and Process Integration

(Sánchez and Montoya 2013). Nevertheless, a single microorganism can produce the enzymes required to hydrolyze both celluloses and hemicelluloses, though with a varied preference for the substrate. Theoretically, consolidated bioprocessing is more energy efficient and cost-effective compared to other configurations (SHF, SHCF, SSF, and SSCF) (Danquah et al. 2011; El-Naggar et al. 2014). In recent years, the CBP process has gained enormous popularity because it has been projected to have potential to reduce the cost of bioethanol production significantly, thus reducing the selling price of ethanol to lower than the US $0.70 per gallon (Van Zyl et al. 2007). There are many advantages associated with the process. Firstly, the minimum number of fermentation units is required for CBP, which reduces the cost of the process. Secondly, the cost of enzymes is reduced along with the requirement of feedstock for enzyme production externally (Jouzani and Taherzadeh 2014; Vohra et al. 2014; Robak and Balcerek 2018). Also, the feedback inhibition of cellulases is avoided because of the consolidation of saccharification and fermentation steps (Jouzani and Taherzadeh 2014). The CBP of biomass can be accomplished either by using a single microorganism with the potential of both hydrolysis and fermentation, or a consortium of microorganisms consisting of microbial strains specialized for cellulase production, biomass hydrolysis, and (co-)fermentation of sugars (Sánchez and Montoya 2013; Saini et al. 2017). The major challenge during the process is to develop such microorganism or the microbial consortia, which can carry out consolidated bioprocessing of lignocellulose with high efficiency. In nature, very few microbes are known to possess the ability of cellulolysis and fermentation of sugars to ethanol. Their application in CBP is limited owing to low titers of end product obtained from them. Often, the single microorganism used for the process is a genetically modified CBP enable strain. There are generally two strategies adopted to develop a CBP microorganism, i.e., genetic engineering of ethanologenic microbes for the production of (hemi-) cellulolytic enzymes or genetic engineering of cellulase producers for ethanol production. Also, the native strains with cellulolytic and ethanologenic abilities are improved by genetic engineering to enhance their CBP potential (Saini et al. 2017). An ideal CBP microorganism or microbial consortia should have some characteristic features including the production of carbohydrate hydrolytic enzymes, tolerance to inhibitors, co-fermentation of C6 and C5 sugars, industrial robustness, faster growth rate, tolerance to high ethanol titers, lower production of fermentation by-products such as acetic acid and lactic acid (Sánchez and Montoya 2013; Ali et al. 2016). Several microorganisms belonging to bacterial, fungal, and yeast groups have been used for CBP research. The cellulolytic filamentous fungi such as T. reesei, Fusarium oxysporum, Neurospora crassa, Monilia sp., Paecilomyces sp., Mucor indicus, Rhizopus oryzae, Aspergillus oryzae, and Neocallimastix patriciarum are known to possess ability of both cellulose hydrolysis and fermentation (Taherzadeh and Karimi 2007; Ali et al. 2016; Saini et al. 2017). However, without genetic improvement, these microbes are inefficient in bioconversion of biomass to ethanol with higher yields. Also, the production of by-products such as lactic acid and acetic acid is seen. Clostridium thermocellum, a thermophilic cellulolytic anaerobic

Ethanol Production from High Biomass Loading

147

bacterium, is a widely used microorganism for CBP. These bacteria possess metabolic pathways for mixed acid fermentation and produce a wide range of saccharolytic enzymes. The cellulases in surface complexed cellulosomes have high specificity. The thermophilic conditions during CBP by C. thermocellum are beneficial in increasing the activity of hydrolytic enzymes, reducing the risk of contamination, and improving the separation of ethanol (Saini et al. 2017). However, genetic improvements are required to enable pentose fermentation and other CBP characteristics to make it a competent CBP microbe. Additionally, Clostridium phytofermentans, Clostridium thermosaccharolyticum, Clostridium thermoshydrosulfuricum, Thermoanaerobacterium, Thermoanaerobacter ethanolicus, Thermoanaerobacter mathranii, Thermoanaerobium brockii, Phlebia sp., and Thermobifida sp. are also potential candidates for CBP of lignocellulose (Vohra et al. 2014; Jouzani and Taherzadeh 2014; Sebayang et al. 2016; Saini et al. 2017; Pang et al. 2018). However, again genetic modification to construct improved strains having most of the CBP characteristics is still needed in native microbes. Among various ethanologenic microbes, S. cerevisiae, Pichia stipitis, Kluyveromyces marxianus, Zymomonas mobilis, and E. coli have been modified genetically to make them CBP enabled microbes (Van Zyl et al. 2007; Vohra et al. 2014; Saini et al. 2017; Bušić et al. 2018). Furthermore, co-culture or consortium mediated approach is also employed to carry out consolidated bioprocessing of lignocellulosic biomass (Lee et al. 2017; Singh et al. 2017b; Saini et al. 2017; Pang et al. 2018). Table 4.4 summarizes the outcomes of various studies on consolidated bioprocessing of various lignocellulosic biomass. Although CBP is economically most attractive process compared to other configurations, being at a developing stage, it requires significant technological improvements to make it a process feasible at industrial levels (Satari et al. 2019).

Ethanol Production from High Biomass Loading The high solid loads during biomass hydrolysis are an important factor in cellulosic ethanol production at large scales. Obtaining high ethanol yields requires high amounts of fermentable sugars in hydrolysate, which further depends on initial solid load in saccharification process (Erdei et al. 2013). The high ethanol production is critical for decreasing the cost of downstream processing, i.e., recovery of ethanol by distillation (Qin et al. 2018). The estimates have revealed that the distillation process is economical when ethanol titer is >4% (w/w), which requires the release of at least 8% w/w glucose from the biomass. Achieving this level of glucose requires initial biomass loading >20% during enzymatic hydrolysis (Liu and Chen 2016; Zabed et al. 2016; Dimos et al. 2019). However, relatively lower loading is required when pentoses from the hemicelluloses are co-fermented (Zabed et al. 2016). Hence, most of the studies on cellulosic ethanol production aim at utilizing the biomass at 10–20% concentration. Nevertheless, operation at high solids loadings causes several technical challenges. The high solid levels may decrease the ethanol yield and productivity

Alkali pretreatment (2% NaOH, 160
C, 1 h, 150 rpm) Hydrothermal pretreatment (at 121
C, for 30 min, 1:10 bath ratio)

None

Dilute acid pretreatment (soaking in 1% w/w H2SO4 and then 0.4% w/w alkali) followed by treatment at 162
C, 5 bar pressure, for 10 min

Rice straw

Cassava aerial parts

Rice straw

Sugarcane bagasse

Sugarcane bagasse

Corn stover

Pretreatment AFEX (ammonia fiber expansion) pretreatment at 140
C, for 15 min, using 1 g/g ammonia to biomass loading and water loading 0.6 g/g of biomass AFEX (ammonia fiber expansion) pretreatment at 140
C, for 15 min, using 1 g/g ammonia to biomass loading and water loading 0.6 g/g of biomass Alkali pretreatment (0.8% wt NaOH, 121
C, 60 min, 1:10 solid to liquid ratio)

Feedstock Corn stover

CBP with Clostridium thermocellum DSM 1313, using 14.16 g/ L rice straw containing 61.67 mM glucose CBP under similar conditions using Clostridium thermocellum DBT-IOC-C19

Same as above

CBP using mixed culture of the four recombinant strains of S. cerevisiae CBP using Fusarium Oxysporum MTCC 1755, at 28
C

Process conditions CBP using Clostridium phytofermentans ATCC 700394 (5% inoculum size), 0.5% w/w glucan loading, at 200 rpm, 30
C, initial pH 7.0 CBP using Clostridium phytofermentans ATCC 700394 (10% inoculum size), at 400 rpm, 30
C, initial pH 6.7; 4% w/w glucan loading, 264 h CBP using Phlebia sp. MG-60, with 20 g/L solid biomass, at 28
C, for 240 h

Table 4.4 Consolidated bioprocessing (CBP) of various lignocellulosic substrates

Khuong et al. (2014)

4.5 g/L ethanol production with 65.7% theoretical yield (equivalent to 210 mg ethanol per gram of untreated bagasse) 14 g/L ethanol production from biomass containing 35 g/L glucan 0.85 g/L ethanol production in 120 h (equivalent to 42 mg ethanol per gram biomass) with 23.43% of theoretical yield 0.83 g/L ethanol production in 144 h (equivalent to 41.5 mg ethanol per gram biomass) with 21.54% of theoretical yield Production of 11.93 mM ethanol, 1.45 mM lactate, and 10.71 mM acetate Production of 14.15 mM ethanol, 2.31 mM lactate, and 9.05 mM acetate

Singh et al. (2017b)

Lee et al. (2017) Nongthombam et al. (2017)

Jin et al. (2012a)

Reference Jin et al. (2011)

48.9% glucan conversion, 77.9% xylan conversion, with 7.0 g/L ethanol production, but with 8.8 g/L acetate production

Outcome 2.8 g/L ethanol titer with 76% glucan conversion and 88.6% xylan conversion

148 4 Saccharification Fermentation and Process Integration

Crushed to particle size of 40-mesh screen

None

Corn straw

Microcrystalline cellulose

Co-culture of strains DBT-IOC-C19 (cellulose-degrading) and DBT-IOCDC21 (xylan-degrading) Co-culture of strains DBT-IOC-C19 and DBT-IOC-X2 (sugarfermenting) Co-culture of all three strains CBP using a co-culture of Clostridium thermocellum ATCC 27405 and Thermoanaerobacterium thermosaccharolyticum DSM 571 As above 1.29 g/L ethanol production (98.6% % cellulose degradation) with 26.1% ethanol yield (13.9% more ethanol yield than in anaerobic bottles)

25.28 mM ethanol production 0.45 g/L ethanol production (55.6% cellulose degradation) with 11.2% ethanol yield (28.2% more ethanol yield than in anaerobic bottles)

20.49 mM ethanol production

22.13 mM ethanol production

Pang et al. (2018)

Ethanol Production from High Biomass Loading 149

150

4 Saccharification Fermentation and Process Integration

by increasing the viscosity, concentration of inhibitors, and mass transfer resistance, and decreasing the nutrient transfer and cellulase adsorption (Erdei et al. 2013; Liu and Chen 2016; McIntosh et al. 2017; Gao et al. 2018). High viscosity makes mixing difficult and increases the power requirements in the process involving stirred tank bioreactors (Paschos et al. 2015). It should be noted that several factors govern the efficiency of the process at high solid loads, which include nature and composition of the biomass, the efficiency of the pretreatment, efficiency and concentration of hydrolytic enzymes, and the fermentation configuration. Various strategies can be employed to increase ethanol production from high biomass loads. Paschos et al. (2015) found that a pre-hydrolysis (liquefaction) step before SSF in the P-SSF (pre-hydrolysis simultaneous saccharification and fermentation) process in an in-house free-fall mixing reactor can be effective in decreasing the viscosity and increasing the mixing of the slurry. Liu and Chen (2016) have observed that fed-batch mode of SSCF at high solid loads of corn stover (steam exploded) can increase the concentration of ethanol by reducing the initial viscosity, which ensures the mixing of contents. Gao et al. (2018) have suggested the effectiveness of fed-batch SSF process in reducing the viscosity or mixing and diffusion limitations through the liberation of free water in the process. Dimos et al. (2019) while working on bioethanol production from cotton stalks utilized PSSF at high solid loads considering the role of pre-hydrolysis in reducing the viscosity of the solid–liquid mixture.

On-Site Cellulase Production for Enzyme Cost Reduction The cost of the enzymes (cellulases) is a major impediment in the success of the cellulosic ethanol production process. The cost is contributed by both the quality of the enzyme as well as the cost of each unit of the enzyme to the end user, including the cost of transport (Ellilä et al. 2017). Therefore, other than many strategies known for improving cellulase production, the on-site production of cellulases is also being suggested to be effective in increasing the cost efficiency of the bioethanol production process (Khokhar et al. 2014; Liu et al. 2016) and is, therefore, being tested widely to evaluate its significance. Barta et al. (2008) in their study concluded that capital investment and electricity are among major contributors to the cost of enzyme production. Based on their work on softwood based ethanol plant, they proposed that on-site enzyme fermentation can account for 9–11% of production cost for ethanol. They found that enzymes with high activity and productivity are required for producing more ethanol or reduce the cost of the process (Barta et al. 2010). Rana et al. (2014) produced cellulases from T. reesei RUT-C30 and Aspergillus saccharolyticus for utilization in bioethanol production from wet-exploded corn stover and loblolly pine. Using the in-house cellulases, they achieved ethanol yields comparable to those obtained by using commercial enzymes. Ellilä et al. (2017) engineered T. reesei strain for higher productivity and developed an inexpensive medium, based on soybean hulls and sugarcane molasses, for on-site cellulase production. The low-cost enzyme production was beneficial in hydrolysis of

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5

Microbial and Plant Genetic Engineering for Efficient Conversions

Abstract

Lignocellulosic derived bioethanol is an appealing, sustainable solution to energy crisis in fossil fuels. For viable and inexpensive ethanol generation processes and required economically viable agro-industrial substrates are essential. The handling and exploitation of lignocellulosic substrate is multifaceted, varying in many facets from crop-based ethanol production. The most important prerequisite is a potential microorganism capable to ferment a range of five- and six-member ring sugars as well as to withstand various stress conditions. The concept of metabolic engineering has various components such as enzyme and pathway identification, pathway pipelines, and overall optimization of the strain. Through metabolic engineering, various microbial strains have been assembled which showcase traits that are beneficial for bioethanol production utilizing lignocellulose biomass. There is another type of continuing attempts to advance their traits, development of the efficient fermentation approaches is one of the key factors that requires to be entirely optimized and incorporated to obtain a viable lignocellulose bioethanol plant. Various metabolic engineering approaches to address the above stated problems have been selected, for promising commercial possibilities to generate cellulosic ethanol. Keywords

Plant feedstock · Plant modification · Cell manipulation · Feedstock improvement · Sugar

Introduction Last decades are the phase of growing industrial biotechnology sector mainly producing microbial products such as bioethanol for sustainable environmental solutions. Lignocellulosic derived bioethanol is an appealing, sustainable solution # Springer Nature Singapore Pte Ltd. 2020 D. Sharma, A. Saini, Lignocellulosic Ethanol Production from a Biorefinery Perspective, https://doi.org/10.1007/978-981-15-4573-3_5

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Fig. 5.1 General concept of the metabolic engineering

to energy crisis in fossil fuels. Lignocellulosic bioethanol can add to a greener environment and with the execution of environmental shield in various nations. For viable and inexpensive ethanol generation processes and required economically viable agro-industrial substrates are essential. Recent bioethanol production approaches using agricultural residues like sugarcane bagasse and corn waste are well-documented; though, consumption of an inexpensive substrate such as lignocellulose could make ethanol much economical as compared to the fossil fuels. The handling and exploitation of lignocellulosic substrate is multifaceted, varying in many facets from crop-based ethanol production. The most important prerequisite is a potential microorganism capable to ferment a range of five- and six-member ring sugars as well as to withstand various stress conditions. The concept of metabolic engineering has various components such as enzyme and pathway identification, pathway pipelines, and overall optimization of the strain (Fig. 5.1). Through metabolic engineering, various microbial strains have been assembled which showcase traits that are beneficial for bioethanol production utilizing lignocellulose biomass. After various series of strain modification, some of the key microbial strains such as Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli have occurred and they have functioned nicely at small scale fermentations. There is another type of continuing attempts to advance their traits, development of the efficient fermentation approaches is one of the key factors that requires to be entirely optimized and incorporated to obtain a viable lignocellulose bioethanol plant.

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Engineered Yeast to Produce Cellulosic Biofuels Microbial fermentation of sugars obtained from lignocellulosic substrates to ethanol is carried out by Saccharomyces cerevisiae globally. The huge interest for the utilization of the lignocellulosic biomass as a sustainable substrate to produce bioethanol is due to its availability in significant amount, which does not compete with the existing food vs. fuel controversy (Caspeta et al. 2014; Tsai et al. 2015; Kumar et al. 2015). The common holdup linked with lignocellulosic biomass utilization to produce bioethanol is the fermentation economics, which is actually reliant on the potential of the yeast cells, as the yeast strain used should be tolerant to the rising concentrations of toxic substances, intermediate chemicals, and other incumbent cultural process conditions. Saccharomyces cerevisiae has been the driving strain to obtain ethanol at industrial scale. Saccharomyces cerevisiae can produce bioethanol from hexoses at efficient rates and quantity, but also display required robustness for industrial processes, such as fermentation of high gravity substrates, swift anaerobic metabolism, and uncommonly resistance to phage lysis. To model an idyllic fermentation process at industrial scale, all these characters and robustness are quite significant and desirable to obtain bioethanol from cellulosic biofuels (Fig. 5.2). In extension to that, first, an effective and fast xylose-fermenting trait needs to be hosted by fermenting microorganisms as lignocellulosic sugar hydrolysates composed of xylose up to 40% of total metabolizable sugars. Subsequently, glucose repression of xylose fermentation microorganisms to be improved to increase productivity. Third, tolerance to intermediate toxic inhibitors existing in lignocellulosic hydrolysates needs to be enhanced for fermenting low-cost hydrolysates. In the end, removal of produced bioethanol intermittently is highly beneficial to convert sugar into ethanol maximally. Various metabolic engineering tactics to discourse the above stated matters have been assumed for periods, leading to execution of promising commercial possibilities to produce cellulosic ethanol.

Engineering of Cell for Efficient Conversions There is an instant need to minimize the generation of carbon dioxide emissions, and the utilization of bioethanol as an energy source over fossil fuel has been a virtuous substitute to petroleum solutions. The first-generation bioethanol production that has been optimized in Northern America can be generated at low cost (Sharma 2016). The global biofuels market is expected to rise from $82.7 billion in 2011 to $185.3 billion in 2021. Genetic engineering paves its way towards efficient biomass conversion into biofuel through metabolic manipulations (Radakovits et al. 2010). Glucose and sucrose (derived from cane juice, etc.) are the conventional yeast substrates for bioethanol production. However, efforts have been made to exploit other carbon sources as yeast substrates like lignocellulose and glycerol (by-product of biodiesel synthesis) for biofuel production (Sharma and Dhanjal 2016).

Fig. 5.2 Possible metabolic routes for the conversion of the lignocellulosic biomass to ethanol (Courtesy)

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Genetically engineered yeast (Saccharomyces cerevisiae) easily assimilates pentose sugar through alternative pathways. US biotechnologists developed a pathway of simultaneous breakdown of xylose and to convert acetic acid (toxic for yeast cells) into fuel (Nissen et al. 2000). During alcoholic fermentation by yeast, all sugars are not converted into ethanol; instead some amount is also lost in the form of glycerol, thus reduces the efficiency of bioethanol production. However, scientists overcome this problem through genetic engineering of yeast through introduction of RUBISCO enzyme (from a CO2-fixating bacterium) and spinach gene for efficient utilization of CO2 and reduction in the formation of glycerol. The toxic effect of high ethanol doses on yeast is the biggest limitation on cost-effective bioethanol production. Scientists progressively worked on increasing the production of biofuels through various strategies like by increasing the yeast cells tolerance using potassium and an acidity-reducing compound (Caspeta et al. 2015). Scaling up such type of techniques would result in reduction of biofuel cost. However, efforts are required to translate such laboratory accomplishments to an industrial setting with large-scale batch fermentation.

Engineering of Substrate Utilization The production of first-generation ethanol is chiefly carried out utilizing edible starchy agricultural residues or sugarcane. The utilization of the first-generation agricultural substrates is too expensive and ends to food vs. fuel debate. Lignocellulosic waste is the utmost plentiful biomass available on earth, which is a desirable substitute feedstock for ethanol production. Lignocellulosic residues include cellulose, hemicellulose, and lignin as chief constituents (Zaldivar et al. 2001). Further the breakdown of lignocellulosic biomass certainly yields fermentable sugar such as glucose and breakdown of hemicellulose generates a mix of hexoses and pentoses sugars. The potential utilization of the lignocellulosic waste to bioethanol meets diverse challenges not only technical as well as economic viability which needs to be addressed. So, selection of the efficient and adaptive strain for the utilization of the lignocellulosic waste is thus a critical strategy. The overall efficiency of the fermentation conversion can be improved by genetic manipulation of conventional strains for hydrolysis of both hexose and pentose sugars (Dien et al. 2003; Jeffries and Jin 2004) or by taking out co-culture process of specific strains (Bader et al. 2010; Chen 2011). This strategy is exceptionally beneficial as it simultaneously utilizes both hexose and pentose fractions. The metabolic pipelines or networking are now accessible for various cells and the ease of use of these models presents new tactics to enhance the insight of complex cell regulations. Co-culture fermentation seems to be beneficial over axenic culture strategy for ethanol production using lignocellulosic biomass due to the possibility of synergistic conversion of metabolic abilities of interested microbes (Chen 2011). The dominance of co-culture of substrate-selective cell (such as modified Escherichia coli and

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parent strain of Saccharomyces cerevisiae) on single culture in refining the consumption of glucose/xylose combinations for improved ethanol production in batch process has been documented (Hanly and Henson 2013; Hanly et al. 2012). In recent times, Lisha and Sarkar (2014a, b) examined the effect of different genetic engineering schemes documented by Bro et al. (2006) on Saccharomyces cerevisiae for its effectiveness in improving the bioethanol yield in the perspective of batch/fed-batch process using co-culture and single culture process. Simulations fermentation were carried out with a variety of hexose/pentoses combinations and, for the 50/50 hexose/pentoses (%/%) combination. The batch co-culture process using genetically altered S. cerevisiae (utilize only glucose) and engineered E. coli strain (utilized only xylose) which improved the ethanol efficiency by 40.7% as compared to the single culture of S. cerevisiae process. The improvement in bioethanol yield of simultaneous fermentation system of substrate-selective strain is due to the concurrent conversion of hexoses and pentoses sugars, elevated substrate conversion rate, and decreased process time equated to single culture process of S. cerevisiae strain. They have reported that genetic alteration on xylose-selective E. coli strain should also be investigated as a substitute method for improved ethanol production from concurrent system. Ethanol fermentation prospective of Scheffersomyces (Pichia) stipitis using hexoses and pentoses mixes has been examined (Hanly et al. 2012; Parambil and Sarkar 2014). E. coli has the ability to hydrolyze both glucose and xylose into ethanol via a heterofermentative pathway (Liu and Khosla 2010). Though, the natural bioethanol production route is suboptimal as of other fermentation metabolites like acetate, formic acid, lactic acid, and succinic acid. Conveying the flow of carbon entered to cell mass or above-mentioned by-products in the direction of ethanol will rise the ethanol quantity. Trinh et al. (2008) demonstrated the genetic manipulation for E. coli cell which can convert sugar to bioethanol most effectively from glucose and xylose sugars. Kim and Reed (2010) demonstrated various genetic manipulation approaches for improved bioethanol production by E. coli using hexoses and pentoses and the approaches include removal of double, triple, quadruple, and quintuple genes associated. During anaerobic fermentation S. cerevisiae yields four different products from sugar, i.e., cell biomass, bioethanol, carbon dioxide as by-product, and glycerol accumulated during the process. Some of the metabolic engineering strategies for imparting carbon flux after glycerol to bioethanol involves modification of pathways. Bro et al. (2006) demonstrated various approaches for the redox engineering of the cell metabolism in yeast for the improved bioethanol yields. In silico assessment of the influence of a couple of metabolically modified approaches on E. coli has been reported in the framework of batch co-culture process. The co-culture fermentation includes parent strain of S. cerevisiae that naturally uses only glucose and engineered E. coli strain that utilize only xylose. The removal of the pfl gene only accounts to improve the 31.7% in 50:50 ratio of glucose and xylose. That happened because of the redirection of the carbon flux. Genome level metabolic engineering tactics are time exhausting and the detection of suitable genetically engineered cells is a crucial task. Hence, the findings of in silico

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analysis give important advice for directing in vivo setup and which can reduce the frequency of such costly and time-consuming examinations. Various attempts in distinct areas have been taken out in relative to ethanol production, like substrate expansion, which is planned to improve the productivity and execution of cell during fermentation (Matsutani et al. 1992). Bioethanol production was enhanced by removing the GPD2 gene (glycerol 3-phosphate dehydrogenase) and by overexpressing the gene responsible for the glutamate synthase, i.e., GLT1. in yeast. Present improvement was accomplished by reducing glycerol production and by expanding the conversion flow of NADH to NAD+ (Kong et al. 2007). In one more approach, the gene GPD1, an NAD+ dependent glycerol-3-phosphate dehydrogenase, was replaced by a non-phosphorylating NADP+ GPD obtained from Bacillus cereus in a bioethanol-producing yeast. The obtained strain was reported for low concentration of glycerol and displayed a higher bioethanol quantity (Hirasawa et al. 2007). Though, the lower production of ethanol and the deficiency of high-efficient genetic exploitation strategy restrict its usage. A novel transcription activator-like effector nuclease (TALEN) vector including the left and right arms of TALEN was incorporated into Saccharomyces cerevisiae to sequence ADH2 gene and the hygromycin-resistant gene hyg. The TALEN vector and ADH2 PCR product were incorporated into ΔADH2 to complement the ADH2 gene. Results obtained that bioethanol production was enhanced by 52.4
5.3% via the interruption of ADH2 (Ye et al. 2016). Wild strain of S. cerevisiae can only ferment ethanol by exploiting glucose; the drawbacks of low ethanol concentration and inaccessibility of other substrates, such as polysaccharides, avoid the wide consumption of natural S. cerevisiae (Kuyper et al. 2005; Matsushika et al. 2009). The GDP1 gene encrypting glyceraldehydephosphate dehydrogenase was incorporated in the S. cerevisiae to expedite NAPDH regeneration, so supporting ethanol production using xylose via the pentose pathway (Verho et al. 2003). There is an enhanced concern in utilizing thermophilic microorganisms to produce ethanol from polysaccharide biomass due to the higher operating temperatures and wide substrate variety. For production of second-generation bioethanol the thermophilic microorganisms are suitable (Taylor et al. 2009; Chang and Yao 2011). The degradation spectrum of the thermophile’s bacteria is considerably large as compared with S. cerevisiae and Z. mobilis and their growth does not involve broad mixing, cooling, or heating of the production vessel. Moreover, direct ethanol extraction from the production broth is feasible by in situ vacuum distillation. Thermophiles are more equipped machinery to tolerate excesses of pH and salt present during the process with minimum nutritional needs (Taylor et al. 2009). The major problem of most isolates of thermophiles is producing mixed end products subsequently in low ethanol concentrations and the element that extremely ethanologenic microbes are not naturally cellulolytic and vice versa. There are two major strategies to exploit genetically manipulated thermophilic: increasing the ethanol concentration of cellulase producing strain or expressing cellulases in appropriate ethanologenic strain (Shaw et al. 2010). The first tactic includes increasing ethanol concentrations by eliminating other fermentation end products and

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enhancing ethanol tolerance, while the second tactic entails accumulation of cellulolytic genes to the strain of an efficient ethanol producer. Thermoanaerobacterium saccharolyticum was the initial thermophile which was genetically manipulated to improve the bioethanol production (Desai et al. 2004). In the last decade, various other ethanologenic thermophiles have been metabolically manipulated to enhance ethanol concentration and reduce the production of other by-products like acetate and lactate. Removal or deletion of the product formation genes, positively, increases ethanol concentration is the highly apparent path to grow ethanol titers. Clostridium thermocellum ability to hydrolyze cellulose and ferment it to the ethanol has preceded to exhaustive investigations in this area (Xu and Tschirner 2014). Later experiments with Thermoanaerobacterium saccharolyticum comprise an LDH gene knock (Shaw et al. 2008). The deleted hfs gene cluster and LDH gene is responsible for hydrogenase and LDH, respectively. A significant increase in ethanol (44%) concentration was achieved in contrast to wild type strain (Shaw et al. 2009). Overall, attempts to engineer thermophilic anaerobes for enhanced ethanol production have resulted in limited gains in concentration while reducing the establishment of undesirable end products. Potential goals for genetic modification may perhaps involve the inclusion of the cellulolytic pathway of C. thermocellum into well ethanologenic thermophilic strains.

Tolerance Against Inhibitors, Temperature, and Solvents Numerous biological approaches can be utilized to defeat inhibitory impacts of aliphatic acids, furaldehydes, and phenolic substances on yeast utilization in pretreated lignocellulosic substrates. Probable strategies involve detoxification of the substrates prior to fermentation by exploiting certain enzymes like laccases or utilization of the natural or targeted genetically manipulated bio-reduction potential of the fermenting strain that will cleanse the substrate through the fermentation. The potential to disintegrate inhibitors occurs in S. cerevisiae and another strain and we only want to develop or improve this native approach to daze inhibitors in lignocellulosic substrates in several instances across variation and genetic manipulation. The process can be conducted out in a fermentation design like fed batch which will permit for the biological reduction ability of the strain to be utilized. Okuda et al. (2008) examined the microbial detoxification of lignocellulosic hydrolysate by using a thermophilic bacterium, Ureibacillus thermosphaercus. The overall yield of the bioethanol production by this strain detoxification is like that of calcium hydroxide cure. Chromatographic testing proved that U. thermosphaercus removed the furfural appear in the synthetic hydrolysates, and the phenolic substances present in the wood hydrolysates. The strain grows quickly and utilizes fewer than 5% fermentable sugars. Various fungal strain has been reported for the reduction of the furfural such as, Coniochaeta ligniaria was described to degrade furfural corn stover hydrolysate (Nichols et al. 2008). Chromatographic analysis described the absence or removal of the compounds indicating all the substances of inhibitory by-products during the

Genetic Modification of Plants for Bioethanol Production

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growth phase of the fungal growth and increased xylose utilization in consequent bioethanol fermentations. In a similar study, López et al. (2004) isolated several strains, involving 5 bacteria associated with Methylobacterium extorquens, Pseudomonas sp., Flavobacterium indologenes, Acinetobacter sp., Arthrobacter aurescens, and one fungus, C. ligniaria. All the strains were proficient of diminishing toxic substances from specified mineral medium including a blend of ferulic acid and furfural. Though, only C. ligniaria was found useful in eliminating furfural from corn stover hydrolysate. Detoxification of the sugarcane bagasse was achieved using Issatchenkia occidentalis and Iris orientalis (Hou-Rui et al. 2009). Fermentation of the biological detoxification results in a rise of xylitol productivity. Role of the enzymes in the detoxification also plays an important role in the sugarcane bagasse hydrolysate such as laccase enhanced ethanol concentration by Candida shehatae. The ethanol concentration attained after laccase action was similar to the one detoxificated by using chromatographic and activated carbon method (Chandel et al. 2007). The enzymatic treatment brought about a 77.5% if the sugarcane bagasse phenolics with not influencing furans and acetic acid matter. Adaptive modification of the process strain to the lignocellulosic biomass has been indicated as an option to detoxification method. While chemical, enzymatic, and biological detoxification enhances the fermentability of sugar hydrolysates, it is advantageous to build adaptive bioethanol-producing strain that needs minimal treatment. Such adapted strain not only lessens the detoxification price, but also prevents failure of fermentable sugars (Keller et al. 1998; Rivard et al. 1996; Martín et al. 2007). An alternate method would be to genetically modify S. cerevisiae for production of effective laccase. This would permit detoxification and bioethanol fermentation to continue concurrently in one step, thus reducing the detoxification step and decreasing the price of laccase production (Larsson et al. 2001).

Genetic Modification of Plants for Bioethanol Production One of the bottlenecks in the cellulosic ethanol production at commercial levels is reducing the cost of pretreatment. The pretreatment aims at increasing the digestibility of lignocellulose by deconstructing its structure. The costly pretreatment methods pose a problem in the economic feasibility of the biomass bioconversion process. Lignin is the main barrier in this bioconversion. The biomass polysaccharides, i.e., cellulose and hemicellulose, are embedded in a matrix along with lignin. The biochemical nature of lignin accounts for the recalcitrance of the whole lignocellulose structure. The final yield of fermentable sugars depends on the carbohydrates content of the lignocellulose. Biomass with high polysaccharides level and low lignin level is desirable for obtaining high yields of ethanol in the bioconversion process. Also, the presence of hemicellulose can reduce the accessibility of cellulose to the cellulases enzymes, consequently decreasing the saccharification yields. The genetic modification of the plants can be aimed at reducing the lignin level and

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Fig. 5.3 Approaches Fig. 1 ! Fig. 5.1 Fig. 2 ! Fig. 5.2 Fig. 5 ! Fig. 5.3 to enhance cellulosic ethanol production by plant genetic engineering

increasing the cellulose content of the biomass (Sticklen 2008; Wang and Zhu 2010). Also, the genetic manipulation systems can be employed to produce cell wall hydrolytic enzymes in the feedstock crops themselves. In case of energy crops, the genetic engineering can be done to obtain plants with fast growth, high yields, low recalcitrance, low lignin content, low nutrients requirements, enhanced water use efficiency, etc. (Yang et al. 2011). Thus, different approaches can be used, which may ultimately enhance the bioethanol production from lignocellulosic biomass using plant genetic engineering (Fig. 5.3). The genetically modified plants have been produced using two techniques primarily, i.e., gene transfer using Agrobacterium tumefaciens or gene-gun method (Lee et al. 2008; Sticklen 2008). However, new methods are being continuously developed and explored to generate efficient transgenic plant systems.

Reduction and Modification of Lignin Decreasing the lignin biosynthesis or modifying the chemical structure of lignin may reduce the cost of pretreatment required for biomass bioconversion. The lignin is made up of three main monolignols, i.e., p-coumaryl, coniferyl, and sinapyl alcohols. The pathway for biosynthesis of all precursors is not understood completely. The phenylpropanoid pathway is most popular among biologists. The genes tangled in lignin synthesis, regulation, or polymerization can be overexpressed, downregulated, or suppressed. Franke et al. (2000) overexpressed the ferulate 5-hydroxylase (F5H) gene from Arabidopsis in tobacco and poplar trees. The enzyme is involved in the sinapyl and syringyl lignin biosynthetic pathway in the hydroxylation of ferulic acid, coniferyl alcohol, and coniferaldehyde. The modified plants showed an alteration in their lignin composition. Fu et al. (2011) downregulated the caffeic acid O-methyltransferase (COMT) gene in switchgrass, which resulted in decreased lignin content and altered composition of lignin in the plant. The genetically modified plant required less severe pretreatment and lower

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enzyme loads for biomass saccharification, compared to unmodified plant. Cai et al. (2016) expressed an engineered 4-O-methyltransferase in Populus wood, which modifies the monolignols and prevents their incorporation into lignin. The transgenic plant had lignin with altered structure and lower content. Lee et al. (2017) have successfully overproduced disinapoyl esters (DSEs, resembling sinapyl alcohol monolignol), in Arabidopsis, which could function as hydrolysable subunits of lignin. The downregulation of the genes involved in the biosynthetic pathway reduces the lignin content considerably. Shafrin et al. (2017) downregulated cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT) genes in jute fiber crop. The study showed the 13–14% reduction in the fiber lignin content and an increased level of cellulose content in the transgenic plants. The reduced recalcitrance and increased cellulose level resulted in enhanced sugar release during enzymatic saccharification of the modified plants. Various other studies also revealed that downregulation of cinnamyl alcohol dehydrogenase (CAD) enzyme, COMT, and CCoAOMT (S-adenosyl-methionine caffeoyl-CoA/5hydroxyferuloyl-CoA-O-methyltransferase) genes in alfalfa has resulted in a reduction in lignin content and an alteration in the relative content of monolignols in the transgenic plants (Hisano et al. 2011).

Increase in Cellulose Content The presence of cellulose, the glucose polymer, in higher proportions in plant matter is a desirable attribute for bioethanol production. The cellulose content in the plant biomass can be increased using different mechanisms. The inhibition of lignin biosynthesis generally increases the growth and cellulose biosynthesis in plants. Wei et al. (2005) have generated high cellulose accumulating transgenic loblolly pines, by downregulating 4-coumarate: coenzyme A ligase (4CL) gene. The synthesis of cellulose is carried out by plasma membrane-associated rosette-like structure consisting of cellulose synthase (CesA) enzymes. In addition to CesA genes, several other genes, such as those for membrane-associated endo-1-4β-glucanase, sucrose synthase, and UDP-glucose: sterol glucosyltransferase, are also involved in cellulose biosynthesis. Zhang et al. (2013) have demonstrated that overexpression of UDP-glucose pyrophosphorylase gene (involved in cellulose synthesis) resulted in an increased height and cellulose content in jute. Sumiyoshi et al. (2013) have shown increased cellulose accumulation and enhanced saccharification yields in transgenic rice plants by overexpression of arabinofuranosidase. The arabinose otherwise carries an ester-linked feruloyl substituent, which forms diferuloyl cross-links between arabinoxylans and also bonds to lignin polymers. The overproduction of the enzyme decreased the arabinose content of the transgenic plant and a consequent increase in glucose in the saccharification process.

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Increase in Biomass Content Because cellulosic ethanol production relies on plant biomass, therefore, increasing the plant biomass by genetic engineering is a straightforward approach to enhance bioethanol production from lignocellulose. The overall plant biomass yields can be increased by modifications involving single or specific genes. The alteration of various genes such as those for photosynthesis, transcription factors, phytohormones control, metabolism, cell cycle regulation, and the microRNAs have been found to be effective in increasing the biomass yields in different plants (Rojas et al. 2010; de Freitas Lima et al. 2017). Sakhno (2013) has discussed genetic engineering in various plants such as tobacco, Arabidopsis, Triticum, rice, Brassica, and alfalfa, etc. resulting in increased biomass in the modified plants under different environmental conditions. A study by Simkin et al. (2017) was based on the idea of enhancement of photosynthesis and plant biomass synthesis with the increase in the level of photosynthetic enzymes. They overexpressed Rieske FeS protein (PetC) in Arabidopsis thaliana. Rieske FeS protein is a component of the cytochrome b6f (cyt b6f) complex. The overexpression of protein influenced the quantum efficiency of PSI and PSII, thus, electron transport in the genetically modified plant. This resulted in an increase in the biomass and yield of seeds in plants. Biswal et al. (2018) generated transgenic switchgrass and poplar plants by reducing the expression of a pectin biosynthesis gene (galacturonosyl transferase 4, GAUT4). The results demonstrated up to six folds increase in biomass yield of the modified Switchgrass plants in a 3-year field trial. Fan et al. (2020) generated the transgenic poplar plant for overproducing brassinosteroids by overexpressing the DEETIOLATED2 gene. They observed increased plant growth rate and biomass in the transgenic plants, as evidenced by increase in xylem and deposition in cell wall polymer.

Synthesis of Cell Wall Hydrolytic Enzymes by Transgenic Plants The cell wall hydrolyzing enzymes used for feedstock saccharification are produced at commercial levels using microbial systems. The microbial genes for these hydrolytic enzymes can be introduced in the plants to construct transgenic plants. The energy inputs required for growing genetically modified plants are lower than the production of enzymes using microbes (Sticklen 2008). This is because establishing microbial enzyme production facilities requires investments. An additional advantage is that the propagation of modified plants can scale up enzyme production (Lee et al. 2008). However, the expression or overproduction of glycosyl hydrolases in plant systems may interfere in normal development of the plant. Therefore, different strategies are employed to reduce the negative impact of hydrolytic enzymes (Willis et al. 2016; Xiao et al. 2016). One such strategy is the temporal separation of enzyme production and plant growth using inducible expression systems, which allow protein expression only in the presence of a chemical or environmental signal. Another approach is based on attaching specific target peptides to the C- or

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N-terminus of proteins for subcellular targeting to organelles including apoplast, endoplasmic reticulum, and vacuoles. The subcellular targeting of enzymes in different compartments shows advantages such as correct folding and activity, glycosylation, minimized degradation, and enhanced stability (Sticklen 2008). Also, the cellulolytic enzymes from thermophilic sources can be expressed, which do not disrupt the biomass structural integrity because they are inactive at normal conditions of the plant growth (Xiao et al. 2016). The heterologous expression of glycosyl hydrolases in plants for enhanced cellulosic ethanol production was proposed in the 1990s. The production of various bacterial and fungal enzymes has been tested in plants. Willis et al. (2016) have recently emphasized the potential of insect-gut derived hydrolytic enzymes for producing transgenic plants. Harrison et al. (2011) generated genetically modified sugarcane plants producing three cellulases, i.e., fungal cellobiohydrolase I (CBH I), CBH II, and bacterial endoglucanase (EG) using maize PepC promoter for controlling the gene expression. They accumulated the recombinant enzymes in leaves using subcellular targeting. Harrison et al. (2014) expressed the cellobiohydrolase (CBH, with 96% amino acid sequence similarity with CBH I from Penicillium occitanis) in corn and used the recombinant enzyme from corn stover leaf extracts for enzymatic saccharification of sugarcane bagasse. The study showed that the enzyme preparation from transgenic plants enhanced the performance of commercial cellulases (Celluclast 1.5 L and Cellic CTec2) nearly four times at the pilot scale. Hussain et al. (2015) have also generated transgenic wheat co-expressing the endoglucanase 1 (E1) gene of Acidothermus cellulolyticus and cellobiohydrolase 1 (CBH1) gene of Trichoderma reesei, using Rubisco small subunit promoter (RbcS) of wheat. The E1 endoglucanase enzyme from Acidothermus cellulolyticus has been used widely for producing transgenic plants because of its hyperthermophilic nature. It does not interfere in plant’s growth as it remains inactive at temperatures favoring the growth of the plant (Willis et al. 2016). Li et al. (2018) used transgenic rice plants overproducing Trichoderma reesei β-1,4-D-glucosidase (BGL I) and achieved higher biomass enzymatic saccharification in the non-pretreated biomass of transgenic plants compared to wild type. The enhanced digestibility was attributed to several factors such as increased porosity of biomass, reduced cellulose crystallinity, and increased level of arabinose and lignin H-monomer, in the modified plant. Huang et al. (2019) overexpressed endo-β-1,4-glucanases (glycoside hydrolase family 9, GH9) in rice plants. The transgenic plants showed altered lignocellulose composition and overexpression of cellulases. The mild alkali pretreatment followed by subsequent enzymatic saccharification resulted in higher ethanol yields from the modified plants.

Decrease in Cellulose Crystallinity In the plant cell walls, the cellulose is organized as highly ordered crystalline and less ordered amorphous regions. The rate of cellulose hydrolysis is faster in the amorphous regions. Therefore, one approach to enhance the digestibility of

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lignocellulosic biomass is to reduce the degree of crystallization and cellulose polymerization in the plant cell walls. Maloney and Mansfield (2010) downregulated β-1,4-endoglucanase genes in the poplar plant and evaluated the changes in chemistry and ultrastructure of cellulose in transgenic plants. They produced trees with less crystalline cellulose having low xylose content. Fan et al. (2017) genetically modified rice plants by expressing OsSUS3 genes driven by Arabidopsis AtCesA8 promoter. The transgenic plants showed higher enzymatic saccharification and ethanol production, primarily as a result of reduced cellulose crystallinity of the biomass. Li et al. (2018) constructed genetically modified rice plants by overproduction of Trichoderma reesei β-1,4-D-glucosidase (BGL I) and observed reduced cellulose crystallinity and degree of polymerization in transgenic plants. Huang et al. (2019) also reported a decrease in biomass crystallinity and cellulose degree of polymerization in transgenic rice plants produced by overexpression of endo-β-1,4-glucanases. In another study by Fan et al. (2020), the transgenic poplar plant generated by overproducing brassinosteroids phytohormone showed reduced crystalline index and degree of polymerization of cellulose. Also, the hemicellulose xylose/arabinose ratio was decreased. All these factors together resulted in increased biomass porosity and accessibility, which consequently enhanced the enzymatic saccharification and bioethanol yield from the appropriately pretreated biomass of transgenic plants.

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Wei, T., Nelson, A., & Johnson, E. (2005). Increasing cellulose production and transgenic plant growth in forest tree species. Journal of Forestry Research, 16(1), 67. Willis, J. D., Mazarei, M., & Stewart, C. N., Jr. (2016). Transgenic plant-produced hydrolytic enzymes and the potential of insect gut-derived hydrolases for biofuels. Frontiers in Plant Science, 7, 675. Xiao, Y., Poovaiah, C., & Coleman, H. D. (2016). Expression of glycosyl hydrolases in lignocellulosic feedstock: An alternative for affordable cellulosic ethanol production. Bioenergy Research, 9(4), 1290–1304. Xu, L., & Tschirner, U. (2014). Immobilized anaerobic fermentation for bio-fuel production by clostridium co-culture. Bioprocess and Biosystems Engineering, 37, 1551–1559. Yang, X., Li, T., Weston, D., Karve, A., Labbe, J. L., Gunter, L. E., . . . Tschaplinski, T. J. (2011). Innovative biological solutions to challenges in sustainable biofuels production. In Biofuel production-recent developments and prospects. London, UK: IntechOpen. Ye, W., Zhang, W., Liu, T., Tan, G., Li, H., & Huang, Z. (2016). Improvement of ethanol production in Saccharomyces cerevisiae by high-efficient disruption of the ADH2 gene using a novel recombinant TALEN vector. Frontiers in Microbiology, 7, 1067. Zaldivar, J., Nielsen, J., & Olsson, L. (2001). Fuel ethanol production from lignocellulose: A challenge for metabolic engineering and process integration. Applied Microbiology and Biotechnology, 56(1–2), 17–34. Zhang, G., Qi, J., Xu, J., Niu, X., Zhang, Y., Tao, A., . . . Lin, L. (2013). Overexpression of UDP-glucose pyrophosphorylase gene could increase cellulose content in Jute (Corchorus capsularis L.). Biochemical and Biophysical Research Communications, 442(3–4), 153–158.

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Bioethanol: Product Separation Methods

Abstract

Bioethanol can be generated from various feedstock like corn waste, sugarcane residues, paddy and wheat residue, and other starchy materials. In recent times, ethanol comprising 99.5 wt % alcohol or extra is in huge demand for fuel purposes. Various methodologies that have been adopted to dehydration of bioethanol are pervaporation, extraction from fermentative broth, molecular mesh adsorption process, and fractional distillation. The recovery and downstream processing of the systems depends upon the different compositions which is affected by the presence of azeotropy. The ethanol amelioration outside the azeotropic mixture is not possible by conventional fractional distillation, precise replacements need to be working. Various methodologies to recover the ethanol from its azeotropic behavior are categorized as extractive distillation, adsorption. Further, improvement of membrane-based separation and simultaneous removal of ethanol will improve the obtained yield. Purity and ease of separation technologies makes ethanol more convenient, viable, and costeffective. Keywords

Separation · Downstream processing · Distillation · Molecular sieves · Pervaporation

Introduction Bioethanol is the utmost capable fuel of the forthcoming as it is produced from agricultural feedstocks and is of ecofriendly nature due to non-emissions of the greenhouse gases (GHG). Bioethanol can be generated from various feedstock like corn waste, sugarcane residues, paddy and wheat residue, and other starchy materials. First-generation fuels comprise ethanol and biodiesel which are # Springer Nature Singapore Pte Ltd. 2020 D. Sharma, A. Saini, Lignocellulosic Ethanol Production from a Biorefinery Perspective, https://doi.org/10.1007/978-981-15-4573-3_6

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directly obtained from a feedstock (Lee and Lavoie 2013). Second-generation biofuels are classified as fuels obtained from an extensive selection of various feedstocks, particularly but not inadequate to lignocellulosic feedstocks (Brennan and Owende 2010). Third-generation biofuel of algal fuels originated as of algae typically depends on the lipid accumulation by the cell (Hannon et al. 2010). Bureau of Indian Standards (BIS) declared that 10% ethanol can be used as to blend the ethanol in automotive fuel, in the year 2004 (Sakthivel et al. 2018). Blending of 10% ethanol works as antifreeze agent in the cold climate. Ethanol improves the efficacy of the engine and is an outstanding cleaning agent (Kaminski et al. 2008). In recent times, ethanol comprising 99.5 wt % alcohol or extra is in huge demand for fuel purposes. Various methodologies have been adopted to dehydration of bioethanol are pervaporation, extraction from fermentative broth, molecular mesh adsorption process, and fractional distillation. The ethanol amelioration outside the azeotropic mixture is not possible by conventional fractional distillation, precise replacements need to be working (Frolkova 2000). Various methodologies to recover the ethanol from its azeotropic behavior are categorized as extractive distillation, adsorption. The diverse approaches employed to obtain anhydrous ethanol for use as fuel are described below.

Membrane-Based Approaches Pervaporation Method Pervaporation is an approach for the separation of fermentative mixtures from their liquid state using fractional vaporization over a membrane. Pervaporation is regarded as an appropriate and active membrane technology to perform the recovery of comparable boiling point substances confined in an “azeotropic combination.” The advantage of utilizing the membrane-based process for such determinations is because of its high selectivity, competence, and low-energy necessities (Crespo and Brazinha 2015; Luis and Van der Bruggen 2015). Currently, pervaporation is regarded as an ecofriendly approach as a substitute for conventional approaches such as fractional distillation (Figoli et al. 2015). Pervaporation is not only useful to remove the ethanol but also be useful for several other molecule separation such as alcohol, tetrahydrofuran, acetonitrile, ethylene glycol, acetone (Premakshi et al. 2016; Wang et al. 2017; Ji et al. 2018; Han et al. 2015; Penkova et al. 2018; Khayet et al. 2008; Moulik et al. 2018; Guo et al. 2006; Khoonsap and Amnuaypanich 2011). Pervaporation is quite useful for the various other allied industries like food, healthcare, pharmaceutical, and chemical industries. In this approach of separation, a binary or multicommodity combination is parted by fractional vaporization utilizing a porous or non-porous membrane. The liquid mixture containing water and ethanol is in straight interaction with the discriminating cross of the membrane, though the infuse is in a vapor stage, which is augmented

Reverse Osmosis Based Separations

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with higher affinity. The permeating liquid is separated using the driving force of vacuum pump, sweeping gas, or temperature change and then condensed and separated (Crespo and Brazinha 2015). Various features distress the pervaporation effectiveness as temperature, feed quantity of flow rate, pressure of permeate flow, cutoff of membrane (Valentínyi et al. 2013). The selection of hydrophilic or hydrophobic membrane depends upon the feed concentration (Khalid et al. 2019). The permeate pressure is a critical factor, lower permeate pressure outcomes in enhanced routine by improving the flux and separation degree (Bello et al. 2012; Jiraratananon et al. 2002; Khalid et al. 2019). Likewise, the membrane area also upsurges when the stage cut value rises (Khalid et al. 2019). The membrane matrix is additional vigorous factor which determines the pervaporation efficiency (Khalid et al. 2019). Polymeric and inorganic ingredients are mostly utilized in this respect. Polydimethylsiloxane is the most important polymeric class material used to remove low concentration of ethanol (Peng et al. 2010). The Si-O bond freeness due to advanced ethanol passing capacity due to diffusion pays to the outstanding selectivity (Mohammadi et al. 2005; Miyata et al. 1996). Mixed membrane containing inorganic fillers type, biochar core shell particles is the perfect solution to tackle such problems (Te Hennepe et al. 1987; Zhuang et al. 2016; Naik et al. 2016a, b; Li et al. 2017; Zhu et al. 2017; Lan et al. 2017; Khan et al. 2018). Though, there is huge space in relations to the separation agent and relative flux. In last decades, various polymers have been used as choice of matrix in membrane-based pervaporation claim, which are presently utilized in the preparation of assorted matrix membranes for bioethanol separation and ethanol dehydration. The incorporation of the zeolites into membrane provides better solution for product seperation-based solutions. Zeolites are mainly aluminosilicate solids showing a -ve charge outline of microscale pores for separation (Rhodes 2010). Carbon nanotubes are also important with specific electromagnetic and mechanical characteristics. Additionally, silica can also be useful to fabricate hydrophilic membranes for ethanol separations. Though, the surface hydroxyl functional clusters of silica also effect the separation efficiency (Wan et al. 2010). Pervaporation is an efficient energy conserving substitute to facilitate the distillation and evaporation. The pore size should be of enough size which allows water molecules to permit through and hold any other impurities. As a outcome, a molecular based sieve of a pore size of approx. 4 Å is attained. The most commonly used material for such kind of operations are zeolites. The other membrane-based separation methods are.

Reverse Osmosis Based Separations The process of reverse osmosis (RO) has been utilized to develop the broth at the end of production process. Reverse osmosis is a kind of process where the osmotic pressure is applied to the membrane for separation of ethanol from production media. As a result, water molecules pass through contrary to the natural gradient,

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parting behind a suspension improved in ethanol purity. Reverse osmosis is an important step before performing the fractional distillation-based separation of the ethanol (Wenten 2016). Modified cellulose with reverse osmosis has been practiced purifying the bioethanol feed (Choudhury et al. 1985). But the use of elevated pressure in the separation procedure is one of the major bottlenecks in reverse osmosis approach. Lee et al. (1985) tried to block this matter by deploying the osmotic pressure incline. It was described that this approach could deliver several folds much energy saving as compared to the traditional distillation method. Though ethanol purification can be attained with reverse osmosis, the clumsiness of this approach incumbers investigation. The reasonableness is largely restricted by the elevated pressure, extreme case of fouling, inability of treatment higher concentrated bioethanol, and damage or deterioration of the membrane (Jiang et al. 2017). The surface alteration by means of reduced graphene oxide, silver nanomaterial has revealed better water desalination performance (Safarpour et al. 2015; Faria et al. 2017). Though, for industrial claims in bioethanol separation, reverse osmosis must be added to discover the above-discussed limitations. A dephlegmator is a kind of maneuver utilized to partly condense a multi-variant vapor (Haelssig et al. 2012). Dephlegmator cannot be explored for fuel rating enrichment; yet, it can be also utilized to the reflux stream directed to the fractionation and correcting columns (Haelssig et al. 2011; Bernhard et al. 1986). For alcohol concentration, dephlegmation can be explored to advance the effectiveness of pervaporation approach (Baker 2012).

Liquid–Liquid Separation Membrane liquid extraction is a combined approach where the traditional liquid– liquid separation has been achieved in a solo component and takes place. In this strategy, different nonmixing liquids, i.e., feed and solvent, are separated using a microporous membrane based on the difference in solubility (Núñez-Gómez et al. 2014; Groot et al. 1990). A general three-fold upsurge in the substrate ingesting by product parting was demonstrated by different batch and fed-batch cycles. Though, this hybrid approach is not so far been extensively sightseen for bioethanol separation. Snochowska et al. (2015) examined the utilization of membrane extraction. A hollow fiber filtration approach was used having inlet on the tube direction and the ionic liquid on the shell adjacent was settled. The optimal extraction productivity of 20.2% was attained with a feed quantity as low as 1 wt %. The maximal activity was reported using advanced feed flow rate with higher dilution concentration of the material. Consequently, an approach with microporous hollow fibers may deliver high mass transfer per unit volume with huge surface area. Though, membrane fouling, inadequate considerate of system of the separation and mass transfer, and the difficulty of the project are the key issues constraining the effective claim of this method (Aslam et al. 2017). A handful of information is only accessible in the information and additional inclusive research requests to be directed.

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Vapor Permeation Approach Vapor permeation approach is a practical method to be united with ethanol fermentation as an in situ mixture separation approach which could evade the ethanol inhibition. Vapor permeation means the transport of molecules over a membrane from a vapor feed mix solution to a vapor permeate. Vapor permeation is carefully connected to gas permeation contrary only in that a vapor mixture comprises substances that are condensable at typical conditions while a gas combination comprises only permanent gases. In the instance of a membrane of non-porous behavior, vapor permeation is connected to pervaporation, where feed phase is different (Kujawski 2000). In one case, combination of the vapor permeation assembly after distillation was demonstrated for additional improvement (Suematsu et al. 1989). Enrichment simulation concept with 5% wt ethanol feed was demonstrated (Vane et al. 2010; Vane and Alvarez 2008). The necessity of the extraction features on the working conditions (i.e., temperature, pressure, composition, superheating) is established with investigational results for designated mixtures and membranes.

Adsorption Methods The adsorption process is grounded on the intrinsic features of the adsorbing material. The fundamental process and superiority of the adsorption rate progression based on the traits of adsorbents. Various studies on many adsorbent materials of chemical and natural origin have been reported. Natural adsorbents are observed as a competitive material to be considered as an effective adsorbent (Banat et al. 2000; Al-Asheh et al. 2004; Lalik et al. 2006; Karimi et al. 2014). After distillation about 95% wt purity ethanol has been attained so, water based adsorbing materials are the molecule of choice. Natural adsorbents are further classified based on their origin and nature such as natural minerals and starchy adsorbents. Adsorbents of agricultural origin have enormous applications. Adsorbents originated from different seeds, types of cereals, beans, and other feedstocks (Sun et al. 2007; Al-Asheh et al. 2004; Baylak et al. 2012; Niu et al. 2014; Kong et al. 2014; Samiran et al. 2016; Khatun et al. 2017; Karri and Sahu 2018; Liew et al. 2018; Chang et al. 2006a, b; Quintero and Cardona 2009; Wang et al. 2010; Kim et al. 2011; Lam et al. 2017, 2018) are categorized as category I. While natural clinoptilolite (Ivanova et al. 2009, 2010; Karimi et al. 2016) and Phillipsite (Colella et al. 1994; Al-Asheh et al. 2009) are classified under category II. The main variances amid normal and chemically prepared adsorbents are the pore size and their circulation. Adsorbents working in the liquid phase procedure are type 1 (Huang et al. 2008). In some dehydration strategies the water–ethanol mixture is condensed by concentration, the ethanol is then dehydrated by molecular based sieves, and the adsorbent is renewed by desorption. Some disadvantage of this approach is the huge volume of liquid received at the molecular absorbent sieve revival phase. Performing out this approach in the vapor phase rejects the wetting of the molecular

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sieves. Investigators’ present approaches are being attentive on investigational strength of many zeolites for extracting ethanol–water (Ladisch et al. 1984; Lu et al. 2007). Carbon is an additional effectual water adsorbent. Performing out the procedure in an adiabatic carbon adsorbent established that the adsorbent is steady over 85 adsorption–desorption sets using hot air or nitrogen (Simo et al. 2008). The current commercial gage process called pressure-swing adsorption is directed in an adsorber–desorber complex (Pucci 1989). The inherent restrictions upsetting adsorbtion are adsorbent particle dimensions, pore magnitude, type of phase, temperature conditions, flow rate, and concentration (Karimi et al. 2019). In the active adsorption method, flow and holding time perform an important part in uttering the intrinsic adsorption mechanisms. Any upsurge in the flow rate improves the film mass transfer constant.

Extraction Methods If the mixture phase is replenished to the bioreactor, the removing agent must be together harmless and inert to cells (Offeman et al. 2008). It is relevant to use separation for aqueous solutions comprising 2–5 wt % bioethanol. Separation with paraffin oil is directed at 105  C, and the material is then placed at 30  C. The subsequent ethanolic material comprises 4–5 wt % shorter hydrocarbons and can be further straight to fuel. Extraction of water–ethanol suspension with supercritical CO2 has not been industrialized. However, supercritical CO2 extractant is utilized in the pharmaceutical formulations as an environmentally friendly approach (Rodrigues et al. 2006). CO2 is easy to utlised as it is gladly discrete from ethanol during distillation (Güvenç et al. 1999; Knez et al. 2008). Budich and Brunner (2003) observed the quantities of energy essential for gaining ethanol (kJ/l) by different methods: pressure swing, distillation with pentane, extraction with supercritical CO2. The probable of supercritical carbon dioxide for extraction of bioethanol is now being examined. In specific, the subsequent issues in batch extraction are reflux ratio, bioethanol quantity in the starting mixture, and extraction circumstances and time. Some of the researchers demonstrate the use of the supercritical CO2 for the extraction of the dilute or low quantity fermentation keys of ethanol (0.1–1.7 wt %). It was established that rising the pressure, temperature, and ethanol quantity in the primary mixture is promising for extraction (Pucci 1989; Budich and Brunner 2003; Schacht et al. 2008). As the aqueous mixture obtained from fermentation media containing 4–15% ethanol, they can be right extracted by pervaporation, or pervaporation or preconcentrated close to azeotropic conformation by distillation (Rodrigues et al. 2006; Simo et al. 2008; Schacht et al. 2008). As we have discussed above, currently the approach to extract azeotropic mixtures is based on concentration field rearrangement among separation sections. The major and primary component of this

Extractive Distillation

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azeotropic extraction comprise separation, each performing its specific function (Serafimov and Frolkova 1997; Frolkova 2000).

Distillation Distillation process is defined as the taking the fermented media containing ethanol and water, which is heat to distinct them-typically seperate in an elevated column. As ethanol evaporates quicker than water, the ethanol upsurges through a tube, gathers and condenses into added container. The water from the fermentation media is left behind. Extractive distillation (Ma et al. 2016; Gao et al. 2017; Jaime et al. 2018; Zhang et al. 2018; Sun et al. 2019; Hu et al. 2019; Yang et al. 2019a), pressure-swing distillation (Liang et al. 2017; Zhang et al. 2017; Ma et al. 2018; Yang et al. 2019b; Wang et al. 2019), azeotropic distillation (Mishra and Kaistha 2018; Dai et al. 2019; Han et al. 2019; Pla-Franco et al. 2019; Yang et al. 2019c; Li et al. 2019) are often used in petroleum, chemical industry, and pharmacological formulations to extract azeotropic solutions.

Extractive Distillation Extractive distillation is a fractional vaporization method, in the existence of a non-volatile and high boiling point extracting mixture that it is generally known as separating agent, which is supplementary to the azeotropic solution to change the relative volatility of the important constituent deprived of extra azeotrope creation (Fig. 6.1). With a suitable high boiling of extracting agent, the separation is consuming minimum energy demands (Timofeev et al. 2003). All-purpose necessities and traditional means for agents were defined by Kogan (1971). The current methods to estimate the separation require a deeper understanding of the extractive systems, Fig. 6.1 Flowsheet for the extractive distillation column (Courtesy by: Gil et al. 2014)

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with the mutual preparation of specific manifolds in the concentration (Serafimov et al. 2008). A reasonably innovative approach that was not been extensively utilized in the commercial extraction of water ethanol azeotropes is using solo column with ethylene glycol as the catalyst for the efficient separation (Brito et al. 1997; Chao et al. 2007; Kotai et al. 2007). Extractive distillation is capable for carrying out small batch distillation in different sectors. Brito et al. (1997) performed dehydration in the existence of ethylene glycol in single column with 20 holding trays. Solution with a known concentration is poured to the tray on 12th place, and nearly pure water is strained from the tray on serial position 6. The column can be snug with a second reboiler (Chao et al. 2007). The standard extractive distillation plan confirms the similar product superiority and ethylene glycol concentration, but it contains binary columns. The first column is for the ethanol extraction column. The second column is the water extracting column, with 20 trays. This kind of separation model is much energy and investment rigorous. Bioethanol dehydration knowledge engage not only traditional separation catalyst, but also liquified salts, combinations of liquid extraction catalyst and salts, liquids, and extremely branched polymers (Seiler et al. 2003; Huang et al. 2008). Extractive distillation approach with varied different salts concentrations is going to be a new option to get high purity products. This development cartels the conventional extractive distillation. With this collective approach, it is possible to solve various difficulties of transport, dissolution and corrosiveness.

Pressure-Swing Distillation Azeotropes of non-ideal component mixture are quite difficult to extract by utilizing usual distillation. Though, parting of non-ideal components is an actual routine process in various industries. In direction to attain better purity of the integral components, improved methods are active which in turn reduce the distillation which is extremely expensive. Of the commonly employed enhancement methods, pressure-swing distillation approach is the modest and most inexpensive method (Winkle 1967). A precondition for this approach is that the conformation of azeotrope must be changed suggestively with the variation in pressure (L’vov 1960). This approach is utilized in the dehydration of ethanol originated after production (Arifeen et al. 2007). A well-organized method of separating ethanol from the fermentation broth is by pervaporation, which accommodates the ethanol quantity of up to 40 mol %, surveyed by distillation in changeable swing pressure. In the present method, exploiting the heat of the process can decrease the operating expenditures for ethanol separation. That has engrossed special consideration from investigators as it has the excessive benefit of not needing any extra solvent to be presented into the system. Because of that, it is considered as an alternative approach to commonly applied azeotropic or extractive distillation (Fulgueras et al. 2016; Kumar et al. 2010; Lladosa et al. 2011). Because to its working difficulty, the process maximum output of a distillation column can produce investments of 20–50% of

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total energy utilized (Kiss et al. 2012). Relative studies demonstrate that the pressure-swing distillation is an improved choice than extractive distillation from the position of both financial reflections and product limpidness (Luyben 2013; Wang et al. 2015).

Azeotropic Distillation Separation In azeotropic type of distillation approach, a mixture establishing consistent azeotropes due to the presence of the early combination is acquaint with the column. The extraction of the azeotropic separation needs a distinct determination method to be used. It is usually considered that such method in furthermost situations is expensive in order of energy as the azeotrope mixtures experience numerous evaporations. Homo-azeotropic distillation is suitable when the solution of the azeotrope forming substances which is practical deprived of getting separated. The commonly used separating agent is ethylene diamine (Timofeev et al. 2003). Such distillation is usually utilized in different to distinct boiling point but closely placed for mixes and azeotropes by utilizing an extra entrainer to change the comparative volatility. Ethanol–water combination as a characteristic azeotropic classification has stayed widely reported by numerous professionals for its multifaceted features of conditional sensitivity, many steady conditions, long transient, and non-direct subtleties (Fig. 6.2). It is the azeotropic approach of distillation where the mixture is extracted intentionally in 2 stages by presenting a heteroazeotrope creating substances (L’vov 1960; Kogan 1971; Hilmen 2000; Vasconcelos and Wolf-Maciel 2000; Offeman et al. 2008). As the hydrolysis of vegetable residues feedstocks, waste, polysaccharides discard and rest materials of biological origin give dilute ethanol yields, it is suitable to perform traditional distillation to attain azeotropic conformation before heteroazeotropic distillation.

Adsorption–Distillation Ethanol can be extracted from water by uniting distillation and adsorption process. The column is functioned at an advanced pressure than desorption system. The catalyst is well dehydrated ethanol. After which the compression of the columns is altered due to which the adsorber converts the desorber and so on. The heat generated during the process is utilized to heat the distillation column. The distillate originated from this column, is near to the azeotropic conformation, which ultimately transferred to the vaporizer. The accumulated product at bottom comprises only hints of ethanol. An examination of the works done previously established that the expansion and industrialization of hybrid systems uniting membrane and column for distillation and membrane parting is among the importance areas today. Water– ethanol combinations are typically parted by pervaporation or permeation. Different ethanol hybrid dehydration systems with dissimilar briefings of mass transfer

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Fig. 6.2 Outlines of conventional heterogeneous azeotropic distillation (Courtesy by: Sun et al. 2011)

procedures for numerous feedstock configurations have previously been industrialized (Hilmen 2000; Koczka et al. 2007). The probable ethanol hybrid dehydration processes can comprise 1–2 distillation columns with the option of pervaporation. The presentation of all the discussed hybrid options can be enhanced by increasing the membrane surface area due to change in refining its geometry (Kafarov et al. 1996; Szitkai et al. 2002; Gomez et al. 2008).

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7

Lignocellulosic Waste Valorization and Biorefineries Concept

Abstract

Lignocellulosic waste is the extremely encouraging renewable feedstock for sustainable energy production and fine chemicals. Lignocellulosic biomass is structurally a complex mixture of the various compounds. Lignocellulosic biomass is not hydrolyzed to product like glucose or other simpler sugar. The hydrolysis of the lignocellulosic biomass yields hexoses and pentoses sugar, various by-products like organic acids, carbon dioxide, plant fiber, and lignin residues. The concept of biorefinery or circular bioeconomy is upsurging solution to make profit and maximal utilization of the lignocellulosic waste. The valorization of lignin and cellulose fractions into energy or fine chemical is depending on the usefulness of discerning depolymerization of the pretreatment scheme which usually involves harsh pyrolytic and solvothermal practices aided by corrosive acids or alkali. The existing solution to valorize lignocellulosic biomass is enzymatic hydrolysis due to its less energy necessity and fewer extent of pollution caused, but the major constraint is the low availability of cellulose due to its stiff association with lignin. So, to develop an efficient biorefinery or circular bioeconomy system which is commercially viable and fulfill upcoming expectation in converting lignocellulosic substrates into fuels. The conclusive aspects in a feasible lignocellulose biorefinery system will be the substrate availability and raw material supply. Devoted tools to care the application of advanced technologies and make the market acceptance of new stuff will be desirable to hasten the evolution. Keywords

Biorefinery · Waste valorization · By-products · Metabolites · Yield

# Springer Nature Singapore Pte Ltd. 2020 D. Sharma, A. Saini, Lignocellulosic Ethanol Production from a Biorefinery Perspective, https://doi.org/10.1007/978-981-15-4573-3_7

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Background and Introduction Greenhouse gasses emission and accumulation due to domestic and industrial emissions leads to buildup of large amount of carbon dioxide mainly due to the economy propelled by fossil fuels. On the contrary, fossil fuel exhaustion has been recognized as a upcoming challenge (Kumar et al. 2015). Lignocellulosic waste is the extremely encouraging renewable feedstock for sustainable energy production and fine chemicals (Sharma 2016). Lignocellulosic biomass is structurally a complex mixture of the various compounds. Lignocellulosic biomass is not hydrolyzed or converted to product like glucose or other simpler sugar. The hydrolysis of the lignocellulosic biomass yields hexoses and pentoses sugar, various by-products like organic acids, carbon dioxide, plant fiber, and lignin residues (Sharma and Dhanjal 2016). As it is evident that a low level of bioenergy obtained from lignocellulosic waste as compare to agricultural feedstock like starch and sugarcane. Secondly, due to high cost of production including pretreatment actions to deconstruct biomass components via disruption of the intractable structure of native rigid polymers; low effectiveness of enzymatic hydrolysis of recalcitrant feedstock poses a major task. The concept of biorefinery or circular bioeconomy is upsurging solution to make profit and maximal utilization of the lignocellulosic waste. The valorization of lignin and cellulose fractions into energy or fine chemical is depending on the usefulness of discerning depolymerization of the pretreatment scheme which usually involves harsh pyrolytic and solvothermal practices aided by corrosive acids or alkali. Such practices end up with the nonselective degradation of the lignin into several products that may not be valuable as a chemical constituent or energy solution and in many cases it has end up as toxic agents accumulated during the process. Investigating milder, selective, and greener industrial chemistry approach, consequently, has grown into a crucial focus of study for the valorization of lignocellulosic biomass. Recent economic and policy expansions in United Nations and various countries are keen to generate alternative energy over to fossil fuels drawbacks: • Finite or nonrenewable supply • Greenhouse gasses production and global warming • Increasing price and unexpected fluctuations. All these limitations have reinforced the concern in replacements, renewable, green, and reasonably viable energy solutions like ethanol. Additionally, ethanol can be either blended with gasoline or can be utilized as a lone energy source using devoted engines; besides, it has higher intensity of vaporization and octane number as contrasted to gasoline. Agricultural residues such as starchy crops, sugarcane bagasse, and corn stover are ideal feedstock to produce the first generation of bioethanol which are not costeffective or industrially viable yields. But, the second-generation fermentations include lignocellulosic biomass, which are inexpensive, not debatable as fuel vs. food, abundant, and renewable. Lignocellulosic waste mainly composed of

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cellulose, hemicelluloses, and lignin in a complex structure, which is obstinate during decomposition. The existing solution to valorize lignocellulosic biomass is enzymatic hydrolysis due to its less energy necessity and fewer extent of pollution caused, but the major constraint is the low availability of cellulose due to its stiff association with lignin. So, to develop an efficient biorefinery or circular bioeconomy which is commercially viable for converting lignocellulosic materials into fuels.

Lignocellulosic Biomass Valorization Presently, our society mind setup is changed from our tradition values and concept of the “Collection, processing and throwing away make” to “recycle and recovery” of resources to gain “green environment and well-being of biodiversity and species health with socio-economic wealth.” Lignocellulosic feedstock is utmost lucid biomass obtained from plant kingdom. Lignocellulosic biomass appears as sustainable substitute renewable substrate to the traditional nonrenewable substrates such as fossil fuel (Fig. 7.1). The advantageous features of the lignocellulosic biomass make it most suitable renewable source to be used as potent energy solution because of waste nature and availability, inexpensive, stable price, rich source of carbohydrates, adaptable, and non-competitive to food vs. feed debate. However, conversion of plant biomass into ethanol is still complex and it limits its commercialization scale up and production at viable quantity. Even though different integrations of specific technological modification have been inspected and out of that few are quite prominent like solid state fermentation (SSF), submerged fermentation, biorefinery concept, and circular bioeconomy for competent conversion of lignocellulosic hydrolysate into ethanol. So, integration of the different approaches and the process optimization is still a key issue which is essential to be addressed in coming years for industrial scale up. The common challenges of the current lignocellulosic conversion to bioethanol are the low yield of fermentation product, residual sugar, and other toxic compound accumulated during the process. Even the nature and composition of the lignocellulosic contents of biomass significantly change with respect to the source and type of biomass. The cause for low fermentable sugar concentration is also due to partial biodegradation of lignocellulosic biomass during hydrolysis. For effective research practices should be stimulated to overwhelm such kind of technical hurdles concerning execution on commercialization scale. One of the key alternatives to this problem is enzymatic hydrolysis of the lignocellulosic biomass through evolving potential inexpensive of cost-effective enzyme cocktail as a promising choice. Consequently, an inclusive approach would be more suitable to appraise the degree of effects of such factors on the effectiveness of the process and ethanol titer. Fermentation of glucose and xylose sugars fractions is a key methodical barrier for scale up of ethanol production from lignocellulosic biomass. Such bottleneck can be daze through screening out naturally accessible potent microorganisms with a

Fig. 7.1 Levels and generations of the valorization of the lignocellulosic biomass

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potential of hydrolyzing different types of sugars and can stand to the inhibitors. Alternatively, for lignocellulosic biomass valorization, specific engineered strain using system biology approach, metabolic and evolutionary engineering approaches for strain improvement are required. Further, pentose utilization, overexpression of present pathways (Pentose phosphate pathway), down regulation of by-product pathways leading to toxin accumulation.

Biorefinery Concept Circular bioeconomy or biorefinery approach is a facility that incorporates biomass transformation practices and equipment to generate clean fuels, power, and valueadded chemicals from waste. Biorefinery is like existing petroleum refinery, which generates multiple fractions of the fossil fuels according to their properties and fractions. By generating various products, a biorefinery holds benefit of the many fractions in waste and their intermediates, thus expanding the value derived from the lignocellulosic feedstock. Biorefineries not only solve the energy crises but may also settle the problems of waste management and mitigation of greenhouse gases. Lignocellulosic wastes can be transformed, via suitable enzymatic and chemical approach, into either gaseous or liquid fuels. Such kind of biorefinery solutions generates products such as paper-pulp, solvents, organic acids, resins, laminates, adhesives, flavors, activated carbon, bioethanol, residual sugars, etc. which commonly remain unexploited in the conventional processes (Fig. 7.2). The appropriateness of this process is further improved from the point that it can use a range of biomass asset of plant origin. The idea of the biorefinery for valorization of biomass was conceptualized by Cherubini (2010). The notion of the biorefinery was utilized to competently yield value-added materials from various feedstocks like lignocellulosic waste (Özdenkçi et al. 2017), algal feedstock (Bastiaens et al. 2017), food wastes (Esteban and Ladero 2018), biologically remediated feedstock (Mohan et al. 2016), and compost (Chen et al. 2005).

Fig. 7.2 General outline of biorefinery concept and levels

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The International Energy Agency “Bioenergy Task 42” termed biorefining as “the sustainable sort out of biomass into a variety of bio-based materials grouped in food, feed, value added chemicals and other materials and bioethanol. Biorefineries can be a tool to fractionate multiple chemicals or products by fractioning to various intermediates (hexoses and pentoses, proteins and triglycerides) that can be additionally converted into value-added compounds. Every step of an ideal biorefinery is also described as a “cascading phase.” The classification of the biorefinery depends upon the type and level of the processing or valorization of the biomass. Substantial research exertions are still essential in order to stand the lignocellulosic biorefinery a justifiable and economically viable process. So far, different generations of the biorefinery notion have been expressed and documented (Clark and Deswarte 2008). Different phases of the biorefinery have been worked out and formulized as: • Stage I biorefinery (single feedstock, simpler process, and major product) • Stage II biorefinery (single feedstock, multilevel processes, and many end products) • Stage III biorefinery (complex feedstocks, integrated processes, and multiple end products) There are a number of constrains associated with the construction and plan of biorefinery process such as: • Constraints to attain the maximal efficiency with upgraded designs as well as incorporation of conversion stages, i.e., upstream and downstream processing. • Constraints to handle extensive range of feedstock and workout the regional and suitable regional solutions over global homogenization or uniformity. • Constraints to take all the magnitudes of the biorefinery design factors such as feedstock features, feedstock quality, and availability; trade-offs between energy consumption for feedstock and product circulation, production, and product market prices.

Utility of a Lignocellulosic Substrates Lignocellulosic substrate is the substance of interest for claims that value from its furniture to home construction or paper and craft production. Lignocellulosic biomass primarily comprises various natural polymers occurring in form of cellulose, hemicellulose, and lignin. In addition to this, lignocellulosic substrates include varying quantities of moisture, proteins, and various minerals depending on its source (Dahmen et al. 2019). But, the viability of this segment actual hinge on the unpredictable oil costs and the financial backing. In adding, the growth of bioenergy globally is impeded by the enduring serious debate on the sustainability of plant biomass availability (Lewandowski 2015). It is anticipated that the utilization of lignocellulosic feedstock to get value-added chemicals and other metabolites will

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rise, since feedstock is the only resource of renewable carbon and because of its comparative availability and appropriateness. The going on debate on suitability of lignocellulosic biomass in Europe and even other regions on contesting biomass utilization and potential source has directed to the subsequent standards for “sustainable” feedstock source (Lewandowski 2015): • The lignocellulosic feedstock is not utilized for nutrition or animal feed uses. • The lignocellulosic feedstock is not essential to sustain environmental purposes, like soil humus matter or to substitute nutrient removal to sustain ecological balance. • The lignocellulosic biomass should be grown and cultivated on marginal land (Elbersen et al. 2018). • The appropriateness and selection of the regional lignocellulosic biomass should be promoted. Regional availability and utilization of the lignocellulosic biomass should be promoted to minimize the transport cost and environmental impacts. It has been established that the transport distances of up to 50 km for the conversion to bioethanol are only feasible in terms of cost and other operational viability. So, for sustainable biorefinery establishment, the approach should be carefully evaluated for its sustainability, life cycle assessment, environmental impact, and socioeconomic distribution. Lignocellulosic biomass production may ensure income for communities and small farmers in tribal and rural areas (Lewandowski and Faaij 2006). One reason promoting the usage of lignocellulosic substrates that is frequently referred to is the point that it does not contend straight with food source (Nanda et al. 2015; Memon et al. 2018). It is expected that nearly 40% of agrarian biomass need to stay on the field and an extra 20–30% are redirected into several on-farm usages, chiefly food (Daioglou et al. 2016).

Biorefinery of Lignocellulosic Biomass Lignocellulosic feedstock is considered as a renewable carbon rich substrate, presenting multivariant paybacks to produce bioenergy and compounds. The consumption of substrate as a foundation of energy is partial by its availability and the maximal translation amount (Basu 2018). The justifiable cultivation and obtaining of lignocellulosic biomass is the major apprehension on the use of agricultural produce (Gavrilescu 2014). In view to increase the usage of feedstock, comprising the feedstock produced from the numerous translation pipelines, and alter it to valued added products, the combined biorefinery is well-thought-out. In a biorefinery infrastructure process in which different conversion technologies like thermochemical, combustion, and microbial conversions are combined to produce sustainable value-added products (Table 7.1). To create cost-effective process of lignocellulosic substrate hydrolysis biomass, the merging of saccharification and production processes is an advantageous

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Table 7.1 Value-added chemicals and fractions potentially derived from lignocellulosic biorefinery (Adopted from: Menon and Rao 2012) Lignocellulosic biomass

Cellulose

Hemicellulose

Lignin

Polymer Levulinic acid Ethanol Lactic acid Propanoic acid Itaconic acid Glutamic acid Glucuronic acid Succinic acid Xylitol, ethanol Hydrogen 2,3 butane diol Ferulic acid, lactic acid Furfural Chitosan Xylo-oligosaccharide Syngas Hydrocarbons Phenols Oxidized products Macromolecules

approach, but it needs the potential strain capable of cellulose/hemicellulose hydrolysis with desired products. For establishing such type of process, various prerequisites such as engineered strain able to produce cellulolytic enzymes, with extended substrate spectrum, tolerance to intermediate toxic compound, and productivity titer (Menon and Rao 2012; Mussatto and Teixeira 2010). Lignocellulosic biomass is a viable source of feedstock, taking into consideration the extensive range of plant possibilities and high accessibility in humid weathers. Out of total lignocellulosic biomass produced only 3% are used for chemical production, biofuels, and other stuff (Baruah et al. 2018). Primarily, feedstock is obtained from barley waste, coconut shell, corn, trimming fruit cluster, rice straw, sugarcane waste, sorghum shoots, wheat straw, and other waste (Zhang 2008; De Bhowmick et al. 2018). Biorefinery approach to produce the bioethanol using non-woody lignocellulosic feedstock of Miscanthus sinensis L. (Chinese silver grass) to get cellulose has been developed. A cohesive process composed by ethanol organosolv pretreatment pursued by a membrane ultrafiltration process was used to obtain various fractions. In the end, production expenses of the ultrafiltrated lignin were approximately 52D/tonne of lignin. The “lignocellulose feedstock biorefinery” requires “naturedry” raw substance like wood, crop straw, forest liter, and agricultural lignocellulosic remains. It is estimated that 10–50 billion dry tonnes of lignocellulosic remains

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Fig. 7.3 Dispensation of lignocellulosic biomass by various thermochemical and biochemical conversions (Courtesy by: Menon and Rao 2012)

are available as a raw material which can be utilized for bioethanol production. Combining organosolv approach with coupled separation method, i.e., membranes ultrafiltration, an efficient and fundamentally economic system can be used with tangible industrial applications. Liquid bioenergy agents like bioethanol, pyrolysis oil, and various fractions of gaseous fuels are the different forms of energy which can be obtained from lignocellulosic biomass by its by thermochemical or biochemical handling (Fig. 7.3). The pretreated substrate can be managed using range of process designs like distinct hydrolysis and fermentation, real-time sugar extraction and fermentation, concurrent saccharification and fermentation by different strains, and combined biomass handling (Fig. 7.4). Integrated or combined processing of the lignocellulosic waste lowers the production and capital cost.

Sustainability Assessment of Biorefineries For a successful second-generation biorefinery it is crucial to have an efficient process and cost-effective supply chain of biomass-to-chemical production. Zhang and Wright (2014) resolute the optimum plant sizes, sites, and product distributions from an integrated rapid pyrolysis biorefinery supply chain. The major products of the biorefinery are bioethanol, value-added chemicals, and lignin. We cannot forget to include type and availability and uniformity of the biomass available, buying

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Fig. 7.4 Multistage fermentation and consolidated biomass processing (Courtesy by: Menon and Rao 2012)

prices, market requirements, logistics costs, and processing knowhows have also been the crucial of biorefinery sustainability (Hu et al. 2017). The major challenges in biorefinery system is sustainability process facility and logistics support (Borghesi and Gaudenzi 2013). Based on the findings from the literature, most of the hazards recognized are largely linking to operational risks, fermentation and distribution, political driven, social economic risks, money flow, delay and interruptions in supply chain (Ho et al. 2015). Majority of the experiments were entirely examined with a solo measure in mind, i.e., economic prospects or environmental risk, wherein the potential multifaceted effects of various factors were generally overlooked (Tuazon and Gnansounou 2017). The evaluation of bioprocess can be evaluated grounded on the three-way lowest outline which involves the environmental, economic, and social attributes (Liew et al. 2014). The data obtained in the study advocates bioethanol production mainly related to the greenhouse gas emissions and its advantageous effect on the suburban population. Though, obstacles like working and investment flow sideways with the availability of the feedstock impede the industrialization of bioethanol production (Azapagic 2014).

Biorefinery Life Cycle Assessment Life cycle assessment (LCA) is a critical approach of understanding and assessing the ecological influence of product formed in a process. LCA assessment of any biorefinery process consists of four key approaches. • • • •

The objective and scope The life cycle catalogue or inventory Assessment of the impact Results interpretations

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LCA experiments have been done to see the impacts on environment due to various biorefinery arrangements. LCA system broadly undertook the assessment of many kinds of biorefineries like lignocelluloses biomass and algae as case model (Gnansounou and Pandey 2017). Different case studies of LCA of various feedstock such as sugarcane, vetiver, fruit bunches, microalgae have been demonstrated and studied (Dufosse et al. 2017; Vaskan et al. 2017; Raman and Gnansounou 2017; Montafia and Gnansounou 2017; Gnansounou and Raman 2017). Implementation of the LCA evaluation of any biorefinery is critical to design and recognizing the improvement of functioning and possible environmental impact of the same. Nizami et al. (2017) emphasized that LCA evaluation of the biorefinery in terms of fortitude of the environmental loads in biobased product lifespan which may creates constrains in the investigation due to facts accessibility. LCA assessment of the sustainable sugarcane biorefinery was established and evaluated by Silalertruksa et al. Sugarcane biorefinery is growing attention as an encouraging option for improving the advantages of biomass in Asian countries. Silalertruska et al. (2016) established the eco-friendly sustainability of sugarcane based biorefinery approach in terms of possible environmental influences. The biorefinery approach involves sugarcane growing, milling, and value-added product use, i.e., sugarcane bagasse for power generation, molasses for bioethanol, and stem trimming for fertilizer and soil improvement. The findings of the study discovered the progress of sugarcane farming methodologies. The possible influences on climate change, process of acidification, photo-oxidant creation, particulate matter, and fossil reduction could be diminished by about 38%, 60%, 90%, 63%, and 21%, correspondingly. Suggestions for efficient execution of the planned sugarcane biorefinery system to Thai sugarcane and sugar trades are debated. In continuation, Nieder-Heitmann et al. (2019) also described the LCA with multi-criteria assessment of sugarcane biorefinery system especially in South African sugar industries. The valorization of sugarcane biomass is a possible option to guarantee the sustainability. In different setup, an itaconic acid biorefinery system (setup 1), (setup 2), polyhydroxyalkonates (PHA) biorefinery system (setup 3), succinate and PHA production biorefinery (setup 4), succinate biorefinery (setup 5). The LCA assessment of environmental impact was performed to classify possible delinquent or “hot spot” sectors in the individual procedures and to assess the ecological effects of the products to each other, along with fossil fuel as a control reference product. In eLCA assessment the carbon footprint and water paucity influence were involved as the dual key eLCA factors, collected with one social economic life assessment (sLCA) factor. The LCA was controlled and weighed in a multi-criteria assessment which can be a tool to regulate the most sustainable explanation for completing the task. The readiness level of any biorefinery system is also quite critical for the assessment of the chances of success. In general, the Technology Readiness Level (TRL) approach, initiated by NASA, which was utilized to imply how effective expertise can be applied on industrial scale (Booysen et al. 2016). The technology readiness levels varies from a TRL 1–9. The initial 5 levels (TRL 1–5), which actually are the different steps in research and invention phase and comprise key

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Fig. 7.5 Different levels of technological readiness in scientific domains

research, technology claim, practicality demo, and pilot scale evaluation in TLR 5. The concluding stages (TRL 6–9) form steps of the manufacturing stage and involve demo of the expertise in TRL 6–8, with industrial execution and use or field applications in TRL 8–9 (Fig. 7.5).

Technology and Socioeconomic Analysis of Biorefinery (TEA) The technological economic sustainability of lignocellulosic biorefineries can be substantially better by extracting value-added chemicals in sync with conventional biofuels like bioethanol. TEA intents to assess the scientific and financial attributes of trade (Lauer 2008). TEA contains the yield of the investment and running prices contemplating the different technologies aspects of biorefinery. Capital expenditure was assessed by power law correlations centered on unit capability. Data related to biorefinery systems were reported by various studies (Hamelinck et al. 2005). TEA was used formerly to inspect several kinds of biorefineries systems like corn waste

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(Luo et al. 2010), sugarcane (Silalertruksa et al. 2017), glycerol (D’Angelo et al. 2018), lignin-based substrates (Nielsen et al. 2017), rice straw (Elsayed et al. 2018), food remains (Solarte-Toro et al. 2019), and variety of lignocellulosic residues (Klein-Marcuschamer et al. 2011). Monte Carlo analysis of the uncertain biorefinery has been planned by Hytönen and Stuart (2011). They planned to execute TEA with hazard study in the development layout stages of the biorefinery system. Kapanji et al. (2019) analyzed the TEA of chemically catalyzed lignocellulosic biomass biorefineries in a conventional sugar factory with value-added by-product such as sorbitol or glucaric acid and electricity production (Fig. 7.6). The TEA of energy self-sufficient biorefineries system in sugarcane based lignocellulosic biomass into value-added sorbitol or glucaric acid along with electricity generation was investigated. The most efficient sorbitol biorefinary setup using dilute acid pretreatment with a financial investment cost of US dollar 3.96/L, which is slightly commercially viable (Fig. 7.7). Out of all the approaches, different energy self-sufficient lignocellulosic biorefineries system via chemical modification or pretreatment and occupied to a conventional sugar mill was achieved and examined. Chemical pretreatment setup was found most economically feasible than the steam eruption approach in TEA analysis. However, the high investment cost of the combined heat and power plant made them only slightly cost-effective. Few handfuls of the reports on socioeconomic analysis of lignocellulosic biorefineries has been documented. Hasenheit et al. (2016) emphasized that the social and financial influence is not limited solitary to the valorization of the biomass and ethanol generation in the biorefinery but also broadens to the ranches where the lignocellulosic feedstock is cultivated and handled. The social and economic outcomes on the generation of different bioenergy commodities were classified as positive, impartial, or negative effects. Rakotovao et al. (2018) assessed a countryside biorefinery by using a social and economic context regional attachment of economic events inside the area. Social life cycle assessment was incorporated as a key criterion to investigate the social facet of the pastoral biorefinery. Information on the social facets of biorefineries is still in its infancy and need to be covered in the techno-economic assessment of the developed biorefinery or circular bioeconomy framework.

Regulations for Forthcoming Biorefineries To establish a lignocellulosic biorefinery for efficient ethanol productions and valueadded products with techno socioeconomic aspects assessment, there should be some guidelines which needs to be fix. A biorefinery, likewise to what appears in oil refinery and it should be centered on feedstock modernizing processes, where a raw substrate is constantly upgraded and processed. It means that a biorefinery should divide all the feedstock elements, and take the lead, to a superior concentration of extra pure value-added chemical. As a significance, an agricultural feedstock cannot be directly burnt without any earlier treatment, since the purpose of a

Fig. 7.6 TEA analysis of the sugarcane industry based biorefinery system (Courtesy by: Kapanji et al. 2019)

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Fig. 7.7 Sorbitol/glucaric acid biorefinery and combined heat and power plant (Courtesy by: Kapanji et al. 2019)

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biorefinery is to increase the value of the diverse biomass. This concept leads to the following remarks: • An ideal biorefinery should generate at least one soaring value-added chemical/ material product, in addition to low vale and high yield products. • An ideal biorefinery should generate at least one energy source in addition to heat and electricity; the production of either bioethanol (liquid) or methane (gaseous) biofuel. In a successful biorefinery system, all the energy needs of the numerous biomass conversion processes should be supplied internally by the generation of heat and electricity from burning of biomass residues. For example, in a lignocellulosic ethanol unit plant, lignin, after extraction from cellulose and hemicellulose, can be utilized to provide the heat and electricity needed by the unit. Likewise, solid, liquid, and gaseous residues should be reduced. This goal can be attained in two behaviors: by means of all the dissimilar biomass fractions for producing an extensive spectrum of numerous products at one site, or setting up industrial “bio-clusters,” where biomass flow contacts among diverse plants are endorsed in order to convert a downstream deposit of a plant into an upstream raw substance for one more plant.

Case Studies: Lignocellulosic Valorization Through Biorefinery Lignocellulosic agriculture residue is known as agricultural feedstock that prevents the possible dispute with the land distribution and food produces (Ma et al. 2019). Comprising of hexose and pentose, and another polysaccharide which are composed of ample monomer renowned as xylose (Ma et al. 2019). To exploit on lignocellulose the process must be created on its important structure to efficiently produce various bioenergy end products. Agreeing to the report of the European Union, the use of lignocellulosic residues via enzymatic hydrolysis to generate biofuels is expected to be a 15-standard expertise by 2020 as part of the expansion of biorefinery. Meighan et al. (2017) demonstrated the dual-stage separation of sugarcane waste by autohydrolysis and glycerol organosolv delignification in a biomass biorefinery system. Bioethanol generation from lignocellulose waste presents a solution to existing environmental questions instigated by fossil fuels while fulfilling the biorefinery idea. Among the key pretreatments frequently used for sugarcane bagasse processing and fractionating, autohydrolysis (AH) and autocatalyzed organosolv delignification are contained by the more environmentally beneficial methods for the elimination of hemicelluloses and delignification (Baêta et al. 2016; Novo et al. 2011). In their study, 2 stage fractionations, i.e., autohydrolysis followed by glycerol organosolv delignification of sugarcane waste was established using experimental designs. Cheali et al. (2015) established various approaches to upgrade lignocellulosic biorefineries for generation of value-added chemicals. Initially, current

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superstructure indicating the lignocellulosic biorefinery model network is expanded to include the routes for catalytic conversion of bioethanol to value-added byproducts. Secondly, the optimization challenge for process improvement is formulated and resolved for two different purposes (a) techno-economic criterion (b) the sustainability criterion. Results suggest first that there is a substantial possibility of increase of operating profit for biorefineries generating bioethanol or other value-added chemicals. Next, the optimal models for upgrading bioethanol achieved also improved with respect to sustainability associated with the petroleumbased processes. The multi-product biorefinery offered a stronger and risk-aware upgrading approach studying the ambiguities that are normal for a long-term financing possibility. Tran et al. (2020) demonstrated the simultaneous hydrolysis of hexose and pentose by yeast for effective lignocellulosic biorefinery. Saccharomyces cerevisiae, a capable industrial host for biorefinery concept, has been established to develop its product outline. Though, the subsequent and slow fermentation of xylose into desired products rests one of the key tasks for efficient biorefinery. They have developed a powerful mixed-sugar co-fermenting yeast with advanced xylose conversion capability during concurrent glucose/xylose co-fermentation. To strengthen xylose catabolism, the overexpression of PPP pathway was chosen using a DNA assembler approach and overexpressed ever-increasing pentose intake and ethanol titer by twofold. The execution of the newly engineered strain with increased xylose hydrolysis was further than increased by elevating process temperature and thus substantially condensed the co-fermentation time by half. Due to its excellent co-fermentation potential and capability of further modification has possible as a stage in a lignocellulosic bioeconomy. Alriols et al. (2010) exhibited integrated organosolv and ultrafiltration lignocellulosic circular bioeconomy system. Non-woody lignocellulosic residues were fractionated by a biorefinery system to attain cellulose, lignin fractions, and a hemicellulos supplemented liquor after economically and environmentally viable measures. A combined process comprised by ethanol organosolv pretreatment pursued by a membrane ultrafiltration option was utilized to remove the various fractions. Production expenses of the attained ultrafiltrated lignin were approximate causing in 52 D/tone of lignin for the examined method circumstances.

Conclusion and Future Perspectives Various biorefinery pathways, from different biomass to product formation, can then be recognized, conferring to the many types of feedstock and products. Biorefinery system is comparable to recent fossil refinery, which produces different fuels and products. A key driver for the expansion and application of biorefineries is the mandate for energy, fuels, and value-added chemicals. Further future research and tools and methods for adoption will result in novel processes, contribute to more sustainable routines related to straight fossil-based systems. The utilization of

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sustainable biomass is not enough to guarantee a prosperous forthcoming for next generation; environmental protection using greener practices is also essential. The source of sustainably feedstock is the criterion for a lignocellulosic material to achieve the goal. It can be achieved using agricultural residues of perennial nature which could be the most promising substrate (Clifton-Brown et al. 2019; Fabbrini et al. 2019; Hoeber et al. 2018; Wagner et al. 2019). The conclusive aspects in a feasible general lignocellulose biorefinery system will be the substrate possible and supply. The description of the suitable measure of conversion size remains as vital question. Subsequently, this will outcome in value-added materials and technical stage inclined on lignocellulosic feedstock. Both syngas and lignocellulosic biorefineries system will show a role in the use of lignocellulosic biomass. Dependable effort on full progression steps is still infrequent, mainly at TRL levels above 4. For bioprocess incorporation, energy and mass flows to be improved along the whole value chain (Budzianowski and Postawa 2016; Nikolakopoulos and Kokossis 2017). Devoted tools to care the application of advanced technologies and make the market acceptance of new stuff will be desirable to hasten the evolution.

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8

Fermentation Economics and Future Prospects

Abstract

The major objective of any successful fermentation process is its ability to produce a fermentation product. Thus, the product must be sold in a way that it should be able to recover all the costs along with the desired profit. The monetary drivers for biotechnology and the enthusiasm to switch towards industrial fermentation are distinct for separate applications, like pharmaceuticals or bioethanol to choose two limits, with ethanol so far being the leading fermentation item for consumption by volume and sales. The economic viability of combining enzyme production in the lignocellulosic bioethanol process varies on the price of the full-scale industrial preparation of cellulase enzyme. To improve the link between off-site and integrated production, more comprehensive and revised data for off-site enzyme production are necessary, as also additional upscaling and enactment of both off-site and combined processes. Keywords

Fermentation economics · Cost · Operational cost · Product recovery · Production efficiency

Introduction Fermentation is utilized to produce an extensive range of metabolites in food and biopharmaceutical sector, which comprise small non-glycosylated proteins like human growth hormone; ethanol; lifesaving antibiotics; immunoglobulins for prevention of the numerous diseases. Fermented foods are in need all over the world and the production of fermented food is essential in many countries in order to deliver income and employment (Sharma 2016; Sharma and Dhanjal 2016). Food production and efficient processing is evidently the most vital source of income and occupation in Africa, Asia, and Latin America. The Food and Agriculture # Springer Nature Singapore Pte Ltd. 2020 D. Sharma, A. Saini, Lignocellulosic Ethanol Production from a Biorefinery Perspective, https://doi.org/10.1007/978-981-15-4573-3_8

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Organization (FAO) of the United Nations has said that value added by advertising and treating raw products can be a lot higher than the value of primary produce (Anon 1995). For example, in sub-Saharan Africa more than 60% of the human assets has been used in the small and medium scale food handling sector, and also among one third and two thirds of enriched manufacturing is founded upon agricultural raw produce (World Bank 1989; Conroy et al. 1995). Fermented cereal food products and alcoholic beverages followed by the fermented dairy products are the 3 extremely vital sectors of economy. Red and white biotechnologies are known as the biggest two applications. The annual sales generated by both technologies (white and red) are about US$ 36 billion which continues to grow strongly. The financial facets of white biotechnology are much varied and possess totally distinct applications and target markets, thus presenting thousands of substantially different products. In last decade, the usefulness of biotechnology as far as synthetic products are affected was anticipated to grow over-consistently to about US$ 1000 billion by 2020, and high growth levels were anticipated for sales of fine chemicals produced by biotechnology (Meyer 2011). The average value created by one unit of installed fermenter volume is two orders of scale lower for white biotechnology. The major objective of any successful fermentation process is its ability to produce a fermentation product. Thus, the product must be sold in a way that it should be able to recover all the costs along with the desired profit. Also manufacturing should be done according to market demand. Various possibilities which could occur for new product will be already existing because either the same or somewhat similar product is sold by some others. Secondly, if some new product is being discovered e.g. in case of new antibiotic in order to get well established it will require market and also approval or permission by FDA (Food and Drug Administration). The other difficulties related to marketing of new products are its demand which could be low or it has quite few uses. So, the patent coverage for a new product with less demand followed by substantial uses could be challenging. Once the new product comes into the market, it must come across through intense competition from exisitng products. So, in order to succeed through that competition, the product should possess low or slightly less price than other products in order to get sold. Briefly, fermentation process as well as its products should be able to compete with other products existing already in market on an economically sound basis. The monetary drivers for biotechnology and the enthusiasm to switch towards industrial fermentation are distinct for separate applications, like pharmaceuticals or bioethanol to choose two limits, with ethanol so far being the leading fermentation item for consumption by volume and sales. The major drivers that are applicable for all claims are given below: • Financial: Substrate cost and availability, production expenses, new business prototypes, etc. • Legal: Directive and legislation, subsidization policies, and so on.

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• Perception: Customer demand for “greenness,” fashion from chemical based to “organic,” and so on. • Innovation: Gigantic gene transfer, computational biology tools, collaboration versions, and so on. In certain cases, fermentation is an inherently messy process, and downstream separations may increase costs still further. In any process, it is important to understand the cost of breakdown, one should be able to determine where the largest potential for savings lie. General cost considerations can be categorized into: equipment’s, maintenance of asepsis, aeration, conversion efficiencies, volumetric productivity, utilities, raw material and media, and also other factors including temperature, dissolved oxygen tension, foaming, instrument purchase followed by installation and microprocessor control of feedback loop.

Factors Affecting Fermentation Economics Market Potential A process is considered commercially successful when it produces high yield of any fermentation product. The selling price of the product should be such that it recovers the production cost with satisfactory profit. On the other hand, the demand or market potential of the product decide to what extent product should be produced. There are two ways to equate demand and supply ratio. First, whether the market for the product may already exist or not and the same product has previously been sold by others or presently under selling. Second, what is the potential of establishment of market for a newly discovered fermentation product which has not been sold previously in the market i.e. it is required to establish a market for that product. Placement of any new fermentation product in the market is a time-consuming and costly affair as it require approval by the government agencies like Food and Drug Administration or others before they introduced in the market. Sometimes, due to fewer uses and less demand of certain fermentation products, it not easy to place it in the market. The market of some products already exists due to consumers’ acceptability and long-term persistence of these products in the market. Market potential, production cost, market demand and sale and competitions among existing products are important aspects to be considered at the time of launching a new product in the market. Many fermentation products are not sold directly in the market but used internally for an industrial concern. On the other hand, a fermentation product can be directly sold to a different industrial use which chemically transforms the fermentation product before sale in the market. Doing so, it is possible to reduce the extra costs for advertising, packaging, and also distribution to retail outlets which apply to fermentation products marketed directly to public. In addition to this, generally it does not require any trademark, proprietary name, and generic name. Foreign sales present an added market potential for many fermentation products. For example,

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exporting product and selling in the foreign marker and on the other hand, manufacturing the same product in foreign country and selling in their market make a wide difference in the profit.

Fermentation and Product Recovery Costs For any fermentation process, the economic position of product is decided by its production and distribution. • Medium constitutes: Usually, the costs of different constituents for the production medium decide the competitive position and potential profits of a fermentation product. For example, inoculum media are usually less expensive because they are designed to promote fast growth of the producer instead of converting a large amount of carbon substrate in the product. At the same time, they are required in less volume. In contrast, production media which is aimed to produce the product required in large volume and may time high cost of a single medium can affect the selling price the fermentation product. As any component of the medium to reduce the cost, one should try to make provision for the alternate low-cost replacement of constituents while formulating the production media. As a matter of fact, every component is directly or indirectly influencing the economics of the fermentation process. For example, because of the world political situation, if the availability and cost of cane black-strap molasses become too high, one cannot afford to use this as a carbon source. Under such situations there should be provision for an alternative carbon source which is cost-effective and made available easily. One should remember that sometimes the use of an alternate medium requires the use of a different fermentation microorganism or strain of the organism. Several media need decontamination to remove some contaminated chemical species as they influence the product accumulation and many times make the recovery process difficult. For example, certain metal ions can be removed by ion-exchange resins. Many media require pretreatment before they are employed. For example, starch must be pretreated with amylase to release fermentable sugars and proteins must be degraded by proteolytic enzymes to release amino acid for yeasts growth. Acid or alkali are required to adjust the pH of the medium during production process. As medium reagents are not so costly, but considerable amount may be required for the adjusting the pH value which many times leads to a considerable high cost. Same way, media rich in protein components produce foam and increase the chances of contamination. Under such condition control of foam either by chemical antifoam agents or with mechanical device also add the cost of production. Recovery and purification of fermentation product is very important step of down step process and markedly increase the overall product cost. Recovery and purification should be fast, easy, and should occupy less step with application of low-cost chemicals.

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• Fermentation incubation period: Fermentation process with short incubation period is less costly compared to processes with prolonged incubation with reference to the inoculum build up and to production. One can harvest more batch in case of short incubation period fermentation with a larger turnover of fermentation equipment. Fermentation requiring long incubation periods requires additional requirement, extra labor cost, and have more possibility of getting contamination. • Contamination and sterilization: Many fermentation processes are more prone to contamination compared to protected fermentation processes. For example, fermentation processes in which foaming is the common problem, prolonged incubation requiring processes, processes in which the product producing organisms poorly compete with contaminants for fermentation nutrient, processes in which contaminants degrade or chemically change the fermentation products, certain fermentation processes sensitive to lytic phage (for example, certain bacterial and actinomycetes fermentations). Those fermentation processes in which media sterilization cost is not affordable certain non conventional methods to control the growth of contaminants should be adapted. These are adjusting low pH of the medium. Selecting a substrate poorly amenable to attack by contaminants, Partial sterilization of the medium, Selecting certain chemicals which retard the growth of contaminants. If the fermentation medium is severely contaminated, then it must be discarded and this adds more cost to the production. Even though the medium is not gravely contaminated and may not be serious enough to discard although affect the overall yield of the product. Many time mutation in genetically unstable, due to which production strain results in low-yielding cells. • Yields and product recovery: High yield and adequate recovery of any fermentation product are of prime importance in any fermentation process. For any fermentation product, high yield with proper recovery and purity affect its position on the open market. The cost of these important downstream processes is very high but extremely important too. • Product purity: The purity of fermentation product decides its future stand and long-term market value. The costs of product are directly associated to its purity. For example, some antibiotic preparations useful for human applications should be sterile and also free from various pyrogens. On the other hand, some products which are being used with other antibiotic preparations are sold in a crude form in order to blend with animal feeds. Some fermentation products can also be marketed at different level of purity and at a more than one concentration. For example, lactic acid is sold at different strengths which may range from approximately 20–85% and also purity levels range from the relatively crude to high purity edible and U.S.P grades and each of these different grades of lactic acid has a place in the market. Purification steps like solvent extraction and followed by crystallizations for a fermentation product significantly contribute to the overall costs. • Waste disposal: The overall production cost of any fermentation product is influenced by outflow of capital used for waste treatment and disposal and it depends on the following two aspects: (i) acceptance of fermentation waste with

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or without pretreatment by municipal waste-treatment facilities or any other local service providers or (ii) maintenance of waste-water treatment plant by the fermentation. As per state and federal regulations, it is not possible to dispose fermentation wastes into rivers, streams, or any other stagnant water bodies for long time. In certain case, for example, waste generated in the fermentation processes which exploit plant or animal pathogens as the fermentation organisms requires an additional expense of sterilization before discarding to avoid biohazards. There are many sources through which waste is generated. There are waste generated from the actual fermentation, wastes from recovery processes, waste from cleaning water, waste from cooling waters, waste from various stages of product recovery and purification, etc. Many times, it is feasible to use waste water for some other purposes, for example, cooling waters can be utilized in media makeup for similar or other fermentations. Many times, it is possible to recover by-products from the fermentation wastes. Labor cost: The cost which incur to pay for non-technically and technically trained personal working in any industry at all level is called labor costs. This includes labor related to cultures handling, Inoculum, Production, Product recovery and purification, Maintenance of product sterility, Packaging of the product, Steam production, Equipment maintenance and cleaning, Administration, etc. This labor costs depends on type of fermentation process (batch of continuous), level of containment to be employed, type of organisms under use (genetically engineered or non-genetically engineered), volume of the product, etc. Research costs: To maintain a competitive commercial stand, the cost picture of a fermentation process which include expenses induced in the research and discoveries for the development of the process should be clear. Many times, high cost research without positive outcome in terms of innovations and novelty is of no use for industries facing financial crisis. Investing in research is a longterm venture. It gives incredible financial success provided it is done in a right direction. Capital expenditure: Commercial production of a newly developed fermentation is expensive and may require outlay of capital. Establishment of expensive fermentation equipment which are in continuous use for the production along need the maximum requirement for capital expenditure with other facilities. Thus, a new fermentation setup requires the pre-empt of existing facilities along with the construction of additional facilities. A similar condition may take place for product recovery and purification facilities. A newly developed fermentation process may have need of the setting up of newly designed fermentation equipment. Installation of this equipment to a great extent adds additional cost to the fermentation costs. Patent position: The profit picture of any fermentation process or product is remarkably influenced by sound patent position. Patent position of process or product helps in reducing the extensive commercial competition which remarkably influences the profit picture. A promising patent position provides greater potential for cost recovery and an adequate profit. The costs for getting a patent are relatively small. However, in case any infringement proceedings are

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instigated, the patents become a costly affair. Patents can yield revenue to the holder through its utilization in the production and marketing of the fermentation product. For getting royalties, it is possible to licensed then patent to other fermentation companies and even competing companies. Even after doing so, the patent holder can still produce the fermentation product. Many times, the patent holder may not have the capital or facilities to produce and market the product. In such cases, it may be to the advantage of the patent holder to license the patent without producing the fermentation product, or to sell the patent outright. • Overhead: General expense incurred for the running of a business is called overhead expenses which include taxes, rent, insurance, heat, light, audit and accounting, depreciation, and other routine office expenses. These all cost leads to contribute the overall economics of the products before they introduce in the market for sale.

Process Assessment The prior deliberations must be evaluated keeping in mind the present and future market conditions to assess the economic potential for any fermentation process development. It is equally important to re-evaluate the process during commercial production. Following aspects should be considered during overall evaluation of any fermentation process. Estimation of present and future availability and price of fermentation substrates, costs of labor, overhead expense, public demand for the product, competition in the market, potential for bettering yields, product recoveries, capability and facilities to meet market demands for the product, consideration of all present and future costs, selling price and desired profit for the product. All these dimensions must be studied thoroughly to decide before the production of the fermentation products. The following conclusions could be drawn from the above given study. The strategy which involves single use upstream equipment will be advantageous in microbial fermentation under the given conditions like: if in short time period we are required to produce a certain number of batches in that case single use equipment will provide us with sharp solution e.g. product is targeted during its development stage, in the production of various clinical examinations, also in certain toxicity study, or in certain cases of vaccine surge situations. On the other hand when higher production rate is required then in that situation, according to this study, a single use strategy will be able to increase batch capacity by 50% in case of single product scenario and in case of multiproduct scenario increase will be 100% as compared to stainless steel strategy. In case of full facility utilization, single use alternative provided higher opportunities of making profit which is about 150 annual batches as compared to stainless steel alternative where it is produced only 100 batches. If in certain cases, facility utilization rate is minimal, then, the capital investment obtained in such case will also be low. This is because of lower upfront investment also. The annual maintenance qualification will be of low cost which will indeed contribute

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towards single use equipment over stainless steel equipment because lower fixed cost will get associated with single use equipment. In order to make bio-products more economically efficient, development of open (unsterile) and continuous processes is paid an increased attention. Continuous fermentation when utilized properly will lead to a reduced cost of industrial by-products. The feeding of raw materials as well as removal of products should be done in continuous manner with reduced or no risk of contamination, even if it is under open (unsterile) conditions. Stability of biological system during long cultivations, yield, and also productivity of the process are some of the factors which play an important role. Microorganisms which are able to survive and grow under extremes of conditions like high or low pH, temperature, osmotic pressure, under the conditions of mixed culture, cell immobilization, and solid state cultivation, are of great interest for developing open and continuous fermentation processes (Li et al. 2014).

Case Studies A scale-up design for bioethanol production which uses sequential batch, simultaneous cold starch hydrolysis, and fermentation indicates that this process can be economically feasible. High levels of income are generated from both the bioethanol and from their by-products such as: food-grade wheat gluten. The payback period is predicted to be 2 years with a discounted cash flow rate of return of 46%. With the increasing trend of bioethanol production and its consumption as an alternative biofuel, particularly in case of automobile sector, analysis is required to be done in order to improve the economic state of bioethanol production by making it cost-effective as well as equivalent to fossil fuel (Sassner et al. 2008). In this regard, production cost of bioethanol needs to be improved by making use of suitable, sustainable, and abundant raw material (Sassner et al. 2008). Analysis was done in order to determine cost required to produce bioethanol from the three crops cultivated: corn, wheat, and triticale. Among the three triticale was the most preferable crop. Consumption of waste crops or their by-products from various industries such as starch processing can also serve as a very rational approach. The efforts and investments involved in promoting the second generation of bioethanol on lignocellulosic biomass should be intensified. In addition, the economy of bioethanol production can be further improved by utilizing the by-products obtained during the production of bioethanol in order to produce animal feed and lactic acid. Various investigations performed on ethanol obtained using various different non-edible substrates have newly gained significant interest because such productions can prevent the dispute between food and fuel. Recently, an attempt was made in order to examine the production of ethanol by utilizing low-cost feedstock, specifically, seaweed solid wastes which were achieved after the extraction of κ-carrageenan (Mojović et al. 2012). The use of seaweed solid wastes will concurrently help to surmount its disposal challenge. Two distinct processes have

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been used: the SHF (separate hydrolysis and fermentation) process and the SSF (simultaneous saccharification and fermentation) process. It was detected that the SSF of seaweed solid wastes with Saccharomyces cerevisiae has various rewards over SHF because the SSF technique is a simple one-step procedure that can save time, and is cost-effective and also reduces energy consumption while achieving a high yield of bioethanol than that of SHF. Bioethanol obtained from lignocellulosic portions of sugarcane (bagasse and leaves), also known second-generation ethanol, has a higher market ability as an automotive fuel, though, the process is still under review on pilot as well as protest scale. From a practice point of view, any upgrades in plant design can lower the production cost, delivering better profitability and effectiveness if the conversion of the whole sugarcane is considered (Tan and Lee 2014). According to the simulations, the 2G bioethanol produced from sugarcane bagasse and leaves in Brazil is already competitive without subsidies with 1G starch-based bioethanol production from Europe. Moreover, 2G bioethanol could be produced at a lower cost if subsidies were used to compensate for the opportunity cost from the sale of excess electricity and if the cost of enzymes continues to fall. Macrelli et al. (2012) described the chronic effects associated with greenhouse gas emissions (GHG) on the atmospheric environment due to excessive use of fossil fuel. There has been growing awareness regarding renewable energy resources due to the adverse environmental effects caused by GHG emissions. In order to reduce net greenhouse gas emissions lignocellulose an agricultural residue provides promising alternatives over the petroleum source for bioethanol production. Lignocellulosic bioethanol production has not been commercialized successfully on a large scale because of certain technological barriers. In order to obtain this goal of economically successful bioethanol production technology which includes pretreatment, fermentation, dehydration, and enzymatic hydrolysis are also required to develop. 80% of cellulose in 40% of lignin from lignocellulose (generally containing 30–46% of cellulose and 18–25% of lignin) can be recovered on pretreatment with ionic liquid. On enzymatic hydrolysis, of recovered cellulose for bioethanol production about 76% of reducing sugar yield is obtained. Techniques such as nanofiltration and ultrafiltration during hydrolysis are able to concentrate 27% of reducing sugar and are able to recover 73% of enzymes which possess 50% of catalytic activities. Methods like ultrafiltration rejects about 100% of yeasts and produces 15ge/l/h ethanol which can be used to produce 99.8 wt % ethanol by pervaporation method which involves membrane-based dehydration. This review mainly focuses on bioethanol via methods which are eco-friendly as well as sustainable. The main motivation behind this study was to understand the economically feasible production process of fuel ethanol via integrated fermentation-pervaporation operation (O’Brien et al. 2000). Total annual costs of fermentation, distillation, and dehydration section were compared between processes involving continuous fermentation-pervaporation system over batch fermentation process and standard corn dry milling process. In brief, detailed capital and operating costs have been compiled for a conventional batch fermentation process in a 50 million gallon/year

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fuel ethanol plant. The hypothetical processes which employed pervaporation coupled with continuous fermentor where parameters were already determined from the data obtained during long-term laboratory experiments. For the above-mentioned hypothetical process, the annual costs were slightly high for the techniques such as fermentation, distillation, and dehydration than that of fermentation pervaporation process. However, analysis of the hypothetical process indicates that smallest improvement either in pervaporation flux or in selectivity may lead such systems to be cost-effective. Significant pilot plant studies are required in order to obtain technical feasibility.

Strategies to Improve Fermentation Economics of Bioethanol • Expansion of biocatalysts with enhanced resistance to hemicellulose by-products accumulation (reduces the requirement for distinct detoxification approaches) (Gubicza et al. 2016). • Alternative of sulfuric acid with the low concentration phosphoric acid (it excludes the necessity for high-priced metals). • Work out the blending and pumping problems associated to high fiber solids loading (streamlines material handling, reduces chances for contamination, and enhances product concentration). • Restricting the utilization of chemicals to those that are nutrients for the biocatalyst and for eventual use as an elevated nitrogen fertilizer (incomplete downstream processing of chemical cost via multiple usage). • Co-fermentation of glucose and pentoses in the same fermenter (removes early liquid solid separation, fiber washing, and detoxification of hemicellulose substrates). • Downstream processing of the accumulated bioethanol using molecular sieve and other membrane-based separation reduces the fermentation cost (Kumar et al. 2015).

Future Prospects A combined procedure could decrease the greenhouse gas emissions when lignocellulose biomass-based bioethanol production, and the expense of an integrated fermentation could be managed by obtaining enzymes produced on off-site. Suck kind of study focused on the environmental and economic evaluation of a combined process, and in directive to improve the comparison to the off-site case, more comprehensive and revised data concerning industrial off-site enzyme production are particularly crucial. The economic viability of combining enzyme production in the lignocellulosic bioethanol process varies on the price of the full-scale industrial preparation of cellulase enzyme. To improve the link between off-site and integrated production, more comprehensive and revised data for off-site enzyme production are necessary, as also additional upscaling and enactment of both off-site and combined

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processes. To make a bioethanol process as an economical approach, we should test our laboratory experiments to pilot scale and subsequently at commercial scale.

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