Handbook of Biofuels Production: Processes and Technologies [3 ed.] 0323911935, 9780323911931

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HANDBOOK OF BIOFUELS PRODUCTION

Woodhead Publishing Series in Energy

HANDBOOK OF BIOFUELS PRODUCTION Processes and Technologies THIRD EDITION Edited by

RAFAEL LUQUE Department of Organic Chemistry, University of Córdoba, Córdoba, Spain

CAROL SZE KI LIN School of Energy and Environment, City University of Hong Kong, Hong Kong

KAREN WILSON Centre for Advanced Materials and Industrial Chemistry, School of Science, RMIT University, Melbourne, VIC, Australia

CHENYU DU Department of Chemical Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom

An imprint of Elsevier

Woodhead Publishing is an imprint of Elsevier 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2023 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-91193-1 (print) ISBN: 978-0-323-91521-2 (online) For information on all Woodhead publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisitions Editor: Peter Adamson Editorial Project Manager: Tim Eslava Production Project Manager: Maria Bernard Cover Designer: Mark Rogers Typeset by STRAIVE, India

Contributors Laura Aguado-Deblas Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, Co´rdoba, Spain Mohamed HM Ahmed Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD, Australia Wouter Arts Centre for Sustainable Catalysis and Engineering, KU Leuven, Leuven, Belgium Luqman Atanda Science and Engineering Faculty, Centre for Agriculture and the Bioeconomy, Queensland University of Technology, Brisbane, QLD, Australia Najihah Abdul Bar Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Nuno Batalha School of Chemical Engineering, The University of Queensland, Brisbane, QLD, Australia Felipa M. Bautista Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, Co´rdoba, Spain Carolina Botella Shell Espan˜a S.A, Madrid, Spain Juan Calero Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, Co´rdoba, Spain Elias Christoforou School of Engineering and Applied Sciences, Frederick University, Nicosia, Cyprus Loris Cottoni Unitelma-Sapienza, Universita` degli Studi di Roma, Roma, Italy Darfizzi Derawi Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Ana Belen Dı´az University of Ca´diz, Ca´diz, Spain Weiliang Dong State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China

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Chenyu Du Department of Chemical Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom Rafael Estevez Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, Co´rdoba, Spain Paris A. Fokaides School of Engineering and Applied Sciences, Frederick University, Nicosia, Cyprus Isabel Lo´pez-Garcı´a Physical Chemistry and applied Thermodynamics, University of Cordoba, Cordoba, Spain Guillermo Garcia-Garcia Department of Chemical and Biological Engineering, The University of Sheffield, Sheffield, United Kingdom Kleio Gioulounta Department of Environmental Engineering, Democritus University of Thrace, Xanthi, Greece Fabio Giudice Unitelma-Sapienza, Universita` degli Studi di Roma, Roma, Italy Elli Heracleous Chemical Process & Energy Resources Institute, Centre for Research and Technology Hellas; School of Science & Technology, International Hellenic University, Thessaloniki, Greece Ernesto Hernandez Canterbury Christ Church University, Canterbury, United Kingdom Jesu´s Hidalgo-Carrillo Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, Co´rdoba, Spain Yunzi Hu Bio-chemical Conversion Lab, Center for Biomass Energy Research, Guangzhou Institute of Energy Conversion, CAS, Guangzhou, China Hessam Jahangiri Department of Engineering and Mathematics, Sheffield Hallam University; School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom Anas Jamil Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD, Australia Min Jiang State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China

Contributors

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Yujia Jiang State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China Muxina Konarova School of Chemical Engineering, The University of Queensland, Brisbane, QLD, Australia Angelos A. Lappas Chemical Process & Energy Resources Institute, Centre for Research and Technology Hellas, Thessaloniki, Greece Hannes Latine Centre for Sustainable Catalysis and Engineering, KU Leuven, Leuven, Belgium Chong Li Kunpeng Institute of Modern Agriculture at Foshan, Chinese Academy of Agricultural Sciences, Foshan; Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Shenzhen Key Laboratory of Agricultural Synthetic Biology, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China Hong-Ye Li Jinan University, Guangzhou, People’s Republic of China Yi Liang Shell Global Solutions US Inc, Houston, TX, United States Carol Sze Ki Lin School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong, People’s Republic of China Clara Lo´pez-Aguado Universidad Rey Juan Carlos, Madrid, Spain Jiasheng Lu State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China Carlos Luna Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, Co´rdoba, Spain Diego Luna Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, Co´rdoba, Spain Rafael Luque Department of Organic Chemistry, University of Cordoba, Co´rdoba, Spain Juan Antonio Melero Universidad Rey Juan Carlos, Madrid, Spain

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Gabriel Morales Universidad Rey Juan Carlos, Madrid, Spain Piergiuseppe Morone Unitelma-Sapienza, Universita` degli Studi di Roma, Roma, Italy Jinhua Mou School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong, People’s Republic of China Thomas Nicolaı¨ Centre for Sustainable Catalysis and Engineering, KU Leuven, Leuven, Belgium Miloud Ouadi School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom Bruno Pandalone Centre for Sustainable Catalysis and Engineering, KU Leuven, Leuven, Belgium Marta Paniagua Universidad Rey Juan Carlos, Madrid, Spain Anshu Priya School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong, People’s Republic of China Xiujuan Qian State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China Deepak Raikwar Centre for Sustainable Catalysis and Engineering, KU Leuven, Leuven, Belgium Noor Azira Abdul Razak Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Antonio A. Romero Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, Co´rdoba, Spain Bert Sels Centre for Sustainable Catalysis and Engineering, KU Leuven, Leuven, Belgium Sivakumar S.V. Shell India Markets Pvt. Ltd, Bengaluru, India James G. Speight CD&W Inc., Laramie, WY, United States Katerina Stamatelatou Department of Environmental Engineering, Democritus University of Thrace, Xanthi, Greece

Contributors

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Nazrizawati A. Tajuddin School of Chemistry and Environment, Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, Selangor, Malaysia Ioanna A. Vasiliadou Department of Environmental Engineering, Democritus University of Thrace, Xanthi, Greece Bo Wang Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Shenzhen Key Laboratory of Agricultural Synthetic Biology, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China Xiang Wang Jinan University, Guangzhou, People’s Republic of China Zhen-Yao Wang University of Technology Sydney, Ultimo, NSW, Australia Karen Wilson Centre for Advanced Materials and Industrial Chemistry, School of Science, RMIT University, Melbourne, VIC, Australia Fengxue Xin State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering; Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing, People’s Republic of China Tang Xu Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Shenzhen Key Laboratory of Agricultural Synthetic Biology, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen; School of Life Sciences, Henan University, Kaifeng, China Wei Yan School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong, People’s Republic of China Wenming Zhang State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering; Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing, People’s Republic of China Dawei Zhou State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China

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Jie Zhou State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China Xinhai Zhou State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China Xiaoyan Zou Department of Chemical Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom; Key Laboratory of Functional Inorganic Material Chemistry, Heilongjiang University, Harbin, Heilongjiang, China

Preface The increasing awareness and concerns about our dependency on fossil resources and the depletion of crude oil reserves caused by industrialization and expansion of transportation in emerging markets, together with political instability in countries with oil reserves, have led to volatility in fuel prices and energy supply. In addition, the appeal for the reduction of greenhouse gas (GHG) emissions has been the hottest topic in recent United Nations climate change conferences. In recent decades, the majority of GHG emissions are contributed by fossil fuel combustion and industrial processes. Therefore, there is an urgent need for the development of a low-carbon economic system to replace the fossil-fuel-based system. These factors have become the driving forces for exploring new alternatives to replace fossil oil. Biomass is a feedstock that can fulfill requirements for fossil oil substitution, as it is a renewable resource whose sustainable supply can be ensured. Besides, biomass has abundantly available stored solar energy, so it could act as a “natural battery” that facilitates energy supply security. Based on the success of the first and second editions, this new edition of the Handbook of Biofuels Production: Processes and Technologies aims to provide an overview of the latest progresses in various technologies for biofuel production. Special emphasis has been given to advanced-generation biofuels, which include biofuels produced from nonfood materials. The significant progress in the bioenergy industry has encouraged further exploration of low-carbon technologies for the production of advanced-generation biofuels (and biochemicals) from low-value waste biomass. Collective efforts from various fields encompassing bioenergy technologies and people including politicians, economists, environmentalists, scientists, and engineers are needed to come up with alternatives, policies, and choices to advance the key technologies for a more sustainable future. This new edition of the handbook is divided into four parts. Part One comprising Chapters 1–4 covers the major issues and assessment of biofuels production. Basic details on biofuels, including their classification, potential feedstocks, production processes, policies regarding their production and use, and socioeconomic and environmental considerations and challenges, are presented in this part. Part Two comprising Chapters 5–11 highlights the latest technological advancements in biofuels production. This part discusses strategies to produce biofuels, such as chemical and biochemical

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conversion processes and technologies. Parts Three and Four comprising Chapters 12–16 provide details on thermal and thermochemical conversion processes, integrated production approaches, and applications of biofuels. As editors of this new edition, we sincerely hope it can serve as a reference text mainly designed for courses at the postgraduate level. The book will be also useful to provide practical information to early-career researchers, practitioners in the field of bioenergy, and consultants to energy agencies that study the feasibility of biomass projects and plan biofuels production in a rational way. We take this opportunity to acknowledge and thank all contributors for their excellent collaboration and timely contributions that have helped to put together this book covering a highly comprehensive range of topics. The coeditors would like to express their sincere appreciation and gratitude to the editorial project managers at Elsevier, namely Tim Eslava, Madeline Jones, Mica Ortega, who patiently and kindly took us through the development of this book over the past 3 years to achieve this impressive final result, which would not have been possible without their support. In addition, we would like to extend our gratitude to Mohanraj Rajendran, copyrights coordinator from the Copyrights Team, who ensured a smooth permission-seeking process. Last but not least, we sincerely thank Helena Chin (11-year-old niece of Carol Lin) for her great effort in preparation of the front cover picture. The picture symbolizes biomass as nature’s way of storing energy originating from the sun, which allows us to use this energy when the sun is not shining. With very best wishes for a successful and enjoyable reading. Rafael Luque Carol Sze Ki Lin Karen Wilson Chenyu Du November 2022

CHAPTER 1

Introduction: An overview of biofuels and production technologies Anshu Priyaa,#, Yunzi Hub,#, Jinhua Moua, Chenyu Duc, Karen Wilsond, Rafael Luquee, and Carol Sze Ki Lina a

School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong, People’s Republic of China b Bio-chemical Conversion Lab, Center for Biomass Energy Research, Guangzhou Institute of Energy Conversion, CAS, Guangzhou, China c Department of Chemical Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom d Centre for Advanced Materials and Industrial Chemistry, School of Science, RMIT University, Melbourne, VIC, Australia e Department of Organic Chemistry, University of Cordoba, Co´rdoba, Spain

1.1 Introduction Unprecedented growth in the global population, coupled with accelerated urbanization, has led to an increase in the demand for all forms of energy and an increase in environmental pollution. An extensive analysis of the demand and supply of energy sources revealed that fossil fuels fulfilled 84% of the world’s primary energy demand in 2019. Major sources of this supply included oil (33.1%), coal (27%), natural gas (24.2%), hydroelectricity (6.4%), renewable energy (5%), and nuclear energy (4.3%) (Fig. 1.1). The carbon emissions from energy production have increased by 0.5% for every 1% gain in global economic output since 2010 (Statistical Review of World Energy, 2020). The global energy demand is predicted to rise by more than 50% by 2025, while the consumption is estimated to increase by over 90% (World Bioenergy Association, 2020). The diminishing global fuel reserves, rising fuel prices, and the environmental impact of fossil fuels have spurred the exploration for sustainable, affordable, renewable, and environmentfriendly energy sources. In this context, the development and utilization of biofuels as alternative energy sources have gained much importance. Biofuels are liquid or gaseous fuels derived predominantly from a variety of biological sources by physical, chemical, biological, or combinatorial methods. #

First authors with equal contribution.

Handbook of Biofuels Production https://doi.org/10.1016/B978-0-323-91193-1.00002-0

Copyright © 2023 Elsevier Ltd. All rights reserved.

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Renewable energy

Nuclear energy

Hydroelectricity Oil

Natural gas

Coal

Fig. 1.1 Distribution of global energy sources.

Fig. 1.2 shows the generalized life cycle of biofuels. Their production and consumption are gaining importance because of their capability to replace fossil fuels and meet global energy requirements, while lessening the contribution of fuels toward global warming. In addition to sustainability, mitigation of environmental pollution, and reduction in greenhouse gas (GHG) emission, biofuels are preferable to fossil fuels due to several other advantages, such as their renewable nature, security of supply in the future, and cost-effectiveness; these advantages are especially notable in the context of continually rising petroleum prices and geopolitical instabilities in major fossil-fuel-producing regions of the world. Biomass can be converted to a variety of biofuels, including liquid fuels, such as biodiesel, biomethanol, bioethanol, and gaseous fuels, such as methane and hydrogen. The major difference between biomass-generated and petroleum-based fuels is the oxygen content. In contrast to petroleum-based fuels, biofuels have oxygen levels of 10%–45%, while petroleum has essentially none, which makes the chemical properties of biofuels different from petroleum-based fuels (Demirbas, 2009). Depending on the chemical nature and complexity of biomass, biofuels are classified into different generations, namely first, second, and third generation of biofuels. The first-generation biofuels are produced from food-based feedstock; this primarily includes the use of crop plants, such as corn, sugarcane, wheat, and oilseeds (canola, soybean, palm, etc.), to produce biodiesel and bioethanol. The production of second-generation biofuels utilizes nonedible lignocellulosic materials as substrates for energy production. The substrates chiefly

Fig. 1.2 The typical life cycle of biofuel.

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include agricultural by-products, such as sugarcane bagasse and cellulosic crop waste, and noncrop plants, such as perennial grass, Jatropha and Pongamia, to produce bioethanol and biodiesel. In South-East Asia, edible palm oil has drawn much attention for biodiesel production, while the potential of nonedible oilseeds of Jatropha is also being explored for biodiesel production. Both first and second-generation biofuels require an abundance of land and other agricultural resources for cultivation. This creates a competition between food and biofuel production, especially regarding land use. In particular, first-generation biofuels have a direct impact on food price as they utilize food crops for fuel generation (Gurjar et al., 2021). Such issues of food-versus-biofuel production, use of land, and agricultural resources were addressed by the development of third-generation biofuels. In this generation, marine macroalgae, seaweed, algal biomass, and cyanobacteria emerged as attractive feedstocks that could be used to produce bioethanol, biogas, and biodiesel. Due to their rapid growth rate; high lipid content; no land requirement; easy cultivation under controlled, artificial, and nutrient-rich environments, such as open ponds or closed photobioreactors; easy harvesting; and lipid extractability, algal and marine feedstocks have emerged as a feasible and sustainable substitute that may provide better fuel security and meet current and future fuel demands (Chhandama et al., 2021). Transportation, construction, industry, and agriculture are the sectors that have the highest demand for fuels. Transportation accounts for the biggest share, 70%, of all fuel consumed globally, which is currently mainly obtained from fossil fuels. Despite environmental, social, and economic challenges, the global demand for fuel by the transportation sector has multiplied over the last few decades and is expected to grow further in the future (Kuo and Chen, 2009). Fossil fuels must be replaced with cleaner and more sustainable biofuels to tackle the rising demand for energy and associated challenges. Taking this into account, the Renewable Energy Directive of the European Union aims to increase renewable energy generation by 27% by 2030 (Osaki, 2019). Similarly, the United States has set a goal to increase the annual production of bioethanol from 56.8 billion liters to 60.6 billion liters by 2022 through the Energy Independence and Security Act of 2007 (Pittock et al., 2015). The biofuel sector has experienced significant improvement with the advent of modern technologies. The potential application of biofuels has expanded beyond their conventional use in the transportation sector; biofuels are now used for generation of electricity and heat, for cooking, and as fuel in the aviation industry. Extensive research is being conducted in

An overview of biofuels and production technologies

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the bioenergy sector worldwide to increase the efficiency of biomass utilization and biofuel production. Different forms of biofuels ranging from biodiesel to bio-jet fuel and biogas have emerged as feasible energy options. Biodiesel is biodegradable and therefore nonexplosive. Moreover, it has a high combustion efficiency, cetane number, and flash point, with a low sulfur and aromatic content, due to which it is extensively used as a transportation fuel, either in its pure form or in combination with gasoline. In addition to biodiesel, bio-aviation fuel is another type of biofuel. Also known as green diesel or bio-jet fuel, bio-aviation fuel is a biomass-derived synthetic paraffinic kerosene. It is widely used in combination with petroleum-derived jet fuel as a sustainable and clean solution to mitigate environmental pollution by the aeronautical industry. Both biodiesel and bio-aviation fuel are potential alternatives to conventional fuels in the transportation sector to power vehicles, motorized engines, and aircraft (Kathrotia et al., 2021). Bioalcohols, such as bioethanol, biomethanol, and biobutanol, and biogas, such as biomethane and biohydrogen, produced through biochemical processing, are also promising green and clean biofuel substitutes for fossil fuels. The environmental and socioeconomic advantages of biofuels for the mitigation of environmental pollution and reduction of carbon footprints have motivated nations to decrease their dependency on fossil fuels and increase biofuel consumption. The highest consumption of biodiesel in 2019 was reported to occur in the United States, followed by Brazil. In 2019,
43 million barrels of biodiesel were consumed in the United States (U.S. Energy Information Administration (EIA), 2020). The United States and Brazil also rank among the largest biodieselproducing nations and accounted for approximately 6.5 and 5.9 billion liters of biodiesel produced, respectively, in 2019 (Ebadian et al., 2020). During 2000–19, the global production of biofuel increased from 187 thousand barrels to 1841 thousand barrels, broadening the overall market size to US$136 billion (Statista, 2020). It is estimated that the biofuel market size will continue to grow and reach US$153.8 billion by 2024 (Statista, 2020). The increase in biofuel production globally is driven by a variety of factors including technology readiness, biofuel-friendly policies, and market demand. The establishment of a competitive market for biofuels will be facilitated by financial incentives, subsidies, and supportive policies. Biomass production for generation of biofuels also supports the agricultural sector by creating employment opportunities, labor, and commercial prospects for domestic harvest. The potential for land restoration by the cultivation of

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biomass and generation of income by production and export of biofuels to industrialized nations are attractive outcomes that may pave the path for further progress. In view of such environmental and socioeconomic advantages, there is a need for sufficient investment and government and legislative support to create an economically competitive global market for biofuels. The aim of this book is to provide a deeper insight into the vital drivers of biofuel production, technological advancements, policies, regulatory issues, environmental and socioeconomic considerations, life cycle assessment (LCA), applications of biofuel production, and associated challenges. The book is divided into four major parts. Part One covers the major issues and assessment of biofuel production. Basic details on biofuels, including their classification, potential feedstocks, production processes, policies regarding their production and use, and socioeconomic and environmental considerations and challenges, are presented in this part. Part Two of the book chapter highlights the latest technological advancements in biofuel production. This part discusses strategies to produce biofuels, such as chemical and biochemical conversion processes and technologies. Parts Three and Four of the book provide details on thermal and thermochemical conversion processes, integrated production approaches, and applications of biofuels.

1.2 Biofuel production processes and technologies 1.2.1 Biofuel production from various feedstocks Biofuels can be produced from diverse feedstocks for various applications with the overarching aim of meeting global energy demand while minimizing environmental impacts. Different generations of biofuels are classified according to the raw materials used to prepare them. Each generation of biofuels has different benefits and drawbacks. First-generation biofuels utilize raw materials from the human food chain, including corn, sugarcane, and oilseeds, such as soybean and peanut. In Europe, biodiesel accounts for more than 80% of the total biofuel production, which is mainly derived from rapeseed oil (European Biomass Association, 2014). As compared with other feedstocks, the most prominent advantage of first-generation biofuels is the high conversion efficiency; these biofuels have high energy yield, and thus, are economically profitable. Fatty acids, sugars, and starch components in crops can be converted to biodiesel and bioethanol through basic biochemical processes. However, firstgeneration biofuels face severe criticism in terms of land use and food

An overview of biofuels and production technologies

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shortage; they are also considered to be a leading factor for increased food prices and deforestation in the Amazon and in Indonesia (Achinas et al., 2019). Second-generation biofuels are generated from nonfood biomass residues, such as agricultural wastes and woody materials. LCA studies have revealed that lignocellulosic bioethanol production is energetically sustainable and contributes more to reduction in GHG emissions than do first-generation biofuels (Angili et al., 2021; Morales et al., 2015). However, the recalcitrant structure of lignocellulose resists enzymatic digestion, therefore leading to relatively low biofuel yields (Table 1.1). Other challenges, including an unstable supply of raw materials and the use of terrestrial water in the production process, also represent great concerns. Conversion technologies for second-generation biofuels are still under development to improve their overall efficiency (Siqueira et al., 2020). In the past few years, the production cost of lignocellulosic waste has been reduced significantly from 157.3–171.2 €/L in 2015 to 81.5–95.4 €/L in 2020 (Achinas et al., 2019). Biofuel produced from microalgae is called third-generation biofuel and has been highlighted as a promising alternative to renewable sources due to its positive environmental and economic impacts. Unlike crop-based feedstocks, microalgae cultivation does not rely on cropland or large quantities of freshwater (Nie et al., 2020). Chlorella is a typical biofuel-producing microalga with a lipid-rich nature (35%–50% lipids) (Shah et al., 2018). Proteins and carbohydrates from algae can be converted to bio-oils and syngas by pyrolysis or gasification (Anto et al., 2020). Some algae can produce biohydrogen and Table 1.1 Yields from first- and second-generation biofuel production methods (in liters per unit of feedstock amount and cropland) (FAO, 2017; Millinger and Thr€an, 2018; Strengers et al., 2016). Generation of biofuel

Type of biofuel

First generation

Bioethanol Bioethanol Bioethanol Biodiesel Biodiesel Bioethanol

Second generation

Bioethanol

Feedstock

Sugar plant Corn Wheat Soybean Rapeseed Barley straw Palm wood

Yield (L/kg)

Yield (L/ha)

0.25–0.5 0.4–0.46 0.4 – – 0.054

5570–6840 3030 2000 530 1570 –

0.03

–

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biogas via anaerobic fermentation. In comparison with conventional feedstocks, biomass from algae has several advantages (Vij et al., 2021): • Faster growth and higher photoconversion capacity; • Batch or continuous cultivation throughout the year, making algae a constantly available source of energy; • Grown on nonagricultural lands using saline water or wastewater; • GHG reduction as the process can be coupled with carbon dioxide fixation. However, low conversion efficiencies based on current systems of production limit biofuel production from algae at an industrial level. Technological barriers in the selection of suitable algal species, cultivation procedures, reactor designs, and downstream extractions have yet to be overcome to make algae-based biofuels economically feasible (Vij et al., 2021). An emerging type of biofuel categorized as fourth-generation biofuel uses genetically modified algae for biofuel production; this type of feedstock is reported to have higher CO2 capture capacity and biofuel productivity (Zhu et al., 2017). For example, modified Phaeodactylum tricornutum sp. presented a 35% increase in lipid content and approximately 1.1-fold increase in triglyceride content (Yang et al., 2016). Moreover, some advanced studies have proposed the increased use of waste materials to produce biofuels. Nwankwor et al. (2021) investigated an approach to synthesize gasoline-range fuels from waste plastic through thermal catalytic cracking reactions, resulting in the highest liquid yield of 89.3% from used polystyrene (Nwankwor et al., 2021). Factory emission such as carbon dioxide can be recycled into gasoline by Fischer-Tropsch reactions to reach productivities as high as 3337 barrels per day (Damanabi and Bahadori, 2017). It has been estimated that biofuel production should be increased from 9.7  106 to 4.6  107 GJ/day between 2016 and 2040 to effectively limit global warming (Correa et al., 2019). First-generation feedstock is currently the main source of biofuels; in view of the issues of global hunger and deforestation, it is vital to develop its substitutes. Therefore, lignocellulosic wastes and microalgae are considered to be the best options currently available to achieve sustainability goals (Mat Aron et al., 2020).

1.2.2 Physical, chemical, and biochemical processes and technologies The production process of biofuels from feedstocks specific to the four generations of biofuels is illustrated in Fig. 1.3. Although the first to fourth generations use different raw materials, their conversion processes are based on similar principles.

An overview of biofuels and production technologies

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Fig. 1.3 An overview of biofuel production pathways using different feedstocks.

Pretreatment methods vary with the structural properties of the feedstock; the aim is to reduce particle size and partially remove recalcitrant components, which subsequently aids in conversion procedures that are mainly dependent on chemical compositions. Oil and lipids are converted to biodiesel through transesterification. Sugar, starch, and proteins are used to produce biofuel and biogas through fermentation and gasification, respectively. Biodiesel comprises C14-C20 fatty acid methyl esters (FAMEs) that are synthesized by transesterification of fatty acids and/or glyceryl esters in oilseeds, vegetable oils, animal fats, and waste cooking oils. Transesterification reactions are accelerated by catalysts, including strong sulfuric acid, strong alkalis, solid acids/alkalis, and suitable enzymes (lipases). Current industrial production methods mainly use alkaline catalysis due to their high conversion efficiency; however, the alkaline wastewater produced from this method causes environmental pollution (Ambat et al., 2018). In comparison, methods that use enzymatic catalysis consume small amounts of alcohol under mild conditions and release small amounts of pollution. Eversa (from Novozymes) can produce FAMEs with a yield of 97% in 16 h with 1 wt% enzyme dose (Mibielli et al., 2019), demonstrating the potential feasibility of enzymatic catalysis for biofuel production. Sugar from starch-rich plants (e.g., corn) and lignocellulosic biomass is released via hydrolysis by concentrated acids or enzymes; this process is termed

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saccharification. In view of the high operational costs of acid recovery and equipment corrosion problems with acid hydrolysis, enzymatic hydrolysis is the preferred method. Various recalcitrant structures in different types of lignocellulosic biomass require enzyme cocktails to perform a specific synergistic action (Lopes et al., 2018), which poses the main technical obstacle in achieving efficient saccharification. In recent years, Novozymes (Denmark) has developed a new technology based on tailored enzymatic cocktails for specific feedstocks to deliver the best performance in hydrolytic conversion processes (Lopes et al., 2018). On-site cellulase production is another strategy to encourage such feedstock-specific enzymatic degradation (Siqueira et al., 2020). Bioethanol can be produced from fermentable sugars by yeast, fungi, or bacteria. Current advances focus mainly on simultaneous saccharification and fermentation to improve the production efficiency and prevent substrate inhibition. Finally, after separation and purification, bioethanol can be used as a fuel independently or as a blend with gasoline. Another strategy is to convert biomass into a hybrid compound. In this case, different components do not need to be isolated through hydrolysis or extraction. Syngas fermentation is an attractive biochemical conversion route to produce H2 and CO or CO2 from biomass, followed by methanation, anaerobic fermentation, or Fischer-Tropsch reaction to produce biomethane, bioethanol, or biodiesel. Some companies, such as LanzaTech, are deploying commercial ethanol-production facilities to utilize syngas from orchard wood and nutshells (Liakakou et al., 2019). Co-gasification of coal and biomass is emerging as a clean fuel technology that can achieve high thermodynamic efficiency with relatively low CO2 emission (Kamble et al., 2019). At this stage, the most widely applied conversion method is anaerobic digestion (AD), by which all organic compounds can be metabolized using a group of diverse microorganisms to produce a gaseous mixture, i.e., biogas, mainly consisting of methane and carbon dioxide. This method can be applied to various types of substrates, such as sewage sludge and municipal solid waste, for energy generation. Moreover, AD should be highlighted as a cost-effective approach, especially for substrates containing high water content, which makes volume reduction and incineration difficult (Patel et al., 2016). Considering the slow reaction process (several days to weeks), numerous innovations have been proposed to improve biogas productivity, from new types of equipment to detailed operation conditions (Tabatabaei et al., 2020). Furthermore, the integration of AD into biorefinery frameworks has been considered as a potential strategy to upgrade biogas production systems (Budzianowski, 2016). The pros and cons of different biomass conversion technologies are summarized in Table 1.2.

Table 1.2 Summary of different biomass conversion technologies (Tabatabaei et al., 2020). Technology

Hydrolysis and fermentation Gasification

Conversion conditions

30–50°C; pH 4.5–6.0 350–1800°C

Main products

Advantages

Disadvantages

Sugar, bioethanol, CO2 Gas (CO, CO2, H2, CH4)

•

Large-scale application

• •

• • •

Compact Low cost High efficiency (40%–50%)

• • •

Anaerobic digestion

35–55°C; anaerobic

Gas (CO2, CH4)

• •

Commercially proven Applicable to organic waste with high water content

• • •

Complexity High cost (especially with lignocellulosic biomass) Lab- or pilot-scale High emissions of NOx, CO2, and ash Complexity (especially lignocellulosic biomass) Bad odors Low substrate levels inside the reactor Long reaction process

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1.2.3 Microbes involved in biofuel production processes Microbes play important roles in biochemical conversion processes by functioning as the source of lignocellulosic material, enzyme producers, or metabolic factories. As stated in Section 1.2.1, the third and fourth generations of biofuel are obtained from microalgal oils. Approximately 40,000 microalgal species have been identified to date; many of these can accumulate up to 20%–80% of lipid by total biomass content (Khan et al., 2018; Mobin and Alam, 2017), where mixotrophic microalgae can produce 69% higher lipid content than heterotrophic microalgae. Table 1.3 lists some intensively studied microalgae species, including the commonly occurring Chlorella sp. The yield of lipids from microalgae is sensitive to cultivation conditions, including light intensity, temperature, and carbon source, among others. The lipid content increases in Isochrysis galbana and Scenedesmus obliquus at high temperatures, while the opposite is true for Chlorella vulgaris (Ruangsomboon, 2012). Therefore, the evaluation of a specific strain and understanding of its optimum growth environment is essential to harvest the highest amount of lipids. Genetically modified microalgae employed in fourth-generation biofuels exhibit improved adaptability for oligotrophic environments, such as wastewater (Abdullah et al., 2019), along with enhanced lipid productivity and carbon fixation (Table 1.4). Microbes function as enzyme producers in the hydrolysis of starch plants and lignocellulosic biomass. Amylase, glucoamylase, and cellulase cocktails are essential catalysts to convert starch and cellulose into fermentable sugars. Aspergillus sp., Trichoderma reesei, and bacteria, including Bacillus licheniformis, are employed as host strains for industrial enzyme production.

Table 1.3 Microalgae species for oil production (Mat Aron et al., 2020). Species or genus

Oil composition (%)

Lipid productivity (mg/L/day)

Ankistrodesmus Botryococcus braunii Chlorella protothecoides Chlorella sorokiniana Chlorella vulgaris Dunaliella tertiolecta Nannochloropsis Scenedesmus

28–40 34–75 40–55 22–24 35–45 33 35–47 34

459 – 1209–3701 420–550 200–1100 – 290–321 820

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Table 1.4 Lipid production and fixation of GHGs by genetically modified microalgae. Process

Species

Achievements

Biofuel production

Chlamydomonas reinhardtii

• •

Phaeodactylum tricornutum

• •

Chlamydomonas reinhardtii Synechococcus elongatus

•

GHG fixation

•

1.5-fold increase in lipid content 20% increase in triglyceride content 35% increase in lipid content
1.1-fold increase in triglyceride content 50% increase in photosynthesis efficiency 41% increase in carbon uptake

1.3 Technoeconomic and environmental assessment The potential of biofuels in the field of energy security and mitigation of environmental pollution has led to their entry into the energy market. Beyond energy benefits and pollution control, the development of biofuels also aids in the creation of employment and strengthens the economy. The generation of bioenergy is reported to have several technoeconomic, social, and environmental advantages and disadvantages for sustainable development. Positive attributes of biofuels include mitigation of environmental pollution, energy security, provision of an alternate source of clean and green fuel, regional growth, and rural development. Contrarily, there are several serious concerns associated with biofuel production, including competition with food cultivation that leads to an increase in food prices, high production costs, energy extensive processes, reduction in land and water quality by extensive use of agrochemicals for enhanced crop production for biofuels, loss in biodiversity, and deforestation. Although biofuels have entered the global market despite these shortcomings, their acceptance and commercialization proceeding much slower than expected. The main obstacles in their commercialization include the requirement of large capital investments, operational challenges, extensive resource utilization, spatial distribution, and variability in biomass feedstock (Anderson et al., 2020). Governmental support and policies are direly needed to address technical, socioeconomic, and environmental issues related to biofuel production and to promote it sufficiently to reach ambitious production targets. The governments of various developed and developing countries have attempted to do so by introducing incentives for development of biofuel production

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units. The production of biofuels can further be facilitated by regulatory directives for biofuel use, provision of financial aids, subsidies, promotion of policies supporting national/international collaborative ventures, and grants and policies that strengthen research and development. Furthermore, sustainable development efforts should also focus on the LCA of biofuels. The LCA of biofuel production is an efficient cradle-to-grave approach for the determination of the environmental effects of the product, production process, and technology involved. Dedicated software and inventory databases efficiently facilitate LCA calculations. Several LCA studies focusing on GHG emissions, carbon footprint, global warming potential, and energy balance have been conducted to assess the impact of feedstock, the production process, and the biofuel supply chain. The LCA can generate distinct results for apparently analogous systems because of the influence of system boundaries, variation in background data, assumptions, models, and the direct and indirect factors affecting the study (Wang et al., 2021). To bring consistency in LCA analysis, studies are being conducted around the globe for the development of inventories and databases, as well as guidelines and standards, for curating average data for fuel production systems (Chandrasekaran et al., 2021). A comparative LCA between biofuels and fossil fuels will facilitate an efficient evaluation of the environmental performance of the two types of fuels. Furthermore, the potential of biofuels to fully replace fossil fuels in major sectors, such as transportation, can be evaluated by taking into consideration the LCA results and other technoeconomic factors. Among major biofuels, bioethanol and biodiesel have the widest acceptability in the transportation sector. They are safe to handle, biodegradable, renewable, and cost-effective. The current scenario of energy crisis has necessitated the promotion of biofuels as an alternative fuel for use in motorized vehicles. Biofuels have the potential to substitute conventional fossil fuels in diesel engines as they are very similar to conventional diesel. Biodiesel offers improved viscosity, cetane number, energy content, and phase changes. Furthermore, on account of its high flashpoint of
150°C, biodiesel is a viable and safe alternative to conventional fuels and can be efficiently used in existing engines. Additionally, biodiesel provides better lubrication than conventional diesel, which enhances engine performance and prolongs engine life. Biodiesel has also been reported to contribute to reduction in vehicular emissions, particularly the emission of hydrocarbons, particulate matter, carbon dioxide, sulfur dioxide, and carbon monoxide; however, it generates slightly higher NOx emissions (Shahid et al., 2021). Most of

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the vehicles currently on the market can efficiently use a blend of 10% biofuel and more. Biofuels must comply with the prescribed quality standards to ensure optimal engine performance so that they can be used in engines and vehicles either in pure form or as a blend. However, certain limitations, such as NOx emissions, fuel freezing at low temperature (cloud point), corrosiveness to brass and copper, pumping and atomization problems due to high viscosity, and degradation under extended storage periods, need to be addressed for their efficient application in engines (Niculescu et al., 2019). As an alternative to liquid fuels, electricity-based engines have been developed as another feasible option for use in the transportation sector. The major advantage of electric vehicles is that there is no direct pollutant emission. Despite this, the current transportation sector favors liquid fuels over electricity due to several advantages that the latter offers. In comparison with electric vehicles, fuel-driven vehicles have greater mileage and faster and easier fuel refilling. The continuous supply of conventional fuels, along with ease of transportation, handling, and storage, encourages the transportation infrastructure to continue in its current fuel-based mode. However, with the development of modern technologies and alternatives, the complete replacement of fuels with electricity is now a possibility.

1.4 Challenges, opportunities, and future prospects in biofuel production 1.4.1 Challenges: Socioeconomic and technological challenges While many politicians, financial institutions, and scientists are confident that biofuels could be promising alternatives to fossil fuels, we must remember that no energy-generation technology is perfect: Everything has two sides, and the benefits and drawbacks will wax and wane in prominence as external conditions change. The feedstocks used for current commercial biofuel production, such as corn and palm, are grown in “high-input low-diversity” systems, which are typified by large-scale monocrops with extensive usage of pesticides, fertilizers, and irrigation. Due to various flaws, these systems have been criticized extensively. First, current biofuel production only utilizes a portion of the biomass, such as seeds or fruits, leaving a significant amount of biomass energy unused in straws or leaves. Maintaining such a system, which favors monoculture of energy crops, necessitates large areas for crop production, creating a competition for space and resources, such as food and livestock

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feed. Change in land use for monocultural ecosystems may even increase soil erosion and decrease soil fertility. Furthermore, this system may increase the net GHG emissions over its life cycle, as intensive farming practices that require reclamation, irrigation, fertilization, and pest and weed control to achieve high yields may promote soil respiration and increase CO2 emissions. Consequently, land degradation and groundwater and air pollution occur, which adversely affect human health. Monocultural agriculture also has the potential to diminish regional habitat diversity. In addition, an inappropriate introduction of energy crops may result in biological invasion issues. Cellulose-based biofuel production is garnering much scientific and industrial attention as it is more profitable than crop-based biofuels. Pretreatment, solid-liquid separation, hydrolysis, saccharification, fermentation, distillation, and purification are all required in the production of ethanol from cellulose. The major technological challenge is the pretreatment process, which transforms cellulose into fermentable components. Presently available methods are prohibitively costly for commercialization. The enzymes that convert cellulose to ethanol are plant-specific and may exhibit different effects on cellulose in different plants. The pretreatment process may become more complicated if a range of plants are mixed and utilized as feedstock. Therefore, the discovery of novel enzyme proteins that can effectively degrade cellulose is a major focus of biofuel research (Horn et al., 2012). Microbial species are a key component of biofuel production as microorganisms can utilize straw and other biomass as raw materials for biotransformation into biofuels. The commercialization of biofuel requires the use of engineered or artificial microorganisms with characteristics superior to those of wild species. Thus, integrating genetic and metabolic engineering with synthetic and systematic biology is key to building cellular factories that can improve the production economics of biofuels. However, the elaborate regulatory systems in microbes are difficult to manipulate as they have evolved over millions of years. It is challenging to find suitable enzyme targets and metabolic pathways for modification or replacement, and the insertion or deletion of certain genes might affect the expression of other genes in the strain. In addition, engineered strains may face issues, such as strain degeneration, that do not affect naturally evolved strains. Biofuels from algae/microalgae have the potential to become important renewable energy sources in the future considering that algae have the characteristics of broad geographic distribution, high oil content, strong environmental adaptation, short growth cycles, and high yield as compared with nonfood biomass

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(Chowdhury and Loganathan, 2019). However, the difficulty with thirdgeneration biofuels is their inability to replicate the productivity achieved in laboratory cultures on large industrial scales due to limiting factors such as undesired growth rate, insufficient light for photosynthesis, and inefficient harvesting and extraction technologies (Leite et al., 2013). Although genetic modifications might alleviate the inefficiency of biological functions, the question of whether it is worth risking an invasion of fisheries and water resources by a possibly harmful algal species is still being debated. Costs and profits are another major concern in matching biofuel technologies with currently available fuels in the market. The primary raw materials of biofuels are still corn and sugarcane, and the future development in the crop-based biofuel sector is unclear due to changes in crop supply and demand. The switch from starch/sugar-based biofuels to second-generation cellulose-based biofuels would be advantageous as raw materials are available and abundant, which ensures food security. Nevertheless, the conversion technology for cellulose-based biofuel is still plagued by issues such as raw material collection, high production costs, and influence of oil price. Second-generation biofuels based on lignocellulosic materials may have great promise, but for the time being, they can only replace a tiny fraction of the world energy supply. Moreover, given the present agricultural production conditions and biofuel processing technologies, biofuel production in many countries is not economically feasible without subsidies. The competitiveness of different biofuels, feedstocks, and production sites, and thus, and their economic feasibility, may vary with time due to changes in the market and technical advancements in industry. Biofuel businesses are flourishing due to policy interventions, such as subsidies, and the obligation to blend biofuels with fossil fuels. Current biofuel policies, however, should be evaluated, and their costs and effects should be analyzed thoroughly. In both developed and developing countries, many policies have resulted in substantial economic, social, and environmental consequences. Interactions between agriculture, biofuels, and policies frequently disfavor biofuel feedstock producers in developing countries, exacerbating the hurdles to the establishment of production and export businesses in these counties (Saravanan et al., 2018).

1.4.2 Opportunities and future prospects in biofuel production: Replacement of fossil fuels Sustainable alternatives to fossil fuels have superior environmental impacts and economic benefits and thus, will satisfy long-term energy needs. The essential value of biofuels is their ability to capture solar energy via green

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Handbook of biofuels production

plants as a low-cost energy storage medium. The benefits of replacing traditional fossil fuels with biofuels in terms of environmental protection include lower GHG net emissions and air pollution, less contamination of soil and groundwater, and better waste management (assuming that waste streams, such as household waste, are used to produce biofuels). Biofuels can replace certain fossil fuels in the power or transportation sectors, decreasing reliance on fossil fuel supplies from the standpoint of energy security and national security. Biofuels can boost agricultural, forestry, and industrial development by promoting and supporting the production of energy crops and trees, generating job opportunities, and thus, contributing to social stability and development (Demirbas, 2008; Fargione et al., 2008). To address the aforementioned impacts and challenges of biofuel production, it is important to expedite the development of technologies for the production of biofuels using the second generation of biomass raw materials. The utilization of nonfood feedstocks, such as agricultural waste, municipal, and forest waste, as well as fast-growing energy crops, such as switchgrass, enhances the promise of biofuels as a sustainable technology (Skaggs et al., 2018). Microbial cell factories, which are the core of biorefining, also pave a green road for biofuel production. Combining metabolic engineering and synthetic biology technologies to construct an efficient microbial cell factory has demonstrated promising results and a large market opportunity (Choi et al., 2019). Microbes can help improve biofuel production; however, owing to high production costs and policy changes, such as subsidies, many companies are unwilling to continue to engage in technological research and development, which is critical for the biofuel industry. Technical advancements can lower the cost of agricultural production and biofuel processing and thus, make biofuels an economically and environmentally sustainable energy source in the future. Research and development of second- and third-generation technologies, in particular, might significantly improve the future applicability of biofuels (Rodionova et al., 2017). Biofuel production from cellulose biomass and next-generation biofuel conversion technologies will be able to compete with conventional fossil fuels without the need for government subsidies as scientific research and industrial technology advances. Based on biofuels’ history and current scenarios, their production should have a bright future despite the numerous challenges faced by this industry. However, it is undeniable that the global economic system and patterns are evolving in tandem with the advancement of civilization and technology. We must recognize that the growth of biofuels is a century-long issue that

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21

must be addressed on a scientific basis and with prudence. Rather than naively chasing short-term economic gains, it is important to comprehend the associated technological, social, and environmental challenges faced in biofuel production. Biofuels should be developed as part of broader regional and national sustainable development strategies and not only in the context of satisfying energy requirements. To achieve this goal, the primary task is to promote sustainable research and application of biofuels in collaboration with scientists, decision-makers, governments, and stakeholders. This will aid in making positive contributions to ecosystem management, selection of suitable energy crops, environmental assessment, and ecological monitoring.

1.5 Conclusion Biofuels have the potential to ensure energy security and are considered to be a substitute for depleting fossil fuels. Biofuels promise a clean, green, and sustainable source of energy. It is now apparent that there is a need to explore means of synthesizing bioenergy from locally accessible, reliable, affordable, and low-cost feedstocks that can meet the energy demand. The use of biomass as feedstock is a lucrative option for biofuel production as it ensures continuous production and regional cultivation by supporting the valorization of local production chains. Owing to the several benefits of biofuels and an increase in the demand for fuels, the world has witnessed an expansion in the biofuel market, which has driven nations to design and frame policies that encourage and boost biofuel production at the domestic level. Biofuel-producing countries heavily rely on domestic feedstock for biofuel production, which is influenced by factors such as climate, region, and market trends. The overall price of biofuels largely depends on the type of biofuel, feedstock, geographical location, resources required, such as land or water, competition with food crops, the efficiency of production technology, and the global crude oil price. To encourage the development of biofuel industries, governments should devise financial incentives, subsidies, and platforms for research and development to ensure the accomplishment of socioeconomic and environmental goals with a scope for international trade. For sustainable biofuel production, there is a need for further research exploring new avenues, such as identification of efficient biomass sources, technological development, and genetic modification of producer microorganisms.

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Horn, S.J., Vaaje-Kolstad, G., Westereng, B., Eijsink, V., 2012. Novel enzymes for the degradation of cellulose. Biotechnol. Biofuels 5, 1–13. Kamble, A.D., Saxena, V.K., Chavan, P.D., Mendhe, V.A., 2019. Co-gasification of coal and biomass an emerging clean energy technology: status and prospects of development in Indian context. Int. J. Min. Sci. Technol. 29, 171–186. Kathrotia, T., Oßwald, P., Zinsmeister, J., Methling, T., K€ ohler, M., 2021. Combustion kinetics of alternative jet fuels, part-III: fuel modeling and surrogate strategy. Fuel 302, 120737. Khan, M.I., Shin, J.H., Kim, J.D., 2018. The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microb. Cell Fact. 17, 1–21. Kuo, N.W., Chen, P.H., 2009. Quantifying energy use, carbon dioxide emission, and other environmental loads from island tourism based on a life cycle assessment approach. J. Clean. Prod. 17, 1324–1330. Leite, G.B., Abdelaziz, A.E., Hallenbeck, P.C., 2013. Algal biofuels: challenges and opportunities. Bioresour. Technol. 145, 134–141. Liakakou, E.T., Vreugdenhil, B.J., Cerone, N., Zimbardi, F., Pinto, F., Andre, R., Marques, P., Mata, R., Girio, F., 2019. Gasification of lignin-rich residues for the production of biofuels via syngas fermentation: comparison of gasification technologies. Fuel 251, 580–592. Lopes, A.M., Ferreira Filho, E.X., Moreira, L.R.S., 2018. An update on enzymatic cocktails for lignocellulose breakdown. J. Appl. Microbiol. 125, 632–645. Mat Aron, N.S., Khoo, K.S., Chew, K.W., Show, P.L., Chen, W.H., Nguyen, T.H.P., 2020. Sustainability of the four generations of biofuels—a review. Int. J. Energy Res. 44, 9266–9282. Mibielli, G.M., Fagundes, A.P., Bender, J.P., Vladimir Oliveira, J., 2019. Lab and pilot plant FAME production through enzyme-catalyzed reaction of low-cost feedstocks. Bioresour. Technol. Rep. 5, 150–156. Millinger, M., Thr€an, D., 2018. Biomass price developments inhibit biofuel investments and research in Germany: the crucial future role of high yields. J. Clean. Prod. 172, 1654–1663. Mobin, S., Alam, F., 2017. Some promising microalgal species for commercial applications: a review. Energy Procedia 110, 510–517. Morales, M., Quintero, J., Conejeros, R., Aroca, G., 2015. Life cycle assessment of lignocellulosic bioethanol: environmental impacts and energy balance. Renew. Sust. Energ. Rev. 42, 1349–1361. Niculescu, R., Clenci, A., Iorga-Siman, V., 2019. Review on the use of diesel– biodiesel–alcohol blends in compression ignition engines. Energies 12 (7), 1194. Nie, J., Sun, Y., Zhou, Y., Kumar, M., Usman, M., Li, J., Shao, J., Wang, L., Tsang, D.C.W., 2020. Bioremediation of water containing pesticides by microalgae: mechanisms, methods, and prospects for future research. Sci. Total Environ. 707, 136080. Nwankwor, P.E., Onuigbo, I.O., Chukwuneke, C.E., Yahaya, M.F., Agboola, B.O., Jahng, W.J., 2021. Synthesis of gasoline range fuels by the catalytic cracking of waste plastics using titanium dioxide and zeolite. Int. J. Energy Environ. Eng. 12, 77–86. Osaki, K., 2019. US Energy Information Administration (EIA): 2019 edition US Annual Energy Outlook report (AEO2019). Haikan Gijutsu 61, 32–43. Patel, M., Zhang, X., Kumar, A., 2016. Techno-economic and life cycle assessment on lignocellulosic biomass thermochemical conversion technologies: a review. Renew. Sustain. Energy Rev. 53, 1486–1499. Pittock, J., Hussey, K., Dovers, S., 2015. Climate, Energy and Water. Cambridge University Press.

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Rodionova, M.V., Poudyal, R.S., Tiwari, I., Voloshin, R.A., Zharmukhamedov, S.K., Nam, H.G., Zayadan, B.K., Bruce, B.D., Hou, H., Allakhverdiev, S.I., 2017. Biofuel production: challenges and opportunities. Int. J. Hydrog. Energy 42, 8450–8461. Ruangsomboon, S., 2012. Effect of light, nutrient, cultivation time and salinity on lipid production of newly isolated strain of the green microalga, Botryococcus braunii KMITL 2. Bioresour. Technol. 109, 261–265. Saravanan, A.P., Mathimani, T., Deviram, G., Rajendran, K., Pugazhendhi, A., 2018. Biofuel policy in India: a review of policy barriers in sustainable marketing of biofuel. J. Clean. Prod. 193, 734–747. Shah, S.H., Raja, I.A., Rizwan, M., Rashid, N., Mahmood, Q., Shah, F.A., Pervez, A., 2018. Potential of microalgal biodiesel production and its sustainability perspectives in Pakistan. Renew. Sustain. Energy Rev. 81, 76–92. Shahid, M.K., Batool, A., Kashif, A., Nawaz, M.H., Aslam, M., Iqbal, N., Choi, Y., 2021. Biofuels and biorefineries: development, application and future perspectives emphasizing the environmental and economic aspects. J. Environ. Manag. 297, 113268. Siqueira, J.G.W., Rodrigues, C., Vandenberghe, L.P.D.S., Woiciechowski, A.L., Soccol, C.R., 2020. Current advances in on-site cellulase production and application on lignocellulosic biomass conversion to biofuels: a review. Biomass Bioenergy 132, 105419. Skaggs, R.L., Coleman, A.M., Seiple, T.E., Milbrandt, A.R., 2018. Waste-to-energy biofuel production potential for selected feedstocks in the conterminous United States. Renew. Sust. Energ. Rev. 82, 2640–2651. Statista, 2020. https://www.statista.com/statistics/274163/global-biofuel-production-inoil-equivalent/. Statistical Review of World Energy, 2020. https://www.forbes.com/sites/rrapier/2020/06/ 20/bp-review-new-highs-in-global-energy-consumption-and-carbon-emissions-in2019/?sh¼4035f0cc66a1. Strengers, B., Overmars, K., Kram, T., Ros, J., 2016. Greenhouse gas impact of bioenergy pathways. PBL Planbureau voor de Leefomgeving. Tabatabaei, M., Aghbashlo, M., Valijanian, E., Kazemi Shariat Panahi, H., Nizami, A.S., Ghanavati, H., Sulaiman, A., Mirmohamadsadeghi, S., Karimi, K., 2020. A comprehensive review on recent biological innovations to improve biogas production, Part 1: Upstream strategies. Renew. Energy 146, 1204–1220. U.S. Energy Information Administration (EIA), 2020. https://www.eia.gov/energy explained/biofuels/use-of-biodiesel.php. Vij, R.K., Subramanian, D., Pandian, S., Krishna, S., Hari, S., 2021. A review of different technologies to produce fuel from microalgal feedstock. Environ. Technol. Innov. 22, 101389. Wang, D., Jiang, D., Fu, J., Hao, M., Peng, T., 2021. Assessment of liquid biofuel potential from energy crops within the sustainable water–land–energy–carbon nexus. Sustain. Energy Fuels 5, 351–366. World Bioenergy Association, 2020. Global Bioenergy Statistics 2019. World Bioenergy Association. Yang, J., Pan, Y., Bowler, C., Zhang, L., Hu, H., 2016. Knockdown of phosphoenolpyruvate carboxykinase increases carbon flux to lipid synthesis in Phaeodactylum tricornutum. Algal Res. 15, 50–58. Zhu, B., Chen, G., Cao, X., Wei, D., 2017. Molecular characterization of CO2 sequestration and assimilation in microalgae and its biotechnological applications. Bioresour. Technol. 244, 1207–1215.

CHAPTER 3

Biofuels: Technology, economics, and policy issues Piergiuseppe Morone, Loris Cottoni, and Fabio Giudice Unitelma-Sapienza, Universita` degli Studi di Roma, Roma, Italy

3.1 Introduction Significant socioeconomic changes are expected to occur in the near future. Most notably, the world population is projected to grow from 7.7 billion in 2019 to around 8.5 billion in 2030, 9.7 billion in 2050, and 10.9 billion in 2100 (United Nations, 2019). At the same time, large and fast-growing economies are projected to experience increasing wealth, with Brazil, Russia, India, China, and South Africa (the BRICS countries) leading the change. The BRICS countries are expected to climb into the group of top-ranking countries by share of global gross domestic product by 2050, with China leading the group ahead of the United States. Other emerging-market countries, such as Indonesia, Mexico, and Turkey, are projected to closely follow this leading group (McManus, 2016). Such demographic and economic trends will lead to a so-called explosion in the global middle class, a phenomenon that has been taking shape over the last 10 years but whose pace of expansion is likely to increase, reaching 5.4 billion people around 2030. International analysts forecast that 2 billion people could join the middle class—defined as those earning between US$6000 and US$30,000 a year on a purchasing-power parity basis—by 2030 (Wilson and Dragusanu, 2008). Socioeconomic growth correlates with increasing consumption and greater demand for processed foods and manufactured goods, adding pressure to the energy supply system. A per capita income of US$6000 has been identified as the entry income-level that causes a rapid increase in energy demand, with a slightly higher threshold—US$8000–9000—leading to increased demand for higher-end consumer durables such as automobiles (Wilson and Dragusanu, 2008). Although fossil fuels accounted for over The opinions contained in this text are expressed entirely in a personal capacity and are in no way attributable to the Ministry of Foreign Affairs and International Cooperation. Handbook of Biofuels Production https://doi.org/10.1016/B978-0-323-91193-1.00012-3

Copyright © 2023 Elsevier Ltd. All rights reserved.

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81.2% of the world’s primary energy consumption in 2020 (International Energy Agency, 2020), the finite nature of these fuels renders them unreliable as long-term resources; it is recognized that currently known reserves may last as little as half a century—depending, of course, on rates of production and consumption. British Petroleum (BP), for instance, issued a 2019 report in which it stated that oil reserves at then-current production rates would last 50 years.a BP’s CEO, Bernard Looney, affirmed that the COVID-19 crisis “is bound to reshape the global economic, political and social environment in which we all live and work. It has the potential to accelerate emerging trends and create opportunities to shift the world onto a more sustainable path” (BP Statistical Review of World Energy, 2020, p. 2). However, he acknowledged that coal is still the single largest source of power generation, accounting for 36% of global power generation in 2020. The pandemic caused sharp declines in energy demand—especially during the spring of 2020—of up to 8% for coal, 5% for oil, and 2% for gas, which were mainly due to lockdowns and travel restrictions (IEA, 2020). As reported by the International Energy Agency (IEA), renewables were the only energy source registering growth in demand in 2020, driven by a larger installed capacity and policies promoting priority dispatch of renewables ahead of other generation sources (IEA, 2020). For 2020, the projected contraction in overall energy demand was around 6%, the largest ever in absolute terms (IEA, 2020). For the same year, global carbon dioxide (CO2) emissions were expected to decline by 8% to the same level as 10 years earlier, but as the IEA warns, without investment in green energy, the subsequent rebound in emissions may be larger than the decline. As affirmed by the Organization for Economic Co-operation and Development (OECD), the spread of COVID-19 raised awareness of the consequences of a lack of pandemic resilience and preparedness; in the future, climate change, water pollution, deforestation, and illegal wildlife trade may increase the risk of further pandemics. The OECD stresses the importance of continued investment in economic transformation and technological innovation, notwithstanding the need for swift economic recovery; the stimulus packages that states are implementing might offer a timely opportunity to favor a more sustainable and environmental-friendly economy (OECD, 2020).

a

https://blogs.platts.com/2019/06/25/global-oil-reserves-data-muddled/.

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It appears that growing awareness of the intrinsic unsustainability of the current economic model has contributed to emergence of the idea that modern society should make a paradigm shift toward a biobased circular economy. This conceptualized new economic model would use biomass as the key input for production and would reuse, repair, refurbish, and recycle existing materials and products, turning waste into resources. Taking into consideration the economic, social, environmental, and policy issues that are at stake, research on biofuels is expected to be a part of this new model. In this chapter, we attempt to address this challenging task by trying to understand the current technological and economic standing with regard to this paradigm shift and what might drive this shift in the near future. The remainder of the chapter is structured as follows: Section 3.2 provides a theoretical framework for understanding the change and identifying the key variables involved and looks at alternative development patterns. Section 3.3 provides an overall assessment of various biofuels, examining the comparative levels of innovativeness, sustainability, and readiness of alternative technologies. Section 3.4 reflects upon economic, environmental, and social concerns. Section 3.5 addresses policy and regulatory issues associated with the change. Section 3.6 presents an overall conclusion of the chapter.

3.2 Moving from fossil fuel to biofuels: Insights from sociotechnical transition theory The paradigm shift from a society heavily based on traditional fossil fuels toward a society based on renewable energy will be neither easy nor automatic. Such a shift will involve transitioning to a resource-efficient society that is increasingly based on biomass-derived fuels, chemicals, and materials. Such a major change must entail a sociotechnical transition—from an old production paradigm to a new one—in which social and technological relationships will coevolve. Based on insights from evolutionary economics, scholars have recently developed a heuristic model to study such complex technological changes (van den Bergh et al., 2011). In this model, the innovation process within a given sociotechnical system is characterized by the adaptive capacity of the incumbent sociotechnical regime, by the competitive selection pressures exerted by other regimes, and by new sociotechnical configurations in niches (Rip, 1992; Smith et al., 2005). Moving from the assumption that such processes occur in a multidimensional space, this multilevel perspective (MLP) can be used to examine how

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innovation (re)configures the social and technical elements of a system. The MLP conceives an evolving sociotechnical system as structured along three levels of analysis: the landscape (macro-level), the sociotechnical regime (meso-level), and the niche (micro-level). The landscape and the niches are derived concepts because they are defined in relation to the regime (Geels, 2002). In turn, the sociotechnical regime is defined as a stable configuration of various elements (including institutions, techniques and artifacts, infrastructures, power relations, rules, policies, and competences) that determine the “normal” development and use of technologies. Such technologies are subject to a “lock-in” effect that strengthens the stability of the configuration and braces institutions, social practices, and technological infrastructures (Raven et al., 2010). The dominant rules or modes of thinking that guide approaches and actions within a regime effectively exclude radically alternative innovations. Hence, a regime’s evolution is path-dependent and occurs mostly through incremental innovations (Kemp et al., 1998). However, radical, path-breaking innovation can occur in niches, where rules, institutions, and motives differ from those of the encompassing regimes; these are “protected spaces” where “nurturing and experimentation with the co-evolution of technology, user practices, and regulatory structures” take place (Schot and Geels, 2008, p. 538). In other words, niches are like incubation rooms in which new and emerging technologies are temporarily protected from normal market competition and pressures. Niches follow an evolutionary process that might ultimately lead them to full development status (or maturity, in the terminology of Lopolito et al., 2011). A niche is considered fully developed when there is substantial sharing of knowledge within the niche, when relevant and influential actors are attracted to it, and when those actors converge on a common set of expectations for the future development of niche technology (Lopolito et al., 2011). Niche maturity is a necessary condition for a change to happen but is not sufficient. For a transition to occur, adequate pressure must also be exerted on the regime from the landscape level. Such pressure destabilizes the capability of a regime’s configuration to keep pace with evolving norms and rules (Schot and Geels, 2008), providing an opportunity for change. Building on the MLP, a new strand of literature has focused on the study of sustainability transitions, in which the notion of transition has been applied to fundamental environmental challenges in several sectors, including transportation and congestion (especially with regard to road traffic), air pollution, fossil fuel depletion, and CO2 emissions, as well as various environmental issues associated with agricultural and food systems

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(Geels, 2010, 2011). Such sustainability transitions are challenged by the strong path dependencies and lock-ins that prevail in such sectors (Ahman and Nilsson, 2008; OECD/IEA, 2011; Safarzynska and van den Bergh, 2010; Unruh, 2000). As a case in point, when addressing the transition from a fossil fuel economy to a biofuel economy, one should bear in mind that the fossil fuel technological regime is mostly characterized by the presence of large firms (e.g., car manufacturers, electric utilities, oil companies, food and agriculture companies) that have access to “complementary assets,” such as specialized manufacturing capabilities, experience with large-scale test trials, distribution channels, service networks, and complementary technologies (Rothaermel, 2001). The strength of the incumbent fossil fuel regime implies that the transition to a sustainable economy will require concerted pressure from “technology, policy/power/politics, economics/business/markets, and culture/discourse/public opinion” (Geels, 2011, p. 25). MLP analysis of green innovations has often been conducted without sufficient attention to the roles of power and politics underpinning the development and implementation of specific policies or to the impact of existing regimes and incumbent actors. Policymakers and incumbent firms can be seen as core alliances at the regime level, oriented toward the status quo because of mutual dependencies (Geels, 2014). Moreover, although there is a general vision of the sustainable technologies that will be required for the transition to a biofuel economy (or, more broadly, to a bioeconomy), the emergence of such technologies faces additional barriers, such as the absence of well-defined technological trajectories, long development times, and crucially, uncertainties regarding market demand and social and environmental gains (Markard et al., 2020; K€ ohlera et al., 2019). Significant effort has been invested in scientific research to reduce these uncertainties, combining technological assessments with economic evaluations and environmental sustainability studies. The roles of policy and regulation issues have also been investigated. The following sections review these studies to assess the development status of biofuels as a fundamental step toward a sustainable transition.

3.3 Assessment of first- and next-generation biofuels Biofuels (or agrofuels) are renewable fuels derived from biological feedstock. Differently from fossil fuel production, the production of biofuels does not typically entail the release of hazardous compounds. Biofuels can be solid

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(e.g., firewood), liquid, or gaseous (e.g., methane, biogas, bio-hydrogen) and can be produced by converting biomass to biofuel through chemical, biochemical, and thermal conversion processes. Currently, there are unsolved technological, economic, and policy questions that make the sustainability of biofuels uncertain. The following section addresses these issues, distinguishing between first-, second- (or next-), and third-generation biofuels.b

3.3.1 First-generation: Bioethanol, biodiesel, and other biofuels First-generation biofuels—most notably bioethanol and biodiesel—should no longer be regarded as residing in technological niches; they are part of the sociotechnical regime, and their distribution is widespread and consolidated throughout the world. Around 5% of the world’s arable lands are currently used to grow feedstock for biofuel production using mature technologies.c However, the commercial competitiveness of firstgeneration biofuels usually remains poor compared with fossil fuels due to higher production and processing costs. A partial exception regarding the production of sugarcane ethanol in Brazil is discussed in Section 3.5.1. In addition, the competitiveness of biofuels is strongly linked to oil price fluctuations,d causing uncertainty for producers and investors. When oil prices are high, demand for biofuels can escalate, creating a risk that market demand will exceed supply. However, when oil prices are low and remain low for a long time, the biofuels industry risks unbearable financial losses.e Here, we consider the technical and economic features of the different biofuel types. b

The term first-generation biofuels usually refers to ethanol produced from sugar-rich and starch-rich crops, to biodiesel made from oilseed crops or animal fat, and to pure plant oil (PPO). Typically, these feedstocks can also be used as food for human consumption or as livestock feed. Second-generation biofuels are those made from nonedible and/or lignocellulosic biomass, with typical outputs of lignocellulosic ethanol, biomass-to-liquids, and biosynthetic natural gas. Third-generation biofuels are usually those that do not compete with food crops or for land; algae-based biofuels typically fall within the third-generation category (HLEP, 2013). c http://biodieselmagazine.com/articles/2516887/biofuels-require%20-little-arable-landbut-provide-many-benefits d Biofuel competitors include methane, liquefied petroleum gas, and electricity used for marine, aviation, and agricultural purposes. e These circumstances have induced several national governments to enhance the competitiveness of biofuels by introducing ad hoc public policies, which are discussed in Section 3.5.

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3.3.1.1 Bioethanol Bioethanol (ethylic alcohol, ethanol CH3CH2OH) is currently the most commonly produced biofuel because it can be used in gasoline engines. It is the same organic compound as the alcohol in alcoholic beverages and is mainly produced from the fermentation of crops such as sugarcane, corn, and other plants with a high sugar or starch content.f Nowadays, bioethanol can also be derived from lignocellulosic materials, such as woods (e.g., pine, poplar, and eucalyptus trees) and herbaceous plants (e.g., Miscanthus, switchgrass, and alfalfa), from agroindustrial feedstocks (corn stover, sugarcane bagasse, wheat, and rice straw and from marine biomass materials, mainly algae and seagrasses (Sillanp€a€a and Ncibi, 2017). All these are indeed defined as further generations of bioethanol, as they do not compete with food supplies (Robak and Balcerek, 2018). After its production by microbial fermentation, bioethanol is distilled, dehydrated, and eventually denatured. It can be blended with gasoline to make fuels ranked by ethanol content from E5 (5% ethanol and 95% gasoline) to E100 (100% ethanol). The use of E100 reduces net greenhouse gas (GHG) emissions to 87%–96% when compared with regular gasoline. Bioethanol is mainly used in car engines, but it can also be used in fuel for tractors, planes, and boats. In flexible-fuel (flex-fuel) vehicles, it can be used as an interchangeable biofuel substitute for gasoline. Due to its low freezing point, bioethanol has to undergo specific chemical processes in order to not be used as a jet fuel (Wang and Tao, 2016). The energy density of bioethanol (E100, 22.37 kWh/gallon) is around two-thirds that of gasoline (34.02 kWh/gallon), giving it a gallon-gasoline equivalent (GGE) value of 1.5.g An E85 blend (made with 85% ethanol

f

Common examples are sugar beet, sorghum, grain, barley, hemp, kenaf, potatoes, sweet potatoes, cassava, sunflower, fruit, molasses, corn, stover, grain, wheat, straw, and cotton. Bioethanol from cellulose and algae, agricultural residues, coal, solid waste, and other nonfood feedstock constitutes a second-generation biofuel that is currently being developed and is likely to become commercially viable in the foreseeable future. Moreover, bagasse, Miscanthus, and switchgrass are generally considered to be second-generation biofuel feedstocks. Second-generation biofuels are revisited in Section 3.3.2. g Gallon gasoline equivalent (GGE) is a coefficient that indicates the amount of alternative fuel required to equal the energy content of one gallon of gasoline. The higher the ratio, the lower the energy density of the fuel.

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and 15% gasoline, 24.03 kWh/gallon) reaches 1.39 GGE.h The relative energy densities of these fuels allow the fuel consumption of a vehicle using any fuel to be represented using the transferable metric of miles per gallon equivalent (MPGe) efficiency.i However, with its higher contents of oxygen, chloride ions, halide ions, and aluminum hydroxide, bioethanol is more corrosive to vehicle engine components than gasoline without bioethanol. To be sustainable, biofuels should generally not be transported over long distances, as their lower energy densities make their haulage inefficient, further reducing their competitiveness. In 2019, the largest producer of bioethanol was the United States (with 15.7 billion gallons produced that year), followed by Brazil, the European Union (led by Germany), and China.j Since the 1980s, Brazil has played a pioneering role in the field of biofuel technologies and is currently selfsufficient, with bioethanol accounting for more than half of its domestic transportation fuel market (World Bank, 2005; Stattman, 2019). In Brazil, bioethanol is produced mainly from sugarcane,k whereas in the United States, it is produced mainly from corn—accounting for around one-third of the country’s corn production—and comprises approximately 6% of the total gasoline production. The European Union experienced an initial delay in bioethanol market development. However, in March 2019, the Commission specified the sustainability criteria for biofuels, thereby clarifying the regulatory framework. In 2013, the opening of the Crescentino Biorefinery in the province of Vercelli (north of Italy) marked a turning point for the production of bioethanol from cellulose in Europe. With 49,000 m3 of bioethanol production capacity, this biorefinery was the world’s first commercial-scale cellulosic ethanol plant. Financial issues affecting the owning group caused a temporary suspension of the facility in 2018, but the biorefinery was able to fully resume its operations in 2020.l

h

https://www.treehugger.com/fuel-energy-comparisons-85636. Miles per gallon equivalent (MPGe) is a coefficient that represents the average distance traveled for every GGE unit of energy consumed over the course of a specific duty cycle. The coefficient is expressed in miles/gallon. j https://afdc.energy.gov/data/10331. k Although sugarcane has proved to be more efficient and sustainable than corn for bioethanol production (Dutta et al., 2014), it requires a tropical/subtropical climate, which renders it viable as a feedstock only in the in tropical and subtropical South America. l https://www.polimerica.it/articolo.asp?id¼23225. i

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3.3.1.2 Biodiesel The second most common biofuel is biodiesel derived from vegetable oils or animal fats containing long-chain esters. The chemical structure of biodiesel is different from that of petroleum diesel, as it contains carbon, hydrogen, and oxygen, whereas petroleum diesel is composed of hydrocarbons only (hydrogen and carbon without oxygen). Biodiesel can be used in regular diesel engines, either in pure form or blended with conventional diesel fuel in any proportion. The most common blends by per cent biodiesel content are B2, B5, B20, and B100 (pure biodiesel). Biodiesel can be used not only as fuel for roadgoing vehicles but also as a heating oil and to power railway locomotives. The main feedstocks for biodiesel production are soybeans in the United States and rapeseed globally (Devisscher, 2007; Friedman and Van Gerpen, 2014), in addition to palm oil (which is the feedstock used by Indonesia, the world’s largest producer). However, considering recent technological developments, other feedstocks, such as palm oilm or Jatropha, may soon challenge the predominance of these sources (Richmond-Bryant et al., 2014).n Additionally, biodiesel can be produced using waste cooking oil collected and recycled from industries that use edible oils for cooking or other industrial purposes. Differently from virgin vegetable oil produced from dedicated crops, waste cooking oil is a by-product that would be wasted if not recycled. This makes waste cooking oil a cheap alternative feedstock for biodiesel production, and its low prices can enhance the competitiveness of biodiesel in the overall fuel market. All biodiesel feedstocks are refined through transesterification, a process by which triglycerides in the feedstocks react with alcohol and a catalyst to produce biodiesel (which contains fatty acid methyl esters—FAME), along with glycerol as a by-product. The performance indicators of biodiesel are close to those of regular diesel and are better in some regards. The GGE of regular diesel is 0.88 (37.95 kWh/gallon, 113.64% of gasoline’s energy density). The GGE of the B20 blend (20% biodiesel and 80% regular diesel) is 0.90 (37.12 kWh/gallon, 111.11% of gasoline’s energy density), and the GGE m

For instance, palm oil production is widespread in Malaysia (Timilsina et al., 2011), where its yield is about five times higher than that of rapeseed and 10 times that of soybeans. n A more comprehensive list of feedstocks used for biodiesel production includes rapeseed oil, Pongamia, field pennycress, jojoba, flax, sunflower, coconut, hemp, waste vegetable oil, fungi, coffee grounds, animal fats, lard, algae (both micro- and macro-algae), Salicornia bigelovii, Chinese tallow, and sewage sludge.

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of the B100 blend (pure biodiesel) is 0.96 (34.80 kWh/gallon, 104.17% of gasoline’s energy density).o According to data compiled by Statista,p Indonesia is the world’s largest producer of biodiesel, with 7.9 billion liters produced in 2019, followed by the United States (6.5 billion liters) and Brazil (5.9 billion liters). The European Union (EU), as a whole, produces more biodiesel than any single country worldwide (8.5 billion liters). Biodiesel is also the most important biofuel in the EU and, on an energy basis, represents approximately threequarters of its total biofuel consumption in the transport sector. In 2018, France, Germany, Spain, Sweden, and Italy were the largest biodiesel consumers in the EU, accounting for 63% of total EU biodiesel consumption. China’s biodiesel production (600 million liters in 2019, nearly 158 million gallons) lags far behind its ethanol fuel output (2.7 billion liters). 3.3.1.3 Other biofuels Other biofuels with less significant impact and more limited distribution include biogas, other bio-alcohols (e.g., biomethanol, biobutanol, etc.), firewood, vegetable oil, bioethers, dried manure, and agricultural waste (Guo et al., 2015). Methanol is the simplest alcohol (CH3OH), and like ethanol, it can be used as a fuel. Currently, methanol is predominantly a fossil fuel produced from natural gas. Methanol can also be obtained by gasification of biomass (biomethanol), but its economic and commercial viability is still being assessed. From a technical point of view, biomethanol can be used for several purposes: (1) in internal combustion engines as a substitute for gasoline, even at only half the energy density of the latter; (2) as a substitute of diesel, either when dehydrated to dimethyl ether or as biodiesel produced by transesterification of vegetable oil; (3) in purpose-built biomethanol-powered vehicles or in plug-in-hybrid-electric and hybrid-electric vehicles; (4) for electricity production in gas turbines or fuel cells; and (5) as a domestic fuel (IRENA, IEA-ETSAP, 2013; IRENA, 2021). Butanol (C4H9OH) is an alcohol produced from sugar through ABE fermentation (acetone, butanol, and ethanol). Butanol can be used in unmodified gasoline engines. o p

https://afdc.energy.gov/files/u/publication/fuel_comparison_chart.pdf. https://www.statista.com/statistics/271472/biodiesel-production-in-selected-countries/ #::text¼The%20United%20States%20and%20Brazil,gallons%20of%20biodiesel%20by% 202025.

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Biogas is a mixture of several gases (e.g., methane, CO2, nitrogen, and hydrogen) produced by anaerobic fermentation or anaerobic digestion of organic substances. Like natural gas, biogas can be compressed and used in many applications, including as a fuel for motor vehicles. Differently from biogas, syngas is obtained through partial combustion of organic matter with oxygen present. Before combustion, the feedstock is dried or undergoes pyrolysis. After partial combustion, the resulting biogases are a mixture of carbon monoxide, hydrogen, and other hydrocarbons. Moreover, syngas can be used in the transport sector and for production of heat and electricity. Green dieselq is produced using the same feedstocks as biodiesel (mainly animal fats or vegetable oil); however, its production process differs significantly. Whereas biodiesel is produced through transesterification, green diesel is produced through hydrocracking (a catalytic cracking process at high temperature and pressure in the presence of added hydrogen)r or hydrogenation (a catalyzed chemical reaction with molecular hydrogen). Unlike biodiesel, green diesel has the same chemical properties as regular diesel, meaning it can be used without modification of diesel engines or of existing infrastructure for the distribution of petroleum-based diesel. The remaining first-generation biofuels are biofuel gasoline, which is produced from a genetically engineered strain of Escherichia coli,s and bioliquids, which are liquid fuels obtained from biomass that are used solely for energy purposes other than transportation (i.e., heating and electricity production).

3.3.2 Beyond the first-generation biofuels The first-generation biofuels (mainly manufactured from starch, sugars, and vegetable oil) have some negative aspects and inherent shortcomings from

q

Here we refer to hydrogenation-derived renewable diesel (HDRD), which is also known as green diesel or second-generation biodiesel. On this point, see US Department of Energy Alternative Fuels Data Center http://www.afdc.energy.gov/fuels/emerging_green.html. r Hydrogenation differs from hydrocracking because the former is achieved by reduction of inorganic components (e.g., nitrogen or sulfur) or addition of hydrogen to unsaturated bonds, whereas in the latter process, larger molecules are cracked into smaller ones. Different types of catalysts and different pressure and temperature conditions are used in these processes; for example, hydrogenation occurs at lower temperatures than hydrocracking. s A bacterium that can transform glucose into biogasoline (Koppolu and Vasigala, 2016).

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both an economic perspective and a technological perspective (see Section 3.4). These limitations weaken their competitive position relative to fossil fuels. To address these difficulties, there have been massive investments in recent years to support development of technologies for production of new biofuels and improvement of first-generation biofuels. The main goal of these investments has been to reduce feedstock requirements by improving energy conversion efficiency. A second objective has been to enhance the competitiveness of biofuels by developing more efficient logistical structures. Another major target of innovators and investors has been production of commercially viable biofuels, especially bioethanol, from nonfood crops, in particular lignocellulose biomasses.t Example lignocellulose feedstocks include agricultural by-products, such as cereal straw, sugarcane bagasse, forest residues, and organic components of municipal solid waste.u Experiments to find new methods for production of biofuels by pyrolysis, anaerobic digestion, gasification, enzymatic hydrolysis, and improved incineration are ongoing (Lee et al., 2019). However, production of ethanol from cellulose is difficult because the sugars for fermentation are trapped in a complex chemical structure. Cellulose has high hydrolytic stability and structural robustness deriving from cross-linking between polysaccharides (cellulose and hemicellulose) and lignin via ester and ether linkages. These linkages must be broken to open the cellular structure for hydrolysis. The feedstock (lignocellulose) is far cheaper than the food crops used in first-generation biofuel production. Conversely, the transformation and pretreatment processes are more expensive, requiring high capital expenditures for construction of advanced biorefineries. Another issue for production of ethanol from cellulose is the cost of the enzymes required for biochemical conversion of the sugars that will eventually be fermented to produce bioethanol. These enzymes play an indispensable role in breaking down the cellulose, a process better known as enzymatic hydrolysis. However, these enzymes are costly. Cellulase and hemicellulase, which are used in the production of cellulosic ethanol, are

t

Lignocellulose is composed of cellulose, hemicellulose, and lignin. Other examples include dedicated feedstock, such as vegetative grasses, short rotation forests, and other energy crops (copra, castor seed, sesame, groundnut kernel, mustard seed, sunflower, and cotton seed).

u

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more expensive than their first-generation counterparts. Based on actual purchased price of cellulase enzyme in industrial enzyme market and the conventional ethanol yield, enzyme cost is up to $2.71/gal ethanol, accounting for 48% of the minimum ethanol selling price (Liu et al., 2016). According to Rarbach (2017), together with the costs for feedstock, overhead and insurance and financial costs, enzyme production ranks among the top four cost factors (together making up for more than 80% of full costs). Cellulosic ethanol can also be produced thermochemically (pyrolysis/ gasification at high temperatures of 600–1100 °C); cellulose is transformed into gaseous carbon monoxide and hydrogen, two gases that can then be converted into bioethanol through fermentation (see Dutta et al., 2014). If this innovative extraction method could be coupled with higher productivity of the feedstock (achieved through biotechnology), it could lead to a sustainable technological transition; the predominance of fossil fuels could be challenged, allowing for an eventual shift in the sociotechnical regime. Although thermochemical and biotechnological production methods have similar potential yields in energy terms (liters per ton of feedstock), in practice the yields are different. Yield estimates are complicated by variations between the diverse processes under development and between the biofuel yields from different feedstocks. Hence, wide ranges are quoted in the literature for the overall comparative yields (Sims et al., 2010; Hirani et al., 2018). Differences between thermochemical and biotechnological production methods also extend to the fuels they produce. The latter methods produce ethanol, whereas the former can be used to produce a wider range of longer-chain hydrocarbons from the synthesis of gas, including biofuels suitable for aviation and marine applications. The production of lignocellulosic ethanol could be rendered more efficient if both the C5 (pentose) and the C6 (hexose) sugars released during pretreatment and hydrolysis could be fermented into ethanol. (Biswas et al. (2013)) published a study demonstrating that the native strain Scheffersomyces (Pichia) stipitis CBS6054 is suitable for ethanol fermentation of both glucose and xylose present in hydrolysates of wet exploded bagasse without the need for detoxification, achieving substantial ethanol yields. First-generation biofuels require prime croplands for feedstock production. By contrast, second-generation biofuels require only marginal croplands, as they can be produced from biomasses not grown as arable crops and/or using innovative biotechnologies (see Fig. 3.1). This is a key element of novelty in the second-generation biofuels, and it overcomes, to a certain

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Biomass

First generaon

Starch

Third generaon

Second generaon

Cellulose Hemicellulose Lignin

Triglycerides Fat acids Lipids Proteins

+ Forth generaon

Photosynthec microorganisms

Ethanol

Ethanol Butanol Biomethane Biohydrogen

Syngas Hydrogen

Bio-oil Pyro-gas Char

Alga-oil Biodiesel Aviaon fuel

Electrobiofuels

Fig. 3.1 Beyond first-generation biofuels. Adapted from Nanda S., Mohanty P., Sarangi P. K., 2018. A broad introduction to first-, second-, and third-generation biofuels, in Recent Advancements in Biofuels and Bioenergy Utilization, Springer, pp. 1–25.

extent, the indirect land-use change (ILUC) problem,v which is a core element in current policy and economic debates regarding biofuel sustainability (explored further in Section 3.4). Moreover, biotechnological innovation enables development of genetically engineered crops that grow faster, require less water and fertilizers, and are more resistant to diseases and drought than their non-engineered counterparts (Hielscher et al., 2016). To further reduce reliance on arable lands for feedstock production, innovators are pioneering new technologies to produce biofuels using microalgae as feedstocks (i.e., third-generation biofuels). An increasingly relevant role could be played by bio-jet fuel, as the aviation sector is looking for scalable sustainable alternatives. At the current stage of development, bio-jet fuel can be produced with the following technologies: upgrading of fats, oils, and greases (FOGs) to HEFA-SPK (hydrotreated esters and fatty acids synthesized paraffinic kerosene, which is fully commercialized; Fischer-Tropsch synthesized paraffinic kerosene (FT-SPK) (based on gasification); alcohol-to-jet-synthesized paraffinic kerosene (ATJ-SPK); and catalytic hydrothermolysis jet (CHJ) (Van Dyk and Saddler, 2021). According to IRENA (2021), fuels produced via the HEFA technology cost three to six v

https://www.iscc-system.org/how-to-deal-with-indirect-land-use-change/; https://ec. europa.eu/commission/presscorner/detail/en/MEMO_12_787.

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times more than conventional jet fuel, depending on the current cost of petroleum jet fuel and the lipid feedstock used to make the bio-jet fuel. While in the short- to mid-term conventional jet fuels will remain cheaper, in the long term, they are likely to reach price parity, thanks to technological development and policy support (IRENA, 2021). However, at present, the emergence of technologies that use these low-cost feedstocks is severely limited by high initial costs of the necessary fixed capital investments. Several other new-generation biofuel technologies are being explored, such as biohydrogen and fourth-generation biofuels, which can be produced without burning feedstock. Fourth-generation biofuels are produced (i) by designer photosynthetic microorganisms to produce photobiological solar fuels, (ii) by combining photovoltaics and microbial fuel production (electrobiofuels), or (iii) by synthetic cell (Aro, 2016). The fourth generation represents an evolution of the third generation in that the origin of the carbon atoms remains atmospheric, but the conversion of carbon into biofuel already takes place within the cell, so that the post-processing only concerns the purification and concentration of the biofuel (Lu et al., 2011). Some argue that the direct conversion of carbon dioxide into ethanol using mainly algae and cyanobacteria can achieve remarkable results (Berla et al., 2013). The nature of cyanobacteria makes them quicker and easier to engineer than more complex organisms, increasing their productivity (Lu et al., 2011). According to Moravvej et al. (2019), triglyceride oils produced by microalgae are exactly the same as those produced from oil crops such as canola and sunflower. Therefore, it is easily possible to extract biodiesel from the oils, the content of which is as high as 30%–70% of the dry biomass of the microalgae (Moravvej et al., 2019). Electrobiofuels are produced from hydrogen and carbon dioxide. Hydrogen is produced from water by electrolyzers powered by renewable energy, while carbon dioxide can be captured from the air or from exhaust fumes from industrial processes such as cement or steel production or fossil fuel power generation. The combination of these two compounds leads to the production of methane or methanol and, if necessary, subsequently to the synthesis of other compounds such as diesel or gasoline. (Torella et al. (2015)) demonstrated a novel and scalable integrative bioelectrochemical production system for isopropanol. The solar water splitting catalyst, based on earth-abundant metals, was used to provide energy for growth of a bacterium Ralstonia eutropha.

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Generation First

Second

Benefits

Limitations

Reducing Greenhouse Gas (GHG)

Inefficient production

Facile and economic conversion

Food v. fuel competition

Reducing GHG Feeding food waste as raw material

Expensive processes to purify the lignocellulosic raw material

Eliminating competition between food and fuel

Demanding cutting-edge technology for development

Independent from arable land for growing the raw material

Third

Algae-based biofuel with essay cultivation Significantly advanced rate of algal growth Elimination of competition between food and fuel

Energy-deficient process Low yield in lipid extraction Contamination of biomass in open ponds

Wide variety of growth media, like wastewater and seawater Fourth

Synthetic biology to intensify lipid extraction from algae Enhance CO2 sequestration through the engineered algae Superior productivity

Pond system High cost of photobioreactor High initial investment Research at immature stage

Fig. 3.2 Benefits vs. obstacles. Adapted from Dutta K., Daverey A., Lin J., 2014. Evolution retrospective for alternative fuels: first to fourth generation, Renew. Energy 69, 114–122, https://doi.org/10.1016/j.renene.2014.02.044.

According to several studies (Liew et al., 2014; Cuellar-Bermudez et al., 2015; Aro, 2016), fourth-generation biofuels hold a great potential to definitely substitute previous generations and compete with fossil fuels too. Though, further research centered on synthetic biology is needed to improve their economic and technological yield. Overall, every generation of biofuels is susceptible to further improvement and developments, taking into account their strengths and weaknesses (see Fig. 3.2).

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Biomaterials Biochemicals

Integrated Biorefinery

Pulp Mill Electricity Bioenergy plant Biofuel

Fig. 3.3 The integrated biorefinery. Adapted from Mongkhonsiri G., Charoensuppanimit P., Anantpinijwatna A., Gani R., Assabumrungrat S., 2020. Process development of sustainable biorefinery system integrated into the existing pulping process, J. Clean. Prod. 255, https://doi.org/10.1016/j.jclepro.2020.120278.

3.3.3 Integrated biorefineries: Making biofuel along with other high-added-value products Research and development (R&D) efforts have recently targeted development of a brand new biorefinery concept: the “Integrated Biorefinery” (see Fig. 3.3), an industrial facility that uses many different biomass types as input for production of biofuels, power, heat, chemicals, nutraceuticals, feedstocks, foods, and other high-added-value products and materials (including bioplastics). Use of integrated biorefineries is becoming a cornerstone of sustainable development of a circular and efficient economy, as they can produce replacements for a wide range of petroleum-based products using a diversity of biomasses and avoiding waste (Accardi et al., 2013). According to the EU Commission, biorefineries should adopt a cascading approach to the use of their inputs, giving preference to products that offer the highest added values and resource efficiencies. These metrics favor bio-based products and industrial materials over biofuels. The principle of cascading use is based on single or multiple material uses followed by energy production by burning the remaining end-of-life material. The cascading approach takes mitigation of greenhouse gas (GHG) emissions into consideration; by-products and wastes from one production process are either fed into other production processes or used to produce energy. Biorefineries can thus contribute to the principles of a “zero-waste society” (European Commission, 2012).

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The main goals of this new type of biorefinery are to improve conversion efficiency and avoid waste. These goals are achieved by establishing innovative technologies and creating and increasing value from new and more commercially profitable coproducts. Integrated biorefineries are also crucial for development of next-generation biofuels, as they can improve the overall competitiveness of such biofuels in two ways: by improving yields from the transformation process (e.g., producing more fuels from the same amount of feedstock) and by producing other nonfuel, marketable, high-added-value products, the earnings from which can be used to decrease the price of the produced biofuels, hence enhancing their competitiveness in the overall fuel market. A recent focus of research investment has been to improve the efficiency of biorefineries by putting alternative concepts in competition at the R&D stage and in small-scale demonstration projects (Denny et al., 2017). A biorefinery requires a uniform, year-around, cost-efficient, and reliable supply of biomass feedstocks of the desired quality. To minimize investment risk associated with a biorefinery project, careful assessments of the costs and uncertainties associated with transport, storage, and handling of the required biomass are necessary. Numerous sources of variability affect biomass supply chains, such as weather uncertainty; seasonality; physical and chemical characteristics, geographical distributions, and low bulk densities of biomass feedstocks; structures of biomass suppliers and their willingness to grow biomass crops; local transportation and distribution infrastructures; and supplier contracts and government policies (Sharma et al., 2013). In other words, as discussed in Section 3.2, alternative innovation niches are competing to reach technological maturity, which is one of the preconditions for a transition to occur. The other conditions are economic viability and sufficient pressure from the macro level (the landscape), which should be reflected in policy and regulatory changes. Both conditions are addressed in the remainder of this chapter.

3.4 Economic, environmental, and social issues Section 3.3 covers a broad array of biofuels and assesses their technoeconomic properties and performance. In this section, we deepen our analysis, focusing in more detail on economic, environmental, and social issues. As is widely acknowledged, fossil fuels are associated with many sustainability problems. However, biofuels are not immune to such problems. Hence, when assessing biofuels from economic and environmental

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perspectives, there are sustainability issues to consider. Moreover, there are fundamental social concerns to address because most croplands are in developing countries where local communities may be at risk of exploitation. According to the World Commission on Environment and Development (also known as the Brundtland Commission), development can be deemed sustainable if it meets the needs of the present without compromising the ability of future generations to meet their own needs. Using this definition, it can be easily understood why biofuels present sustainability problems. Examples of the sustainability issues associated with biofuel production include phenomena such as indirect land use change (ILUC), deforestation, and displacement of agricultural production.

3.4.1 Socioeconomic issues Whenever food commodities are used as biofuel feedstocks, there is a risk that croplands will be diverted from food and fiber production to biofuel production. This reallocation of resources could have a negative impact on food supply, causing a general increase of food prices. Taking corn as an example, its use as a biofuel feedstock can trigger a chain reaction: if corn prices increase, milk, beef, pork, and cheese prices will also eventually rise because corn is used to feed livestock. However, there is no consensus on the existence and magnitude of this effect, as the reasons for food price fluctuations are numerous and complex. In general, some of the factors that may contribute to commodity price fluctuations are crop productivity, expectations of consumers and producers, financial speculation, precautionary demand, prices of substitute and complementary goods, adverse weather conditions, energy costs, and inappropriate public policies (Schmieg, 1993; Delle et al., 2017). Several studies have sought to isolate the effects of biofuel development on food prices, with everything else being equal (Gerber et al., 2008; Ajanovic, 2011; Zilberman et al., 2012; Kgathi et al., 2012; Shrestha et al., 2019). A central challenge has been to disentangle and separate all of the other factors contributing to food price changes so that the additional impacts of biofuel development and the resulting effects on food prices can be assessed (HLPE, 2013). In 2018, global biofuel production increased in all major producing regions except Argentina, where biodiesel production decreased to its lowest level in 4 years, mainly due to less favorable export opportunities (OECD/FAO, 2019). Biofuel feedstock prices remained at levels similar

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to those in 2017, with the exception of vegetable oil prices, which dropped to historically low levels (OECD/FAO, 2019). Biofuel-to-feedstock price ratios increased in 2018 for biodiesel, increasing its profitability, whereas profits decreased marginally for ethanol producers (OECD/FAO, 2019). As observed by Sims et al. (2010), estimates of the actual effects of biofuel development on food prices vary: some studies found, on average, that when food price increases were linked to use of biofuels, between 15% and 25% of the total food price increase could be attributed to the impacts of biofuel development. Other studies (Zhang et al., 2009; Du et al., 2011; Ajanovic, 2011) conclude that there is no relationship whatsoever, and some authors argue that biofuel development could be responsible for 75% of an observed increase in food prices (Chakrabortty, 2008). It should be noted that the study of a link between food prices and biofuels development is highly influenced by the timeframe of the analysis and the geographical coverage of the study: the cited papers were all published in the aftermath of the 2008 financial crisis, with relevant disruptions in trade flows and the economic cycle. Moreover, their mostly focus on the American region—especially the USA, but also Brazil—and on Europe, probably since these are among the largest producers. However, considering that less than 2% of the world’s arable land is used for biofuel production, factors other than biofuel production may affect food prices more significantly. Adverse weather conditions, for instance, may curtail food production, necessitating additional use of land and fertilizers to maintain food supplies. This increased demand for resources could induce deforestation, which can lead to increased GHG emissions. According to the OECD and FAO, between 2018 and 2027, feedstock demand linked to biofuel production should stabilize, and annual bioethanol output is projected to rise by only 0.7% (12 bn liters per year), compared with a 3.9% increase over the preceding decade (64 bn liters per year). Biodiesel production is expected to increase by 0.4% (5 bn liters per year), as compared with an increase of 9.5% (29 bn liters per year) over the previous 10 years (OECD/FAO, 2018). Fluctuations in biofuel prices can impact the economically optimal deforestation rate and the amount of land used for agricultural purposes. As with the previously discussed impacts of adverse weather, an “infinite pain-chain” may develop, in which climate change causes overall crop productivity to decrease (because of droughts, storms, etc.), meaning that more

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land is needed for cultivation. Accordingly, increased land exploitation is likely to result in higher GHG emissions (because of deforestation). In addition, increased land use for biofuel feedstock can take away space from food croplands (provided that the amount of cultivated land is fixed), because climate regulations can limit new exploitation of lands purposely conserved to reduce the impact of climate change (Steinbuks and Hertel, 2016). Increased feedstock prices improve the incomes of farmers and raise land values and might contribute to new job creation (especially in developing countries where agriculture remains highly labor intensive) and growth opportunities for underdeveloped areas, thus reducing internal migration (Gustafson, 2013; Dimova, 2015). Developed countries such as the United States are currently net food exporters, but if their domestic demands for agricultural feedstock increase (e.g., for biofuel production), food exports to food-insecure countries could diminish (Nonhebel, 2012). If food prices rise, developing countries that export feedstock for biofuel production could eventually benefit, and the resulting economic growth could outweigh the negative aspects of food price increases connected to the use of land for biofuel production. However, there are several problems linked to this hypothesis, as not all of the people living in developing countries can benefit equally from feedstock production, and some underdeveloped regions are particularly vulnerable to rising food prices. FAO studies have shown that some good practices can accommodate sustainable production of food, bio-based products, and bioenergy (including biofuels). These practices include agroecological zoning and complementing food production with bioenergy generation through sustainable agriculture intensification. There is also good potential for development of integrated food-energy systems, such as mixed food and energy crop systems, and increased use of biomass for energy (e.g., biogas from livestock manure), to optimize land use (FAO, 2017).

3.4.2 Socioenvironmental issues Another important aspect of biofuel production is its environmental sustainability. It is not always easy to assess the ecological footprints of biofuels, as they involve many factors linked in complex ways. In 2017, the Roundtable

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for Sustainable Biofuels (RSB) set 12 criteria for the evaluation of biofuel sustainabilityw: (1) Biofuel production shall follow international treaties and national laws regarding such things as air quality, water resources, agricultural practices, labor conditions, and more; (2) Biofuels projects shall be designed and operated in participatory processes that involve all relevant stakeholders in planning and monitoring; (3) Biofuels shall significantly reduce greenhouse gas emissions as compared to fossil fuels. The principle seeks to establish a standard methodology for comparing greenhouse gases (GHG) benefits; (4) Biofuel production shall not violate human rights or labor rights, and shall ensure decent work and the well-being of workers; (5) Biofuel production shall contribute to the social and economic development of local, rural, and indigenous peoples and communities; (6) Biofuel production shall not impair food security; (7) Biofuel production shall avoid negative impacts on biodiversity, ecosystems, and areas of high conservation value; (8) Biofuel production shall promote practices that improve soil health and minimize degradation; (9) Surface and groundwater use will be optimized and contamination or depletion of water re- sources minimized; (10) Air pollution shall be minimized along the supply chain; (11) Biofuels shall be produced in the most cost-effective way, with a commitment to improve production efficiency and social and environmental performance in all stages of the biofuel value chain; (12) Biofuel production shall not violate land rights. These indicators address a shortlist of sustainability issues related to biofuel production, but not all of them can be estimated using current life-cycle assessment (LCA) methodologies. According to the RBS, large and potentially negative effects—such as indirect land use change (impacting biodiversity, socioeconomic relationships, and greenhouse gas emissions)—can be derived from interactions between food, fodder, fuel, and fiber markets. This specific problem remains a major unsolved factor for assessment of biofuel carbon footprints because it is tightly linked to deforestation, which endangers local habitats and biodiversity. Direct land use change was already covered by RSB’s principles and criteria, so RSB worked with its members and partners to address indirect impacts, creating the Low Indirect Land Use w

http://rsb.org/wp-content/uploads/2017/04/RSB-Guide-to-the-RSB-Standard.pdf.

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Change (iLUC) Risk Biomass Criteria and Compliance Indicators (RSB, 2015). Positive carbon balance, or at least carbon neutrality, is crucial to sustainable biofuel production. However, carbon neutrality (meaning that CO2 released to the atmosphere during combustion is theoretically offset by carbon fixed during the feedstock growth) cannot be concretely achieved because there are additional emissions of CO2 and other GHGs during the production, distribution, and transportation of biofuels. The impacts of ILUC can be mitigated by adopting a payment for environmental service schemex in which landowners are paid to conserve land using carbon offsets. However, competitiveness of these schemes depends largely on the price of CO2, which is hard to estimate precisely but should not be lower than the expected profits from feedstock production using the same land. To grow, feedstocks need fertilizers, herbicides, fungicides, and pesticides. Some of these products, when vaporized, release GHGs to the atmosphere. Moreover, fertilizer production, which commonly requires large quantities of fossil fuels, can contribute to soil erosion and degradation and underground water pollution. Another factor that can undermine the sustainability of biofuels is depletion of water resources. Compared with fossil fuel production, biofuel production can require larger volumes of water for feedstock cultivation and conversion of feedstocks to biofuels. For instance, production of a gallon of bioethanol (excluding cultivation) requires around 4 gal of water, whereas refining a gallon of oil requires only 1.5 gal of water (Phillips et al., 2007). The volume of water consumed in bioethanol production and use varies based on the types of crops used as feedstocks, the climate of the location where the crops are grown and its soil characteristics, the production volume, and how the fuel is used. Moreover, the total water footprint cannot be used as the sole criterion for selecting raw materials for bioethanol production; instead, crops with high green water footprints should be considered where local climates can accommodate the increased water use (Chiu et al., 2016). Although the need for water might be less of a challenge in some regions near the tropics or the equator, where abundant rainfall supplies part of the resources needed for cultivation (such as Brazil, Indonesia, or Malaysia), increased water use remains a significant drawback to biofuel production in most other regions of the world. x

An example is REDD (Reducing Emissions from Deforestation and Forest Degradation); http://www.un-redd.org/.

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Finally, biofuels can be severely polluting, as their production involves not only emission of GHGs but also production of formaldehyde, acetaldehyde, and other aldehydes derived from alcohol oxidation and combustion. Continuous support and establishment of initiatives, such as the Global Bioenergy Partnership and the Roundtable on Sustainable Biofuels, can maintain the development of sustainability criteria or standards with the active collaboration of developing country partners, in concert with training and support for implementation. However, compliance with strict and onerous certification systems may further reduce the already challenged competitiveness of biofuels relative to fossil fuels. These aspects are addressed in the following section, which considers legislative and regulatory issues affecting biofuel development and production.

3.5 Policy actions and regulatory frameworks As previously discussed, biofuels are generally still not economically viable when compared with fossil fuels. Although higher prices for biofuels could be partially sustained by consumers willing to pay an “environmental premium,” policymakers throughout the world have supported biofuel production to enhance their competitiveness in other ways. For instance, tax incentives (Edenhofer et al., 2012) can be applied along the whole biofuel value chain but are most commonly provided to biofuel producers (e.g., excise tax exemptions or credits), end consumers (e.g., tax reductions for biofuels at the point of sale), or both groups. In the United States, for instance, Volumetric Excise Tax Credits for blending fuel ethanol and biodiesel have been provided to biofuel producers under the American Jobs Creation Act since 2004. The Further Consolidated Appropriations Act of 2020 retroactively reinstated and extended several alternative fuel tax incentives. Effective through December 31, 2020, this law reinstated the alternative fuel infrastructure tax credit, the excise tax credit for alternative fuels and alternative fuel mixtures, the tax credit for second-generation biofuel production, the fuel cell motor vehicle tax credit, the special depreciation allowance for second-generation biofuel plant property, the tax credit for small agri-biodiesel production, and the tax credit for qualified two-wheeled plug-in electric-drive motor vehicles. It also reinstated the income and excise tax credit for biodiesel and renewable diesel fuel mixtures, effective through December 31, 2022.y y

https://afdc.energy.gov/laws/key_legislation.

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In the European Union, the Energy Taxation Directive permits exemptions or reductions from energy taxation for biofuels (Directive 2003/96/EC). Although most EU countries do not have differentiated fiscal frameworks for biofuels, Austria, Croatia, Czech Republic, France, Sweden, Slovakia, Denmark, Lithuania, the Netherlands, Slovenia and Portugal provide incentives for biofuel use. In France, E10 and E85 ethanol-gasoline blends are taxed at lower rates than unblended gasoline. In Sweden and Denmark, taxes are reduced for fuels with smaller CO2 footprints. In Finland, transport fuels are taxed based on their energy contents and CO2 footprints. Latvia and the Czech Republic apply a reduced excise duty rate to E85. The same reduction theoretically applies in the Netherlands, but E85 is no longer offered in the Dutch market. Furthermore, the excise duty rate for biofuels is set at 0% in Croatia and Slovenia. In Portugal, only biofuels produced by small producers benefit from an excise tax exemption (ePURE, 2020). Along with tax exemptions, some middle-income countries (such as Malaysia) directly subsidize their agricultural sectors, aiming to increase employment rates in these sectors, boost economic growth, and foster export of feedstocks. However, such policy measures may produce unintended negative consequences, as demand for agriculture-specific fossil fuels, which are usually not taxed, may increase as biofuel demand rises. More generally, most policies intended to enhance biofuel competitiveness, whether implemented globally or locally, can be partially neutralized by phenomena such as the Jevons paradox,z the green paradox,aa or the carbon leakage effectab and the associated race to the bottom side effect.ac Government and public institutions are also investing in R&D to improve and discover new technological niches linked to biofuel z

The green paradox occurs whenever a new technology increases the efficiency with which a resource is used (thus reducing the amount of final product needed per-unit of use), but the overall rate of consumption of that resource rises because of increasing demand (Bauer and Papp, 2009; York, 2006). aa As suggested by Sinn (2008), an environmental policy that becomes greener with the passage of time acts like an announced expropriation for the owners of fossil fuel resources, inducing them to intensify resource extraction and hence accelerate global warming. ab Carbon leakage occurs when there is an increase in CO2 emissions in one region as a direct result of a policy to cap emission in another region. This phenomenon means that domestic climate mitigation policies can be less effective and more costly than intended once overall effects are accounted for (OECD/IEA, 2008). ac https://www.transportenvironment.org/sites/te/files/publications/2016_11_Briefing_ Palm_oil_use_continues_to_grow.pdf.

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production. Here, it is observed that public R&D investments in biofuelrelated technologies are most effective when complemented by other policy instruments, particularly deployment policies that simultaneously enhance demand for such new technologies. Public spending on R&D and deployment policies creates a positive feedback cycle, attracting private sector investments, accelerating learning by inducing private sector R&D, and thus further reducing the production costs of new biofuel technologies (Edenhofer et al., 2012).

3.5.1 Brazilian incentive and regulatory systems During the 1970s, Brazil was the first country to experiment with production of bioethanol on a large scale with financial support from the government. This initiative was brought into action when the 1970s oil crisis brought a sharp increase in oil prices, which—combined with a relatively weak domestic currency and high inflation rates—made Brazil’s oil imports prohibitively expensive. The policy decision was also facilitated by the low price of sugar in Brazil (due to the large stocks of sugar available). Large-scale bioethanol production helped Brazil withstand the oil crisis and simultaneously pursue energy independence. Later, in the 1980s, Brazil’s bioethanol sector was partially deregulated, and direct subsidies were withdrawn. Ethanol production in Brazil is now well-developed due to of decades of supportive policies from local policymakers. The applied agricultural processes and technologies are modern, and the overall efficiency of biofuel production and use is among the highest in the world because the bagasse is always used to produce power and heat. The energy balance (output energy/input energy) is very favorable and, in some cases, can reach a ratio as high as 10.2.ad Since the 1970s (with its National Alcohol Program), the federal government of Brazil has imposed mandatory blending of ethanol with gasoline. In 1993, the blend requirement was fixed (with some exceptions) at 22% ethanol, and in 2007, this was increased to a minimum of 25%. Originally, Brazil’s bioethanol subsidies were intended to be temporary; the government waited for bioethanol to reach competitive parity with gasoline, but when petroleum prices fell at the global level in 1986, the withdrawal of subsidies became problematic. In the 1990s, Brazil’s subsidies were ad

http://www.nytimes.com/2006/04/10/world/americas/10brazil.html?pagewanted1& sqBush%20Brazil%20ethanol&stnyt&scp5&_r; https://www.greenfacts.org/en/biofuels/ figtableboxes/biofuel-brazil.htm.

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withdrawn, and its bioethanol prices were liberalized in 2002.ae Due to oil price and tax legislation, sugarcane bioethanol has remained competitive, even without subsidies, since 2005 (Tokgoz and Elobeid, 2006). When oil prices began to rise again in the early 2000s, Brazil became a net exporter; however, these exports (mainly to the United States) were limited by a US$0.54 tariff imposed by the US federal government on every gallon of imported bioethanol. However, as partial compensation, imported bioethanol was eligible for US$0.45 per gallon ethanol subsidies provided by the United States.af More recently, the Brazilian government imposed a freeze on petrol and diesel prices to prevent energy prices from rising and avoid inflationary pressure.ag However, ex-refinery oil and diesel prices did increase. Nonetheless, domestic petrol and diesel prices remained lower than international prices between 2011 and 2014 (Oliveira and Almeida, 2015). This policy measure was introduced at a time of general crisis for the Brazilian ethanol industry. The industry had experienced poor sugarcane harvests due to unfavorable weather conditions and faced high sugar prices in the world market, which induced a switch to production of sugar rather than ethanol. As a consequence of these pressures, the Brazilian ethanol industry experienced a supply shortage for several months during 2010 and 2011. Prices climbed to the point that ethanol fuel was no longer attractive to owners of flex-fuel vehicles. As a countermeasure, the Brazilian government reduced the minimum ethanol blend in gasoline from 25% to 18% to reduce demand and prevent ethanol fuel prices from rising further. However, for the first time since the 1990s, (corn) ethanol fuel had to be imported from the United States. As a result of a combination of higher ethanol prices and government subsidies to keep gasoline prices lower than their international market value, only 23% of flex-fuel car owners were regularly using ethanol in the early 2000s, as compared with 66% in 2009.ah Fluctuations of ethanol and gasoline prices continue to determine Brazil’s bioethanol consumption, as demonstrated by a recent increase in hydrous ethanol demand in 2019 (Barros and Flake, 2019). ae

Gasoline taxes in Brazil are around 54%, whereas bioethanol taxes are between 12% and 30% (see: http://www1.folha.uol.com.br/mercado/2008/08/438347-imposto-poegasolina-brasileira-entre-as-mais-caras.shtml). af http://www.ethanolproducer.com/articles/4591/brazil-launches-campaign-to-removeethanol-tariff/. ag As the government is Petrobras’s controlling shareholder, it can influence oil product prices and cushion the impact of international fuel price volatility on domestic prices. ah http://info.abril.com.br/noticias/tecnologias-verdes/2013/11/etanol-e-usado-hoje-emapenas-23-dos-carros.shtml.

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Brazil’s current standards require a 27% blend of ethanol in gasoline and have also required a gradually increasing ethanol content in biodiesel blends from 8% bioethanol in March 2017 to 9% in March 2018 and 10% in March 2019. These quotas do not include land-use considerations or other sustainability criteria.ai In 2016, Brazil launched the RenovaBio program within its National Biofuels Policy, which is based on annual carbon intensity reduction targets, certification of the efficiency of biofuels in reducing greenhouse gas emissions, and decarbonization credits. This policy targets compliance with Brazil’s 21st Conference of the Parties (COP21) commitments while promoting adequate expansion of biofuel production and use in Brazil’s national energy matrix and improving the predictability of different biofuels availability in its national market (Barros and Flake, 2019).

3.5.2 United States incentive and regulatory systems In contrast, in the United States, the main goal of national biofuel policies since the 1970s has been for the country to become independent from external fuel supplies. The main pillars of the US regulatory framework are the Clean Air Act and the Energy Policy Act. The Clean Air Act of 1963 was one of the world’s first environmental protection laws. Although it did not concern biofuels, the act (and its subsequent modifications in 1970, 1977, and 1990) created the foundations of the current US environmental regulatory framework. The Energy Policy Act, approved in 2005 (amending the Clean Air Act), not only provided tax incentives and guaranteed loans for production of several types of renewable energy but also defined fuel sustainability standards. Regarding the latter, the most important sections of the Energy Policy Act are those concerning the Renewable Fuels Standard (RFS). The RFS called for the annual use of 7.5 billion US gallons of biofuels by 2012.aj Also, each renewable fuel category within the RFS program must emit lower levels of GHGs in comparison to the replaced petroleum fuels.ak ai

https://www.transportpolicy.net/standard/brazil-fuels-biofuels/#::text¼Brazils% 20current%20standards%20require%20a,considerations%20or%20other%20sustainability% 20criteria. aj http://images1.americanprogress.org/il80web20037/americanenergynow/ AmericanEnergy.pdf. ak http://www.afdc.energy.gov/laws/RFS.

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The 2005 standards were eventually improved and complemented in 2007 by the Energy Independence and Security Act of 2007. This legislation significantly increased the size of the US renewable fuel program and included key changes, such as boosting the long-term goals to annual use of 36 billion gallons of renewable fuel in transportation by 2022, adding explicit definitions for renewable fuels to qualify, creating allowances for volumes from certain existing facilities, and incorporating specific types of waiver authorities.al In 2011, the US Environmental Protection Agency (EPA) amended the RFS to create RFS2. RFS2 contains strict provisions to improve the sustainability of biofuels. Moreover, it provides detailed regulations for next-generation biofuels and defines other measures to improve biofuel sustainability. More recently, the US Federal Government has signaled its intention to access the full potential of American energy production and gain US energy independence. As of 2020, The U.S. Department of Agriculture (USDA) is seeking public input to help with creation of its Higher Blends Infrastructure Incentive Program (HBIIP), a new program that will expand the US availability of domestic ethanol and biodiesel by incentivizing the expansion of sales of renewable fuels.am In May 2020, in the context of the COVID-19 crisis, the U.S. Secretary of Agriculture announced a 100 million dollar allocation to support competitive grants to enhance the availability and sale of renewable fuels.an

3.5.3 European Union incentive and regulatory systems The EU has some of the most complex, detailed, and technically developed biofuel legislation. This legislation is highly integrated, with its main directives and legal frameworks dealing with issues of sustainability, renewable energy, climate policy, trade policy, agricultural policy, state aid, and environmental protection. The complexity of the EU system is made even higher given that the central legislation coexists alongside national and subnational legislations. The European legislative framework defines detailed sustainability standards and criteria, voluntary and support schemes, and action plans for biofuel production. More specifically, EU biofuel legislation foresees the use of al

https://www.congress.gov/bill/110th-congress/house-bill/6. https://www.usda.gov/media/press-releases/2020/01/16/usda-seeks-input-new-ethanolsales-infrastructure-incentive-program. an https://www.usda.gov/media/press-releases/2020/05/04/usda-announces-100million-american-biofuels-infrastructure. am

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a wide range of policy tools such as subsidies, blending mandates, duties, taxes, and incentives. In this framework, the most important related directives are the Renewable Energy Directive (RED, 2009/28/EC); the Fuel Quality Directive (2009/30); the Biofuels Directive (2003/30); the Directive on quality of petrol and diesel fuels and amending Directive 2009/28/ EC on the promotion of renewable energy sources (2015/1513); and the recast of the 2010–2020 RED I Directive (RED II, 2018/2001). Differently from regulations, directives are not self-executing, as they merely identify goals to be achieved, leaving the processes of implementation to the discretion of each Member State. In the European Union, increasing use of biofuels was initially encouraged by policymakers to mitigate the effects of climate change, with a target of 20% of EU energy consumption coming from renewable sources by 2020. In particular, the 2009 RED directive mandated that renewable sources must satisfy at least 10% of final energy consumption in the transport sector by 2020.ao This directive also established that biofuels must produce 50% lower GHG emissions than equivalent fossil fuels and that their feedstocks must not be obtained from high biodiversity value lands or carbon-rich forests. However, GHG emissions are not easy to determine because estimates vary according to the LCA methodology used (Silva Lora et al., 2011). In April 2015, the European Parliament approved the ILUC Directive (2015/1513), which addressed one of the main drawbacks of the previous legislative framework, namely the lack of solutions to indirect land-use change. Although the overall target for biofuel’s share of energy use in the transport sector remained the same—10% by 2020—the new directive mandated that at least 3% of biofuel production should not come from food crops (i.e., it should be based on next-generation biofuels). However, the scheme used to calculate contributions toward the target of 3% from innovative nonfood biofuels is complex.ap ao

Transport sector CO2 emissions represent 23% (globally) and 30% (OECD) of overall CO2 emissions from fossil fuel combustion. The sector accounts for approximately 15% of overall greenhouse gas emissions (OECD/ITF, 2010). ap The scheme defines the following measurement criteria (https://www.uu.nl/sites/default/ files/20150106-iluc_methodology_report.pdf): • Biofuels from used cooking oil and animal fats (counted two times) • Renewable electricity in rail (counted 2.5 times) • Renewable electricity in electric vehicles (counted five times) • Advanced biofuels (counted two times and with an indicative 0.5% sub-target, with options for member states to remain below that target).

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Although relevant, these regulatory frameworks did not prove to be particularly effective in solving key problems associated with biofuel production (Palmer, 2015). In practice, the main policy tools were subsidies for farmers, while other important factors (such as ILUC, impact on food prices, biodiversity and soil loss, and technological upgrades) were partially neglected. In 2018, to cut the link between crop-based biofuels and deforestation, the RED II Directive introduced a change of approach to the ILUC issue with the definition of “high ILUC-risk feedstocks with a significant expansion on land with high carbon stocks.” This legislation capped land use by high ILUC-risk feedstocks in each Member State at 2019 levels until 2023, followed by their gradual elimination by 2030. The Directive also prioritized second- and third-generation biofuels and use of electricity in transport. Incentives provided to biofuel producers create an uneven playing fieldaq and create uncertainty for all market operators that rely on subsidies for their economic survival. Among the motives that have led European policy makers to regulate biofuels have been concerns about GHG emissions and future availability of fossil fuels (and the associated risk of shortages) and, in some cases, the search for energy independence. With the development of fourth-generation biofuels, most of the rationales that drove public policies supporting development of first-generation biofuels are becoming increasingly irrelevant (Foldvary and Klein, 2003).

3.6 Conclusions This chapter assesses biofuels from technological, economic, and policy perspectives, with the aim of understanding the technological maturity level and societal readiness for a sustainable transition toward a biobased economy. This transition from a fossil fuel society to a biofuel-based society entails several concomitant changes. It requires that sufficient pressure be exerted upon the dominant regime (the fossil fuel technological regime) both from micro-level technological niches, where new green technologies are developed and nurtured, and from the macro-level landscape, where a aq

Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee, and the Committee of the Regions. A policy framework for climate and energy in the period from 2020 to 2030 (http:// eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri¼CELEX:52014DC0015& from¼EN).

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vision of the future is shaped and translated into societal expectations, policy actions, and regulatory interventions. The assessment provided in this chapter shows how, from the innovation niche perspective, there are several alternatives and competing biofuel technologies that differ in terms of feedstocks, refining methods, and most importantly, technoeconomic performance and environmental impacts. A clear distinction is made between first- and next-generation biofuels. The latter are more distant from technological maturity but provide possible solutions to the limited economic competitiveness that characterizes firstgeneration biofuels and to the possible environmental drawbacks associated with indirect land-use change. In addition, the Integrated Biorefinery is a new industrial concept being launched by the international scientific community to satisfy growing societal demands for green products and energy sources. To this aim, an integrated biorefinery is defined as a scientific and technical platform on which biomasses otherwise designated as waste products are turned into fuels, energy, and chemicals (including basic chemicals, fine chemicals, and specialized biopolymers and bioplastics) through technologies and processes that produce minimal waste and have limited environmental impacts (Accardi et al., 2013). In November 2019, the US Department of Energy announced a US$14 million investment to build a demonstration-scale integrated biorefinery, which will add to the nearly 30 such biorefineries already present in the country.ar From the macro-level landscape perspective, strong signals are coming from the policy level, where a vision is forming around the need to reduce reliance on fossil fuels by switching to alternative energy sources. This vision has emerged, however, on two different grounds: whereas energy independence has mostly directed US policy, environmental concerns and GHG reduction goals have inspired European policy. Broadly speaking, policy actions to date have mainly involved economic support, development of standards and regulations, and direction of public funds toward R&D investments. Many countries now have biofuel policies in place and are successfully developing a new economic sector and markets. However, the main challenge—making biofuels economically competitive with fossil fuels, even without public support—remains an unaccomplished aspiration.as ar as

https://www.energy.gov/eere/bioenergy/integrated-biorefineries. As discussed earlier, biofuel production has gained momentum and competitiveness in Brazil. This is mainly due to the long-lasting supporting policies described in Section 3.5.1.

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R&D investments and public support directed to development of nextgeneration biofuels and integrated biorefineries play a major role in addressing this challenge. However, sharp decreases in fossil fuel prices (oil prices in particular) due to the COVID-19 pandemic may impair this process and slow the transition to sustainability. Depending on sociopolitical factors, economic concerns may encourage policymakers to either undertake less innovative energy policies or accelerate the transition using economic recovery investments that are already being implemented. Finally, this chapter considers the impacts that biofuel development and policies can have on food security and land competition. Biofuel development has effects that are global and local, positive and negative, and short and long term. Many of these effects are due to increased competition for food, land, and water. Hence, a growing concern when designing biofuel policies is the limitation of their potential negative impacts and strengthening of their potential positive impacts, thus combining economic efficiency with environmental and social sustainability. This is an ambitious but necessary path for change to occur and for the sustainability transition to take shape.

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Further reading Abdullah, B., Syed, A.F.M., Shokravi, Z., Ismail, S., Kassim, K.A., Mahmood, A.N., Aziz, M.M.A., 2019. Fourth generation biofuel: a review on risks and mitigation strategies. Renew. Sustain. Energy Rev. 107, 37–50. https://doi.org/10.1016/j. rser.2019.02.018. Babcock, B.A., 2006. Cheap Food and Farm Subsidies: Policy Impacts of a Mythical Connection. vol. 12 Center for Agricultural and Rural Development. Iowa Ag Review. No. 2. IRENA and Methanol Institute, 2021. Innovation Outlook: Renewable Methanol. International Renewable Energy Agency, Abu Dhabi. Kojima, M., Johnson, T., 2005. Potential for Biofuels for Transport in Developing Countries. ESMAP—World Bank.

CHAPTER 4

Feedstocks and challenges to biofuel development Carolina Botellaa, Ana Belen Díazb, Ernesto Hernandezc, Yi Liangd, and Sivakumar S.V.e a Shell Espan˜a S.A, Madrid, Spain University of Ca´diz, Ca´diz, Spain c Canterbury Christ Church University, Canterbury, United Kingdom d Shell Global Solutions US Inc, Houston, TX, United States e Shell India Markets Pvt. Ltd, Bengaluru, India b

4.1 Introduction Most recent scenarios for addressing climate change prominently feature bioenergy, demand for which is predicted to accelerate in the next three decades. Currently, bioenergy accounts for 70% of the global supply of renewable energy and 10% of the total supply of primary energy. The largest proportion of total bioenergy (modern and traditional) is used by the building sector, for purposes such as cooking and heating (26%). The second-largest proportion is used by the industrial sector (7%), followed by the transport sector (3%), mostly in the form of liquid biofuels made from crops such as sugarcane and corn, and finally, the power sector (2%) (IRENA, 2020). In the 1.5 °C pathways reviewed in the recent Intergovernmental Panel on Climate Change (IPCC) Special Report on Global Warming, biomass (154.1 EJ) is predicted to account for 26.3% (median minimum-maximum range ¼ 10.3%–54.1%) of total primary energy (580.8 EJ) by 2050, which will be an increase from 10.3% in 2020 and 14.4% in 2030 (Rogelj et al., 2018). Energy and fuels derived from biomass will thus play critical roles in limiting increases in global average temperatures to 2°C, or possibly 1.5°C, as stated in the Paris Agreement. However, it is vital to ensure that the demand for biomass is sustainable and does not worsen environmental stressors on water supply, land productivity, and ecosystems in general. A feedstock is defined as any renewable biological material that can be directly used as a fuel or converted to another form of fuel or energy product. This chapter describes different types of feedstocks and their importance

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for the development of liquid and gas biofuels, with a focus on feedstocks currently used in industry, economic and sustainability challenges, and the potential of emerging feedstocks.

4.2 First-generation (1G) feedstocks 1G feedstocks are those derived from food related crops and plants. They are generally edible and are used to produce carbohydrate and lipid-rich streams that are subsequently used as raw materials for biofuels. 1G feedstocks are further classified based on their origin, as follows: (1) sugar feedstocks (e.g., sugarcane, sugar beet, and sweet sorghum); (2) starch feedstocks (e.g., corn, wheat, potatoes, and rice); and (3) edible oil feedstocks (e.g., rape seed).

4.2.1 Sugar feedstocks Sugar feedstocks are derived from agricultural crops. Sugarcane represents 85% of the global sugar crop, and sugar beet accounts for the remaining 15%. It is predicted that by 2030, the annual production of sugarcane and sugar beet will be 1960 Mt and 302 Mt, respectively (OECD/FAO, 2021). Sugar is produced by 71 countries from sugarcane, by 43 countries from sugar beet, and by 9 countries from both types of plant. Brazil is currently the world’s largest sugar producer, and Brazil and India are predicted to produce 21% and 18% of the world’s sugar, respectively, by 2030 (OECD/FAO, 2021). Sugarcane and sugar beet typically comprise around 20% sucrose, 75% water, 5% cellulose, and less than 1% inorganic salts. These sugar feedstock crops have less chemical energy stored as sucrose than starchy crops, which store 10 times more chemical energy in their cereal grains. 1G feedstocks can be transformed into biofuels by either chemical or physical processes, or a combination thereof, which are also known as transformation pathways. Ethanol is one of the most common commercial biofuels produced from biomass. In a traditional system, sugar feedstocks are extracted as juices from sugar crop biomass, which are then fermented to form a broth containing diluted ethanol and other by-products such as cells and debris. Most of the solids are separated from this broth by precipitation, centrifugation, or other processes, and the resulting ethanolic light stream is distilled to produce either hydrated or dehydrated ethanol of higher purity. The traditional production system described above has been improved and used to improve other production systems. For instance, bioethanol

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production has been used to enhance raw sugar extraction in cane sugar mills. A genetically alternated Saccharomyces cerevisiae strain (GYK-10) that uses glucose and fructose, which are usually considered to inhibit sugar production, was used for this purpose. This strain has several advantages: (1) it can be used for food production; (2) it selectively uses the monosaccharides glucose and fructose to produce ethanol and does not use sucrose (which is the target product in sugar production); and (3) it can be easily separated from sucrose-rich solutions due to its flocculent nature (Kato et al., 2016). The development of integrated and optimal designs for the production of ethanol from sugarcane feedstock has been extensively reviewed, and two conventional approaches have been identified for the optimization of sugarcane-processing plants: (1) the production of 1G ethanol and electricity in a conventional distillery and cogeneration plant; and (2) the production of 1G and second-generation (2G) ethanol and electricity in a distillation, hydrolysis, and cogeneration plant (Bechara et al., 2018). Sugar feedstocks result in high production volumes and yields of ethanol, low profit margins, and high final production costs. Thus, small improvements in ethanol yield can have a large effect on bioethanol production economics and can be achieved by strategies involving (1) free-energy conservation; (2) redox metabolism; or (3) the reduction of losses during the processing of sugar feedstocks (Gombert and van Maris, 2015). Strategies (1) and (2) favor the production of more ethanol and less glycerol and yeast biomass; strategy (3) requires process and yeast improvement. One of the challenges of producing ethanol from sugarcane is optimizing the synchronization of the sugarcane harvest with the loading and transport of feedstocks. Lozano-Moreno and Mar
echal (2019) developed a dynamic model to quantify the generation of different products and by-products of a sugar mill and their economic and environmental effects. They concluded that integral harvesting was the best strategy in terms of cost, energy input, and carbon emissions. The integration of 1G and 2G feedstocks to suit a given bioethanolproduction scenario has much potential, as such integration has synergistic effects and can maximize benefits across the technical, environmental, and economic domains (Ayodele et al., 2020). Process integration could also reduce environmental effects and the use of fossil fuels, making biofuel biorefineries more sustainable and leading to 31% increases in ethanol production (Ocampo Batlle et al., 2021). Ethanol usage in the transportation sector is restricted by the 10%–15% blend wall in the USA, which results from technological and infrastructural

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limitations (U.S. EPA, 2012). To overcome these limitations and extend the use of biofuels to other sectors, such as the aviation sector, methods are needed to directly convert ethanol and sugars to hydrocarbon blends. Such methods have recently been developed (Li et al., 2016; Chong and Ng, 2021; Morgan et al., 2019) and are now being commercially deployed by various companies. Brazil has been a key player in this domain with its use of sugarcane ethanol, its leadership in the jet (aviation) biofuel supply chain, and its recent policies recommending regulations to support bioethanol production (Moncada et al., 2019). Global strategies for the sustainable production of aviation fuels via state-of-the-art technologies such as alcoholto-jet (ATJ) and the hydroprocessing of fermented sugars are also being developed (Ng et al., 2021). Chong and Ng (2021) reviewed the different pathways for biojet fuel production and the latest commercial deployments. They noted two main types of pathways for the conversion of sugar to jet fuel: (1) biological-type direct sugar-to-hydrocarbon conversion pathways; and (2) aqueous-phase reforming pathways. The first type of pathways are based on advances in genetic engineering and screening technologies, but remain the early stages of development due to complexities and long residence times, which significantly increase the cost of production. The second type of the pathways involves hydrogenation, condensation, oligomerization, and hydrotreating. These are being developed by different companies, and there is little information in the literature. More efforts are being undertaken to prevent competition between the use of sugar feedstocks to produce biofuels and their use as food for humans and animals. This issue has sparked worldwide “food vs. fuel” debates and concerns that greenhouse gas (GHG) emissions will be increased by direct and indirect land-use changes (Searchinger et al., 2008). New models were devised for the sustainable production of aviation biofuels by assessing 15 pathways that involve strategies to reduce emissions linked to land-use changes (Zhao et al., 2021).

4.2.2 Starch feedstocks Starch is a polysaccharide of glucose monomers that can be hydrolyzed to fermentable sugars, which can then be converted to ethanol or drop-in fuels. The main starch-based feedstocks used for biofuel production are grains, such as corn and wheat, and tubers, such as potatoes and cassava. Corn contains approximately 70% starch (dry weight basis), and nearly 144 million Mt of corn were used for biofuel production in 2020. This

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represents approximately 90% of the feedstock used for ethanol production in the USA and 50% of the feedstock used for this purpose in Europe (OECD/FAO, 2021). Wheat is the third-most widely produced crop worldwide, after corn and rice, and comprises approximately 70% starch (dry weight basis). In 2020, 5.6 million Mt of wheat were used to produce biofuel in OECD countries (OECD/FAO, 2021), and 3.3 million Mt of wheat were used to produce ethanol in Europe (ePURE, 2021). Other grains used for ethanol production are barley, rye, millet, and sorghum. These grains contain less starch than corn but require less nitrogen fertilizer and can grow in marginal-quality soil, and thus have economic and environmental advantages. Tubers such as cassava, with a 40% starch content (dry weight basis), and sweet potatoes, with a 40%–70% starch content (dry weight basis), can be cultivated in tropical or warm regions and offer high ethanol yields (ETIPBioenergy, 2021). Currently, ethanol is the most commonly produced biofuel and represents approximately 80% of the total renewable liquid-fuel produced worldwide. In 2020, ethanol production reached 103.2 billion liters (BL) (a decrease from the 114.6 BL produced in 2019), with US producers of corn-derived ethanol leading the list of ethanol producers (53.3 BL; a decrease from the 59.7 BL they produced in 2019). Production is predicted to decrease by 5% in the USA in 2021, due to lower gasoline demand as a result of the coronavirus disease 2019 (COVID-19) pandemic and the absence of new policy drivers (IEA, 2021). However, this decrease will be partially offset by increased production in India, which announced a plan for 7% ethanol blending in 2021 and is expected to increase production to 3 BL, compared with the 1.8 BL it produced in 2020 (3% of global production). India currently generates ethanol from sugarcane, but these blending targets are expected to significantly increase production from grains, such as rice and corn. Europe produces approximately 5% of the world’s ethanol biofuel (4.7BL in 2020), mainly from starch-based feedstock, such as corn (48.6%) and wheat (21.1%) (ePURE, 2021). China produces approximately 3% of the world’s ethanol biofuel (RFA, 2021), mainly from corn and wheat (both accounting 72%), cassava (19%), and sweet sorghum (1%) (Wu et al., 2021). The production of 1G ethanol from starch-based feedstock involves the enzymatic conversion of starch into glucose by α-amylase and glucoamylase in a two-step process, followed by the fermentation of glucose to ethanol by the yeast S. cerevisiae. The ethanol is then separated and purified, usually by distillation-rectification-dehydration.

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There are two methods for processing corn: wet milling and dry milling. Dry mills usually have a low capacity and are built primarily to produce only ethanol, whereas wet mills can also produce other high-value by-products, such as high-fructose corn syrup, dextrose, and glucose syrup (Vohra et al., 2014). Despite the production of corn-based biofuel more than doubling between 2007 and 2020, its consumption is expected to decrease by 0.5% annually due to restrictive biofuel policies (European Union [EU], Brazil, and the USA), the strengthening of food security and the focus on increased sustainability (e.g., EU). Some eutrophic regions may also see intensive cultivation to establish credible sustainability standards. Moreover, the availability and prices of starch-based feedstocks for fuel production are affected by production yields, and extreme weather events, which can be accentuated by climate change, may also cause higher volatility in the market (OECD/FAO, 2021). Although the conversion of starch to ethanol is a well-established industrial process, technological developments can improve the process and help overcome some of the challenges faced by the industry. For example, the use of immobilized enzymes can reduce the consumption of enzymes and enable recycling, and water usage can be reduced by using solid-state fermentation (Botella et al., 2009). Moreover, a consolidated bioprocess in which enzyme production, starch saccharification, and fermentation are performed in a single step has great potential to streamline biofuel production (Drosos et al., 2021). Furthermore, to reduce energy demands, starch hydrolysis can be performed at lower temperatures: cold starch hydrolysis has been shown to reduce capital and operation costs and increase overall yields (Cinelli et al., 2015). As described in the previous section, ethanol is mainly blended with gasoline for use in road transport. However, new technologies have been developed to convert ethanol to biojet fuel via a pathway comprising dehydration, oligomerization, and hydrogenation. The biojet fuel produced using this pathway is denoted ATJ and was first tested in aircraft in 2012. As alcohol production costs are considered the greatest barrier to the commercialization of ATJ fuels (Guti
errez-Antonio et al., 2017), any improvements to the processes described above will also increase the commercial viability of sustainable aviation fuels. Such developments are important, as the aviation sector requires large amounts of sustainable liquid fuel in the short-to-medium term to decrease their carbon footprint.

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Finally, although not yet commercialized, research on the fermentative conversion of starch into hydrogen appears promising and could potentially be used for mobility or industrial purposes (Lo et al., 2011).

4.2.3 Edible oil feedstocks Edible oils are a 1G feedstock that can be used for the production of biodiesel and renewable diesel. The most used oil feedstocks are derived from palm, sunflower, rapeseed, and soybean crops. Edible oil feedstocks offer advantages over other feedstocks, as edible oils can be produced more efficiently and in higher yields, based on centuries of accumulated agricultural and processing knowledge. However, as in the case of sugar feedstocks, there is competition between the use of edible oil feedstocks to produce biofuel and their use as food for humans and animals. On average, the dry-matter edible oil content of crops ranges from 19% in soybean to 47% in sunflower seeds. Approximately 20–32 Mt of oil seeds are produced by China, the EU, Canada, and Ukraine each year, and more than 86% of this is converted into protein meal, oil for use as food and in oleochemicals, and biofuels (OECD/FAO, 2021). Rapeseed oil is the main feedstock used for biodiesel production in Europe, whereas the palm oil consumed in Europe is typically imported from Malaysia and Indonesia. It is estimated that by 2030, Malaysia and Indonesia will generate 83% and 34% of the world’s palm oil and vegetable oil, respectively; these countries already export 70% of their vegetable oil, which comprises 60% of global oil exports (OECD/FAO, 2021). Homogeneous catalyzed transesterification is used to produce biodiesel from oils, due to the simplicity and low energy consumption of this reaction. This transesterification reaction is thus a fundamental part of the biodiesel production process, together with the steps involving the feedstock, catalyst, and the separation and purification of the product. The seeds are first harvested from crops and then crushed for oil extraction by mechanical, chemical, enzymatic, or thermal processes, or any combinations thereof. The solid biomass by-products are then separated, and the extracted oil is transesterified to a mixture biodiesel, glycerol, and other by-products. The resulting mixture is then fractionated into crude biodiesel and glycerol-rich streams. Crude biodiesel is washed to remove methanol, which affords a semi-pure biodiesel that is then dried to reduce its moisture content.

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Modern biodiesel synthesis has developed following recent advances in oil extraction and conversion technologies. Advanced extraction technologies such as steam distillation, supercritical fluid extraction, microwaveassisted extraction, and ultrasound-assisted extraction were recently reviewed (Mohiddin et al., 2021). Biodiesel can be produced via catalyzed or uncatalyzed reaction systems that are either homogeneous or heterogeneous (Karmakar and Halder, 2019; Mohiddin et al., 2021). Thermalextraction methods, such as steam distillation, and other novel technologies, such as supercritical and superheated processes, offer the promise of better economic feasibility. Supercritical and superheated processes, although costly, can generate high-quality biodiesel from low-to-high-grade feedstock without significantly altering free fatty acid (FFA) and moisture contents. These processes also simplify biodiesel separation, as they do not require catalysts or generate by-products, and involve higher reaction rates than catalyzed processes (Karmakar and Halder, 2019). Greener methods to synthesize catalysts are also being developed; for example, a novel solid acid catalyst comprising sulphonated organosilane carbon nanotubes converted 93.1% of FFAs in kenaf seed oil into biodiesel and other products under moderate conditions (Macawile et al., 2020). Other modern catalysts show promise for use in biodiesel production. For example, biochar is sourced from biomass and is reusable and inexpensive; magnetic catalysts are environmentally friendly, hydrothermally stable, and ferromagnetic, which enables them to be magnetically separated (up to 70% faster than through filtration and centrifugation) and thereby affords a better product; and enzymes such as intra- and extracellular lipases have high reaction stability and selectivity and afford high yields, although they are expensive (Mohiddin et al., 2021). For example, biodiesel yields from an edible oil feedstock were increased to 97% by using a lipase from a mutant Aspergillus oryzae (Chang et al., 2021). The transesterification of vegetable oils and hydroprocessed esters and fatty acids (HEFAs), such as hydrotreated vegetable oils (HVO), is now a well-established technology. HEFAs, such as HVOs, commonly known as renewable diesel fuels, are produced by hydroprocessing of a wide range of oils and fats (Di Gruttola and Borello, 2021). HEFAs/HVOs are considered the leading alternatives to diesel and jet fuel and are already approved by the American Society for Testing and Materials (ASTM; D7566-14). Unlike biodiesel produced by transesterification, HEFAs/HVOs are largely straight-chain paraffinic hydrocarbons with high cetane numbers and are free of sulfur, oxygen, and aromatic compounds. HEFAs/HVOs also generate less NOx when burned and have superior cold-flow properties and

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storage stabilities. These and other advantages have motivated various companies to commercially produce (Technology Readiness Level [TRL] 9) HEFAs/HVOs as sustainable aviation fuels (Ng et al., 2021). The increased use of HEFAs/HVOs has necessitated the deployment of new regulations to ensure there is a level playing field for sustainable air transport (European Commission, 2021). The main challenge of using 1G edible oil feedstocks is that it may lead to a large increase in GHG emissions due to direct or indirect land-use changes. In order to replace all the petroleum used in USA, the total land area required is 330% as a percentage relative to the size of USA to cultivate soya, 75% to cultivate jatropha, and 23% to cultivate oil palm, with all these crops produced to be used for the production of biofuels (Georgianna and Mayfield, 2012). The use of palm oil to produce biodiesel is expected to reduce in the EU and USA due to deforestation and related problems, and the rise of alternative oil feedstocks. Nevertheless, some studies have signaled the way forward for the sustainable production of palm oil for use in biodiesels by analyzing its effects on ecosystem services, the well-being of people, and other sustainability aspects (Ayompe et al., 2021; Dey et al., 2021). Some other challenges that affect 1G biodiesel production are cost, feedstock extraction, undesirable by-products, the near impossibility of recovering homogeneous catalysts, and the limited biodegradability and reusability of homogeneous catalysts (Mohiddin et al., 2021). The latter problems can be overcome by using heterogeneous catalysts, and other improvements can be realized by using new types of reactors (such as spiral reactors), optimizing production costs for industrial scale-up, and adding antioxidants to increase biodiesel shelf-life (Karmakar and Halder, 2019). 1G oil feedstock can also be enzymatically extracted using sustainable and less harmful chemicals to generate by-product feedstocks rich in carbohydrates and proteins for animal food or fertilizers, as part of an oil-production process similar to other methods (Karmakar and Halder, 2019). Process integration can also improve biodiesel conversion yields and reduce energy usage, thus making biodiesel production more sustainable and economically feasible (Ocampo Batlle et al., 2021).

4.3 Second-generation (2G) feedstocks 2G feedstocks for biofuel production are resources that are not used for food, such as lignocellulosic feedstocks and non-edible oils, and have minimal effects on land-use.

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4.3.1 Lignocellulosic feedstocks Lignocellulosic feedstocks include dedicated energy crops (switchgrass, miscanthus, bamboo, sweet sorghum, tall fescue, kochia, and wheatgrass), agricultural crop residues (corn stover, wheat straw, and rice straw), forestry residues, wood-processing residues, and municipal solid waste. These types of biomass are either waste products, or agricultural by-products, or crops grown on marginal land to improve water and soil quality and enhance overall farming productivity (US Office of Energy Efficiency and Renewable Energy, 2021). Agricultural crop residues include biomass materials, which are primarily stalks and leaves that are not harvested or removed from fields for commercial use. Dedicated energy crops are either herbaceous energy crops or shortrotation woody crops. Herbaceous energy crops are perennials that are harvested annually and reach full productivity over 2–3 years. Short-rotation woody crops are fast-growing hardwood trees (such as hybrid poplar, hybrid willow, silver maple, eastern cottonwood, green ash, black walnut, sweetgum, and sycamore) that are harvested within 5–8 years of planting. Forestry residues include biomass that is not harvested or removed from logging sites in commercial hardwood and softwood stands, and material obtained from forest-management operations, such as pre-commercial thinning and the removal of dead and dying trees. In 2016, the US Department of Energy (DOE) issued its latest Billion-Ton Report, which quantified the biomass resource availability in the country for new industry uses, based on various assumptions. Table 4.1 shows that in 2022, in a base-case scenario at a cost of $60/t or less, there may be 742 million tons of agricultural residues, forestry resources, and waste available (U.S. Department of Energy, 2016). This amount is predicted to increase to 1.1 billion tons by 2040, assuming that there are no changes in crop yields and that dedicated energy crops comprise the majority of potential biomass resources. Along with the biomass resource potential assessment, the Billion-Ton Report also released a separate report on the environmental effects of potential biomass production scenarios in the USA. This revealed that biomass production could be substantially increased based on assumed biomass-supply constraints, without adversely affecting the environment (U.S. Department of Energy, 2017). In general, cellulosic feedstocks have fewer environmental effects than conventional food/feed-based feedstocks, with agricultural and forestry residues making the smallest contribution to environmental changesa. Lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin, with ash and extractives comprising a smaller fraction. The a

https://bioenergykdf.net/billionton2016/1/1/table.

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Table 4.1 Current and potentially available agricultural residues, forestry resources, and waste in the USA for new industry uses, based on various different assumptions. Million dry tons Feedstock

2016

2022

2030

2040

154 144 68 365

154 144 68 365

154 144 68 365

154 144 68 365

101 83 101 0 66 268 633

109 88 123 78 67 377 742

97 77 169 239 67 572 937

97 80 176 411 67 751 1116

Currently used resource

Forestry Resources Agricultural Resources Waste Resources Total Potential Resources (Base-Case Scenario)

Forestry Resources (all timberland)a, b Forestry Resources (no federal timberland)a, b Agricultural Residues Energy Cropsc Waste Resourcesd Total (all timberland) Total (Currently Used+Potential) a

Forestry baseline scenario. Forestry resources include whole-tree biomass and residues from non-federal land, in addition to other forest residues and forest-thinning biomass. c Energy crops were first planted in 2019. d The potential amount of biogas obtainable from landfills is estimated to be approximately 230 billion ft3 per year. Note: Numbers may not sum to the indicated totals because of rounding. This table was generated using an interactive tool devised by the U.S. Department of Energy, 2016. 2016 Billion-Ton Report: Advancing Domestic Resources for a Thriving Bioeconomy, Volume 1: Economic Availability of Feedstocks. M. H. Langholtz, B. J. Stokes, and L. M. Eaton. Oak Ridge National Laboratory, Oak Ridge, TN. https://doi.org/10.2172/1271651. http://energy.gov/eere/bioenergy/ 2016-billion-ton-report (Leads), ORNL/TM-2016/160. b

presence of lignin and cross-linking between components gives lignocellulosic biomass a complex spatial structure in which cellulose (a carbohydrate polymer of glucose) is wrapped in a dense “shell” of hemicellulose (another carbohydrate polymer of pentoses [xylose/arabinose], hexoses [galactose/ glucose/mannose], and uronic acids), and lignin (an aromatic complex phenol polymer). The composition of hemicellulose depends on the source biomass and species type. Hardwoods (deciduous trees), annual plants, and cereal mainly contain xylan hemicellulose, whereas softwoods (coniferous trees) mainly contain glucomannan hemicellulose. A comprehensive database (Phyllis) developed by The Netherlands Organization (TNO) contains information on the composition of lignocellulosic feedstocks and residues for individual materials and on average values for groups of materials (TNO, 2021).

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The traditional use of lignocellulosic materials is in combustion to produce heat to make steam and to generate renewable electricity. In contrast, the use of these materials for producing advanced 2G biofuels or in bio-based chemical platforms requires the effective degradation of the lignocellulose structure and the release of monomers from long-chain polymeric C5 and C6 sugars. The technical and economic challenges of these conversion processes are discussed in subsequent chapters. The use of lignocellulosic materials as a feedstock for fuel production is highly encouraged worldwide. The US EPA’s Renewable Fuel Standard Program defines various renewable fuel pathways based on the feedstock type and reduction in overall life-cycle emissions. Cellulosic biofuels generated from cellulose, hemicellulose, or lignin as feedstock qualify for a D3 or D7 (cellulosic diesel) Renewable Identification Number (RIN), which have higher monetary value than other RINs (US EPA, 2012). Similarly, the EU Renewable Energy Directive Recast (RED II, 2018) assigns 0 gCO2e/MJ to the cultivation of agricultural and forestry residue for biofuel production, which lowers the overall carbon intensity of these fuel products. Animal manure has received much recent attention as a sustainable feedstock, both in the USA and EU. In accordance with California’s LowCarbon Fuel Standard, the use of animal manure as a feedstock to generate renewable natural gas (RNG) could decrease the carbon intensity of RNG to 400 gCO2e/MJ. In the EU, the typical carbon intensity of RNG derived from wet manure is approximately 100 gCO2e/MJ, which is much lower than that of fuels produced from other feedstocks (RED II, 2018). Given that the anaerobic digestion of animal manure requires little pretreatment, the cost of making RNG could be lower than the cost of making other fuels. Accessing the sugars in agricultural residues for liquid fuel production requires thermal, chemical, or biological pretreatment. The high cost of such processes has hindered the use of lignocellulosic feedstock to make liquid fuels, with several companies divesting their cellulosic ethanol plants over the past few years. These decisions might have been partially affected by the high cost of feedstock and the complexity of the process. However, there is a success story in this space: the Brazilian energy company Raı´zen S.A. has built a cellulosic ethanol facility that uses sugarcane bagasse as its feedstock and is planning to build a second facility with double the capacity. This would increase the total generating capacity to 120 million liters of cellulosic ethanol per year (Mano, 2021). This has revitalized the industry and may prompt companies to review projects to produce fuel from agricultural

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residues. The increased emphasis on reducing net carbon emissions from industry suggests that lignocellulosic materials will be increasingly used to produce fuels. Agricultural crop residues comprise organic materials that are byproducts of harvesting and processing agricultural crops. They are generated either in the field at the time of harvest or coproduced when crops are processed elsewhere. They typically include stalks and leaves (e.g., corn stover, wheat straw, rice straw, and stubble) and are abundant, diverse, and widely distributed across the world. Large quantities of crop residues are produced worldwide, but significant quantities are either left in fields, or burnt as waste without recovering heat, or plowed back into the soil as fertilizer. The burning of agricultural crop residues poses a threat to the environment and human health, due to the emission of toxic gases and particulate matter (Khare et al., 2021). However, it has become an annual feature of farming in the densely populated agricultural regions of India, China, and Southeast Asia, due to demands for increased agricultural productivity that often results in ecological imbalances in the agroecosystem. An in-depth analysis of the sustainability differences between farming and industry is presented in Maletta and Diaz-Ambrona (2020). The effective management of crop residues, with concomitant leveraging of agricultural resources from existing lands as feedstock for sustainable fuels and chemicals for energy, provides an opportunity to recycle materials that are essential for soil integrity. For instance, making biochar from agricultural residue could be carbon-negative and provide a sustainable method for the utilization of biomass residue. Normally, plants absorb CO2 from the atmosphere during growth and release CO2 upon attaining senescence; this is thus a carbon-neutral process. When biomass is converted to biochar, carbon is sequestered in soil in the form of biochar. Therefore, biochar production is a carbon-negative approach that is considered a safe route for the disposal of organic wastes, such as agricultural residues (Bhandari, 2014). The application of biochar to soil is currently recommended as a costeffective and reasonable strategy for global carbon sequestration (Spokas et al., 2012). Forest residues are a by-product of forest harvesting and are a major source of biomass for energy generation. Forest residues comprise organic matter remaining after the logging of timber (including limbs and tops of trees, culled trees, and components of trees that are unmerchantable) and dead, diseased, and poorly formed trees that are not harvested. Some of this woody debris can be collected and used to produce bioenergy, with

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sufficient debris left to provide habitats and maintain proper nutritional and hydrological features. Alternatively, large amounts of woody biomass can be harvested to reduce the risk of fire and pests, aid in forest restoration, and increase forest productivity, vitality, and resilience. Thus, biomass can be harvested for use in generating bioenergy without negatively affecting the health and stability or ecological structure and function of forests. However, such residues usually have low density and heating values and are expensive to transport. Thus, forest residues and wood wastes for direct combustion are usually obtained from modified forests with larger tree densities. Robust forest-management policies are therefore needed to ensure that this resource is not overexploited. Forests will only remain a sustainable resource for energy if they are regrown as fast as they are consumed.

4.3.2 Non-edible oil feedstocks Non-edible oil feedstocks include waste cooking oil, animal fat, and dedicated oil crops such as jatropha, tobacco, and karanja. International biofuel sectors are strongly influenced by national policies, which have three major goals: (1) to support farmers; (2) to reduce GHG emissions; and/or (3) to increase energy independence. These biofuel policies cause much uncertainty in the vegetable oil sector, as approximately 14% of global vegetable oil supplies are earmarked for biodiesel production. In the EU, policy reforms and the emergence of 2G biofuel technologies will likely prompt a move away from crop-based feedstock (OECD/FAO, 2021). Currently, almost 75% of biodiesel is produced from vegetable oils (20% rapeseed oil, 25% soybean oil, and 30% palm oil) with the remaining mainly produced from waste cooking oils. WCOs or used cooking oils (UCOs) are oils and fats that have been used for cooking or frying in the food processing, restaurant or fast-food industries, or by consumers in households. The European Waste Catalogue (EWC) classifies WCOs and UCOs as municipal wastes (household waste and similar commercial, industrial, and institutional wastes) and as separately collected fractions under the code 20 01 25 (edible oils and fats). UCOs obtained from wastewater-treatment plants are also considered nonhazardous materials under code 19 08 09 (grease and oil mixtures from oil/water separation containing edible oil and fats). Technologies to produce biodiesel and HVOs from waste-oil and animal-fat feedstocks are well established and provided 8% of all biofuel output as of 2018 (IEA, 2020).

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Although UCOs are inexpensive and accessible, they are challenging to convert to biodiesel due to their composition and nature. UCOs contain high concentrations of FFAs and thus must be pretreated before they can be processed by conventional homogeneous, alkaline processes. The typically intense smell of UCOs arises from their chemical composition, especially the volatile fraction containing aldehydes, alcohols, ketones, hydrocarbons, and organic acids. Many of these decomposition products are formed via triacylglycerol oxidation and the Maillard process, which generate nitrogen-containing derivatives from a reaction between the carbonyl units of carbohydrates and the amino groups of proteins. Given that commercial vegetable oil is almost odorless, the chemicals responsible for the aroma of fried oil are likely produced during the frying process, which produce low-molecular-weight volatile compounds. Thus, the removal of acetic, propanoic, butanoic, and hexanoic acids and hexyl acetate from UCOs can decrease their odor (Mannu et al., 2019). Different waste animal fats (WAFs) from animal meat-processing facilities, rendering companies that collect and process animal carcasses, and large food-processing and service facilities can be used to obtain feedstock for biodiesel production. Such fats include beef tallow extracted by rendering the fatty tissue of cattle, mutton tallow from sheep, pork lard from pigs, and chicken fat from the feathers, blood, skin, offal, and trims of chicken (Mora et al., 2020). Animal fats and greases tend to be solid because of their high content of saturated fatty acids (SFAs). Most WAFs contain high concentrations of various SFAs (myristic, palmitic, and stearic acids). Typically, SFA compositions are as follows: tallow and pork lard, 40%; beef tallow, 45.6%; mutton tallow, 61.1%; lard, 39.3%; and chicken fat, 32% (all w/w). The low SFA percentage in chicken fat accounts for its viscous and semisolid nature. Recycled greases are also considered waste greases and are classified based on their FFA content as yellow greases or brown greases. Yellow greases contain less than 15% (w/w) FFAs and are produced by heating animal fats and vegetable oils collected from commercial and industrial cooking businesses. Brown greases contain greater than 15% (w/w) FFAs and are sometimes referred to as trap greases, i.e., materials that are collected in special traps in restaurants that prevent grease from entering the sanitary sewer system (Bankovic-Ilic et al., 2014). Inedible animal by-products are classified by the EU in terms of their risk to human or animal health: (1) highest risk; (2) high risk; and (3) lowest risk and fit for human consumption (although not generally used for human food because of

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inedibility or a lack of commercial viability). Type-(3) by-products are as largely used as animal feeds and pet food, but all types can be used for biodiesel production (some stakeholders have reported that type-(3) yields better-quality biodiesel (Ecofys, 2016)). Biodiesel derived from non-edible oil is considered a 2G biofuel. Jatropha, rubber seed, jojoba, tobacco seed, sea mango, neem, candlenut, mahua, karanja, and yellow oleander are examples of non-edible plant sources that yield 2G feedstocks. As non-edible oil plants are grown mainly on waste land, the oil extracted from these plants can be utilized as an alternative feedstock for biodiesel production, thereby circumventing food vs. fuel challenges. These plants can grow almost anywhere with minimal cultivation effort, even in sandy and saline soils that are not suitable for food crop production. The plantation costs for non-edible oil crops are generally much lower than those for edible crops, as the cultivation of edible crops requires high soil nutrition, good irrigation, and the maintenance of soil nutrients and moisture. Thus, the utilization of non-edible oils as feedstock would minimize the biodiesel production cost due to the low cost of raw materials. However, such sources will also have lower oil yields, as they are grown on waste land without proper nutrition. Although the compositions of non-edible and edible oils are similar, with both containing FFAs, SFAs, and unsaturated fats, edible oils also contain valuable nutrients and antioxidants. Non-edible oils derived from jatropha, sea mango, rubber seed, and candlenut are not suitable for human consumption, as they contain toxic substances. For instance, jatropha seed oil contains the purgative curcas, and rubber seed oil contains cyanogenic glucoside. The percentage oil contents in jatropha seed (40–60wt%), rubber seed (40–50 wt%), sea mango seed (40–50 wt%), candlenut (60–65wt%), polanga (60–70 wt%), and yellow oleander (60–65 wt%) are much higher than those in edible oil crops, such as rapeseed (37–50 wt%), soybean (20 wt%), and palm (20wt%). Oil yields (kg/ha/year) from non-edible oil crops are either comparable with or higher than those from conventional oil seeds used to generate biodiesel (jatropha: 2500 kg/ha/year; candlenut: 1600 kg/ha/year; neem: 2670 kg/ha/year; karanja: 900–9000 kg/ha/year; yellow oleander: 5200 kg/ha/year; sea mango: 1900–2500kg/ha/year vs. soybean: 446 kg/ha/year; rapeseed: 1190 kg/ha/year; palm: 5950kg/ha/year; Shaah et al., 2021). Seed yields and oil contents vary with region and atmospheric conditions, i.e., the regions of harvest can influence the yield. For instance, jatropha planted in arid regions only yields approximately 500 kg/hectare/year of oil (Balat and Balat, 2010).

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Although non-edible oils from such crops are considered to be good alternative feedstocks for biodiesel production, challenges remain. Specifically, effective pretreatments that can address the challenges associated with their composition (high FFA content, high polyunsaturated FA content, and low unsaturated FA content), up-to-date production technologies, longterm plantation, and land-use management policies and community involvement are key to the successful scale-up of biodiesel and renewable diesel production from this resource.

4.4 Third-generation (3G) feedstocks Algae are often referred to as 3G feedstocks. Microalgae (unicellular) and macroalgae (multicellular) have been investigated as potential fuel sources, with their scalability, productivity, and continuous supply of biomass being the most important aspects under consideration. The major components of algae are lipids, carbohydrates, and proteins, and the relative proportions of each within algae depend on the species and cultivation conditions used (Zhu, 2015; Vitova et al., 2015). The composition of algae means that they can be used for direct energy production via lipid extraction (Lakaniemi et al., 2013) and for biofuel production (bioethanol, biogas, biodiesel, and bio-oils) via anaerobic digestion, fermentation, transesterification, and liquefaction. However, compared with fossil fuel extraction and isolation, these methods are more complex and economically expensive (Adeniyi et al., 2018). In particular, processes that require biomass drying are highly energy-intensive, which makes algae unattractive as raw materials for pyrolysis, direct combustion, or gasification (Milledge et al., 2019). Microalgae are microscopic organisms that grow via photosynthesis in freshwater and marine environments. The global potential of this feedstock is limited to areas with enough solar radiation, water, and nutrients. It has been estimated that by 2035, the areas with the highest potential for algae biomass cultivation will be Asia (2.5 EJ/year), Africa (0.75 EJ/year), North and South America (0.75 EJ/year), Oceania (0.2 EJ/year), and Europe (0.15 EJ/year) (Bosˇnjakovic and Sinaga, 2020). There are more than 3 million types of microalgae, and they are divided into carbohydrate-rich and lipid-rich classes. For example, species such as Neochloris oleoabundans (UTEX-1185), Porphyridium aerugineum (UTEX755), Porphyridium cruentum, and Spirogyra sp. contain greater than 30% carbohydrates by dry weight, and species such as Dunaliella, Chlorella, Cylindrotheca, Nannochloris, Crypthecodinium, Isochrysis, Neochloris, Porphyridium,

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Nannochloropsis, Nitzschia, Phaeodactylum, Tetraselmis, and Schizochytriu contain 20%–50% lipids by dry weight (Zhou et al., 2014; Zhou et al., 2015; Jalilian et al., 2020). Chlorella is the most common microalga used for biodiesel production through direct transesterification, due to its high biomass and oil production. The use of microalgae for this purpose is promising, as they can accumulate higher concentrations of oil than feedstocks such as soybean; in fact, the biodiesel production yields from microalgae are 200 times higher than those obtained from plant/vegetable oils. For example, algae with 30% oil in dry biomass can produce 58,700 L/ha/year of biodiesel, which is higher than that obtained from oilseed rape or soybeans (1190 L/ha/year and 446 L/ha/year, respectively; Singh and Singh, 2015). It was also shown that low illumination and nitrogen limitation during cultivation improve FA accumulation and increase biodiesel production yields (Amini et al., 2016). The use of closed photobioreactors rather than open ponds for microalgae cultivation also maximizes biomass productivity and photosynthetic efficiency. However, these systems require high initial investments and cannot be used to grow all microalgae strains (Wang et al., 2012). The high carbohydrate content (mainly in the form cellulose and starch) and low lignin content of some microalgae make them useful as readily available carbon sources for the production of bioethanol, biobutanol, and biogas (Chew et al., 2017; Nagappan et al., 2019). Some studies have shown that the carbohydrate content in microalgae can be increased under stressful conditions. For example, nitrogen depletion increases starch and glycogen concentrations in some microalgae and thus improves biofuel production yields (Cheng and He, 2014). It is also possible to use two-stage cultivation to increase carbohydrate, lipid, and hydrogen concentrations to improve yields; the first stage involves microalgae-growth optimization, and the second stage involves biofuel accumulation (Nagappan et al., 2019). The use of microalgae for biofuel production has some advantages as they can grow in wastewater, partially clean and salty water, and generally require less water to grow than terrestrial plants (O’Connell et al., 2013). Although microalgae appear to be one of the key renewable resources that can meet the demand for biodiesel, transportation demands necessitate the growth of large quantities of microalgae (Demirbas and Demirbas, 2011). Moreover, although microalgae grow faster than terrestrial plants due to their high photosynthetic efficiency (Wang et al., 2017), microalgal cultivation is not currently economically feasible for biofuel production due to temperature, photosynthesis, and harvesting costs ( Jalilian et al., 2020). Harvesting

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techniques are considered the main bottleneck (Yew et al., 2019), with centrifugation and filtration during harvesting accounting for 2% of total production costs (Alam et al., 2017). Furthermore, microalgal cultivation at large scales requires significant volumes of water and fertilizers (Yin et al., 2020). Several studies have revealed that fertilizer production accounts for 50% of the energy consumption and GHG emissions during algal cultivation (Zhu et al., 2017). Moreover, Yang et al. found that the water footprint of microalgal cultivation processes is very large with 1 kg of algal biodiesel requiring 3726 kg of water (Yang et al., 2011). However, post-harvest water recycling could reduce this usage (down to 591 kg of water per kg biodiesel) and the associated costs. Alternatively, urban and livestock wastewater could be used for algal cultivation; this would eliminate inorganic nutrients from wastewater (nitrogen and phosphorus) and allow microalgal biomass production with CO2 sequestration via photosynthesis (Kadir et al., 2018). However, it is important to control the nitrogen: phosphorus ratio in such methods, as successful microalgal growth requires optimal ratios (Salama et al., 2017). Despite the advantages offered by microalgae, biodiesel production from this source is not yet commercially viable. Moreover, the production costs are at least double those for petroleum-based diesel production ( Jalilian et al., 2020). Research in this field has been focused on the development of more efficient technologies, strains with high lipid concentrations, and the induction of lipid production through different methods. For example, lipid production can be increased by metabolically engineering lipid pathways and manipulating cultivation by applying stressful conditions, such as nitrogen starvation and high salinity (Chen et al., 2017). Microalgae can also be used for hydrogen production via biophotolysis or photofermentation with hydrogenases and nitrogenases (Razu et al., 2019). Previous studies have shown that microalgae from genera such as Botryococcus, Chlorococcum, Chlamydomonas, Chlorella, Tetraspora, Scenedesmus, Synechocystis, Anabaena, Nostoc have hydrogenase activity that can be harnessed for hydrogen production (Eroglu and Melis, 2011). However, hydrogen production from microalgae has been successful at the laboratory scale but not at large scales, due to problems such as low light-capturing and CO2-fixation efficiencies and the oxygen sensitivity of enzymes (Razu et al., 2019). The costs of algal cultivation and downstream process need to be reduced to make hydrogen production feasible, which could possibly be achieved by genetically and metabolically engineering the two main enzymes involved in the process (Anwar et al.,

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2019). Indeed, genetic engineering was recently used to increase the hydrogen-production potential of certain microalgae strains (Baebprasert et al., 2011). The push for industrial use of microalgae for biofuel production in the last century, driven by improving sustainability of fuel production and reduction of fossil fuels usage, translated into a high impact research field with high number of patents until 2012 (Li et al., 2020). After that, with the increase in commercialization of shale gas and electric cars, the research interest decreased. One the most cited and validated patents, US20160208243A1 of The Broad Institute of the Massachusetts Institute of Technology (MIT), was filed in 2015. Many of the other patents are older than 10 years and close to expiration, showing the field is currently in period of stasis. The subjects of the most-cited patents are genetic manipulation of microalgae, cultivation to promote biofuel production and downstream biorefining. Macroalgae, also known as seaweed, are classified based on their photosynthetic pigmentation as green macroalgae (Chlorophyta), brown macroalgae (Phaeophyta), or red macroalgae (Rhodophyta) ( John, 2011). Macroalgae have a high content of carbohydrates (25%–77% dry matter) (del Rı´o et al., 2020). In addition, brown macroalgae are rich in alginate and fucoidan, green macroalgae are rich in ulvan and xylan, and red macroalgae are rich in carrageenan, agar, and xylose (Saratale et al., 2018). Their high carbohydrate and low lignin content suggest that they could be cultivated commercially for alcohol-based fuel production ( Jalilian et al., 2020). Anaerobic digestion is also a simple and effective method for converting macroalgal biomass into methane or biogas (a mixture of CO2 and methane), with some commercial advantages as dewatering and drying are not required. Generally, biogas produced through the anaerobic digestion of macroalgae contains 50%–70% methane, 30%–45% CO2, 90%) only for high alcohol-tooil molar ratios, high catalyst concentrations and high temperatures Slower than in the alkaline process Complex, lowgrade glycerol Difficult, the catalyst ends up in the byproducts. No reusable catalyst High, temperature: >100°C Low, but high cost of equipment due to acid corrosion High, wastewater treatment required

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lipases occurs under more gentle conditions and can utilize a wider variety of triglyceride substrates (e.g., raw materials, waste oils, fats) with high levels of free fatty acids (FFAs). The biodiesel separation and purification are also considerably easier, which results in an overall more environmentally friendly process (Talha and Sulaiman, 2016; Tacias-Pascacio et al., 2019).

6.2 Lipases Lipases are highly versatile enzymes and produced in several industrial processes. Lipases are found in animals, plants, and microorganisms and play a key role in the metabolism of oils and fats. Lipases take part in the deposition, transfer, and metabolism of lipids. The microbial lipase market was estimated to be USD 425.0 million in 2018, is projected to reach USD 590.2 million by 2023, and has grown at a compound annual growth rate of 6.8% since 2018 (Chandra et al., 2020). Lipases are hydrolases (EC 3.1.1.3) that catalyze the hydrolysis of long-chain triglycerides by acting on carboxyl ester bonds in triglycerides to produce fatty acids and glycerol. Lipases catalyze this reaction at the lipid-water interface. The lipase structure has a central L-sheet with an active site consisting of a serine on a nucleophilic elbow located in a groove of the structure. This groove is covered by a peptide lid that undergoes conformational changes when the lipase encounters the lipidwater interface to make the active site accessible for the acyl moiety ( Jegannathan et al., 2008). Lipases have both hydrolytic and synthetic activities and can thus take part in various industrially important reactions, such as esterification and transesterification (alcoholysis and acidolysis). Lipases from fungi and bacteria are easily produced in bulk quantities because of their extracellular nature (Yagmurov et al., 2017; Zavarise and Pinotti, 2019). Lipases are widely used to process fats, oils, detergents, degreasing formulations and food, as well as in the synthesis of fine chemicals, pharmaceuticals, paper, and cosmetics (Sharma, 2017; Hasan, 2006). Lipases (EC 3.1.1.3) are powerful tools that, in addition to hydrolysis reactions, also catalyze various synthetic reactions including esterification, transesterification, and aminolysis. Lipases have an excellent catalytic activity and stability in non-aqueous media, and their specificity, regioselectivity, and enantioselectivity can be successfully used for many applications in organic synthesis, including kinetic resolution and asymmetric synthesis (Gonzalo, 2018; K€ uhn et al., 2020).

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Lipases can be divided into three classes based on their specificity and/or selectivity: regio- or positional-specific lipases; specific lipases for a certain type of fatty acid; and specific lipases for a certain class of acylglycerols (mono-, di-, or triglycerides). In terms of regioselectivity, lipases can be divided into three types: sn-1,3-specific (which hydrolyze ester bonds in positions R1 or R3); sn-2-specific (which hydrolyze the ester bond in position R2); and nonspecific (which do not distinguish between the ester bond positions to be cleaved). Most known lipases are 1,3-regiospecific with activity on the terminal positions. Another important aspect is the acyl migration phenomenon that occurs inside the triacylglycerol molecule, as reported in several studies (Peng et al., 2020; Zhou et al., 2021). The substrate specificity of lipases is determined by their ability to distinguish different structural features of the acyl chains, such as the nature of the acyl source (e.g., free acid, alkyl ester, glycerol ester), length, double-bond position and configuration, and the presence of branched groups. Lipase selection is thus one of the most influential factors for biodiesel production using various renewable raw materials. Commercially produced lipases are mostly of microbial origin. Submerged culture methods and solid-state fermentation are widely used for commercial lipase production. Lipase-producing microorganisms (e.g., bacteria, fungi, yeasts) are isolated and screened for their lipolytic activity (Bharathi and Rajalakshmi, 2019). Microorganisms with a high activity are then selected for commercial lipase production. Lipase production depends on a number of factors including carbon and nitrogen sources, pH, temperature, dissolved oxygen, agitation, and metal ions (Salwoom et al., 2019). Lipase production can also be induced by using lipids as a carbon source (Suci et al., 2018). Purification strategies include the concentration of a culture medium via ultrafiltration or ammonium sulfate precipitation followed by further purification using sophisticated techniques such as affinity chromatography, ion exchange chromatography, and gel filtration (Tan et al., 2015). Several novel techniques, such as membrane processes, immune purification, hydrophobic interaction chromatography, and column chromatography, have been applied for lipase purification (Chantal Br€amer et al., 2018). The production and purification schemes of lipases for large-scale applications should be high-yielding, rapid, and inexpensive (Melani et al., 2020; Priji et al., 2021).

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6.3 Enzymatic production of biodiesel 6.3.1 Extracellular and intracellular lipases The two major categories of enzymatic biocatalysts are extracellular lipases and intracellular lipases. The enzyme in the case of extracellular lipases is initially recovered from a live-producing microorganism broth and then purified, whereas an intracellular lipase remains either inside the cell or within the cell walls. The major producing microorganisms for extracellular lipases include Mucor miehei, Rhizopus oryzae, Candida antarctica, and Pseudomonas cepacia (Gog et al., 2012). Previous studies that reported the use of free lipases for biodiesel production principally focused on lipase screening (Geoffry and Achur, 2018) and the factors that influence the reaction rate (Toldra´-Reig et al., 2020b). Soluble lipases have the advantages of an easy preparation procedure and low cost; however, their reuse is limited in many cases owing to inactivation after a single use. Improved immobilization technology has produced lipases with enhanced levels of reusability and operational stability, leading to higher conversion rates and shorter reaction times (Kumar et al., 2020). The major disadvantage of producing biodiesel using extracellular enzymes is the relatively high cost of the lipase due to the complex separation and purification procedures. In contrast, an acceptable ester yield can be achieved at a lower cost by using microbial cells that produce intracellular lipase as whole-cell biocatalysts. However, the use of intracellular lipases implies a slower process than when using extracellular lipases. The main intracellular lipases that have been investigated include biocatalytic systems based on Rhizopus oryzae yeast.

6.3.2 Lipase immobilization Biodiesel production using immobilized lipases (ILs) has attracted great interest in recent years (Zhong et al., 2020), and significant progress has been made in both the immobilization techniques and process development for IL-mediated biodiesel production. ILs offer several advantages over soluble or free lipases in terms of large-scale applications for biodiesel production (Marı´n-Suarez et al., 2019), such as easy recovery and reuse, higher adaptability for continuous operation, fewer effluent problems, greater pH and thermal stability, and higher tolerance to reactants and products. However, the current ILs still suffer from several drawbacks for industrial applications, including (1) enzymatic activity loss during immobilization; (2) high carrier cost; (3) low stability in oil-water systems; and (4) the requirement of novel

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reactors for sufficient mixing and maximizing the oil-to-biodiesel conversion. Several materials have been explored in the literature to immobilize lipases, including various polymer resins, celite, silica, ceramics (Zhong et al., 2020; Aggarwal et al., 2021), carbon nanotubes (Bourkaib et al., 2020), supported ionic liquid phase (Lee et al., 2018; Ullah et al., 2018), magnetic particles (Badoei-Dalfard et al., 2019), and microspheres (Zhang et al., 2020). However, the cost of the carrier material must be low for industrial applications. The immobilization procedure should also be easy to perform with a high active-lipase recovery rate, and the IL activity must be maintained for a long running time. These goals can generally be achieved by (1) improving the immobilization technologies; (2) optimizing the transesterification process; (3) developing novel bioreactors; and (4) intensifying the process integration to reduce the operation cost. Various immobilization methods can be applied for lipases used in biodiesel production, including adsorption, cross-linkage, entrapment encapsulation, and covalent bonding (Ismail and Baek, 2020). Several examples (Zhao et al., 2015) are shown in Fig. 6.4. These approaches can be further

Fig. 6.4 Current techniques used for enzyme immobilization.

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classified into irreversible and reversible immobilization techniques depending on the type of interactions between the enzymes and carriers (Alnoch et al., 2020; Rafiee and Rezaee, 2021). Covalent bonding, entrapment, and cross-linking are the most commonly used procedures for the irreversible immobilization of lipases. Physical adsorption and various forms of noncovalent bonding (e.g., affinity bonding, chelation bonding) are wellknown reversible immobilization procedures. Each lipase immobilization technique has its own merits and inevitably some disadvantages (Nezhad and Aghaei, 2021). 6.3.2.1 Lipase immobilization via physical adsorption Adsorption is a commonly used method to immobilize lipases. Several noncovalent interactions are involved in this approach including nonspecific physical interactions, adsorption, biospecific adsorption, affinity adsorption, electrostatic interaction (also known as ionic bonding), and hydrophobic interaction (Aghaei et al., 2020). Compared with other immobilization techniques, adsorption immobilization is advantageous in the following aspects (Rodrigues et al., 2019): (1) mild conditions and easy operation; (2) relatively low cost of the carrier materials and immobilization procedure; (3) no requirement of chemical additives during adsorption; (4) easy regeneration of the carriers for recycling; and (5) high lipase-activity recovery. 6.3.2.2 Lipase immobilization via ionic bonding versus covalent bonding In the immobilization process using ionic bonding, the enzymes are bound via salt links. The carriers typically contain ion-exchange residues such as polysaccharides and synthetic polymers (Okura et al., 2020). The ionic bonding process can be easily performed, and the interactions between. the lipase and carrier are considerably stronger than in the physical adsorption process. Ionic bonding can be conducted under much milder conditions than the covalent bonding method. The ionic bonding method therefore causes little change in the conformation and the active lipase site and generally retains the lipase activity. However, the bonding forces between enzymes and carriers are less strong than those using the covalent bonding technique, and enzyme leakage from the carrier may occur in substrate solutions with high ionic strength or upon varying the pH conditions (Dong et al., 2019).

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6.3.2.3 Lipase immobilization via entrapment or encapsulation Entrapment immobilization refers to the capture of enzymes within a polymeric. network or microcapsules of polymers, which allows the substrate and products to pass through while retaining the enzyme. After entrapment, the lipase proteins are not attached to the polymeric matrix or capsule, but their diffusion is constrained. The entrapment-immobilized lipases are more stable than physically adsorbed lipases. Entrapment immobilization is simpler to perform than covalent bonding and maintains the lipase activity. However, the conversion rate and stability of the entrapped lipases are relatively low when using entrapped lipases for biodiesel production (Santos et al., 2020). 6.3.2.4 Lipase immobilization via cross-linking Lipase immobilization via cross-linking refers to the process of immobilizing the enzyme by the formation of intermolecular cross-linkages. This can be achieved by the addition of bi- or multifunctional cross-linking reagents such as glutaraldehyde. This immobilization technique is usually supportfree and involves joining enzymes to each other to form a three-dimensional structure (Najeeb et al., 2021). Lipases can be directly immobilized from fermentation broth and recovered as cross-linked enzyme aggregates (CLEAs). The formed CLEAs demonstrate significantly high stability in aqueous solu€ tions within a broad pH and temperature range (Ozacar et al., 2019). Despite the associated advantages, cross-linking reactions are usually performed under relatively harsh conditions, such as involving the use of crosslinking reagents that can change the lipase conformation and potentially lead to significant activity loss (Velasco-Lozano, 2020). Other disadvantages of cross-linking immobilization include low immobilization yields and a lack of desirable mechanical properties. Cross-linking is thus coupled with other immobilization techniques (e.g., adsorption) to address these concerns (Torabizadeh and Montazeri, 2020). 6.3.2.5 Commercialization of immobilized lipase for biodiesel production Commercial immobilized lipases that have been thoroughly studied include Novozym 435 (Ortiz et al., 2019), Lipozyme TL IM (Marı´n-Suarez et al., 2019), and Lipozyme RM IM (Rivero-Pino et al., 2020), all of which are extracellular enzymes. The most widely used lipases are Novozym 435 from Candida antarctica immobilized on a microporous acrylic resin, Lipozyme RM IM from Rhizomucor miehei immobilized on an anionic resin, and

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Table 6.2 Advantages and disadvantages of several enzyme immobilitation methods. Immobilization methods

Physical adsorption

Advantages

Disadvantages

–

Desorption of the enzyme from the carrier: slow and with low efficiency

– – –

Cross-linking

– –

Entrapment

– – – –

Covalent binding

– – – – –

Encapsulation (membrane confinement)

– – – – –

Little affect the enzyme structure Simple and low cost technique Wide applicability No pore diffusion limitations High enzyme loading Little desorption (enzyme immobilized by covalent binding) Low enzyme leakage Wide applicability Loss of enzyme activity is low Mild conditions needed Low enzyme leakage Wide applicability Very strong chemical bond Absence of leakage of the enzyme from the support Simple and reversive method with selective applicability Large quantities of enzymes can be immobilized Strong binding nature Simple method for immobilization Low enzyme leakage Very wide applicability

May cause changes in the active site

Complex method – Mass transfer resistance to substrates and products – Substrate cannot diffuse deeply into the gel carrier

Limitation of pore size: only small substrate molecules are able to cross the membrane

Lipozyme TL IM from Thermomyces lanuginosus immobilized on a granulated silica gel. Table 6.2 collects the advantages and disadvantages of enzyme immobilization methods previously described.

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6.3.3 Variables affecting the enzymatic transesterification reaction The crucial factors that affect the productivity of enzymatic biodiesel synthesis are shown in Fig. 6.5 (Szczesna-Antczak et al., 2009). A suitable choice of raw materials and lipase is required for economic viability. The lipase can be modified to improve the stability and catalytic efficiency. These steps are followed by the selection of an organic solvent and optimization of the substrate molar ratio, temperature, water activity, pH of the enzyme microenvironment, and the highest-permissible glycerol concentration in the reaction products (Moreira et al., 2020; Ostojcic et al., 2020). 6.3.3.1 Lipid source Lipases are more competitive catalysts in comparison with acids and alkalis owing to the wide variety of triglyceride substrates that can be used for the enzymatic synthesis of biodiesel. To avoid possible competition from the use of triglycerides for food or biofuels, only inedible fats or those of waste origin are presently considered for biodiesel production applications. These biofuels are considered second generation and offer greater economic viability (Abomohra et al., 2020).

Fig. 6.5 Crucial parameters that affect the enzymatic synthesis yield of biodiesel.

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Vegetable oils extracted from oleaginous plants are currently the main raw materials used to produce biodiesel. These materials presently account for approximately 70% of the total production cost. The most suitable vegetable oils are thus obtained from crops with the highest productivity per hectare or low-cost oils such as waste oils. High fossil fuel prices, the collapse of crop for biodiesel initiatives, and concerns regarding increased CO2 levels in the atmosphere have all raised awareness regarding the need for alternative fuel solutions. Microalgae have promisingly emerged as a potential lowestcost feedstock for biodiesel production and are considered a thirdgeneration biodiesel (Aliyu et al., 2021). Fats and oils may be characterized according to their physical (e.g., density, viscosity, melting point, refractive index) or chemical properties (e.g., acidity, iodine index, peroxide index, saponification index), which influence the biodiesel quality. This means that the fatty acid profile of oil influences the quality of the produced biodiesel (Maa et al., 2020).

6.3.3.2 Acyl acceptor Various types of acyl acceptors, alcohols (primary, secondary, straight, branched chain), and esters can be used in the transesterification process using lipases as catalysts. The most frequently used acyl acceptors are alcohols, particularly methanol and, to a lesser extent, ethanol. Other alcohols can be used (e.g., propanol, butanol, isopropanol, tert-butanol, branched alcohols, octanol), but the cost is considerably higher. Compared with ethanol, methanol is cheaper, more reactive, and the resulting FAMEs are more volatile than fatty acid ethyl esters (FAEEs). However, ethanol is less toxic and can be considered more renewable because it is easily produced from renewable sources by the fermentation of agricultural feedstock. In contrast, methanol is mainly produced from nonrenewable fossil sources (e.g., natural gas) and also inhibits lipases. FAMEs and FAEEs show very slight differences in terms of their fuel characteristics (Leggieri et al., 2018). A stepwise addition of methanol is the most common strategy to avoid lipase inactivation (Hama et al., 2018). Lipase inactivation can also be avoided using a different acyl acceptor, such as methyl acetate or ethyl acetate (Nguyen et al., 2018). Another strategy for resolving the lipase inactivation problem with methanol is the use of organic solvents (Lotti et al., 2018), but solvent recovery difficulties make these methods less competitive at the industrial scale.

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6.3.3.3 Temperature Enzymatic transesterification is generally performed at a lower temperature than the chemical reaction to prevent lipase activity loss. The optimum temperature of lipase activity is in the range of 20–70°C depending on the lipase source. The moderate temperature requirements for lipase-catalyzed transesterification make this process less energy-intensive. Higher temperatures increase the enzyme activity until reaching the optimum temperature, beyond which enzyme denaturation occurs, thereby decreasing its activity. The initial reaction rate also increases with increasing temperature, thus reducing the required conversion time; however, the final conversion efficiency decreases owing to enzyme denaturation beyond the optimum temperature (Kumar et al., 2019). The factors that influence the optimum temperature of the lipasecatalyzed reaction include immobilization, lipase stability, alcohol-to-oil molar ratio, and the solvent type. Temperature is the key operational factor in the continuous reaction process (Zhang et al., 2019). The optimum temperature for the enzymatic transesterification process results from the interaction between the lipase operational stability and transesterification reaction rate (Khoobbakht et al., 2020). 6.3.3.4 Water content Water is essential to maintain lipase conformation and also increases the interfacial area between the aqueous and organic phases where the lipases act (Yang et al., 2019). The water content in a reaction mixture can be determined by either the water activity or as the weight percentage of the feedstock oil. The water activity is the ratio of the vapor pressure of a given system to that of pure water (Zulkeflee et al., 2020). Excess water takes part in transesterification reactions and leads to hydrolysis, which can reduce the alkyl ester yield. Optimizing the water content for the transesterification reaction is therefore very important. The optimum water content in the reaction depends upon the lipase and feedstock, immobilization technique, and solvent type ( Jahangiri et al., 2018). Water content sensitivity is crucial for transesterification because the optimum water content should have an appropriate balance to minimize the hydrolytic reaction and maximize the lipase activity. Various substitutes for water (e.g., tert-butanol, surfactants) are available as reaction additives, but none can match the yield obtained from the water-added reaction. Water takes part in the subsequent hydrolysis and esterification. Water also dilutes ethanol, which has an inhibitory effect on the lipase, and prevents the

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inactivation of lipase by methanol (Rafiei et al., 2018). Lipases have different responses to water content depending on their source. 6.3.3.5 Inhibition by alcohol Alcohols are popular acyl acceptors for the transesterification reaction to produce biodiesel. Methanol is the most widely used alcohol for transesterification because of its low cost and short chain length, which results in a high biodiesel yield. The methanol-to-oil ratio is thus a critical parameter in optimization studies. Various lipases have been shown to have different tolerance levels for methanol. Most previous studies optimized the methanol-to-oil molar ratio in the range of 3:1–4:1 for the lipase-catalyzed conversion. Some lipases have shown an optimum activity at higher methanol-to-oil ratios. Various alternatives have been suggested to overcome methanol inhibition, including the stepwise addition of methanol and the use of other acyl acceptors, solvents, and methanol-tolerant lipase (Lv et al., 2019). 6.3.3.6 Inhibition by glycerol Glycerol also has an inhibitory effect on lipase activity. Glycerol is a product of the lipase-catalyzed transesterification reaction and drives the reaction equilibrium in the reverse direction. Glycerol molecules also form a hydrophilic environment around immobilized lipase molecules, thus preventing the hydrophobic substrate from contacting the enzyme. The inactivation and inhibition effects of methanol and the glycerol product can be reduced by using a membrane bioreactor (Aghababaie et al., 2019). The continuous removal of glycerol from the reaction mixture and use of solvents are viable solutions for minimizing glycerol inhibition (Sˇalic et al., 2018; Al Basir and Roy, 2019). Polar solvents (e.g., tert-butanol) and novel solvents (e.g., ionic liquids) dissolve glycerol and thus minimize its negative effect. Lipases show good stability and improved yield in such solvent systems (Elgharbawy et al., 2018). Another possible strategy to avoid the inhibitory effect of glycerol is to avoid its formation in the transesterification process. Acyl acceptors, other than short-chain alcohols, which do not lead to glycerol formation in the lipase-catalyzed transesterification process have therefore recently gathered interest (Nguyen et al., 2018; Ribeiro et al., 2018; Nascimento et al., 2019). 6.3.3.7 Pretreatment for improving lipase stability. The stability and activity of lipases can be improved by pretreating the enzyme prior to its application. The pretreatment strategy involves exposing

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the enzyme to a substrate and its analogues, organic solvents, and salts. These pretreatments enhance the catalytic performance by maintaining the active sites in open conformation. Methanol inactivation and high costs are major drawbacks for lipases and their successful use for biodiesel production. Pretreatments improve the lipase catalytic performance, methanol tolerance, and stability (Guldhe et al., 2015; Cavalcante et al., 2021). Ultrasonic emulsification pretreatment of immobilized lipases might also be a potential alternative route for producing biodiesel (Gabriel Murillo et al., 2019).

6.4 New tendencies in the enzymatic production of biodiesel Several strategies can be applied to reduce the production costs of enzymatic transesterification during up-, mid-, and downstream processing, as proposed by Zhao et al. (2015) and shown in Fig. 6.6. In upstream processing, the lipase catalytic stability and activity can be improved by protein engineering, strain optimization, and metabolic engineering techniques (Choi et al., 2020). A further reduction of the running cost of the enzymecatalyzed biodiesel production can be achieved using process-intensification strategies, such as improving the immobilization and optimization of the process design. The immobilization of lipase enzymes has been studied

Fig. 6.6 Unit operations and corresponding work that can be applied to reduce the cost of lipase-catalyzed biodiesel production (Zhao et al., 2015).

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for many years using various carriers. However, only a few types of carriers and immobilization processes have been commercialized. Nevertheless, these commercialized ILs are still exceedingly expensive for biodiesel production (Sheldon and Brady, 2019). Some newly developed immobilization technologies involving magnetic particles and nanoparticles have been reported, but remain far from industrial applicability. One solution to the high cost of lipases for biodiesel production is to increase their lifetime in transesterification (Kumar and Pal, 2021). The reaction media, operation parameters, and reactor development should thus be considered. For example, the stability of ILs in conventional aqueous systems is usually poor due to enzyme leaching from the carriers and the inhibitive effects of methanol and glycerol (Zhao et al., 2015). The reactor design is also important to scale up the IL-catalyzed production of biodiesel; however, the development of a high-efficiency reactor for the IL-catalyzed production of biodiesel is progressing slowly. Commonly used reactors include stirred tank reactors (STRs), packed-bed reactors (PBRs), or a combination of the two. Nevertheless, further improvements are required to further intensify the mass transfer by minimizing the mechanical shear force to avoid damage to the carriers and enzymes. Downstream processing is crucial to obtain a biodiesel product that meets the corresponding quality standards. Simulations are usually performed to obtain the mass and energy balance data and process optimization. Enzymatic catalysis for biodiesel production is a relatively new research field, but has attracted an increasing amount of attention from the scientific community and biodiesel industry. Novel techniques have been recently developed to improve the sustainability and economic viability of enzyme catalysis. These techniques mainly deal with reducing the price of enzymes and improving the transesterification conversion efficiency. Table 6.3 lists various novel techniques and recent trends in enzymatic biodiesel synthesis and their associated advantages and challenges.

6.4.1 Novel immobilization techniques Novel immobilization techniques are being developed to enhance the immobilized lipase performance, solvent tolerance, reusability, stability and to simplify the separation process. The main novel materials for lipase immobilization include protein-coated microcrystals (PCMCs), crosslinked PCMCs, magnetic particle carriers, and electrospun nanofibers, all of which have been studied in biodiesel production. Enzymes have the

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Table 6.3 Novel techniques and recent trends in enzymatic biodiesel synthesis and their advantages and challenges. Technique

Advantages

Challenges

Use of a combination of lipases from different sources

Wide substrate specificity, enhanced yields, reduced reaction time

Ionic liquids as a solvent

Improved stability, selectivity and activity of enzyme Improved diffusion and reaction rate, salvation ability can be engineered, can be used to extract lipids, easy separation from products Reduces the feedstock cost, realizes waste management by biodiesel production

Preparation of enzyme cocktail or genetic engineering is tedious Expensive

Enzyme-catalyzed transesterification in a supercritical CO2 medium

Enzyme-catalyzed transesterification for low-cost and high free-fatty acid feedstocks Solvent-free process

In-situ transesterification of microalgae

Cost-effective, environmentally friendly, safe Reduces solvent use and energy consumption

Expensive, requires sophisticated instrumentation

Need for meticulous collection, logistics issues

Mass transfer limitations in the reaction Cost-effective only when the biomass has a high percentage of lipids

advantage of easier separation after being immobilized on magnetic particles and also become immobilized lipases that can be concentrated at a specific location in a reactor by applying an external magnetic field (Badoei-Dalfard et al., 2019; Khoobbakht et al., 2020; Torabizadeh and Montazeri, 2020; Zhang et al., 2020).

6.4.2 Use of a combination of lipases from different sources Lipases from different sources have different substrate specificities and catalytic activities. Lipases with a narrow specificity are not suitable for biodiesel production. The performance of regiospecific lipases may improve when

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used in combination with nonspecific lipases. Some lipases also show higher hydrolytic activity, whereas others show higher synthetic activity. When used in combination, such lipases can enhance the reaction yield and reduce the reaction time (Pedro et al., 2018). A wide range of feedstocks is used for biodiesel production, including triglycerides, FFAs, and regioisomers of mono- and diglycerides. When used for the transesterification of such feedstocks, combinations of lipases with a distinct specificity and catalytic efficiency have demonstrated an improved performance (Monteiro et al., 2021). However, the preparation of such enzyme cocktails, or the development of a microorganism expressing different lipases via genetic engineering route, could be a very tedious process.

6.4.3 Ionic liquids as a solvent in enzyme-catalyzed transesterification The use of volatile, toxic, or flammable solvents is neither safe nor environmentally sound. Novel solvents such as ionic liquids are considered green solvents because of their nonflammability, low vapor pressure, and high thermal stability. Ionic liquids are composed of anions and cations, which can be altered to design a suitable solvent in terms of their melting point, viscosity, density, hydrophobicity, and polarity (Lee et al., 2018; Elgharbawy et al., 2018; Ullah et al., 2018). Enzymes in ionic liquids at room temperature show higher stability, selectivity and improved activity. Ionic liquids have thus attracted interest in the enzyme-catalyzed transesterification process. Hydrophobic ionic liquids have demonstrated higher yields than hydrophilic liquids. However, ionic liquids are presently expensive, even though they can be recovered and reused (Lozano et al., 2020). Consequently, simpler recovery techniques and cheaper ionic liquids must be investigated to determine whether ionic liquid-assisted biodiesel synthesis can be economically feasible (Ong et al., 2021).

6.4.4 Enzyme-catalyzed transesterification under a supercritical CO2 medium Organic solvents are used extensively in enzyme-catalyzed biodiesel synthesis to avoid mass transfer limitations. Because most of these organic solvents are toxic, volatile, and flammable, the use of supercritical fluids as an alternative reaction medium has gained global interest. Enzyme catalysis can be carried out in supercritical CO2 because of its moderate critical temperature (31.1°C) and pressure (7.38 MPa) (Quintana-Go´mez et al., 2021).

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Supercritical CO2 as a reaction medium in lipase-catalyzed reactions offers the advantage of easy separation by reducing the pressure; its salvation ability can also be altered by controlling the temperature and pressure conditions. Supercritical CO2 has also been simultaneously used to extract lipids and as a solvent where the transesterification process occurs. This somewhat lowers the cost attributed to the reaction process under supercritical conditions (Taher et al., 2020).

6.4.5 Statistical approaches for reaction optimization Lipase-catalyzed biodiesel production is influenced by number of factors such as the temperature, methanol-to-oil molar ratio, enzyme concentration, water content, and flow rate in the case of a continuous process. The optimization of these parameters is thus crucial to obtain maximum yields. Statistical methods such as response surface methodology have been widely to optimize lipase-catalyzed biodiesel production (Verdugo et al., 2011; Luna et al., 2014c; Nguyen et al., 2018; Calero et al., 2019; Calero et al., 2020). Statistical methods offer the advantage of studying a great number of parameters in a fewer number of experimental setups. These methods also provide a better understanding of the parameter interactions and extent of their influence on the reaction.

6.4.6 Enzyme-catalyzed transesterification for low-cost and high free-fatty-acid feedstocks The feedstock contributes a major portion of the biodiesel production cost. Edible oils are currently mostly used as feedstock for biodiesel production. However, edible and non-edible oil crops compete with food crops for arable land, which leads to food security concerns. A large amount of water and fertilizers are used to grow these oil crops, which increases the cost of biodiesel production and carbon debt. The use of low-cost waste cooking oil and animal-derived fats as feedstock is therefore gaining interest (Kumar et al., 2018; Suci et al., 2018; Shaban et al., 2020). The use of waste cooking oil also has a dual purpose: biofuel production and waste management. Used cooking oil provides a cheap feedstock source; however, the collection and logistics of this feedstock from sources such as restaurants and food processing plants must be meticulous to ensure its availability for large-scale biodiesel production. Furthermore, waste cooking oil exhibits a high FFA content owing to oxidation (Santya et al., 2019).

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Many non-edible oils, such as microalgal (Li et al., 2020) or waste cooking oils (Zhong et al., 2020), are known to have high FFA and/or moisture contents. High FFA and moisture contents in the feedstock both hamper the biodiesel yield when applied to the chemical catalysis, whereas lipases have shown good tolerance to these factors (Lee et al., 2018; Jayaraman et al., 2020). Thus, despite the high cost of enzymes, their application in converting low-quality feedstock can improve the economic balance in the overall biodiesel production process (Mohadesi et al., 2019). Animal-derived products are usually by-products of slaughterhouses and meat-processing industries (Marı´n-Suarez et al., 2019). Higher calorific values and cetane numbers are the main attractive features of biodiesel derived from animal fats (Toldra´-Reig et al., 2020a,b). Numerous studies have aimed to reduce the high cost of conventional lipases (Luna et al., 2018), and promising results have been attained with the microbial lipase Lipopan 50 BG (Novozymes AS, Denmark) (Calero et al., 2015; Verdugo et al., 2011), a low-cost purified lipase from the microorganism Thermomyces lanuginosus, which is typically used as a bread emulsifier (Moayedallaie et al., 2010). Likewise, Rhizopus oryzae lipase from Biolipase-R can be immobilized on sepiolite and serve as an inorganic support (Luna et al., 2014c). Some microbial lipases from a range of wild strains in lipophilic microorganisms (e.g., olive oil press, animal fats) have been selected to improve the viability and competitiveness of the enzymatic process (Escobar-Nin˜o et al., 2014). These new microorganisms and their lipolytic enzymes can thus notably reduce the cost of enzyme production to more economically produce biofuel.

6.5 Biofuels similar to biodiesel produced using acyl acceptors other than methanol A series of alternative methods have been considered to improve the atom efficiency and avoid the associated problems of glycerol generation in the conventional process. Biofuel production has been studied using new biofuels that integrate glycerol as a derivative product, which is miscible with the FAMEs or FAEEs obtained in the same transesterification process. Therefore, only one reaction is needed. This is possible by using alternative acyl acceptors (mainly esters) instead of the alcohol typically used in the conventional process. Corresponding glycerol esters are thus obtained together with FAMEs (or FAEEs) in the interesterification process. The mixture of reaction products comprises lipophilic compounds that are completely

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miscible with fossil fuels in a new biofuel reaction process that avoids the presence of free glycerol, which is a dangerous compound for engines, and uses a substitute that operates like a fuel (Hama et al., 2018; Nguyen et al., 2018; Ribeiro et al., 2018; Nascimento et al., 2019). This methodology thus avoids the separation of glycerol prior to its transformation, which simplifies the process (Calero et al., 2014a,b; Calero et al., 2015; Calero et al., 2020; Esan et al., 2020) These biodiesel production methods not only prevent waste generation but also increase the process yields to consistently higher values than the typical 12 wt%, by incorporating glycerol derivatives into the reaction products in addition to all of the typical reactants. The highest atom efficiency (almost 100 wt%) is thus obtained because almost every atom is incorporated into the reaction product. Novel methodologies to prepare esters from lipids using different acyl acceptors that directly afford alternative co-products are currently under development (Okoye and Hameed, 2016; Estevez et al., 2019). The interesterification processes can be performed using the same catalysts as those applied in the transesterification processes (e.g., homogeneous or heterogeneous, acidic or basic catalysts, and lipases under supercritical conditions). Most of these processes presently use lipases when applied to biofuel production (Caballero et al., 2009; Verdugo et al., 2010; Verdugo et al., 2011; Luna et al., 2012; Luna et al., 2014a,b,c). Basic catalysts such as CaO, which are capable of selectively (trans)esterifying triglycerides, are also used to obtain glycerol derivatives that are soluble in FAME mixtures (Tang et al., 2015; Calero et al., 2014a; Calero et al., 2020). Instead of using methanol, the lipase-catalyzed synthesis of fatty acid alkyl esters can also be performed using alternative alcohol donors. In this respect, methyl (or ethyl) acetate can be used, as well as dimethyl (or diethyl) carbonate. These mixtures, including glycerol derivative molecules, have favorable physical properties for use as novel biofuels (Sakdasri et al., 2021). In some cases, even the unused reactants are capable of being directly used as biofuel (Hurtado et al., 2019).

6.5.1 Biodiesel produced together with glycerol triacetate in the same transesterification process as oils and fats Mixtures of FAMEs and glycerol triacetate (triacetin) are produced by the interesterification reaction of triglycerides with methyl acetate in the presence of strong acid catalysts (Fig. 6.7). All of the products generated in the above process can be used as components of a patented novel biofuel, which greatly improves the economics of biofuel production (Calero et al., 2015).

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O

O R

O

O

R

O

R

+

3

CH3 H3C

O

O

O

O

Catalyst

H3C

O

O

Triglyceride

CH3

O

O

O

CH3

+

3R

OCH 3

O

Methanol

Diglyceride

Fatty acid methyl ester (FAME)

Fig. 6.7 Gliperol is a novel proprietary biofuel of the Research Institute of Industrial Chemistry Varsow (Poland) that is formed by a mixture of 3 mol of FAMEs and 1 mol of triacetin and obtained by the interesterification of triglycerides with methyl acetate under strong acidic conditions (Calero et al., 2015).

The mixture, termed Gliperol, has been claimed to exhibit fuel characteristics comparable with those traditional biodiesel fuel (Kijenski et al., 2007). Gliperol is composed of a mixture of three FAME molecules and one triacetin molecule. Gliperol is composed of a mixture of three FAME molecules and one triacetin molecule, obtained by the interesterification of 1 mol of triglycerides with 3 mol of methyl acetate. An oil/methyl acetate molar ratio in the range of 1:3–1:9 and temperatures in the range of 40–200°C are usually applied. Most studies have applied lipases as catalysts in solvent-free systems (Nguyen et al., 2018), supercritical conditions (Quintana-Go´mez et al., 2021), or ultrasound-assisted interesterification (Kashyap et al., 2019). Heterogeneous catalysis has also been applied (Ribeiro et al., 2018; Tian et al., 2018; Dhawan et al., 2020; Wong et al., 2020). Despite the greener character of ethyl acetate, this acyl acceptor has received less research attention than methyl acetate ( Jazie et al., 2020; Akkarawatkhoosith et al., 2020), even though previous results indicate its similar behavior to methyl acetate in the interesterification with lipases. However, the corresponding FAEE (instead of FAME) is obtained, together with triacetin, in this case. Many studies have considered ethyl acetate as a viable solution for upgrading the residual glycerol obtained in conventional biodiesel synthesis with respect to the influence of triacetin on engine performance (Zarea et al., 2018). Triacetin is an anti-knocking additive when used with biodiesel in a direct-injection diesel engine, which improves the performance and reduces the tail pipe emissions (Saleem, 2019). In this respect, the interesterification of triglycerides with methyl or ethyl acetate may be a viable methodology to obtain conventional biodiesel (FAMEs or FAEEs) by including some amount of a well-recognized additive such as triacetin.

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6.5.2 Biodiesel produced with fatty acid glycerol carbonate esters in the same transesterification process as oils and fats Dimethyl carbonate (DMC) can be used as a transesterification reagent for making esters from lipids, which directly achieves alternative soluble co-products in the biodiesel solutions. The reaction is attractive because DMC is reputed as a prototypical green reagent due to its lack of negative health and environmental effects (Nascimento et al., 2019; Esan et al., 2020; Esan et al., 2021). Fuels produced using DMC and vegetable oils or animal fats as raw materials are considered to be promising alternatives that can be fully derived from renewable resources. DMC operates as an alternative acyl acceptor that is pH-neutral, cheap, and nontoxic. The reaction between triglycerides and DMC produces a mixture of FAMEs and cyclic fatty acid glycerol carbonate esters (FAGCs), which constitutes a novel biodiesel-like material patented under the name DMC-BioD (Fabbri et al., 2007). The interesterification reaction of triglycerides with DMC can generate a mixture of FAMEs, FAGCs, and also glycerol carbonate (GC), as is indicated in Fig. 6.8, (Calero et al., 2015). These mixtures,

Fig. 6.8 DMC-BioD is a new biodiesel-like proprietary biofuel of Polimeri Europa (Italy) that is obtained by reacting oils with dimethyl carbonate under alkaline conditions and avoids the co-production of glycerol by obtaining a mixture of 2 mol of fatty acid methyl esters and 1 mol of fatty acid glycerol carbonate. The latter can be decomposed, which generates glycerol decarbonate and glycerol carbonate as a variable extension (Calero et al., 2015).

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including glycerol derivative molecules, have suitable physical properties for use as a new patented biofuel with improved atom efficiency because all of the atoms involved in the reaction form a part of the final mixture. In addition to its reputation as a prototype of green reagents (Fiorani et al., 2018), the use of DMC also avoids the co-production of glycerol. The main distinguishing feature of DMC-BioD compared with biodiesel produced from vegetable oil and methanol is the presence of FAGCs in addition to FAMEs. The composition of DMC-BioD and its physical and rheological properties relevant for use as a fuel have been studied to some extent (Calero et al., 2015). In summary, with respect to the benefits and drawbacks of using DMC as an alternative reagent for the interesterification of oil and fats to produce biofuel from renewable resources and alternative co-products (GC and glycerol dicarbonate (GDC)), it should be mentioned that DMC is less toxic than methanol and can currently be manufactured from CO2 and renewable resources using environmentally safe industrial methods. GC and its derivatives are also characterized by low toxicity, and the remaining unreacted DMC does not need to be separated from the reaction products because it is an effective additive for diesel engines due to its high oxygen content (Abdalla and Liu, 2018; Rounce, 2010). Furthermore, the fabrication process is highly simplified compared with the process by which conventional biodiesel is obtained from methanol.

6.5.3 Biodiesel produced together with monoacylglycerol in the same transesterification process as oils and fats A protocol was recently developed to prepare a new kind of biodiesel that integrates glycerol into its composition via the 1,3-regiospecific enzymatic transesterification of sunflower oil using free (Caballero et al., 2009; Verdugo et al., 2010) and immobilized (Luna et al., 2012, 2013) pig pancreatic lipase. The patented Ecodiesel-100 (Luna et al., 2014a) is a mixture of two parts FAEEs and one part monoacylglycerides (MAGs) that integrates glycerol as a soluble derivative product (MGs) in the diesel fuel. However, the specific reagents (e.g., DMC or methyl acetate) employed for the production of Glyperol or DMC-BioD are more expensive than ethanol. The procedure takes advantage of the 1,3 selective nature of lipases, which allows it to retain the process in the second alcoholysis step by obtaining a mixture of 2 mol of FAEEs and 1 mol of MGs as products, as shown in Fig. 6.9. This strategy is based on obtaining incomplete alcoholysis by the application of 1,3-selective

Biotechnological production of biofuels

O

O R''

O

R'

O

R'

O

O

+

2 CH3CH2OH

177

Catalyst

OH

R''

O

OH

+

2 R'

OCH2OH

O Triglyceride

Ethanol

Monoglyceride (MG)

Fatty acid ethyl ester (FAEE)

Fig. 6.9 Ecodiesel-100 is a biofuel obtained by enzymatic technology patented by the University of Cordoba (Spain) that incorporates glycerol and is formed of 2 mol of ethyl esters of fatty acids (FAEEs) and 1 mol of monoglyceride (MG).

lipases; thus, the glycerol remains in the form of monoglyceride, which avoids the production of glycerol as a by-product and reduces the environmental impact of the process (Chen et al., 2020). Ecodiesel exhibits similar physicochemical properties to those of conventional biodiesel. Furthermore, MG has been shown to enhance biodiesel lubricity (Knothe and Razon, 2017; Wadumesthrige et al., 2009; Xu et al., 2010). Ethanol unused in the enzymatic process remains in the reaction mixture after the reaction to form a product blend that can be directly used as fuel. In this respect, some studies (Razzaq et al., 2020; Venu et al., 2019) have shown that blends of diesel fuel and ethanol with biodiesel slightly reduce the maximum power output with respect to regular diesel. No significant differences in the emissions of CO2, CO, and NOx have been observed between regular diesel and biodiesel and ethanol and diesel blends. However, the use of such blends leads to a reduction of particulate matter (Yilmaz, 2014). Consequently, these blends can be used in diesel engines without requiring modifications, which reflects the limited changes compared with pure diesel. The term Ecodiesel is thus currently applied to any blend of fatty acid alkyl ester and ethanol, with or without diesel fuel (Sandoval et al., 2017; Penconek et al., 2013; Kowalewicz, 2006). The production of Ecodiesel with different lipases and several biocatalytic systems, as well as the main reaction parameters, has been studied, and the results are summarized in Table 6.4. Table 6.5 summarizes the pros and cons of the existing methodologies for obtaining biofuels that integrate glycerol as a derivative. This approach enables them to operate as a combustible, together with FAMEs or FAEEs, thus avoiding the presence of free glycerol. In this respect, the production of biodiesel-like biofuels via the interesterification of vegetable oils with methyl acetate or methyl carbonate used as acyl acceptors is clearly simpler than conventional biodiesel production, as is shown in Fig. 6.10, regardless

Table 6.4 Different enzymatic systems studied for the biocatalytic production of Ecodiesel. Crucial reaction parameters ANOVAa

OVATb

Biocatalyst (Lipase)

Form of use

Oil/ EtOH

T (°C)

Biocatalyst weight (g)

Reuses

Reference

PPL (commercial pancreatic lipase)

Free

1:2.6

45

0.01

0

Physical adsorption

1:10.3

45

0.5/0.01

11

Covalent inmob1 Covalent inmob2 Free

1:4 1:4 1:3.5

40 40 20

0.5/0.01 0.5/0.01 0.02

40 25 0

Verdugo et al. (2010) Caballero et al. (2009) Luna et al. (2013)

Free Commercial immobilized Free Physical adsorption Covalent inmob1 Covalent inmob2

1:6 1:6

30 40

0.015 0.04

0 12

1:6 1:6 1:6 1:6

20 30 30 30

0.02 0.5/0.01 0.5/0.01 0.5/0.01

0 9 – 9

Lipopan (Thermomyces lanuginosus) MML (Rhizomucor miehei) Lipozyme RM IM (Rhizomucor miehei) Biolipase-R (Rhizopus oryzae)

Verdugo et al. (2011) – Calero et al. (2014a,b) Luna et al. (2014c) Luna et al. (2014b) (Continued)

Biocatalyst (Lipase)

Origin

Crucial reaction parameters ANOVAa OVATb Oil/ Biocatalyst EtOH T (°C) weight (g)

Reuses

1:6

30

0.02

0

1:6

30

0.05 0.1

10

Commercial immobilized

1:6

30

0.5

16

Free

1:6

30

0.5

10 10

Form Free

CALB Commercial lipases

Enzymatic extracts

N435 ©

Wild strains

Standard strain a

Physical adsorption C. antarcticac

Oil environment

G. terribacillus

Animal fat environment

G. bacilluse

Free

1:6

30

0.5

CALB (CECT)f

C. antarcticac

Free

1:6

30

0.5

Analysis of variance. One-factor-at-a-time method. c Candidaantarctica. d Gram-positive and aerobic genus of bacteria from the family of Bacillaceae. e Gram-positive, rod-shaped bacteria, a member of the phylum Firmicutes. f Candidaantarctica type B (Coleccio´n Espan˜ola de Cultivos Tipo). b

d

MS 3030 PMO

Table 6.5 Comparison of the main characteristics of the different technologies available to produce renewable liquid fuels from vegetable oils. Type

Biodiesel EN 14214

Name

Biodiesel

Gliperol

DMC-Biod

Ecodiesel

Reagent Catalyst

Methanol or ethanol NaOH or KOH

Methyl carbonate Bases or lipases

Ethanol Lipases

Products

3 FAMEs or 3 FAEEs

Methyl acetate Acids, bases or lipases Glycerol triacetate +3 FAMEs

Monoglycerides +2 FAEEs

Byproducts Separation process and cleaning Investment in facilities Free fatty acids and/ or water in the starting oil Catalyst cost Environmental impact

Glycerol Complex

No waste Not needed

Fatty acid, glycerol carbonate+2 FAMEs No waste Not needed

No waste Not needed

Medium

Low

Low

Low

Free fatty acids are transformed to soaps

Free fatty acids are transformed to biofuel High Low

Free fatty acids are transformed to biofuel High Low

Free fatty acids are transformed to biofuel High Low

Low High. Alkaline and saline effluents are generated. Wastewater treatment is needed.

Biodiesel-like biofuels

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Fig. 6.10 Production process of biodiesel-like biofuels via the interesterification of vegetable oils with methyl acetate or methyl carbonate used as acyl acceptors.

Fig. 6.11 Production of biodiesel-like biofuels via the selective ethanolysis of vegetable oils using lipases as the biocatalyst.

of the use of chemical catalysis or enzymes. However, biofuels obtained by the selective ethanolysis of vegetable oils using lipases as biocatalysts are even simpler, as shown in Figs. 6.10 and 6.11.

6.6 Industrial biodiesel production using enzymes Most laboratory-scale IL-catalyzed biodiesel production operations use batch reactions in stirred flasks. However, the reactor must be specially designed for larger-scale operations. Several types of reactors have been studied for industrial biodiesel production, such as STRs (Castillo-Go´nzalez et al., 2020), PBRs (Zik et al., 2020), fluidized bed reactors (Dhanalakshmi and Madhu, 2021), and bubble column reactors (Naira et al., 2020).

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However, only a few of these reactors are suitable for the industrial enzymatic production of biodiesel. To reduce operational costs, enzymatic biodiesel must be produced in continuously operated plants. Several possible solutions obtained at the laboratory scale include Continuous Stirred Tank Reactors (CSTRs), Packed Bed Reactors (PBRs), fluid beds, an expanding bed, recirculation or membrane reactors (Zhao et al., 2015). PBRs are highly applicable for continuous biodiesel production, but their main disadvantage is that the resulting glycerol remains at the bottom of the reactor. This glycerol can be deposited on the surface of the support-immobilized lipase, which reduces the catalytic efficiency. The glycerol must therefore be continuously eliminated in a timely manner during the enzymatic reaction process. Several studies have reported the successful application of PBRs for enzymatic biodiesel production using different setups: a single PBR used with a stepwise addition of methanol (Zhao et al., 2015); a single recirculating PBR (Galeano et al., 2017); a two-stage packed bed reactor with a glycerol extraction column (Ramos et al., 2017); three PBRs in series with intermediate glycerol removal and methanol addition (Zhao et al., 2012); and nine PBRs in series with a hydrocyclone set after PBR to separate the glycerol (Cheirsilp et al., 2008), thus reducing the catalytic efficiency. Although many processes have been developed for lipase immobilization at the laboratory scale, only a few techniques have been successfully commercialized. In this respect, the major bottleneck to technical transfer is the high cost of the lipase immobilization steps. This explains why the market price of Novozym 435, one of the most commonly supported lipase systems, reaches USD 1000/kg (Zhao et al., 2015). The immobilization process should sufficiently recover proteins as much as possible while still effectively retaining their enzymatic activities. The obtained ILs should also have high stability to avoid enzyme leaching or activity loss. The first industrial plant for the enzymatic production of biodiesel was built in China in 2006 with a capacity of 20,000 tons/year. Tert-butanol was selected as the reaction medium and immobilized lipases, such as Lipozyme TL IM and Novozyme 435, were used in this plant as enzymatic catalysts (Zhao et al., 2015). A technoeconomic evaluation is vital to estimate the production cost and determine the most costly units for further optimization (Collac¸o et al., 2020). An economic evaluation usually consists of several steps: the development of process flow sheets; time charts and equipment lists; followed by estimations of the equipment cost and plant and manufacturing costs (Alves et al., 2013).

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The economic feasibility of the enzymatic production of biodiesel depends on a series of factors. These factors mainly include (1) the raw material costs, such as the prices of oil feedstock, alcohol, and enzyme; (2) the process parameters, such as oil-to-biodiesel conversion ratio, retention time for transesterification, biodiesel recovery yield, lipase lifetime, and solvent loss (if used); (3) process design regarding water recycling and heat integration; and (4) the by-product value. It has been found that the lipase cost contributes a large part of the total production cost (Rezania et al., 2019). The extensively used IL Novozym 435 has a high price per kilogram, which indicates that a very high productivity is required for the process to be cost-effective (Wancura et al., 2020; Nielsen and Rancke-Madsen, 2011). The reusability of ILs is therefore important to reduce biodiesel production costs. As shown in Fig. 6.12, the number of IL reuses has a significant influence on the enzyme cost for the IL-catalyzed production of biodiesel. It can be estimated that to make the enzyme cost less than 0.1 USD/kg of biodiesel, the IL should be reused for more than 320, 210,

Fig. 6.12 Effect of number of IL reuses on the estimated lipase cost for different enzyme prices. IL loading: 2% based on raw oil feedstock; oil-to-biodiesel conversion: 95% (Zhao et al., 2015).

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160, 50, and 20 batches without enzyme activity loss for a lipase price of 1500, 1000, 750, 200, and 100 USD/kg, respectively. Technoeconomic and life cycle analyses are very important to provide directions for the successful commercial-scale implementation of this technology. However, there are few available studies on this topic. It is also imperative to compare such alternative technology with the conventional technique. Jegannathan et al. (2011) investigated the economics of biodiesel production processes using an alkali catalyst and free and immobilized enzyme catalysts, considering a batch process and production capacity of 103 tons. The lowest biodiesel production cost was found to be 1166.67 USD/ton for an alkali catalyst. Among the biocatalysts, the immobilized enzyme was shown to have a lower biodiesel production cost of 2414.63 USD/ton compared with the free enzyme (7821.37 USD/ton). The conventional alkali catalyst price is thus substantially lower than the enzyme catalyst price. Among biocatalysts, the immobilized enzymes showed a lower price because of their reuse potential. A life cycle analysis study by Harding et al. (2008) compared chemical catalysis and enzyme catalysis for biodiesel production purposes and showed that the biological route has an advantage over the chemical route in terms of a simplified purification process and energy savings. This study also showed that the biocatalytic route is more environmentally friendly. The effects of global warming, acidification, and photochemical oxidation in the case of enzyme catalysis were reduced by 5%. The reduction in freshwater aquatic toxicity was approximately 12%, while the reduction in marine aquatic toxicity and human toxicity was almost 10%. The reduction in terrestrial ecotoxicity was over 40%, mainly due to skipping the neutralization step, which requires acids. The authors suggested these results are mainly due to the lower steam requirements for the enzymatic process. Despite the higher cost of enzyme catalysis, this approach provides environmental benefits over the conventional process. Using the latest novel strategies, enzyme prices can be cut down by improving their catalytic performance and stability. Both technoeconomic and life cycle analyses thus suggest the promising potential of enzyme catalysis for biodiesel production at commercial-scale production plants.

6.7 Conclusions Numerous lipases have been applied for biodiesel production using a wide variety of triglyceride substrates and acyl acceptors. This approach has been

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successfully used to convert waste fats and oils and eliminate the main problems of traditional alkaline transesterification. However, some precaution must be taken to avoid lipase inhibition when using methanol. The obtained results verify that a high productivity in terms of yield, reuse numbers, and low reaction times can be achieved using enzymes. Further improvements can make industrial enzymatic biodiesel production a viable option for the future. The lipase-catalyzed production of biodiesel has recently attracted considerable attention due to merits such as mild reaction conditions, environmental friendliness, and wide adaptability for feedstocks. The immobilization of lipase facilitates enzyme recovery and increases the enzyme stability. This technique shows great potential for the industrial-scale production of biodiesel. Various approaches have been developed for lipase immobilization, mainly including physical adsorption, ionic bonding, covalent bonding, entrapment, and cross-linking. Nevertheless, only a few of these techniques seem to be economically feasible. Each immobilization technique has its own advantages and disadvantages, and lipase immobilization is usually performed using a combination of two or more approaches. Most commercial ILs are prepared by the adsorption of free lipase on polymeric materials owing to the simplicity of this process, and the carrier is relatively easy to obtain at a low cost. However, the IL stability requires further improvement, especially to strengthen the interaction between the lipase and carriers to prevent enzyme leaching. The cost of lipases continues to be the main obstacle for exploiting their potential; lipase reuse is therefore essential, which can be achieved using immobilized lipases. The industrial use of immobilized lipases requires different qualities and characteristics of the biocatalyst depending on the specific applications. A continued effort within immobilization technology is therefore necessary to obtain solutions for each application. Several operation parameters have been identified that affect the biodiesel yield and IL stability. These parameters mainly include the acyl acceptor type and concentration, water content, enzyme loading, alcohol-to-oil ratio, temperature, and reaction media. Parameter optimization is important to obtain a high biodiesel yield and maximize the enzyme reuse. However, the optimum conditions are highly dependent on the selected oil feedstock and IL. A technoeconomic evaluation is important for the IL-catalyzed production of biodiesel. Lipase cost contributes a significant part of the total production cost. This expenditure can be reducing by decreasing the lipase

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loading (i.e., increasing the lipase’s specific activity) or increasing the IL reusability. However, the entire optimization process should be performed in consideration of water and heat inputs to further reduce the production cost. In summary, it is crucial that the following issues be considered to improve the economic competitiveness of the IL-catalyzed production of biodiesel in the near future. (1) Increased IL stability during transesterification. This can be achieved by preventing lipase from leaching off the carriers and denaturing, which reduces the activity, and can be caused by the accumulation of alcohol and/or glycerol or the sheer force of stirring. (2) Further investigation of process integration and optimization. Process integration should consider water and heat recycling because the total production cost is dependent on the entire process. The optimization of the entire process should be performed with the production cost as the final objective function. (3) Development and maturation of new technologies to avoid the generation of glycerol as a by-product. Biofuels are applicable to diesel engines in a similar way as biodiesel, but without generating glycerol as an inconvenient waste product, which avoids the need for a cleaning process that adds high water and energy costs. (4) Development of more economical enzyme preparation procedures.

Acknowledgments The authors are thankful to MINECO-ENE2016-81013-R (AEI/FEDER, EU), MICIIN (Project ref. PID2019-104953RB-100), Consejerı´a de Transformacio´n Econo´mica, Industria, Conocimiento y Universidades de la Junta de Andalucı´a (UCO-FEDER Project CATOLIVAL, ref. 1264113-R, 2018, and Project ref. P18-RT-4822), and FEDER Funds for financial support.

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Xu, Y.F., Wang, Q.J., Hu, X.G., Li, C., Zhu, X.F., 2010. Characterization of the lubricity of bio-oil/diesel fuel blends by high frequency reciprocating test rig. Energy 35, 283–287. https://doi.org/10.1016/j.energy.2009.09.020. Yagmurov, E.R., Kozlov, G.V., Pushkarev, M.A., 2017. Lipase purification: the review of conventional and novel methods. Journal of hygienic engineering and design, review paper UDC 577.152.311, 60-69. Fuel 228, 390–404. https://doi.org/10.1016/j. fuel.2018.05.004. Yang, X., Wang, Y., Bai, R., Ma, H., Wang, W., Sun, H., Dong, Y., Qu, F., Tang, Q., Guo, T., Binks, B.P., Meng, T., 2019. Pickering emulsion-enhanced interfacial biocatalysis: tailored alginate microparticles act as particulate emulsifier and enzyme carrier. Green Chem. 21, 2229–2233. https://doi.org/10.1039/c8gc03573c. Yilmaz, N., 2014. Investigation of CI engine emissions in biodiesel–ethanol–diesel blends as a function of ethanol concentration. Fuel 115, 790–793. Zarea, A., Bodisco, T.A., Nabi, M.N., Hossain, F.M., Ristovski, Z.D., Brown, R.L., 2018. A comparative investigation into cold-start and hot-start operation of diesel engine performance with oxygenated fuels during transient and steady-state operation. Fuel 228. Zavarise, J.P., Pinotti, L.M., 2019. Progress in the production of fungal lipases by submerged fermentation. Int. J. Adv. Eng. Res. Sci. 6, 367–372. https://doi.org/10.22161/ ijaers.612.38. Zhang, M., Gao, Z., Zheng, T., Ma, Y., Wang, Q., Gao, M., Sun, X., 2018. A bibliometric analysis of biodiesel research during 1991–2015. J. Mater. Cycles Waste Manag. 20, 10–18. https://doi.org/10.1007/s10163-016-0575-z. Zhang, H., Liu, T., Zhu, Y., Hong, L., Li, T., Wang, X., Fu, Y., 2020. Lipases immobilized on the modified polyporous magnetic cellulose support as an efficient and recyclable catalyst for biodiesel production from yellow horn seed oil. Renew. Energy 145, 1246–1254. https://doi.org/10.1016/j.renene.2019.06.031. Zhang, H., Sang, J., Zhang, Y., Sun, T., Liu, H., Yue, R., Zhang, J., Wang, H., Dai, Y., Lu, F., Liu, F., 2019. Rational design of a Yarrowia lipolytica derived lipase for improved thermostability. Int. J. Biol. Macromol. 137, 1190–1198. https://doi.org/10.1016/j. ijbiomac.2019.07.070. Zhao, X., Peng, F., Du, W., Liu, C., Liu, D., 2012. Effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides and preparation of biodiesel by enzymatic transesterification of the lipid. Bioprocess Biosyst. Eng. 35, 993–1004. https://doi.org/10.1007/s00449-012-0684-6. Zhao, X., Qi, F., Yuan, C., Du, W., Liu, D., 2015. Lipase-catalyzed process for biodiesel production: enzyme immobilization, process simulation and optimization. Renew. Sust. Energ. Rev. 44, 182–197. https://doi.org/10.1016/j.rser.2014.12.021. Zhong, L., Feng, Y., Wang, G., Wang, Z., Bilal, M., Lv, H., Jia, S., Cui, J., 2020. Production and use of immobilized lipases in/on nanomaterials: a review from the waste to biodiesel production. Int. J. Biol. Macromol. 152, 207–222. https://doi.org/10.1016/j. ijbiomac.2020.02.258. Zhou, H., Zhang, Z., Lee, W.J., Xie, X., Li, A., Wang, Y., 2021. Acyl migration occurrence of palm olein during interesterification catalyzed by sn-1,3 specific lipase. LWT Food Sci. Technol. 142, 111023. https://doi.org/10.1016/j.lwt.2021.111023. Zik, N.A.F.A., Sulaiman, S., Jamal, P., 2020. Biodiesel production from waste cooking oil using calcium oxide/nanocrystal cellulose/polyvinyl alcohol catalyst in a packed bed reactor. Renew. Energy 155, 267–277. https://doi.org/10.1016/j.renene.2020.03.144. Zulkeflee, S.A., Sata, S.A., Rohman, F.S., Aziz, N., 2020. Modelling of immobilized Candida rugosa lipase catalysed esterification process in batch reactor equipped with temperature and water activity control system. Biochem. Eng. J. 161, 107669. https://doi.org/ 10.1016/j.bej.2020.107669.

CHAPTER 7

Biodiesel production from microbial lipids using oleaginous yeasts Xiujuan Qian, Xinhai Zhou, Dawei Zhou, Jie Zhou, Fengxue Xin, Weiliang Dong, Wenming Zhang, and Min Jiang State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China

7.1 Introduction Biodiesel is a renewable, biodegradable, and nontoxic liquid transportation fuel that can be used to displace petroleum-derived diesel fuel without requiring significant modifications to existing engines or fuel distribution networks (Gebremariam and Marchetti, 2017). However, the current industrial production of biodiesel relies mainly on vegetable oils, which may result in a shortage of edible oils in food markets and an increase in their cost (Rosillo-Calle, 2012). Moreover, the current low crude oil price of USD0.29/L makes biodiesel production unprofitable, even for the most efficient producer in the USA (Mizik and Gyarmati, 2021). Considering the foreseeable depletion of crude oil; the highly controversial “food versus fuel” debate regarding vegetable-derived biodiesel; and the noncompetitive production costs, which depend on the price of vegetable oils, lipids produced by oleaginous microorganisms are potential substitutes for crude oil and plant oils. Microbial lipids are commonly defined as intracellular storage lipids that accumulate in oleaginous microorganisms under certain conditions (Huang et al., 2013a). In general, oleaginous microorganisms, including yeast, fungi, bacteria, and microalgae, are able to accumulate intracellular lipids at more than 20% of their dry cell weight (Huang et al., 2013a). As a new lipid resource, microbial lipids are in competition with traditional petroleumand plant-based oils in the energy, food, and chemical industries, due to their advantages listed below.

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(1) Microbial lipid production is less dependent on the season, climate, and location (Madani et al., 2017). (2) Microbial lipids can be produced from various low-value carbon sources without competing with food for land use, and the composition of microbial lipids synthesized by eukaryotic yeast, fungi, and microalgae is similar to the composition of vegetable oils (Vald
es et al., 2020). (3) Microbial lipid production requires a short cultivation period, and it is easy to achieve continuous large-scale production. (4) The fatty acid profile of microbial lipids varies depending on the oleaginous microorganism and the cultivation conditions used, making them highly suitable for diverse industrial applications. Further, the production of polyunsaturated fatty acids (PUFAs), such as gamma linoleic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), through biological fermentation may prevent overfishing and avoid the presence of contaminants found in fish-derived oils (Maria et al., 2020). Among the oleaginous microorganisms, oleaginous yeasts have advantages over fungi, bacteria, and microalgae because they can quickly grow to high densities, they have a high lipid content, and the production process is less affected by seasonal or weather conditions (Sitepu et al., 2014). The conditions of yeast cultivation are more easily scaled up in an arable landindependent and controllable manner (Sitepu et al., 2014). Moreover, it is relatively easy to genetically modify yeast to improve lipid production or increase the production of specific high-value PUFAs (Li et al., 2017). Moreover, their ability to utilize a large number of renewable substrates and the low cost of materials required for their cultivation make the use of oleaginous yeasts economically viable (Shi and Zhao, 2017). In addition, as the synthesis of acetyl-CoA is efficient in oleaginous yeasts, they may also serve as cell factories to produce other acetyl-CoA derivatives, e.g., poly-3hydroxybutyrate (Li et al., 2016b) and terpenoids (Vickers et al., 2017). The aim of this review was to provide a broad perspective on the use of yeast-derived lipids for the production of biodiesel and high-value PUFAs. The topics discussed in detail in this review include an introduction to oleaginous yeasts, an analysis of lipid metabolism pathways, technologies developed for oleaginous yeast metabolic engineering, the low-value substrates exploited for lipid fermentation, PUFA synthesis, and the challenges faced in the scale-up of yeast-derived lipid production.

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7.2 Oleaginous yeasts More than 600 yeast species have been isolated and identified; however, fewer than 30 species accumulate lipids at more than 20% of their dry weight (Madani et al., 2017). Most of these 30 species are from the genera Rhodotorula, Rhodosporidium, Lipomyces, Cryptococcus, Yarrowia, and Trichosporon (Vald
es et al., 2020). Screening experiments of oleaginous yeasts are still being performed, leading to the identification of novel oleaginous strains (Lamers et al., 2016; Schulze et al., 2014). Among them, Rhodotorula sp. (Maria et al., 2020), Rhodosporidium sp. (Sitepu et al., 2014), Lipomyces sp. (Li et al., 2017) Cryptococcus curvatus (Zhiwei et al., 2015), and Yarrowia lipolytica (Shi and Zhao, 2017) accumulate lipids up to greater than 60% of their dry cell weight through genetic modification or cultivation under specific conditions. Therefore, these species show great potential for commercial single-cell oil (SCO) production. The lipid content and fatty acid profiles of some representative oleaginous yeast species are presented in Table 7.1. The characteristics of these oleaginous yeast species are listed below: • Rhodotorula sp. and Rhodosporidium sp. are industrial workhorses for SCO production due to their high lipid content, wide substrate spectrum, and excellent tolerance of inhibitors present in biomass hydrolysates (Hu et al., 2009). In addition, Rhodosporidium is a good producer of carotenoids, terpenoids, and blue pigments (Wen et al., 2020). • Lipomyces starkeyi is an excellent candidate for lipid production because it has a broad sugar range (including glucose, xylose, and L-arabinose) and accumulates lipids to levels >70% of its dry cell weight (Takaku et al., 2020). • Y. lipolytica has high glycerol-utilization capabilities, and a series of genetic and molecular tools (including pattern chassis cell, vectors, promoters, selection markers, multiple integrations, and signals for secretion) for heterologous protein expression, and secretion and genome modification have been established (Li et al., 2016b). Therefore, this species is attractive as a cell factory for SCO and oleochemical production. • C. curvatus can tolerate and utilize various low-value substrates, such as lignocellulose, acetate, and activated sludge, for SCO production, and therefore, shows potential for use in the development of economically viable processes for commercial SCO production (Zhiwei et al., 2015; Zhiwei et al., 2014; Liu et al., 2016).

Table 7.1 Lipid accumulation and fatty acid profiles of normal oleaginous yeasts. Major fatty acid residues (% w/w) Species

C14:0

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

C20:0

Reference

Cryptococcus curvatus

1.2

29.4

0.6

18.4

44.1

1.5

–

–

Cryptococcus albidus

0.3–3.5

21.1–24.5

0.3–0.4

3.5–5.2

62.3–62.7

1.2–2.8

1.3–2.7

0.6–3.6

Lipomyces starkeyi Rhodotorula glutinis

–

18.9

4.0

5.0

67.9

2.9

0.6

–

1.1

21.4

1.4

4.9

58.6

3.6

2.2

2.2

8.9

14.2

6.6

19.7

41.4

9.2

–

10.6

22.1

6.4

12.4

36.5

11.2

–

–

–

16

7

7

43

18

–

–

(Zhiwei et al., 2014) (Zlatanov et al., 2014) (Probst, 2014) (Zlatanov et al., 2014) (Yaguchi et al., 2017) (Shen et al., 2017) (Carsanba et al., 2020)

Trichosporon oleaginosus Rhodosporidium toruloides Yarrowia lipolytica

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•

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Cryptococcus albidus and Trichosporon oleaginosus are known to use a variety of carbon sources and can be grown without supplementation with costly nutrients (Rosentrater and Muthukumarappan, 2006; Lee et al., 2017).

7.3 SCO metabolism in oleaginous yeasts 7.3.1 SCO synthesis Lipid synthesis mechanisms have been studied in Y. lipolytica (Stylianos, 2016; Ga´lvez-Lo´pez et al., 2019), L. starkeyi (Takaku et al., 2020), and Rhodosporidium sp. (Alvarez et al., 2019). Y. lipolytica is widely considered as a model species of oleaginous yeast due to its fully sequenced genome, well-known metabolic pathways and “generally recognized as safe” certification. Lipid synthesis in Y. lipolytica can occur via de novo synthesis and ex novo synthesis (Fabiszewska et al., 2019). De novo synthesis refers to the direct synthesis of lipids from hydrophilic carbon sources such as glucose or xylose. The lipid synthetic pathway from glucose can be divided into four modules: (1) acetyl-CoA formation, (2) fatty acid chain formation, (3) fatty acid elongation and desaturation, and (4) triacylglycerol (TAG) synthesis (Fig. 7.1). First, a nutrient imbalance (normally insufficient nitrogen) in the culture medium results in the inhibition of isocitrate dehydrogenase (IDH) activity. Gradually, increasing amounts of citric acid are transported to the cytoplasm and converted into oxaloacetate and acetyl-CoA by ATP citrate lyase (ACL). ACL is a key enzyme that catalyzes de novo lipid synthesis, as non-oleaginous yeasts, such as Saccharomyces cerevisiae, do not have this enzyme (Rodriguez et al., 2016), and the inactivation of ACL1 in Y. lipolytica results in a 60%–80% decrease in lipid synthesis (Dulermo et al., 2015). Acetyl-CoA is then irreversibly converted to malonyl-CoA by acetylCoA carboxylase (ACC), which primes fatty acid synthesis. This is a rate-limiting step in oleaginous yeast (Engelking, 2015). Subsequently, acetyl-CoA and malonyl-CoA are condensed by fatty acid synthases (FASs) to fatty acids in the cytoplasm and mitochondria by FAS I and FAS II complexes, respectively (Hu et al., 2019). In oleaginous yeasts, the FAS I complex is responsible for de novo fatty acid synthesis. The FAS I complex is a multifunctional protein with a hexameric α6β6 structure. It has an αFAS subunit consisting of acyl carrier protein, ketoacyl reductase, ketoacyl synthase and phosphopanthetheine transferase domains, and a βFAS

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Fig. 7.1 De novo synthesis of lipids from glucose by Y. lipolytica. Major enzymes involved: IDH, isocitrate dehydrogenase; ACL, ATP-citrate lyase; ACC, acetyl-CoA carboxylase; MDH, malate dehydrogenase; ME, malic enzyme; CMT, citrate-malate acid transferase; FAS II complex, fatty acid synthase complex; E, elongase; DS, desaturase; GPAT, glycerol-3-phosphate acyltransferase; PAP, phosphatidic acid phosphatase; LPAT, lysophosphatidate transferase; DGAT, diacylglycerol acyltransferase.

subunit consisting of acyl transferase, enoyl reductase, dehydratase, and malonyl palmitoyl transferase domains (Rigouin et al., 2018). These eight functional domains catalyze all of the reactions required for fatty acid synthesis in oleaginous yeasts, namely activation, priming, multiple cycles of elongation, and termination (Rigouin et al., 2018). The FAS enzymatic complexes produce acyl-CoA using acetyl-CoA as an initiation molecule and malonylCoA as elongation unit, adding two carbons to the fatty acid backbone each cycle. In addition, two molecules of NADPH are required in each elongation cycle. NADPH is primarily generated during the conversion of malate to oxaloacetate catalyzed by malic enzyme (Ga´lvez-Lo´pez et al., 2019). Palmitic acid (PA) has a 16-carbon fatty acid backbone that can be synthesized after seven elongation cycles in the cytoplasm. PA is then transported to the endoplasmic reticulum (ER) for further elongation and desaturation. Fatty acids up to C26 can be synthesized by different elongases encoded by elo1, elo2, and elo3. Elo1p functions as the main elongase,

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whereas Elo2p and Elo3p are predominantly responsible for the assembly of very long-chain fatty acids for sphingolipid synthesis (Lisa Klug, 2014). After a series of elongation reaction cycles, fatty acyl-CoAs with different carbon chain lengths are formed, but mainly C14, C16, and C18 saturated acyl-CoAs. Unsaturated fatty acids (UFAs) in Y. lipolytica are synthesized aerobically by respective desaturases. The Δ9 desaturase is responsible for introducing the first double bond into C16:0 and C18:0 at the Δ9 position, to form the monounsaturated fatty acids (MUFAs) palmitoleic acid (16:1) and oleic acid (18:1). The Δ12 and Δ15 desaturases are responsible for introducing additional double bonds into the Z12 and Z15 sites of the MUFAs, to form linoleic acid (C18:2) and γ-linolenic acid (C18:3). The most common UFAs in yeasts are C16:1, C18:1, C18:2, and C18:3 (Beopoulos et al., 2010). Finally, various acyl-CoA products (with different lengths and degrees of unsaturation) are condensed with glycerol to form TAG via a series of reactions. TAG synthesis in oleaginous yeasts requires the formation of two key intermediates, phosphatidic acid, and diacylglycerol (DAG). Glycerol-3phosphate acyltransferase catalyzes the first acylation of glycerol-3phosphate at the sn-1 position to form lyso-phosphatidic acid (LPA), which is then catalyzed by LPA acyltransferase at the sn-2 position to yield phosphatidic acid. Phosphatidic acid is further dephosphorylated by phosphatidate phosphatase to form DAG. Finally, DAG is acylated, mainly by Dga1p and Lro1p, to yield TAGs (Sorger and Daum, 2003). TAGs are stored in subcellular compartments termed lipid droplets or lipid bodies (Friedlander et al., 2016). In the ex novo lipid synthetic pathway, hydrophobic substances, such as alkanes or fatty acids, are degraded and hydrolyzed at the surface and thereafter, incorporated in the form of CoA-thioesters, to feed into the lipid synthetic pathway to generate TAG (Be Opoulos et al., 2011). Ex novo synthesis can modify extracellular and intracellular lipid composition to satisfy the requirements of the food or chemical industries (Chao Huang et al., 2017).

7.3.2 TAG and fatty acid degradation When the carbon source in the medium is exhausted or the uptake rate decreases in the late fermentation period, as a general rule, cells will consume their stored lipids to survive (Papanikolaou and Aggelis, 2011). In yeast, the

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degradation of neutral lipids involves six steps: fatty acid formation, activation, oxidation, hydrolysis, dehydrogenation, and thiolysis (Fig. 7.2). Intracellularly stored TAG is hydrolyzed to fatty acid by intracellular triacylglycerol lipase (TGL). In yeast, various isoenzymes of TGL have been identified. Four genes (Sctgl2–5) encoding intracellular lipases are known to be involved in TAG catabolism in S. cerevisiae, and two genes, Yltgl3 and Yltgl4, have been shown to encode intracellular lipases for TAG degradation in Y. lipolytica. The inactivation of one or both of these genes greatly increases the lipid accumulation capacity of the cells (Dulermo et al., 2013). In addition, many microorganisms can secrete lipase into the culture medium to hydrolyze and assimilate lipid substrates (Najjar et al., 2011). Fatty acids are degraded via the β-oxidation pathway. In yeast, β-oxidation is restricted to peroxisomes, whereas in mammals, β-oxidation can occur in both mitochondria and peroxisomes (Beopoulos et al., 2008). In yeast, fatty acids are converted to acyl-CoA esters by acyl-CoA synthetases (ACSs) and then enter the next four β-oxidation steps (oxidation, hydrolysis, dehydrogenation, and thiolysis). Each cycle results in the loss of two carbons of acetyl-CoA, the formed acetyl-CoA will enter central metabolism via the glyoxylate cycle (Fickers et al., 2010).

7.3.3 Regulation of TAG and fatty acid synthesis Fatty acids and TAG are high-energy and reducing-equivalent-intensive chemicals. Therefore, their synthetic pathway is extensively regulated at multiple levels and at many nodes. Lipid accumulation is triggered by a nitrogen imbalance in the culture medium. Under these conditions, the activity of IDH decreases due to the depletion of AMP, resulting in the accumulation of citrate within the mitochondria. Citrate is then transported to the cytosol and cleaved to oxaloacetate and acetyl-CoA by ACL. As ACL is not found in non-oleaginous species, it is thought to be essential for lipid accumulation (Rodriguez et al., 2016). Fatty acid synthesis is regulated at multiple levels, including transcriptional regulation, posttranslational modifications, and feedback inhibition (Silverman, 2015). For example, the expression levels of acc1, fas1, and fas2 are controlled by a UASINO element (UASINO is a cis-acting inositol-sensitive upstream activating sequence) and co-regulated by the ER-associated transcriptional repressor, Opi1, which senses changes in phosphatidic acid concentration (Henry et al., 2012). In de novo lipid synthetic pathways, all membrane phospholipids are synthesized from phosphatidic acid. Blocking lipid flux toward phospholipids has been shown to increase neutral lipid accumulation (Gaspar et al., 2008). In addition, Snf1

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Fig. 7.2 The mechanism of intracellular lipid degradation. TGL, triacylglycerol lipase; FAA, fatty acyl-CoA synthetase; AOX, acyl-CoA oxidase; DBP, d-bifunctional protein; THI, thiolase.

kinase has been shown to participate in the regulation of glycogen accumulation, peroxisome proliferation, and phospholipid synthesis in yeast, in addition to its central role in the response to glucose availability (Shirra et al., 2001). Accordingly, yeasts with the hyperactive Acc1S1157A mutant, which lacks a phosphorylation site for Snf1 kinase, display a shift toward

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a longer average acyl chain length, along with increased FA synthesis and TAG accumulation (Hofbauer et al., 2014). Posttranslational modifications of constitutively expressed intracellular lipases, such as Tgl4, by cyclindependent kinase 28 may also be needed to regulate their activity (Silverman, 2015). The product of the FAS complex, palmitoyl-CoA, causes feedback inhibition of Acc l activity, as do other fatty acyl-CoAs (Sheng and Feng, 2015). Moreover, NADPH provides reducing power during elongation of the fatty acid chain. In addition to lipid synthesis, NADPH participates in many other biological reactions, such as cholesterol and amino acid synthesis (Sheng and Feng, 2015). The regulation of NADPH supply has a significant effect on lipid synthesis. In conclusion, the mechanism regulating lipid synthesis in oleaginous yeast is complex and remains unclear. Further investigation is urgently required to identify methods for enhancing lipid synthesis.

7.4 Oleaginous microorganism engineering The synthesis pathway for SCO accumulation is long and involves a variety of enzymes and regulatory genes. Using metabolic engineering to enhance the SCO synthetic pathway, block competitive pathways, regulate the SCO metabolic network, and introduce a low-value feedstock utilization pathway can efficiently improve SCO production and yield.

7.4.1 Enhancing the synthetic pathway Prior work using “push-and-pull” strategies (increasing the pull on cytoplasmic acetyl-CoA supply and the push on fatty acid synthesis) has led to the construction of efficient lipid synthetic cell factories (Tai and Stephanopoulos, 2013). As discussed above, ACL plays a key role in lipid production in oleaginous yeasts. To prevent the secretion of citrate into the culture medium, so as to increase acetyl-CoA levels for lipid synthesis, the acl from Mus musculus has been overexpressed in Y. lipolytica. The lipid content in the engineered strain was found to double, without a significant effect on cell growth (Zhang et al., 2014). Recently, a new cytoplasmic route for acetyl-CoA formation via non-oxidative glycolysis was introduced into Rhodosporidium azoricum by overexpressing phosphoketolase and phosphotransacetylase. The engineered strain exhibited an 89% increase in the final lipid titer (Silvia Donzella et al., 2019).

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In addition, by introducing a mitochondrial pyruvate dehydrogenase (PDH) bypass into T. oleaginosus ATCC20509, pyruvate is converted to acetaldehyde by pyruvate decarboxylase and is subsequently converted to acetate by acetaldehyde dehydrogenase. The acetate is then converted to acetyl-CoA by ACS to provide the precursor for lipid synthesis. Using this technique, the lipid synthesis capability was enhanced in the engineered strain. The introduction of a PDH bypass contributes to alternative routes to cytoplasmic acetyl CoA and compensates for the consumption of NADPH. As a result, the engineered strain achieved a lipid yield of 0.27 g/g on xylose, approaching the theoretical maximum yield of 0.289 g/g (Kari et al., 2018). ACC catalyzes the first rate-limiting step in de novo fatty acid synthesis. Overexpressing ACC1 in Y. lipolytica results in a twofold increase in lipid content over the control (Tai and Stephanopoulos, 2013). In addition to the overexpression of ACC1, the direct synthesis of malonyl-CoA from malonate by malonyl-CoA synthetase (MCS) also leads to increased malonyl-CoA levels (Chen and Tan, 2013). Moreover, diacylglycerol acyltransferase (DGAT) catalyzes the last rate-limiting step of fatty acid synthesis, overexpression of DGA1 induced by the intron-containing translation elongation factor-1α promoter results in a fourfold increase in lipid production by Y. lipolytica (Tai and Stephanopoulos, 2013). The dual overexpression of DGAT1 and ACC1 increases the lipid content to 61.7% (w/w), with a yield of 0.195 g/g from glucose and a productivity of 0.143 g/L/h (Tai and Stephanopoulos, 2013). Moreover, simultaneous overexpression of Δ9 stearoyl-CoA desaturase, ACC1 and DGA1 in Y. lipolytica results in rapid cell growth and a lipid production of 55 g/L, which is 84.7% of the theoretical maximal yield from glucose (Qiao et al., 2015).

7.4.2 Blocking competitive pathways Competitive pathways of lipid synthesis include the β-oxidation pathway, the transformation of glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP), and the phospholipid synthesis pathway. The β-oxidation pathway is the main pathway for fatty acid degradation in yeast (Chen et al., 2014). By blocking the β-oxidation pathway (Δfaa1 Δmfe), enhancing flux through neutral lipid formation (dga2 overexpression), and mimicking the bacterial pathway for free fatty acid synthesis (tgl4 and Kltgl3 overexpression), engineered strains can produce total lipids (intracellular

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+extracellular) of 92.4% by dry cell weight (Ledesma-Amaro et al., 2016). A strain of Y. lipolytica has been engineered with multiple modifications, including complete blockage of the β-oxidation pathway (by deleting the six pox) and inactivation of the native TAG acyltransferases (Dga1p, Dga2p, and Lro1p) and the △12 desaturase (Fad2p). The expression of hydroxylase CpFAH12 from Claviceps purpurea and a native PDAT acyltransferase (Lro1p) in this strain results in the production of 43% of its normal total lipid levels and more than 60 mg/g dry cell weight of ricinoleic acid (Beopoulos et al., 2014). To redirect carbon flux toward lipid synthesis, the gut2, which codes for the G3P dehydrogenase isomer, has been deleted in Y. lipolytica. This Δgut2 mutant strain demonstrates a threefold increase in lipid accumulation compared with the wild-type strain (Beopoulos et al., 2008). Additional inactivation of the β-oxidation pathway (deletion of pox1–6) in the Δgut2 strain leads to a fourfold increase in lipid content. In addition, the G3P shuttle pathway provides an alternative approach to increase intracellular G3P concentration, and combined with the inactivation of the β-oxidation pathway, the mutant Y. lipolytica strain accumulates up to 65%–75% of its dry cell weight as lipids (Dulermo and Nicaud, 2011). To regulate carbon flux between neutral lipids and phospholipids, a culture medium without the addition of phosphate has been used. The fermentation results showed that the phospholipid component of the total lipid content was significantly reduced from 8.3% to 1.4%, while the TAG content increased from 6.5% to 39.3% in the alga Monodus subterraneus (KhozinGoldberg and Cohen, 2006). However, this strategy has not been reported in oleaginous yeast.

7.4.3 Lipid synthesis regulation As mentioned hereinbefore, two molecules of NADPH are required in each acetyl-CoA elongation cycle. Therefore, enhancement of NADPH supply is the common but significant strategy for SCO production. Currently, three pathways were reported involved in NADPH regulation in oleaginous yeasts (Fig. 7.3): ① pyruvate/oxaloacetate/malate (POM) cycle, which is the crucial source of NADPH for lipid synthesis in most oleaginous fungi and some oleaginous yeast, such as R. toruloides. In this cycle, cytosolic malic enzyme (ME) catalyzes the oxidative decarboxylation of malic acid to pyruvic acid with concomitant reduction of NADP+ to NADPH. For example,

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Glucose NADP+

NADPH

ZWF

Glucose-6P

Ribulosec-5P

Ribose-5P

PPP Fructose-6P Xylulose-5P Glyceraldehyde-3P

Pyruvate

NADPH

POM cycle

ME

NADP+ Malate

Pyruvate

Oxaloacetate

Acetyl-CoA Oxaloacetate

Citrate

Citrate

Acetyl-CoA

Fatty acid

Isocitrate IDCH

Malate

NADP+ NADPH

α-ketoglutarate Mitochondria

Fig. 7.3 NADPH regulation responsible for lipid accumulation in oleaginous yeasts.

overexpression of native ME led to 24% increase of lipid titer in R. toruloides IFO0880 (Zhang et al., 2016). ② Pentose phosphate pathway (PPP), NADPH supply in the absence of cytosolic ME in oleaginous yeasts such as Y. lipolytica and L. starkeyi is met by PPP (Wasylenko et al., 2015; Tang et al., 2010), where NADPH is generated through NADP+-dependent glucose-6-phosphate dehydrogenase (ZWF1). Only overexpression of zwf1 can suppress cell growth, co-expression of zwf1 with acyl-CoAbinding protein increased the saturated lipid content to 30% of dry cell weight in Y. lipolytica (Yuzbasheva et al., 2017). ③ NADP+-dependent isocitrate dehydrogenase (ICDH), transcription of icdh is upregulated by 4.8-fold in R. toruloides under nitrogen limitation (Zhu et al., 2012), heterologous expression of icdh from R. toruloides also results in obvious increase of intracellular lipid content in engineered S. cerevisiae (Fan et al., 2012). In addition, the role of glyceraldehyde-3-phosphate dehydrogenases (GPD)

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in NADPH production and lipid accumulation in oleaginous filamentous fungus Mortierella alpine has been confirmed (Wang et al., 2020); however, overexpression of NADP+-dependent GPD in Y. lipolytica could also increase its lipid yields (Qiao et al., 2017). Moreover, some transcription factors (TFs) were explored to regulate lipid synthesis. In Y. lipolytica, some TFs have been studied involved into lipid metabolism, including Por1 (Poopanitpan et al., 2010), Mig1 (Wang et al., 2013), the basic helix-loop-helix, Opi1-like TFs (Kobayashi et al., 2016; Endoh-Yamagami et al., 2007), etc. Recently, Christophe et al. have screened 38 positive TFs that can enhance lipid accumulation in Y. lipolytica from 148 putative TFs (Christophe et al., 2018). In addition, SNF2 was reported as a regulator of phospholipid synthesis, lipid content shows 3.8fold increase in 4snf2 disruptant (Kamisaka et al., 2006). However, the deep regulatory mechanism for lipid accumulation is not clear at present, more analytical research is needed in further study.

7.5 Feedstock for SCO production The cost of fermentation substrates (40%–50%) is a major contributing factor to the cost of biodiesel production using oleaginous microorganisms (Kumar et al., 2019b). Currently, glucose and starch still play the main roles in SCO production, and they account for up to 80% of the total material cost (Koutinas et al., 2014). Therefore, it is important to explore low-value substrates for SCO production.

7.5.1 Lignocellulose Lignocellulosic biomass, such as agricultural residue and woody crops, is a promising renewable substrate for SCO production due to its abundance, low cost, and high content of assimilable sugars (up to 75%), mainly hexoses and pentoses (Lee et al., 2017). Lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin. After pretreatment, a hydrolysate rich in five- and six-carbon sugars is obtained for microbial cultivation. For example, using an automated online sugar control feeding mode, a dry cell weight of 54 g/L and a lipid content of 59% (w/w) have been obtained with Rhodosporidium toruloides DSMZ 4444 using corn stover hydrolysate as the sole carbon source. This corresponds to a lipid yield of 0.29 g/g and a productivity of 0.4 g/L/h (Fei et al., 2016). Lipid titers of 13.1 g/L, with high lipid content (60% w/w) and high lipid yield (0.29 g/g), have been achieved

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with T. oleaginosus using enzymatic sorghum stalk hydrolysates (Lee et al., 2017). More representative research findings are described in Table 7.2. However, some species of oleaginous microorganisms can only utilize limited types of monomer sugars. For example, the inefficiency of xylose catabolism in Y. lipolytica hinders its use for commercial SCO production from lignocellulosic biomass. To solve this problem, an efficient isomerase-based xylose-utilization pathway was constructed in Y. lipolytica (engineered strain, YSXID), resulting in lipid titres up to 12.01 g/L, with a maximum yield of 0.16 g/g, from Miscanthus hydrolysates (Sang et al., 2020). In addition, the metabolism of other sugars is always inhibited when glucose is present in the fermentation medium. The sequential and slow conversions of these suboptimal sugars remain the main challenges in creating an efficient lignocellulosic biorefinery (Tran et al., 2020). However, the hetero-matrix structure and macromolecular composition of lignocellulose make it hard to depolymerize, and special pretreatment methods, including chemical, physical, or biological methods, are required (Lee et al., 2014). These harsh pretreatment conditions generate numerous small-molecule inhibitors, such as acetic acid, furan aldehydes, and 5-hydroxymethylfurfural, which significantly inhibit cell growth and lipid accumulation (Vald
es et al., 2020). Therefore, oleaginous yeast candidates should have good tolerance to the inhibitors present in complex hydrolysates. Recently, R. toruloides-1588 was reported to be a suitable SCO producer using lignocellulose hydrolysates and was found to be tolerant to several toxic compounds present in hydrolysates. Maximum biomass concentrations of 17.09 and 19.56 g/L, with lipid accumulation of 36.68% and 35.24%, were obtained by R. toruloides-1588 from undetoxified hardwood and softwood hydrolysate, respectively (Saini et al., 2020). In addition, biotechnological techniques, such as adaptive evolution and metabolic engineering, are efficient at improving cell fitness in inappropriate environments. Using adaptive laboratory evolution with formic acid as a single inhibitor or with an inhibitor cocktail, strains of the oleaginous yeast Metshnikowia pulcherrima show increased cell growth rates and a decrease in the lag phase under inhibiting conditions (Hicks et al., 2020). An L. starkeyi mutant generated by ultraviolet irradiation shows a 15.1% increase in cell weight and a 30.7% increase in lipid production compared with the control strain when cultivated on a mixed carbon source (xylose+glucose) (Eulalia et al., 2012). Moreover, applying detoxification methods to remove inhibitors from lignocellulose hydrolysates before fermentation is another commonly used approach. The techniques used for the detoxification of

Table 7.2 Oleaginous yeasts that convert lignocellulose into SCO. Cell weight (g/L)

Lipid content (%, w/w)

Lipid production (g/L)

Microorganism

Substrate

Pretreatment

Fermentation mode

Cryptococcus curvatus ATCC 20509 Trichosporon fermentans

Wheat straw

Dilute acid

Batch flask

17

34

5.8

(Yu et al., 2011)

Wheat straw

Batch flask

9.6

47–49

4.5

(Ren et al., 2016)

24.6

67

16.5

24.3

75.0

18.2

13.9

34.5

4.8

(Niehus et al., 2018) (Poontawee et al., 2018) (Ruan et al., 2012)

Reference

Yarrowia lipolytica

Agave bagasse

Seawater ionic liquid NA

Rhodosporidiobolus fluvialis DMKU-SP314 Mortierella isabelline ATCC 42613 Trichosporon cutaneum AS 2.571 Rhodotorula graminis

Sugar cane

Dilute acid

Corn stover

Dilute acid

Batch bioreactor, 3L Batch bioreactor, 2L Batch flask

Corn stover

Dilute acid

Batch flask

19.4

39.2

7.6

(Hu et al., 2011)

Corn stover

Dilute acid

42.3

34.0

14.4

Corn stover

Dilute acid

Fed-batch bioreactor, 20 L Batch bioreactor, 3L

26

31

8.1

(Galafassi et al., 2012) ( Juan et al., 2016)

T. cutaneum ACCC 20271

Rhodosporidium toruloides Trichosporon dermatis C. curvatus

54

59

31.9

Dilute acid

Fed-batch bioreactor Batch flask

36.4

59

21.2

Corn stover

Dilute acid

Batch flask

11.3

60.8

6.9

C. curvatus ATCC 20509

Corn stover

Dilute acid

Batch bioreactor

33.9

63.1

21.4

R. toruloides DSM-4444

Corn stover

Dilute acid

Batch bioreactor

38.3

60.8

23.3

Trichosporon guehoae UCDFST 60–59 Trichosporon coremiiforme

Corn stover

Dilute acid

Batch bioreactor

29.4

48.3

14.2

Corn cobs

Dilute acid

Batch flask

20.4

37.8

7.7

Corn cobs

Dilute acid

18.7

60.3

11.3

Corn cobs

Dilute acid

Batch bioreactor, 5L Batch flask

17.2

47.0

8.1

Corn cobs

Dilute acid

75

47.2

34

Rhodotorula taiwanensis AM2352 Lipomyces starkeyi CH010 Rhodotorula glutinis CGMCC2.703

Corn stover

Dilute acid

Corn stover

Fed-batch bioreactor, 5L

(Fei et al., 2016) (Yu et al., 2020) (Zhiwei et al., 2016) (Violeta et al., 2018) (Violeta et al., 2018) (Violeta et al., 2018) (Huang et al., 2013b) (Miao et al., 2020) (Huang et al., 2014) (Liu et al., 2015)

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lignocellulose hydrolysates include the use of chemical additives, enzymatic treatment, heating and vaporization, liquid-liquid extraction, liquid-solid extraction, and microbial treatment ( J€ onsson et al., 2013). However, no single method is capable of completely removing all inhibitors from the hydrolysate broth (Sjulander and Kikas, 2020). More efficient and economic detoxification processes are required.

7.5.2 Nonfood biomass In addition to lignocellulose, other types of nonfood biomass, such as chitin, cassava, and Jerusalem artichoke, are good raw materials for microbial lipid production. Chitin is a polymer of acetylglucosamine and is abundant in arthropods and mollusks. N-acetyl-D-glucosamine and glucosamine generated by the hydrolysis of chitin provide good feedstock for SOC production. The oleaginous yeast species C. albidus, C. curvatus, and Trichosporon fermentans have been reported to accumulate lipids at more than 28% of dry cell weight from N-acetyl-D-glucosamine (Wu et al., 2010; Zhang et al., 2011). Cassava is widely cultivated in China. Using cassava starch as a feedstock for SCO production is gaining increasing attention. For example, Rhodotorula mucilaginosa TJY15a has been shown to accumulate 52.9% (w/w) of neutral lipids from cassava starch hydrolysate through fed-batch cultivation (Li et al., 2010). Moreover, heterotrophic Chlorella protothecoides accumulates 41.0% (w/w) of fatty acids and 58.4% (w/w) of neutral lipids from cassava bagasse hydrolysate through fed-batch fermentation (Chen et al., 2015). The Jerusalem artichoke is a perennial herbaceous plant that contains high concentrations of inulin, which can be easily processed to provide a substrate for biorefineries (Qiu et al., 2018). When fed hydrolysates, 39.6 g/L of lipids and a lipid content of 56.5% (w/w) were achieved by R. toruloides Y4 (Xin et al., 2010). The Jerusalem artichoke is superior to traditional grain crops, as it can be harvested three times a year (Li et al., 2016a). The development of these nonfood types of biomass as feedstocks for biorefineries provides a promising solution to the controversial issue of food versus fuel.

7.5.3 Industrial and agricultural by-products Industrial by-products, such as glycerol and starch, have been identified as the most promising feedstocks for the establishment of bio-based industrial biorefineries (Gavrilescu, 2010). Crude glycerol produced as a by-product of biodiesel has to be effectively utilized to contribute to the viability of various value-added biochemicals, including biodiesel (Garlapati et al., 2016).

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When Y. lipolytica strain JMY4086 is cultured with crude glycerol as the carbon source, a lipid content of 40% (w/w) and a lipid productivity of 0.43 g/L/h are obtained, with a coefficient of lipid yield to glycerol consumption of 0.1 g/g (Rakicka et al., 2015). Starch is one of the most abundant carbohydrates in nature. When alpha-amylase and glucoamylase enzymes are overexpressed in Y. lipolytica, the engineered strain grows and synthesizes up to 27% of its total lipid content directly from soluble starch (Ledesma-Amaro et al., 2015). Biorefineries using agricultural by-products or residues, such as lignocellulosic, starchy, and other active compounds, have attracted considerable attention globally (Philippini et al., 2020). Starchy materials, in addition to starch itself, usually contain abundant quantities of protein, lipids, and other micronutrients (Philippini et al., 2020). Chaturvedi et al. (2019) (Chaturvedi et al., 2019) studied several starchy types of biomass, including wheat bran, corn residue, potato peel, and cassava peel, for the growth of the oleaginous yeasts C. curvatus, L. starkeyi, Trichosporon cutaneum, R. mucilaginosa, Rhodotorula glutinis, and Saccharomyces pastorianus. The highest lipid content of 52.04% (w/w) was obtained when L. starkeyi was cultivated with rice residue. The cultivation of R. mucilaginosa with wheat bran resulted in the highest cell biomass production of 61.0 g/L, but a lipid content of only 4.92% (w/w). In addition, due to the diverse and complex properties of agro-industrial residues, adequate pretreatment is always required to bioremediate agricultural waste to release the nutrients required for fermentation processes (Evans, 2018).

7.5.4 Industrial and urban wastewater The acceleration of industrialization and urbanization has resulted in an increase in the discharge of untreated wastewater, leading to serious environmental pollution. Industrial and urban wastewater often contains abundant quantities of sugars and organic acids. The development of wastewater as a cheap raw material for microbial fermentation is of great significance for both cost-effective biomanufacturing and environmental governance (Yadav et al., 2019). R. glutinis CGMCC No. 2258 has been used to treat cellulosic ethanol wastewater, yielding a maximum biomass of 11.31 g/L, a lipid content of 18.35%, and a lipid yield of 2.08 g/L under optimal conditions (Zhang et al., 2018). In addition, chemical oxygen demand (COD), total organic carbon, NH+4 -N, total nitrogen, and total phosphorus removal rates of

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83.15%, 81.81%, 85.49%, 70.52%, and 67.46% were reached, respectively, when T. fermentans CICC 1368 was used to treat refined soybean oil wastewater, without sterilization, dilution, or nutrient supplementation. Under optimal conditions, 7.9 g/L of biomass, 43% (w/w) lipid content, 94.7% degradation of COD, and the removal of 89.9% of oils were achieved (Yu et al., 2018). Volatile fatty acids (VFAs) are short-chain fatty acids in the range of C1–C6 (mainly acetic, propionic, and butyric acid). They are routinely generated during the acidogenic fermentation of various organic wastes. VFAs can reach a relatively high concentration of 10–40 g/L in food waste or animal or human feces (Yin et al., 2014). Developing VFA-rich wastewater as a feedstock for SCO production has gained increasing attention in recent years. Undissolved VFAs under acid or neutral pH conditions lead to the severe inhibition of cell growth and metabolism. An initial pH of 8 was found to be optimal for VFA utilization by Y. lipolytica, with a maximum biomass of 37.14 g/L and a lipid titer of 10.11 g/L from 70 g/L of acetic acid (Gao et al., 2020).

7.6 SCO production techniques Effective SCO production requires the selection of suitable media composition and cultivation conditions, to allow the maximum conversion of the carbon substrate into stored lipids by the oleaginous microorganism. Lipid accumulation is triggered by an excess of carbon and one limiting nutrient, usually nitrogen (Katrin et al., 2016). Typical carbon-nitrogen (C/N) ratios favoring lipid accumulation are in the range of 50:1–150:1 (Calvey et al., 2016). A lipid content of 55% and a lipid titer of 10 g/L have been obtained using L. starkeyi with a C/N ratio of 72:1, while these values are halved when the initial C/N ratio is lowered to 24:1 (Calvey et al., 2016). Growth on glycerol results in the highest specific growth and lipid productivity for R. toruloides CCT 0783 at C/N ratios between 60:1 and 100:1 (Lopes et al., 2020). pH, temperature, inoculum size, glucose concentration, agitation, aeration rate, and fermentation mode all play important roles in biochemical fermentation, and lipid synthesis is no exception. After optimizing the process parameters of pH and temperature, lipid productivity by Saitozyma podzolica DSM 27192 can be enhanced by 40% (Gorte et al., 2020). Under conditions optimized for the overall parameters, Rhodotorula kratochvilovae SY89

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produces a maximum biomass of 15.34 g/L, a lipid yield of 8.60 g/L, and a lipid content of 56.06% in batch bioreactor fermentation ( Jiru et al., 2017). Various models have been designed to simplify the optimization process. For example, model-based optimization using a control vector parameterization approach has been performed to improve lipid productivity. This configuration allows the efficient regulation of the C/N ratio during the process, leading to lipid productivity exceeding that obtained using a single flow rate by more than 40% (Mun˜oz-Tamayo et al., 2014). Similarly, computational response surface methodology has been used to guide and streamline the experimental media optimization matrix with 12 nitrogen sources and 10 carbon sources. Using this optimization strategy, a maximum biomass of 18.4 g/L and lipid content of 49.74% (w/w) were produced from lactose by Cutaneotrichosporon oleaginosus ATCC 20509, with yeast extract as the nitrogen source at a C/N ratio of 120:1 (Awad et al., 2019).

7.7 High-value-added polyunsaturated fatty acids PUFAs such as GLA, AA, DHA, and EPA have attracted increasing interest in recent years due to their unique benefits in improving human health (Fig. 7.4) ( Jovanovic et al., 2021). Lipids from fish are a readily available source of long-chain PUFAs (Sahena et al., 2010). However, the risks of depleting the fish population due to overfishing and the possible presence of contaminants (such as heavy metals) require the development of safer and more economically viable methods of PUFA production (Maria et al., 2020). Vegetable oils are an alternative source of linoleic GLA, ALA, and AA, but long-chain PUFAs, such as DHA and EPA, are not synthesized by plants (Ward and Singh, 2005). Hence, the discovery of PUFAs in oleaginous microorganisms is of great biotechnological importance (Patel et al., 2020). Although microalgae are currently considered to be the most promising source of artificially produced long-chain PUFAs (Kumar et al., 2019a), oleaginous yeasts also exhibit great potential for high-value PUFA synthesis. An oleaginous yeast strain, Candida guilliermondii FO726A, has been shown to have the capability to accumulate EPA and DHA at 7.7% and 18.3% of the total intracellular lipid content, respectively (Guo and Ota, 2000). Through heterologous expression of hybrid PUFA biosynthetic gene clusters from myxobacteria, adapted for Y. lipolytica, a remarkable docosahexaenoic acid (DHA) content of 16.8% of the total lipid content has been achieved (Gemperlein et al., 2019). Through heterologous expression of

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Fig. 7.4 PUFAs synthetic pathway.

several fatty-acid-modifying enzymes (Δ9 elongase of IgASE2 from Isochrysis galbana, Δ12/ω3 desaturase Fm1 from Fusarium moniliforme, and linoleic acid isomerase PAI from Propionibacterium acnes) in T. oleaginosus ATCC 20509, the recombinant yeast accumulates GLA to 21%, eicosatrienoic acid (C20:3) to 16%, and eicosadienoic acid (C20:2) to 9% of the total cellular fatty acid content (Christian G€ orner et al., 2016).

7.8 Conclusions and future prospects Microbial lipid production using oleaginous yeast is technologically feasible; however, high production costs limit its industrialization. The use of inexpensive substrates, such as lignocellulose and nonfood sugar substances, is the most effective approach to reduce the feedstock cost. However, these

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inferior substrates often have a complex composition and contain a large number of inhibitors, making it difficult for the microorganism to effectively use the carbon elements in these substrate hydrolysates (Vald
es et al., 2020). To improve the tolerance of oleaginous yeast to these inferior substrates and their ability to transform the substrates into lipids, it is necessary to integrate the development of substrate pretreatment technology, identify more robust strains, and construct versatile yeast factories through genetic engineering. Low lipid yield is another challenge for the industrialization of microbial lipid production. Hence, a greater understanding of the metabolic networks, modules, components, and mechanisms regulating lipid synthesis is required. An efficient genetic engineering technique can then be applied to improve the total lipid titer, yield, and productivity. However, it is difficult to perform genetic modifications in oleaginous yeast species other than Y. lipolytica, for which there are many genetic engineering tools. Future studies should focus on the development of new strategies and tools to engineer oleaginous yeasts that have naturally high levels of lipid accumulation, a wide substrate spectrum, or the ability to accumulate high-value fatty acids. In addition to increasing lipid production through complex metabolic processes, the production of microbial lipids may become more economically feasible if the biorefinery concept of the co-production of different value-added products is applied. The co-production of microbial lipids with pigments, organic acids, alcohols, or PUFAs has been explored in recent years (Chen et al., 2021). Especially, co-cultured oleaginous yeast with microalgae seems to be a promising approach for wastewater treatment and nutrients recycle (Qian et al., 2020). In this co-culture system, the mineral salt and some organic matter can provide yeast and microalgae nutriments for cell growth cell and metabolism. In addition, microalgae can release organic compounds and O2 during photosynthesis, which can be used as carbon and energy sources by oleaginous yeast. In return, yeast can provide CO2 and growth promoting factors for microalgae, such as vitamins and siderophores. However, it should be noted that lipids are intracellular products, and the co-produced chemicals are also stored inside the cell. Therefore, it may be not easy or economically viable to separate the multiple products. Hence, the development of efficient downstream processes is an important precondition to evaluate the viability of a co-production system. Moreover, lack of cost-effective and efficient extraction of lipid is another major constraint in microbial lipid technology (Hwangbo and Chu, 2020). Lipids extracted from dried biomass have been widely used for analysis due to the higher lipid yields. However, drying the biomass is

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economically prohibitive for large-scale microbial lipid-based biodiesel applications due to the tremendous energy demand (Dong et al., 2016). Direct lipid extraction from wet cell-biomass might be favored since it eliminates the need for costly dehydration. Compared with microalgae, less energy is needed for cell harvest, but the complex cell membrane and thick and rigid cell wall of oleaginous yeast still request cell disruption before lipid extraction (Dong et al., 2016). In addition, solvent extraction is the most commonly used lipid extraction method, which imposes several limitations for microbial lipid large-scale extraction (Hwangbo and Chu, 2020): ①large consumption of toxic organic solvent, resulting in environment pollution and costly solvent investment; ②additional energy and process are needed to separate lipid from solvent. Hence, novel lipid extraction technologies with low energy input, less toxic solvent consumption, and high lipid yield are crucial for large-scale production of microbial lipid.

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Zhang, X., Liu, M., Xu, Z., et al., 2018. Microbial lipid production and organic matters removal from cellulosic ethanol wastewater through coupling oleaginous yeasts and activated sludge biological method. Bioresour. Technol. 267, 395–400. Zhang, H., Zhang, L., Chen, H., et al., 2014. Enhanced lipid accumulation in the yeast Yarrowia lipolytica by over-expression of ATP:citrate lyase from Mus musculus. J. Biotechnol. 192, 78–84. Zhiwei, G., Hongwei, S., Wengting, Z., et al., 2015. Efficient conversion of acetate into lipids by the oleaginous yeast Cryptococcus curvatus. Biotechnol. Biofuels 8, 1–9. Zhiwei, G., Hongwei, S., Xiaobing, Y., et al., 2014. Lipid production from corn Stover by the oleaginous yeast Cryptococcus curvatus. Biotechnol. Biofuels 7, 158. Zhiwei, G., Wenting, Z., Hongwei, S., et al., 2016. Co-fermentation of acetate and sugars facilitating microbial lipid production on acetate-rich biomass hydrolysates. Bioresour. Technol. 207, 102–108. Zhu, Z., Zhang, S., Liu, H., et al., 2012. A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nat. Commun. 3, 1112. Zlatanov, M., Pavlova, K., Antova, G., et al., 2014. Biomass production by Antarctic yeast strains: an investigation on the lipid composition. Biotechnol. Biotechnol. Equip. 24 (4), 2096–2101.

CHAPTER 8

Biochemical production of bioalcohols Jiasheng Lua, Wenming Zhanga,b, Carol Sze Ki Linc, Yujia Jianga, and Fengxue Xina,b a

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People’s Republic of China b Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing, People’s Republic of China c School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong, People’s Republic of China

8.1 Introduction Gasoline and diesel are the two primary transportation fuels. Global gasoline consumption has been increasing continuously in recent decades. However, nonrenewable gasoline applications result in environmental issues, such as air pollution and global warming. Bioalcohols, including bioethanol and biobutanol, are considered to be sustainable alternative biofuels, and they have gained much attention for industrial applications (Sittijunda et al., 2013). Bioethanol is considered to have the greatest potential as an alternative biofuel. It is mainly produced by certain yeasts, such as Saccharomyces cerevisiae (Town et al., 2014). Additionally, biobutanol, a straight-chained fourcarbon alcohol, is used as an advanced biofuel due to its greater intersolubility, higher heating value, greater safety, easier handling, higher viscosity, and lower corrosiveness than other biofuels (Xin et al., 2014; Zheng et al., 2015). Solventogenic Clostridium species, such as C. acetobutylicum and C. beijerinckii, are commonly used biobutanol producers ( Jiang et al., 2017; Li et al., 2016). The development of bioalcohol technology is often associated with unexpected energy crises or increases in oil prices. For example, food crops, such as sugarcane, were first used to produce bioethanol in Brazil in 1975 (Goldemberg et al., 2004). Corn, rice, and wheat were later used to produce bioalcohol in many countries, resulting in the controversy of competition between first-generation biofuels and the food market. Instead of using annual crops, if perennial herbaceous energy crops can be used efficiently, they have the potential to decrease the competition between food and fuel, the risk of land-use change, and other environmental threats ( Jiang et al., 2017; Rettenmaier et al., 2010). Handbook of Biofuels Production https://doi.org/10.1016/B978-0-323-91193-1.00005-6

Copyright © 2023 Elsevier Ltd. All rights reserved.

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Biomass is becoming extremely important due to a decrease in the abundance of fossil carbon sources and increasing environmental and social concerns related to their extensive utilization (Harnos et al., 2012). Biomass-derived alcohols are considered to be the sustainable alternative for internal combustion engines. Cellulosic alcohol may be produced from different types of lignocellulosic biomass (Cai et al., 2016; Qureshi et al., 2008). However, due to the complex structure of lignocellulose, most solventogenic microorganisms cannot directly use it. Therefore, pretreatment and hydrolysis techniques are required to hydrolyze lignocellulose to simple sugars. The high costs of the pretreatment process have hindered the industrial application of these bioalcohols (Cai et al., 2016). Consolidated bioprocessing (CBP) is a promising strategy to achieve bioalcohol production from lignocellulosic feedstock. The process combines enzyme production, cellulose saccharification, and microbial fermentation in a single bioreactor (Xin et al., 2019). Previous studies have comprehensively investigated bioalcohol production from lignocellulose using single microbes. However, the resulting overwhelming metabolic stress places a burden on the metabolic performance of these microbes, leading to a relatively low efficiency of lignocellulose conversion. Co-cultivation systems offer an alternative strategy, as they provide a division of labor between different microbial species, which allows the completion of more complicated tasks and tolerance to more variable environments.

8.2 Types of bioalcohols Methanol, ethanol, 2-propanol, and butanol have the general chemical formula CnH2n+1OH, where n¼1 for methanol, n¼2 for ethanol, n¼3 for 2-propanol, and n¼4 for butanol, and low heating values of 20.094, 26.952, 30.680, and 34.366 MJ/kg, respectively (Wright et al., 2006). These alcohols can be generated in biorefineries from different biomass substrates. The heating value of a biofuel increases with an increase in the number of carbons in the chain. In the bioconversion system, monosaccharides, such as glucose, mannose, galactose, xylose, and arabinose, are key ingredients for the sustainable production of bioalcohols through fermentation.

8.2.1 Biomethanol Biomethanol is typically generated through the thermochemical pathway, with or without catalysts, although certain biological conversion approaches have also been used (Shamsul et al., 2014). The feedstock for methanol

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production may be any type of concentrated carbonaceous material, such as biomass, solid waste, coal, or even carbon dioxide. During the process, carbonaceous feedstock is converted into biogas through gasification and then used to synthesize methanol after purification and processing (Nakagawa et al., 2007). The key benefits of biomethanol are that it is a distributed energy source for power generation (Suntana et al., 2009) and can be easily broken down into carbon dioxide and water vapor after combustion (Shamsul et al., 2014). Biomethanol has been successfully produced in lab-scale reactors, but the requirement for high temperatures and the low conversion efficiency (it requires a large amount of biomass) has prevented wider application of the technology at the industrial scale (Shamsul et al., 2014).

8.2.2 Bioethanol Bioethanol is currently the most commonly used biofuel globally (Ullah et al., 2015). It has many desirable features as an alternative to petroleum (Akhlaghi et al., 2015), which may facilitate a smoother transition from petroleum to bio-based industries (Chundawat et al., 2007). Unlike other bioalcohols, which are still under investigation, bioethanol has emerged as a potential transportation fuel and has been used as an oxygenated compound to replace methyl tertiary butyl ether. Currently, the majority of bioethanol is generated from food crops (Singh et al., 2014). It is expected that new-generation biorefineries, which aim to use waste-derived feedstocks, may reduce the need for food-crop-based bioethanol in the near future. To ensure that second-generation bioethanol production is commercially feasible, many efforts have been made to decrease the capital and operating costs of the biorefinery processes (Pasha et al., 2007). For example, the pretreatment process of softwood forestry residues has been improved to remove only a limited amount of lignin, while simultaneously modifying the surface properties of lignin residues in the fiber (Leu and Zhu, 2013). The cooking temperature has also been decreased to limit the production of growth-inhibiting compounds (Zhang et al., 2014), and thermotolerant yeast has been used in simultaneous saccharification and fermentation (SSF) processes to reduce the negative effects of different temperature optima on ethanol yield (Krishna et al., 2001). Bioethanol is produced from the hydrolysates of lignocellulosic substrates after pretreatment and enzymatic hydrolysis. After the conversion

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of carbohydrates, hexose and pentose can be fermented by commercial or engineered S. cerevisiae or other microorganisms. Depending on the feedstock and pretreatment processes, various types of growth-inhibiting compounds have been found to inhibit the fermentation process. For example, hydroxymethylfurfural (HMF) and furfural formed from the dehydration of mono-sugars inhibit enzymatic activities and glycolysis, resulting in decreased ethanol yield and a longer lag phase for the yeast (Almeida et al., 2007). Synergistic effects have also been reported when HMF and furfural coexist in the hydrolysate, and furfural has been demonstrated to be more toxic than HMF (Taherzadeh et al., 2000). Carboxylic acids generated by the deacetylation of hemicelluloses and HMF may cause anion depletion, reduce the uptake of aromatic amino acids, and thereby reduce biomass conversion and ethanol yield (Almeida et al., 2007). The acids that participate in and form a synergistic effect with water molecules to accelerate the hydrolysis reaction are often hydrophilic small molecular acids, including acetic acid, formic acid, and levulinic acid (Lundgaard et al., 2004). Synergistic effects have not been found among acetic acid, formic acid, and levulinic acid, but formic acid, the product of HMF degradation, has been found to be more toxic than levulinic acid and acetic acid, possibly due to its smaller particle size (Larsson et al., 1999). Phenolic compounds originating from lignin have been found to reduce biomass conversion and growth rate, but they have no significant effect on ethanol yield. Other studies have shown that furfural and phenolic compounds may damage the cell membrane, and many inhibitors can unwind or break DNA and RNA (Almeida et al., 2007; Ibraheem and Ndimba, 2013). Several techniques have been developed to remove inhibitors from hydrolysates based on two approaches. The physiochemical approach aims to remove inhibitors using physical techniques or chemicals, namely evaporation (Cantarella et al., 2004), pH modification (Millati et al., 2002), filtration, adsorption, or ion exchange (Krishna et al., 2001). The physical techniques are effective, but some of them are costly. Biological detoxification techniques include yeast adaptation (Huang and Yuan, 2015), in situ microbial detoxification using fungi and other species (Okuda et al., 2008), and enzymatic treatment (Palmqvist and Hahn-Hagerdal, 2000). Among the biological techniques, continuous yeast adaptation may be an applicable alternative due to the benefits of low cost, high yield, and process stability.

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8.2.3 Biobutanol Biobutanol has a key benefit of a higher heating value (similar to gasoline) than bioethanol, due to its longer chain. Biobutanol is commonly produced through acetone-butanol-ethanol (ABE) fermentation by Clostridia, using a process that can be traced back to the 1940s and 1950s (Ibrahim et al., 2017; Jones and Woods, 1986). Solventogenic Clostridium species, such as C. acetobutylicum and C. beijerinckii, are commonly used biobutanol producers ( Jiang et al., 2017; Li et al., 2016). Recently, an adapted C. acetobutylicum strain was shown to produce 19.1 g/L of butanol from glucose in a batch fermentation, which is close to the peak butanol tolerance of Clostridia (Xue et al., 2012). Product inhibition of fermenting microorganisms is a key challenge that has hindered progress in the commercialization of the butanol fermentation process. Efforts have been continuously made to overcome this challenge through biochemical approaches. For example, a thin polydimethylsiloxane layer improves butanol separation and efficiently relieves the toxicity of butanol to microorganisms and improves butanol production in continuous butanol fermentation processes (Chen et al., 2019). In another study, a final yield of 122.4 g/L of butanol was obtained using a membrane-based continuous fermentation process from cassava as the feedstock (Li et al., 2014).

8.2.4 Biopropanol Biopropanol is another bioalcohol that is similar to biobutanol and has a high heating value. Isopropanol may be dehydrated to produce propylene, a product that can be used to esterify fats and oils for the production of biodiesel. Isopropanol can be produced by Clostridium or genetically modified Escherichia coli strains. Commercial isopropanol production from biomassderived glucose is highly desired. However, the highest concentration of biopropanol obtained from fermentation experiments is 4.9 g/L (Atsumi and Liao, 2008), which is still much lower than the yield of bioethanol. Further research is expected to increase the efficiency and yield of this process before its widespread application.

8.3 Bioalcohol production from lignocellulose hydrolysate Cellulose, hemicellulose, and lignin make up 38%–50%, 23%–32%, and 15%–25%, of lignocellulose, respectively (Gottumukkala et al., 2013;

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Rajendran et al., 2018; Wen et al., 2014a). One of the most essential parameters affecting the economics of the bioconversion process is the chemical composition of the biomass and the molecular structure of its components (Mika et al., 2015). The cell wall of lignocellulosic biomass is a three-dimensional multi-layered structure consisting of three basic components—lignin, cellulose, and hemicellulose—and other minor elements. The physiochemical structure of the plant cell wall has been extensively studied (Sjostrom, 2013). In short, cellulosic fibers serve as the backbone of the cell wall; hemicellulose provides the connection between different fibers and lignin; and lignin is a set of natural aromatic compounds that form a complex structure to cover the fibers. The chemical compositions of selected types of biomass are listed in Table 8.1. Herbaceous biomass contains a higher amount of ash, extractives, and xylan than woody biomass. A higher lignin content provides extra protection for woody structures and prevents biodegradation or enzymatic hydrolysis, thus increasing the cost of the bioconversion process. In a biomass-to-biofuel biorefinery, cellulose and hemicellulose are the major sources of carbohydrates for fermentation, and lignin is a by-product of the biomass-to-bioethanol process. Lignin residues are commonly used as solid fuels and can be combusted for heat and energy (Zhu and Zhuang, 2012), but they also serve as a valuable source of renewable aromatic compounds.

8.3.1 Processing of biomass Biomass may be converted into bioalcohol via two main types of processes: thermochemical and biochemical/biological conversion (Huang and Yuan, 2015). Table 8.1 Chemical compositions of selected types of biomass. Feedstock

Ash

Extractives Lignin Glucan Xylan Mannan Reference

Straw

2–12

11.46

Bagasse Corn stover Lodgepole pine Douglas fir Douglas fir

14

41.4

26.0

0.9

2.0–5.0 5.59

19.5

41.8

24.8

0.93

5.80

13.54

18.46 31.24

16.70 –

–

–

29.1

39.8

6.8

10.1

0.4 0.8

– –

32.0 32.3

44.0 37.7

2.8 6.3

11 8.2

Carvalho et al. (2015) Carvalho et al. (2015) Ouellet et al. (2011) Luo et al. (2010) Russell (1984) Leu et al. (2013)

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The bioethanol production processes for different types of biomass are shown in Fig. 8.1. The processes typically contain four major steps, i.e., feedstock pretreatment, hydrolysis (or saccharification), fermentation, and separation. The pretreatment process is a crucial step to accelerate the saccharification process (Ntaikou et al., 2021). To remove as much lignin as possible and increase the availability of lignocellulose to enzymes, many pretreatment strategies have been developed, including acid or alkali hydrolysis, steam explosion, and organic solvent pretreatment processes (Wang et al., 2015). Depending on the type of biomass feedstock used, each of the unit processes may differ significantly in terms of treatment conditions or the

Fig. 8.1 Bioethanol production from different types of biomass.

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chemicals used. For example, steam explosion (Kaar et al., 1998) and ammonia fiber explosion (Alizadeh et al., 2005) are suitable approaches for the pretreatment of agricultural residues as agriculture residues have lower lignin content and “looser” physical structure. Organosolv and sulfite pretreatment for robust saccharification have been used to treat woody biomass because of the superiority of these methods for delignification (Pan et al., 2005). The hydrolysis of cellulose is usually performed by one of two approaches, i.e., acid hydrolysis or enzymatic hydrolysis. Concentrated acid is used to dissociate the biomass completely, but it is not widely used in industry anymore due to high operational costs, the need for acid recovery, equipment corrosion, and the decomposition of the sugar products. Diluted acid is more widely used in the hydrolysis of cellulose. Furthermore, diluted acid-based hydrolysis process not only requires the correct equipment but also results in by-products, such as furfural, HMF, and ferulic acid, which are major inhibitors of microbial growth and fermentation (Baral and Shah, 2014; Kumar and Murthy, 2011; Rasmussen et al., 2014). For example, in a fermentation using a semi-defined medium made with 60 g/L of glucose, C. beijerinckii P260 produced 21.06 g/L of ABE, with a productivity of 0.31 g/L/h. In contrast, an obvious decrease in yield is observed when dilute sulfuric acid pretreated barley straw hydrolysate is used as the feedstock, with only 7.09 g/L of ABE obtained at a productivity of 0.10 g/L/h (Qureshi et al., 2010). Compared with acid hydrolysis, enzymatic hydrolysis is a preferable approach to convert biomass into fermentable sugars. Cellulose and hemicellulose can be hydrolyzed by an enzyme complex generated from fungi or other microorganisms. The efficiency of enzymatic hydrolysis may be affected by many factors associated with both the substrates and the enzymes. For example, the accessibility of the cellulase to cellulose directly affects the effective binding of the reagents and the catalyst (Leu and Zhu, 2013). Nonproductive binding of the enzyme to lignin and the loss of enzyme activity may also result in lower yield and production rates of the final products (Zhang and Lynd, 2004). The degree of polymerization and cellulose crystallinity also affect the final efficiency of enzymatic hydrolysis. Generally, enzymatic hydrolysis is combined with a simple chemical pretreatment that disrupts the cross-linked lignocellulose matrix and improves the efficiency of enzymatic hydrolysis (Singh et al., 2016). For example, 17.32 g/L of sugar has been achieved from 38.1 g/L of wheat straw using enzymatic hydrolysis with enzymes derived from Trichoderma viride (Wang et al., 2013).

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The resulting sugars were then fermented by C. acetobutylicum ATCC824 to produce butanol, with a final butanol concentration, yield, and maximum productivity of 7.05 g/L, 0.155 g/g, and 0.141 g/L/h, respectively. Additionally, to avoid the substrate inhibition effects on enzymatic hydrolysis, the pretreatment process and fermentation steps can be separated using a separate hydrolysis and fermentation process (SHF). Alternatively, simultaneous saccharification and fermentation (SSF) can also be used, which has been widely used in ethanol production (Bertacchi et al., 2022).

8.4 Bioalcohol production from lignocellulose via CBP using single microbes CBP is a multistep process performed in one bioreactor. It involves complete hydrolase production, enzymatic hydrolysis, and microbial fermentation. Currently, two strategies are used for CBP: the “recombinant strategy” and the “native strategy” (Fig. 8.2). The recombinant strategy uses microorganisms that produce a high yield of bioalcohol and cannot directly use cellulose, but can be genetically engineered to degrade cellulose. Meanwhile, the “native strategy” involves microorganisms that hydrolyze cellulose naturally, but with low bioalcohol productivity. Due to the complexity of cellulosic degradation systems, the native strategy is preferred. Compared with some mesophilic microorganisms, thermophilic microorganisms show great potential in CBP for the direct production of bioalcohol from lignocellulose, due to their high lignocellulose hydrolysis rate (Fu et al., 2016). In general, microbial contamination is an important problem in bioalcohol

Fig. 8.2 Two strategies to achieve consolidated bioprocessing (CBP) in mono-culture systems. EMP, Embden-Meyerhof-Parnas pathway, also called glycolytic pathway; PPP, Pentose Phosphate pathway assemblies of cellulases.

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production, especially in industrial processes. This can be avoided with the relatively high temperatures used in thermophilic fermentation processes. Moreover, compared with 37°C, higher temperatures have lower gas solubility, which further helps maintain an anaerobic environment. In addition, thermophilic fermentation reduces cooling costs (Tukacs-Hajos et al., 2014) and facilitates the recovery of downstream products (Bhalla et al., 2013).

8.4.1 Bioethanol production from cellulose via CBP Few wild-type bacterial strains, mainly thermostable anaerobic bacteria and Clostridium spp., metabolize lignocellulose to produce bioethanol. C. thermocellum is a potential thermophilic CBP host and has been widely used for bioethanol production. C. thermocellum degrades acid-pretreated hardwood and crystalline cellulose at elevated temperatures (e.g. 50–60°C), with up to 90% conversion of the substrate to ethanol through continuous culture (Taylor et al., 2009). The complete genome sequence of C. thermocellum was first reported by Feinberg et al. (2011), and the first successful transformation of this species was performed in 2006 (Tyurin et al., 2006). Akio et al. (2014) used wide-type C. thermocellum to ferment pretreated Hinoki cypress and Eucalyptus hardwoods under anaerobic conditions and achieved ethanol production rates of 79.4 and 73.1 mg/g substrate, respectively. With increasing research in recent years, more natural lignocellulosic hydrolysis strains have been discovered and applied in the production of ethanol. For example, Sigurbjornsdottir and Orlygsson, (2012) successfully isolated Thermoanaerobacterium sp. AK54 from hot springs in Iceland. This strain metabolizes substrates, such as cellulose, and produces ethanol and biohydrogen, with production reaching 1.13 and 0.11 g/L, respectively (Sigurbjornsdottir and Orlygsson, 2012). Fungi have the ability to hydrolyze cellulose and chitin, and they also have good tolerance to many inhibitors, such as carboxylic acids and aldehydes. During the construction of various cellulose-metabolizing genetically engineered strains, various cellulose hydrolase genes from fungi are often regarded as the most suitable candidate genes. Therefore, the use of filamentous fungi for bioethanol production provides more options to expand the available substrates and microbial species used in CBP systems. Inokuma et al. (2013) identified a natural strain of Mucor circinelloides that metabolizes N-acetylglucosamine (N-GlcNAc) and chitin. This strain was found to metabolize 50 g/L of N-GlcNAc to produce 18.6 g/L ethanol after 72 h

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of fermentation under anaerobic conditions, with a productivity of 0.75 g/L/h (Inokuma et al., 2013). It is also worth noting that other species of filamentous fungi have applications in CBP fermentation. For example, engineered Aspergillus oryzae is well developed and used in traditional Japanese bio-based chemical fermentation plants and, more recently, in research on ethanol production via CBP (Hossain, 2013). Although some wild-type microbes possess strong hydrolase production capability, most of them have relatively poor ethanol synthesis ability and ethanol tolerance. In contrast, some microbes have an excellent ability to produce ethanol, but the introduction and successful expression of heterologous hydrolases are major obstacles. Therefore, the construction of an ideal engineered strain for cellulose fermentation via CBP has become a focus of research. Georgieva et al. (2008) used continuous immobilization reaction system technology to study ethanol fermentation under high-temperature conditions via CBP and found that the tolerance of the anaerobic thermophile Thermoanaerobacter mathranii (Δldh) to acetic acid and inhibitors was improved to 10 g/L, which greatly improved the stress resistance and inhibitor tolerance of the strain. Concurrently, the substrate conversion rate reached 68%–76%, and the yield of ethanol reached 0.39–0.42 g/g, when monosaccharides were used as the carbon source (Georgieva et al., 2008). In addition, Brown et al. (2011) found that mutating the alcohol dehydrogenase (adhE) gene of C. thermocellum promoted ethanol tolerance in this strain, resulting in an increase in ethanol concentration by 5–8 times to 50-80 g/L. Metabolic engineering of ethanol-producing strains to utilize lignocellulosic biomass remains a research hotspot. For instance, Munjal et al. (2015) constructed an E. coli strain capable of metabolizing cellulose by overexpressing the endoglucanase (Endo5A) and glucosidase (Gluc1C) genes from a heterologous Paenibacillus sp. This strain was able to produce ethanol under anaerobic conditions, at a conversion rate of 85% of the maximum theoretical value (Munjal et al., 2015). Moreover, the overexpression of three hemicellulose hydrolases and three key xylose metabolism enzymes in S. cerevisiae through surface display endowed the engineered strain with the ability to metabolize hemicellulose in one step and enabled the production of ethanol, with a productivity of 0.37 g/(L h) (Sakamoto et al., 2012). It has been reported that Mascoma Corporation in the United States has made a major breakthrough in ethanol production from cellulose via CBP (Anonymous, 2011). The CBP process is beneficial in reducing the cost of the biotransformation process and is the future direction for development in the field.

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8.4.2 Biobutanol production from lignocellulose via CBP Much effort has also been made to enhance the production of butanol from lignocellulose via CBP. C. cellulolyticum is a mesophilic cellulose-degrading bacterium capable of secreting cellulosomes to efficiently degrade cellulose, but it lacks a butanol synthesis pathway. The introduction of a CoAdependent pathway may realize the synthesis of butanol from cellulose via CBP. The thiolase (atoB), 3-hydroxybutyl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA hydrogenase (bcd), and butyraldehyde/butanol dehydrogenase (adhE2) genes from C. acetobutylicum were introduced into C. cellulolyticum, resulting in the synthesis of 0.12 g/L of butanol (Lin et al., 2015). However, due to the complexity of the multi-enzyme switching system, the synthesis of high concentrations of butanol by introducing a butanol synthesis pathway into C. cellulolyticum still poses major challenges. C. cellulovorans secretes a variety of hydrolases, including cellulases, xylanase. More importantly, compared with C. cellulolyticum, which requires the introduction of multiple related genes to achieve butanol synthesis, C. cellulovorans can be transformed by adhE in one step to realize the conversion of butyrate to butanol. Therefore, C. cellulovorans is a better candidate than C. cellulolyticum for the synthesis of butanol from lignocellulose via CBP. By introducing adhE2 from C. acetobutylicum into C. cellulovorans, 1.42 g/L of butanol was synthesized, while the concentration of butyric acid was reduced by 80%. However, the production of butanol in this process was still relatively low, and there was a high level of acetic acid accumulation (1.60 g/L). To further increase the production of butanol, the ctfA, ctfB, and adc genes were further overexpressed to reutilize acids for the production of alcohol, and 2.27 g/L of butanol was obtained ( Jang et al., 2014; Lee et al., 2009; Lehmann et al., 2012). Metabolic engineering is simpler in C. cellulovorans than in C. cellulolyticum, and it provides a better basis for the realization of CBP. The potency of butanol can be further increased by removing by-products, including acids and ethanol. Thermoanaerobacterium spp. are excellent hemicellulose degraders that have been reported to directly synthesize butanol from xylan. Over the past decade, using T. thermosaccharolyticum as a microbial host for bioalcohol production from hemicellulose has attracted increasing attention. T. thermosaccharolyticum W16, which was isolated by Ren et al. (2010), grows in corn stover hydrolysate (containing glucose and xylose) at 60°C and produces 0.074 g/L of butanol. Newly isolated Thermoanaerobacterium sp. strain M5 produces 1.17 g/L of butanol from xylan via CBP after the optimization

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of the culture conditions. In general, butanol production by Thermoanaerobacterium sp. still occurs at a low level, and further genetic modification and process optimization are needed to improve the final butanol yield.

8.5 Bioalcohol production from lignocellulose via CBP through co-cultivation Mono-culture fermentation to achieve bioalcohol production from lignocellulose via CBP remains a challenge due to the heavy metabolic burden. With the continuous development of synthetic biology, research on alcohol production via CBP using microbial co-culture systems has made great progress ( Jiang et al., 2018) (Fig. 8.3). A co-cultivation system should possess stable metabolic bioalcohol production capacity through utilizing different types of mixed sugars hydrolyzed from lignocellulosic biomass. For example, co-cultivation of the cellulolytic bacterium C. thermocellum, which only metabolizes hexose, with other sugar-metabolizing microorganisms effectively reduces substrate competition between different microorganisms to maximize ethanol production. Xu and Tschirner (2014) co-cultured the cellulolytic strain C. thermocellum, and C. thermolacticum, which metabolizes pentose, and found that the co-cultivation system reduced the fermentation time and increased the ethanol yield compared with the mono-culture systems. To further realize the direct conversion of lignocellulose to ethanol, Shin et al. (2010) constructed an E. coli co-cultivation system for the direct conversion of hemicellulose to ethanol, resulting in a final ethanol concentration of 2.84 g/L, which is 55% of the theoretical yield. In this co-cultivation system, an E. coli strain was engineered to hydrolyze hemicellulose to xylooligosaccharides by the co-expression of two hemicellulase genes. Another E. coli strain was engineered to overexpress xylo-oligosaccharide-utilizing enzymes for the conversion of xylo-oligosaccharides to ethanol. This co-cultivation system distributed the metabolic burden through the extracellular and intracellular expression of different functional enzymes, resulting in increased ethanol production compared with the mono-culture systems. In addition, cellulase systems may also be established in microbial co-cultivation systems. For example, a novel dual microbial Bacillus/yeast system was developed for cellulosic ethanol production. Recombinant B. subtilis used in this system carried eight cellulosome genes derived from C. thermocellum, including one scaffold protein gene (cipA), one cell surface

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Fig. 8.3 Alcohol production from lignocellulose via consolidated bioprocessing through microbial co-cultivation.

anchor gene (sdbA), two exoglucosidase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). The partner Kluyveromyces marxianus KY3-NpaBGS carried the glucosidase (NpaBGS) gene from a rumen fungus. Using this system, 9.5 g/L of ethanol was obtained from 20 g/L of cellulose (Ho et al., 2012). Likewise, Zuroff et al. (2013) further established a co-cultivation system comprising C. phytofermentans and S. cerevisiae, which were responsible for cellulose hydrolysis and ethanol production, respectively. Overexpression of the glucosidase gene in S. cerevisiae resulted in the intracellular hydrolysis of cellodextrin, and the expression of an intermediate cellodextrin transporter in S. cerevisiae, downstream in the process, facilitated the connection of separate pathway modules. This co-cultivation system produced 22 g/L of ethanol from 100 g/L of cellulose. Considering the complexity of lignocellulosedegrading enzymes, co-cultivation of cellulolytic microorganisms with ethanologenic microorganisms is a facile and flexible method to produce ethanol from lignocellulose via CBP. Although cellulolytic C. thermocellum is a model organism for CBP, its application is limited due to low ethanol production. Hence, another way to increase ethanol production and maximize biomass utilization is to co-cultivate C. thermocellum with other ethanol-producing species. All currently reported co-cultivation systems show higher ethanol production than mono-culture systems. C. thermocellum has been co-cultured with many other thermophilic bacteria, including T. thermosaccharolyticum, C. thermohydrosulfuricum, T. ethanolicus, Geobacillus stearothermophilus, and

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T. brockii (Taylor et al., 2009). Considering its potent cellulose-degrading ability, He et al. (2011) co-cultured C. thermocellum with non-cellulolytic Thermoanaerobacter strains (X514 and 39E), resulting in efficient ethanol production. After co-cultivation, ethanol production increased significantly by 194%–440%, to 7.56 and 6.59 g/L for strains X514 and 39E, respectively. In this co-cultivation system, C. thermocellum performs cellulolysis, while Thermoanaerobacter sp. is responsible for ethanol production due to its efficient ethanol production capacity. The interaction between the two strains mainly occurs through the exchange of intermediate metabolites. Furthermore, co-cultured engineered C. thermocellum (with the hpt, ldh, and pta genes knocked out) and T. saccharolyticum produce a high ethanol titer of 38.1 g/L from 92.2 g/L avicel, which is approximately 80% of the theoretical maximum value (Argyros et al., 2011). As mentioned above, most Clostridium spp. cannot directly utilize complex substrates, such as lignocellulose, due to the lack of polysaccharidedegrading enzymes. Therefore, the construction of microbial consortia may be an ideal strategy to realize the direct production of butanol from renewable feedstock. For example, the co-cultivation of C. thermocellum with other butanol-producing microorganisms provides an alternative method to obtain butanol from lignocellulose via CBP. Chimtong et al. (2014) showed that T. thermosaccharolyticum NOI-1 can be co-cultured with C. thermocellum NKP-2 at 60°C. This was the first report on the co-culture of these two thermophilic microorganisms. The butanol content was approximately 2.5 mM, which was increased by 2.1 times compared with mono-culture (Chimtong et al., 2014). Another co-cultivation system using a combination of C. saccharoperbutylacetonicum and C. thermocellum also successfully produced butanol (Nakayama et al., 2011). In this system, C. saccharoperbutylacetonicum strain N1-4 was inoculated after C. thermocellum was cultivated at 60°C for at least 24 h. A final butanol concentration of 7.9 g/L was obtained from 40 g/L Avicel after 9 days at 30°C. In addition, Wen et al. (2014b) co-cultured C. thermocellum and C. beijerinckii to produce butanol from an alkaline extract of corncob and achieved a butanol titer of 10.9 g/L (i.e., 19.9 g/L ABE) after 200 h of fermentation. In this co-cultivation system, sugars accumulated from the C. thermocellum-mediated hydrolysis of alkali-extracted corncob and were then utilized by both strains without the addition of butyrate (Wen et al., 2014b). The butanol synthetic pathway is more complex than the ethanol production pathway. Introducing a butanol synthesis module into model microorganisms, such as E. coli, may aggravate their metabolic stress.

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Recently, Saini et al. (2015) constructed an E. coli-E. coli system in which the butanol biosynthetic pathway was divided into butyrate production and butyrate conversion modules and incorporated into separate E. coli strains. This co-cultivation system was able to reduce metabolic stress and maintain the desired redox balance, resulting in a final production of 5.5 g/L butanol, which was two times more than the amount produced using a mono-culture (Saini et al., 2015). It is worth mentioning that the by-product acetate could freely cross the cell membrane and circulate between the upstream and downstream E. coli strains to facilitate the interconversion of butyrate and butyryl-CoA. In conclusion, in addition to being constructed using the same species, microbial consortia may be constructed using species of different genera, such as fungus-bacterium consortia. Microbial consortia have the advantages of realizing the direct conversion of renewable resources into bioalcohol, improving substrate utilization, increasing production and yield, and reducing process costs. However, although microbial consortia exhibit great advantages for use in CBP, they remain far from being ready for industrial applications, due to the large differences in growth conditions of different strains, the existence of competition between strains, and unclear cooperation mechanisms. Therefore, as an immature but promising technology, more research is needed to develop more robust and stable microbial communities for bioalcohol production.

Acknowledgments This work was funded by the National Key R&D Program of China (2018YFA0902200), the National Natural Science Foundation of China (22008113, 21978130, 21978129, and 22078151), and the Jiangsu Province Natural Science Foundation for Youths (No. BK20200683).

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CHAPTER 9

Production of biogas via anaerobic digestion Ioanna A. Vasiliadou, Kleio Gioulounta, and Katerina Stamatelatou Department of Environmental Engineering, Democritus University of Thrace, Xanthi, Greece

9.1 Introduction Anaerobic digestion (AD) is a biochemical process performed by the concerted action of several types of microorganisms, which convert, under anaerobic conditions, the organic wastes into a gaseous mixture consisting mainly of methane and carbon dioxide (biogas) (Kleinsteuber, 2018; Batstone et al., 2002). AD occurs naturally in many environments devoid of oxygen, such as the bottom of lakes, swamps, landfills, and the intestine of animals. However, the term “anaerobic digestion” is usually used to describe the accelerated process of the naturally evolved bioprocess in an artificial environment of a closed vessel. The first anaerobic digester demonstrating the anaerobic technology was built in Bombay, India, in 1859 (Meynell, 1976). In Exeter, England, in 1895, the biogas recovered from a sewage treatment plant was used to fuel street lamps (McCabe, 1957). In the 1930s, the development of microbiology tools brought up further improvement in the AD technology through identification of the anaerobic bacteria and the conditions favoring and/or limiting the process efficiency (Buswell and Hatfield, 1936). Since then, numerous anaerobic applications have been developed worldwide, in order to cost-effectively reduce the volume of the biomass such as sewage sludge, organic fraction of municipal solid waste, manure, and lignocellulosic biomass and to recover renewable energy in the form of biogas (Weiland, 2010). AD stabilizes the waste biomass providing significant environmental benefits, such as reduction of air and water pollution or contamination from manure pathogens, prevention of biomass putrefaction and acidification, and mitigation of greenhouse gas emissions (Cu
ellar and Webber, 2008). The produced biogas consists of methane (CH4 50%–70%), carbon dioxide (CO2 30%–50%), and small amounts of other components (i.e., ammonia, water vapor, hydrogen sulfide, carbon monoxide) and can be used for the

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production of electricity and heat (Angelidaki et al., 2018). The removal of CO2 from biogas increases the CH4 content and enhances the calorific value of biogas (Awe et al., 2017). The methane-rich biogas (95% v/v) can be directly used as a fuel in vehicles or injected into the natural gas grid (Petersson and Wellinger, 2009). On the other hand, the semisolid residual of AD conversion, the digestate combines fertilizing and conditioning properties and is used as an amendment in soil. Therefore, there is an increased interest in AD application in many countries particularly in European Union (EU), which is supported by the national legislations. There was a steady growth in the number of biogas plants with an average rate of 18% by the end of 2016 in the EU. In 2017, the total installed electric capacity from biogas plants reached a total of 10,532 MW grown by 5% (European, 2018; European Biogas Association, 2016). In 2018, the share of renewable energy in transport and the share of biomethane in gas-fuelled cars accounted for 8.6% and 17% in EU, respectively (European Biogas Association, 2020). The European biomethane sector is developing rapidly, since the number of biogas separation units to biomethane has risen in recent years, from 187 plants in 2011 up to a total of 540 plants in 2017, while at the same year the biogas plants operating in Europe were 17,430(European, 2018). Based on European Union’s strategy for biofuels (EU Commission, 2014), the compressed/liquified natural gas is projected to be at a 5% of transport fuels in 2025, and biomethane, which already accounts for 25% of CNG/LNG fuels, is expected to exceed 60% by 2030. In order to achieve maximum efficiency of AD, it is imperative to optimize the process parameters, to develop efficient digesters, to improve the control and monitoring methods, and to appropriately examine/handle the feedstock’s characteristics (Rawoof et al., 2021; Castellano-Hinojosa et al., 2018). Moreover, an insight on the microbial community involved in waste conversion to energy as well as its response to several process parameters offers the opportunity to control, engineer, and optimize the biogas process (Hassa et al., 2018). By ensuring the stability and sustainability of biogas systems, the multifunctionality of AD process, which interlinks wastes’ treatment, environmental protection and electricity, heat and biofuel production, perfectly fits into the circular economy concept (Fagerstr€ om et al., 2018). The purpose of this chapter is to critically review the traditional approaches and recent developments and perspectives for the AD. Therefore, the factors affecting the process, such as temperature, pH, feedstocks, etc., are extensively reported. The main equations and principles of

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mathematical modeling approaches used as control and monitoring tools of the process are presented. Biogas bio-upgrade applications and reactor’s technological improvements used for enhancing the efficiency of AD are also included. Finally, the transition of AD and biogas production into circular energy economy is discussed.

9.1.1 Process steps of anaerobic digestion AD is considered a reliable waste-to-energy process relying on complex metabolic pathways of several types of facultative or strict anaerobic microorganisms (Kleinsteuber, 2018). Hydrolytic and fermentative bacteria, syntrophic bacteria, and methanogenic archaea, which have different growth rates and sensitivity to environmental conditions (pH, temperature, etc.), closely interact through distinctive and consecutive biochemical stages/steps toward the degradation of organic waste materials to methane and carbon dioxide (biogas). The AD process involves the steps of disintegration, hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Fig. 9.1) (Batstone et al., 2002; Saha et al., 2020). Disintegration: The complex biomass (e.g., sewage sludge) is disintegrated into organic polymers such as carbohydrates, proteins, and lipids. Disintegration includes several steps such as lysis, nonenzymatic decay, phase separation, and physical breakdown (Batstone et al., 2002). Hydrolysis: The insoluble organic polymers (carbohydrates, proteins, lipids) are hydrolyzed to their respective soluble monomers and oligomers (mono- and oligosaccharides, amino acids, long-chain fatty acid, peptides) (Kleinsteuber, 2018; Saha et al., 2020). During this step, hydrolytic fermentative bacteria (i.e., Clostridium, Bacillus, Bacteroides, Fusobacterium, Hydrogenoanaerobacterium, Anaerosalibacter, Eubacterium, etc.) secrete extracellular hydrolytic enzymes (i.e., cellulase, xylanase, protease, lipase, etc.) to facilitate the nutrient transport through the cell membrane. In order to improve hydrolysis, pretreatment schemes can be applied on the feedstocks. Pretreatment processes make the complex substrate matrix more amenable to biological hydrolysis. Hydrolysis is considered to be the primary rate-limiting step of AD since it evolves at a slow rate determining the substrate availability to the subsequent stages. Acidogenesis: It is conducted by acidogenic fermentative bacteria (i.e., Clostridium, Bacillus, Tissierella, Streptococcus, Hydrogenoanaerobacterium, Enterobacteriaceae, Eubacterium), which are able to convert the hydrolyzed monomers and oligomers into short-chain carboxylic acids and fatty acids,

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Fig. 9.1 Process steps of anaerobic digestion and metabolic pathway of involved groups of microbes (Kleinsteuber, 2018; Saha et al., 2020; Pasalari et al., 2021).

alcohols, hydrogen, and carbon dioxide (Kleinsteuber, 2018). Acidogenesis is considered to be the fastest step in AD process (Saha et al., 2020). Since acid formation from sugars is usually accompanied by hydrogen production, carbohydrate-rich substrates can yield biogas with a high hydrogen content (e.g., >30%) under specific conditions (Diamantis et al., 2013; Stamatelatou et al., 2011). Acidogenesis occurs via hydrogenation and dehydrogenation depending on the microbial metabolism (Salama et al., 2019). The acidogenic bacteria grow at a high rate and can tolerate low pH values (Buswell and Hatfield, 1936; Weiland, 2010). As a result of their rapid

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growth, if the acidic products cannot be converted in the subsequent step, the pH may be decreased and the whole AD process may be inhibited. Acetogenesis: Acetate is produced via several pathways. Apart from the acidogenesis step, it is produced during the oxidation of volatile fatty acids (VFA) other than acetate (such as valerate, butyrate, propionate), alcohols, and other fermentation products by syntrophic proton-reducing bacteria that belong to Clostridiaceae, Syntrophomonadaceae, Syntrophaceae, Enterobacteriaceae, and Bacteroidia families. Long-Chain Fatty Acids (LCFAs) produced during lipid hydrolysis are also oxidized to acetate by syntrophic fatty acid oxidizers (SFAOs, Clostridium ultunense, Syntrophomonas, Syntrophus). The hydrogen is also formed in these reactions, but it is thermodynamically unfavorable. This means that the acetate production through oxidation can occur at low concentrations of hydrogen (10
6–10
4 atm), otherwise the other VFAs (valerate, butyrate, propionate) will accumulate and inhibit the process by lowering the pH. The hydrogen level can be kept at the desired level if it is scavenged by methanogens mainly, which interact with the acetogens in a syntrophic mode. Another type of acetogenesis is homoacetogenesis, which involves the CO2 reduction by H2, a conversion performed by the homoacetogenic bacteria (bacteria performing reductive acetogenesis). Homoacetogenesis seems to be favored in case of high levels of hydrogen and low levels of CO2 and alkaline environments (Agneessens et al., 2018); however, it is not a dominant pathway for hydrogen consumption in the conventional anaerobic environment of a biogas plant (Saha et al., 2020). Acetogenesis takes long time since acetogenic bacteria are slow compared with acidogens and generally they have long doubling times, i.e., days. Methanogenesis: Methanogenesis is the strict anaerobic respiratory pathway of AD performed by archaea affiliated to seven orders of Euryarchaeota (Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanocellales, Methanopyrales, Methanomassiliicoccales). Acetate, alcohols, H2, and CO2 produced during acidogenesis/acetogenesis constitute the substrates for methanogenesis. CH4 can be produced via three methanogenic pathways: (1) the reduction of carbon dioxide to methane through hydrogen utilizing methanogenic archaea (hydrogenotrophic methanogenesis), which use hydrogen as electron donor; (2) the oxidation of acetate to methane and carbon dioxide by acetoclastic methanogenic archaea (acetoclastic methanogenesis); and (3) the reduction of methyl compounds, such as methanol or methyl sulfides, to methane by methylotrophic methanogenic archaea (methylotrophic methanogenesis) (Kleinsteuber, 2018; Liu and Whitman, 2008). Acetoclastic methanogens are responsible for producing the 70%

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of methane in the biogas, being the most important methanogens. They are slow-growing microorganisms (doubling time is in the order of days) and are particularly sensitive to a number of parameters such as pH, O2 level, nutrient, and trace element concentrations. In cases of inhibition (e.g., at high ammonia concentration, (Bayrakdar et al., 2017)), acetate can be oxidized into H2 and CO2 by syntrophic acetate oxidizers (SAOs) in syntrophy with hydrogenotrophic methanogens (Kleinsteuber, 2018; Saha et al., 2020), directing methane production through hydrogenotrophic methanogenesis. The methane content of biogas depends on the substrate composition, particularly the oxidation state of the organic carbon found in the substrate; the more reduced the carbon in the initial substrate is, the more methane will be generated. However, on average, the biogas contains 60% of methane.

9.2 Factors affecting the AD process The anaerobic consortium consists of several microorganism groups with different physiology and response to environmental changes that coexist syntrophically or antagonistically. As a consequence, when the activity of one of the microorganism groups is inhibited, the growth rates of other microorganisms are affected, changing the population balance, and often causing a decrease in the process efficiency or even failure. It has been recognized that the most important factors affecting the anaerobic digestion process are the temperature, the pH, the presence of toxic or inhibitory substances, and the nature of the feedstock (composition, nutrients).

9.2.1 Temperature As in any biochemical reaction system, temperature strongly influences the microbial growth rate, the intracellular enzyme activity, and the syntrophic interaction of microorganisms and consequently, the methane yield (Liu et al., 2018; Wang et al., 2019; Ryue et al., 2020). Four different temperature ranges have been reported in the case of AD: psychrophilic (9–25°C), mesophilic (25–35°C), thermophilic (35–55°C), extreme thermophilic (55–70°C) (Pasalari et al., 2021). Although conventional AD is performed at mesophilic temperature ranges, the application of higher temperatures in the range of thermophilic levels accelerates the microbial growth rate and metabolism further (decreasing the retention time required in the digesters), enhances the bacterial diversity, improves hydrolysis and acidogenesis of recalcitrant feedstocks, and promotes pathogen inactivation (Ryue et al., 2020; Tian et al., 2018). However, compared with mesophilic conditions,

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thermophilic digesters require higher energy consumption and are less stable due to the higher sensitivity of microorganisms at this temperature level, the more severe inhibitory effect of ammonia on methanogens, the faster acidogenesis and accumulation of VFAs decreasing the pH (Pasalari et al., 2021; Ryue et al., 2020). The enzymes developed in a microorganism, after proper adjustment, can be tolerant to temperature changes. As a result, there are bacteria that can grow in more than one temperature range. However, in conventional thermophilic digesters, the methanogenic population has lower diversity, and daily temperature fluctuations of higher than 1°C may imbalance the syntrophic interactions risking the stability of the process (Westerholm and Schn€ urer, 2019). Hyper-thermophilic AD has been studied for the enhancement of H2 production (Schuchmann et al., 2018), the improved biogas production from co-digestion of kitchen waste and sludge (Lee et al., 2009) or waste activated sludge and fat, oil, and grease (FOG) (Alqaralleh et al., 2016). It can also be used as a pretreatment step for recalcitrant feedstocks such as sewage sludge and grass (Wang et al., 2014). Moreover, there are many novel hyperthermophilic archaea discovered, which can grow at temperatures higher than 100°C. These microorganisms have unique mechanisms of adaptation, effective DNA repair and replication mechanisms, and proteins, which are composed by a large number of small amino acids and large polar amino acids reinforcing their tertiary structure and enhancing their resistance to aggregation and unfolding (Nie et al., 2021). Psychrophilic temperatures are often imposed due to local climatic conditions in order to reduce the energy required for heating the bioreactor and the operating cost. However, the psychrophilic anaerobic digesters are unstable. They also have low biogas productivity due to the slow microbial activity and the extended methane losses with the effluent, as a result of the increased methane solubility in water. The process could be improved through the application of a high inoculum to substrate ratio using a well temperature-adapted seed culture as inoculum (Meher et al., 1994; Wei et al., 2014). The development of reactor technology enabling the retention of the microorganisms also contributes into making psychrophilic AD feasible (Stuckey, 2012).

9.2.2 pH, volatile, and long-chain fatty acids, free ammonia The pH affects the dissociation of weak acids and bases and, therefore, the formation of undissociated acids and bases, which can easily penetrate the cellular membrane changing the internal pH of the cells. The pH also

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influences the function of the extracellular enzymes and has an impact on the hydrolysis rate. In most cases, the anaerobic transformation of organic matter is achieved most efficiently at a neutral pH; however, many species though can grow at lower or higher pH values. Methanogenesis occurs in a pH range between 6 and 8.5 and is dramatically inhibited when the pH falls out of these limits. On the other hand, acidogens can grow and continue to produce acids at low pH values (Buswell and Hatfield, 1936; Weiland, 2010), intensifying the inhibitory conditions to the methanogens (Laiq Ur Rehman et al., 2019). However, it is known that methanogenesis can occur in extreme environments where very low or high pH values prevail such as swamps, hot springs, etc. Nutrient-rich feedstocks can be easily acidified into volatile fatty acids (VFAs) by acidogenic and acetogenic bacteria. VFAs’ accumulation occurs through the unbalanced coupling of the acid producers and consumers. In the case of low buffer capacity, the build-up of VFAs results in a decrease in the pH and promotes the reactor’s acidification further, causing inhibitory conditions for the methanogens and deteriorating the process (Akuzawa et al., 2011). High concentrations of acetic and butyric acid affect the activity of methanogens to less extent as compared with propionic acid. As a result, in anaerobic digesters with low buffering capacity, pH can be a reliable parameter for assessing the reactor’s state. However, in the case of high buffering capacity, the pH changes are small and cannot be associated with the acid level; therefore, VFAs are the only indicator to be taken into account for an effective process monitoring (Franke-Whittle et al., 2014). It is common that the acidogens producing a mixture of metabolic products switch their metabolism toward the formation of alcohols to avert any further pH decrease (Lowe and Zeikus, 1991; Huang et al., 1986; Gottschal and Morris, 1981). LCFAs, the product of lipid hydrolysis, are adsorbed onto the microbial surface even at very low concentrations and affect the cell wall or membrane functionality in terms of the molecule transfer and the protection mechanisms of the cells (Palatsi et al., 2009). Moreover, flotation and washout of biomass can occur as a result of the adsorption of LCFA (Sousa et al., 2009). Although thermophilic anaerobes are more susceptible than mesophiles to LCFA inhibition, they have the ability to recover faster due to their faster growth rates (Hwu and Lettinga, 1997). High levels of ammonia, produced by the degradation of protein, amino acid, and urea, mainly in nitrogen-rich substrates, can cause instability and low methane production in anaerobic digesters (Bonk et al., 2018; Lv et al.,

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2019). Total ammonia nitrogen (TAN) is needed at low levels (ca. 200 mg N L
1) for microorganisms’ growth, while its buffering capacity stabilizes the pH (Pasalari et al., 2021). However, TAN concentration higher than (>3000 mg NH4-N L
1) can cause inhibition to AD at any pH value. TAN is composed of ammonium ion (NH+4 ) and free ammonia nitrogen (NH3), while their proportions depend on pH and temperature. Ammonia inhibition is mainly caused by the nonionized form of ammonia, free ammonia, which can diffuse through cell membrane. Specifically, inside cell’s membrane, free ammonia is converted to NH+4 , absorbing protons, changing the intracellular pH. In order to balance this change of pH, efflux of K+ occurs via a K+/H+ exchange reaction, leading to energy consumption and inhibition of microorganism’s growth and inactivation of specific enzyme reactions (Sprott and Patel, 1986; Rajagopal et al., 2013). The methods studied to overcome ammonia inhibition are ammonia stripping and scrubbing, ultrasonication, microwave radiation, hollow fiber membrane contactor as well as the addition of biochar, zeolite, and/or trace metals (Krakat et al., 2017; Molaey et al., 2018; Kougias et al., 2013). However, stable biogas production at high ammonia concentrations is possible in continuous systems if accumulation of VFAs occurs, which would counterbalance the ammonia effect and maintain the pH at neutral levels (Banks et al., 2011a). It is noteworthy that, although, high TAN concentrations result in operational difficulties, microorganisms may adapt on these conditions by developing mechanisms of resistance (Kalamaras et al., 2020).

9.2.3 Feedstock composition Feedstock composition is another crucial parameter influencing the stability and the performance of AD (Laiq Ur Rehman et al., 2019). Feedstocks rich in proteins provide nutrients to the digestate, but may cause ammonia inhibition as a result of protein degradation. In carbohydrate-rich materials, the carbon-to-nitrogen (C/N) ratio may become too high for microbial activity. Complex carbohydrates due to low degradability of lignocellulosic materials can pose additional challenges, while easily accessible carbohydrates can cause acidification due to fast acidogenesis step (Westerholm and Schn€ urer, 2019). The C/N ratio of the feedstock is crucial for its biodegradability. A C/N ratio of 20–30 has been reported to be ideal for AD (Go´mez et al., 2006; Shi et al., 2018). Lipid-rich substrates are often used to boost biogas production since the biomethane potential of the lipids (1014 L/kg VS) is much higher than carbohydrates (370 L/kg VS) but

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may risk the stability of the process due to the LCFA inhibitory effect (Palatsi et al., 2009).

9.2.4 Trace elements Μacronutrients (P, N, K, Na, Mg, and Ca), as well as micronutrients or trace elements (TE: Mn, Zn, Fe, B, Co, Ni, Cu, Mo, Se, Al, V, and W), are vital for the growth and metabolism of anaerobic microorganisms. Τrace elements such as cobalt (Co), nickel (Ni), iron (Fe), molybdenum (Mo), manganese (Mn), and selenium (Se) are fundamental for anaerobic microorganisms’ enzymatic activity, especially for acetogens and methanogens. For instance, Fe can act as electron transport as Fe-S (Fe2S2, Fe3S4 clusters), keeping the balance of hydrogen sulfide production. In addition, Frh (F420-reducing hydrogenase) enzyme complex with an Fe-Ni active site elevates metal requirements (Wintsche et al., 2018; Yu et al., 2016). Likewise, Ni binds to the center of a porphyrin, which is unique to methanogens, while Ni-Fe enzymes are present in hydrogenases which oxidize H2 (Laiq Ur Rehman et al., 2019). Manganese (Mn) is an electron acceptor at the respiration process controlling the methyltransferase in methanogenic bacteria. The lack of Selenium (Se) seems to restrict the action of some methanogens and to decrease the anaerobic process, while Molybdenum (Mo) boosts the AD process (Chen et al., 2008; Myszograj et al., 2018). The speciation of TE may result in their low bioavailability, and this further influences the bioprocesses (Fahlbusch et al., 2018; Cai et al., 2018). Any deficit in the TEs can severely limit microbial activity and cause accumulation of fatty acids, resulting in process instability and lower methane production (Moestedt et al., 2016). The bioavailability of TE is mainly affected by the presence of carbonates and sulfates as well as the operational temperature (Cai et al., 2019). Sulfide produced through protein degradation forms complexes with metals, which decrease the uptake of TE by microorganisms. The most attractive strategy to overcome trace element deficiency in anaerobic digestors is via the external addition of micronutrients in the form of dissolved salts or nanoparticles (Laiq Ur Rehman et al., 2019; Antwi et al., 2017). However, the appropriate concentration of TE depends on temperature conditions, the type of digestion (single- or co-digestion), and the substrate (animal manures, solid substrates, organic substrates, etc.). Trace elements have a strong effect on the microbial composition and diversity of anaerobic digesters. For example, methanogenic communities

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abundant in Methanosaeta, Methanosarcina, and Methanomicrobiales, as well as bacterial phyla, such as Bacteroidetes, Firmicutes, Proteobacteria, and Chloroflexi, have been reported to be dominant in anaerobic digesters amended with TE (Pasalari et al., 2021; Westerholm and Schn€ urer, 2019).

9.3 Feedstocks used for biogas and fertilizer production Biogas can be produced by a variety of feedstocks, such as sewage sludge, food wastes, agro-industrial wastes, lignocellulosic biomass, and algae biomass. The availability and selection of the feedstocks may not always be optimal influencing the energy yield and the nutrient value of the digestate generated. It is well known that co-digestion of different materials achieves a more stable nutrient level and may balance the problems associated with different substrates, such as ammonia inhibition, foaming, and acidification (Xu et al., 2018).

9.3.1 Sewage sludge Sewage sludge is a semisolid residual from industrial or municipal wastewater treatment plants containing high organic load, microorganisms, and parasites. AD is widely applied for sewage sludge valorization and stabilization, reducing the risk of health problems caused by pathogens (Neumann et al., 2016). However, microbial cells in sewage sludge form a structure of complex flocs, making hydrolysis the rate-limiting step in AD systems. To overcome poor digestion efficiency, long solid retention time (>20 days) as well as sludge pretreatment is required. During pretreatment, flocs are disintegrated and cells are ruptured, releasing exploitable intracellular and extracellular materials (Xu et al., 2020). Pretreatment methods such as thermal, alkaline, ultrasonic, and biological treatment have been employed individually or in combination, in order to maximize CH4 yield toward the theoretical range (0.45–0.6 L CH4 gVS-1) (Mostafa et al., 2020). The selection of the pretreatment method should satisfy energy and environmental criteria. They are energy-consuming methods, and although they result in organic matter solubilization and increase of the rate and yield of methanization, it is not evident they are viable within a sewage treatment plant. Full-scale application of ultrasonic pretreatment showed it is an energy self-sufficient technology. Moreover, thermal pretreatment processes may require more energy, but they mostly consume thermal energy recovered from combined heat power engines. In this way, they are fully integrated in the sewage treatment plant (Cano et al., 2015; Carre`re et al., 2010). Apart from the energetic aspects, the overall feasibility of any pretreatment process should take into

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account more factors, such as the sludge disposal options, the environmental impact of consuming energy versus chemicals, etc. (Carballa et al., 2011).

9.3.2 Food waste Food waste (FW) disposal has become a major environmental problem all over the world. FW derives mainly from the production, transportation, storage, distribution, and consumption of foods. It is also a major component of the organic fraction of municipal solid waste (OFMSW). It is estimated that approximately one-third of all food produced for human consumption ends up in waste (Morone et al., 2019). Specifically, huge amounts of wastes coming from 26 to 50 million tons of food end up in landfills or combustion facilities in the United States and EU (Wang et al., 2018). The degradation of the environment and the stress brought by the growing global population combined with the increasing global demand for energy, chemicals, and materials make imperative to develop sustainable management schemes to reduce food loss and take advantage of unavoidable food waste (Lin et al., 2013). Despite the efforts made for reducing the preventable FW, there is a growing interest to valorize the unavoidable FW, which is rich in starch, fat, protein, and cellulose while its water content is high (up to 80%). Improvement of the methane yield can be achieved through thermal pretreatment resulting in the reduction of the lignocellulosic content and the solubilization of the carbohydrates. The high concentration of sugars favors fermentative conditions and hydrogen production. This was observed in the case of a two-stage system, where the increased hydrogen production due to thermal pretreatment occurred in the first stage. However, the methane yield taken place in the second stage was not affected, since the extra sugars produced over thermal pretreatment are consumed in favor of hydrogen production (Pagliaccia et al., 2019). Combinations of pretreatment methods have been studied extensively using batch tests and showed the increase in methane yields (Parthiba Karthikeyan et al., 2018). FW has high content of proteins whose conversion leads to toxic levels of ammonia. In addition, the lack of some trace elements at FW leads to the VFAs accumulation. In order to achieve a stable operation of AD, low organic loading rates (OLR) are usually performed during the process (Tampio et al., 2014).

9.3.3 Agro-industrial wastes Agro-industrial wastes are of agriculture and industrial origin and are usually treated using anaerobic co-digestion schemes (Suhartini et al., 2020;

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Rahman et al., 2019). Agricultural residues can be produced either in the field (e.g., parts of the plants, such as stalks, leaves, stems, and seed pods, remaining after crop harvesting) or during the crop process (e.g., husks, molasses, bagasse, seeds, roots, etc.) (Sadh et al., 2018). Industrial wastes are produced by the food processing and livestock industries. Food processing industries producing juice, chips, oil, confectionery goods, and fruit products generate wastes consisting of fruit and vegetable peels, coffee skin, vegetable oil cakes, etc. Fruit industrial wastes may have different compositions of carbon, nitrogen, cellulose, hemicellulose, lignin, and moisture (Rahman et al., 2019). Livestock industries produce huge amounts of animal manures, which require proper disposal. In European Union (EU), about 1.2 billion tons of manure are generated every year (Li et al., 2021). Animal manures, mainly from cattle, swine, and chicken industry, can be easily treated via AD, because they already contain the microbial groups participating in the process and usually have high buffer capacity. Cattle manure is characterized by low C/N ratio, high moisture, and high lignocellulosic content (50% in dry matter). Due to their low C/N ratio (7.2–7.7) (Ning et al., 2019), the monodigestion of animal manure often leads to ammonia toxicity. For instance, chicken manure (CM), which consists of manure and bedding materials (wood shavings, rice hulls, straw), has high level of proteins and amino acids, whose degradation during digestion can cause ammonia toxicity and volatile acids inhibition (Zahan et al., 2018). Slaughterhouse wastes, after an appropriate hygienization process (rendering), are considered to be a suitable substrate for AD with high methane potential. However, their high content in lipids and proteins leads to the production of LCFAs and ammonia from lipid hydrolysis and amino acid degradation (Spyridonidis et al., 2018).

9.3.4 Lignocellulosic biomass Agricultural/forestry residues, energy crops, woody crops, and grass comprise the nonfood lignocellulosic biomass, which is abundant worldwide and is commonly used for biogas and biofertilizer production via AD. Lignocellulosic biomass consists of 10%–25% lignin, 20%–35% hemicellulose, and 35%–50% cellulose, containing also small amounts of other organic and nonorganic compounds (Sawatdeenarunat et al., 2015; Isikgor and Becer, 2015). The holocelluloses (cellulose, hemicelluloses) are embedded in a lignin network making a recalcitrant complex, difficult to biodegrade (Monlau et al., 2013; Zhurka et al., 2020).

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Cellulose is a polymer formed through the condensation of monomers (β-1,4-linked glucopyranose) joined together by glycosidic oxygen bridges. Each unit of glucopyranose has three hydroxyl groups, which have the ability to form strong hydrogen bonds and impart some characteristic properties to cellulose such as hydrophilicity, crystalline structure, biodegradability, etc. ( Johnsy and Sabapathi, 2015). In contrast to cellulose, hemicelluloses are polysaccharides composed by beta-(1!4)-linked backbones, including xyloglucans, xylans, glucomannans, etc. These polymers are made of pentoses (xylose, arabinose, rhamnose), hexoses (glucose, mannose, galactose), and uronic acids (D-glucuronic, D-galactouronic). Their structure may vary among different plant species; however, the main role of hemicelluloses is to strengthen the cell wall by linking cellulose with lignin (Scheller and Ulvskov, 2010). Finally, lignin is a three-dimensional polymer of monomeric phenylpropanoid units, such as p-hydroxyphenyl, guaiacyl, and syringyl. Lignin acts as cellular glue, providing the compressive resistance to the plant tissue, the stiffness the cell wall, and the resistance against pathogens and insects. Cellulose, hemicellulose, and lignin are not uniformly distributed within the cell walls, while their structure and the quantity vary depending on the plant species, tissues, and maturity (Isikgor and Becer, 2015). The AD of lignified plant material is hampered by the fiber structure of biomass, in which cellulose and hemicellulose components are protected by lignin polymers, affecting their biodegradability to the hydrolytic bacteria. Hence, the pretreatment of this nonaccessible structure is essential, in order to increase the accessibility of holocelluloses to bacteria and to improve the efficiency of AD. Pretreatment methods, such as physical (size reduction), thermophysical (steam explosion, ultrasound), thermochemical (wet oxidation, ammonia fiber explosion), chemical (alkali, acid, organo-solvents), and biological (enzymes, microbial) processes have been applied to lignocellulosic residues. The selection of the most appropriate method mainly depends on the specific characteristics of the biomass (plant species) and the operating and environmental cost of the method. After the anaerobic digestion process, the nutrient-rich by-product can be used directly as a fertilizer, achieving similar effects as the commercials chemical fertilizers without adding chemicals (Karimi, 2015).

9.3.5 Algae biomass Algae biomass is a third-generation feedstock for biogas production. Algae are entities of different photosynthetic and heterotrophic phylogenetic groups and are divided into microalgae and macroalgae. Both of them have

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high moisture content (80%–90%), and their composition in lipids, carbohydrates, and proteins varies according to the species, the season, and environmental conditions (Milledge et al., 2019). Specifically, microalgae contain 1%–60% lipids, 8%–70% carbohydrates, and 15%–85% proteins on a VS basis (Xia et al., 2015). The typical C/N ratio of microalgae biomass is ca.10, which is much less than the optimum value of 30 for AD. Furthermore, the cell walls of algae contain recalcitrant polymer compounds, such as lignin, alganeans, ceratenoids, glucosamine, etc., which are resistant to hydrolysis. Therefore, pretreatment through microwave, ultrasonic, chemical, or biological processing is needed to improve the biogas production (Saratale et al., 2018). The residuals of anaerobic digestion process of microalgae can be utilized as fertilizer. On the other hand, macroalgae or seaweeds are classified into green (xylan, ulvan), brown (alginate, fucoidant), and red algae (agar, xylose, carrageen) ranging from millimeters to tens of meters. Macroalgae contain abundant carbohydrates (67%–87% of VS), proteins (9%–25% of VS), and negligible lipids ( 250°C), which, when directly applied to bio-oil under continuous conditions, leads to severe catalyst deactivation by coke deposition and char production plugging the reactor (Wang et al., 2013).

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Fig. 12.1 van Krevelen plot for the elemental compositions (dry basis) of the produced oils by mild (250 K, 100 bar, 4 h) and deep HDO (350 K, 200 bar, 4 h) with various catalysts and products by HPTT (high-pressure thermal treatment). (Copy with permission from Wang, H., Male, J., Wang, Y., Recent advances in hydrotreating of pyrolysis bio-oil and its oxygen-containing model compounds, ACS Catal. 3 (5) (2013), 1047–1070.)

The formation of such products is directly related to the presence of highly reactive compounds, such as aldehydes, olefins, and ketones, in bio-oil, which polymerize at higher temperatures (Mortensen et al., 2011; Venderbosch et al., 2010). Consequently, the bio-oil needs to be stabilized through a preliminary and mild stage (T below 250°C) before deep HDO can proceed. Such stabilization occurs via a first HDO stage dedicated to eliminating coke and char precursors in bio-oil, preventing undesirable side reactions. It is essential to mention that, even though oxygen removal takes placed during this first HDO stage, deep oxygen elimination from biooil requires harsher reaction conditions, demanding at least one extra HDO stage. Indeed, the van Krevelen plot for multiple HDO catalyst shown in Fig. 12.1 clearly demonstrates that deed HDO conditions, i.e., high temperature, are necessary for significant oxygen removal. Even though multistage HDO can minimize some of these issues, another critical challenge for the continuous operation of fixed-bed HDO processes is the low stability of the bio-oil during storage and mild heating. The modification of oil properties, such as viscosity, pH, solid content, and water content,(Cai et al., 2019) implies harsher HDO processing

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conditions, such as higher temperature and hydrogen pressure, translating into faster catalyst deactivation. Due to the aforementioned challenges, the HDO of pyrolysis vapors, before condensation, to hydrogenate reactive intermediates and stabilize the oil has been gaining significant attention with the development of processes combining pyrolysis and HDO in one or two stages. The combination of pyrolysis and HDO, often referred to as hydropyrolysis, consists of hydrogen gas during the pyrolysis process (Resende, 2016). Consequently, under hydropyrolysis, biomass is pyrolyzed under a reductive atmosphere, opposite to the inert conditions characteristic of standard pyrolysis. In the presence of hydrogen and often a catalyst, the biomass pyrolysis reactive volatiles are partially hydrogenated, which prevents polymerization reactions improving bio-oil stability and improving the quality of the final product. An overview of reactor configurations for combined pyrolysis and HDO is given in Fig. 12.2. Much like pyrolysis, hydropyrolysis is commonly performed in a fluidized bed reactor, for fast biomass conversion, in the presence of a bed material, which acts as a heat transfer material and catalyst. Optionally, the fast hydropyrolysis reactor can be coupled to a second fixed-bed HDO reactor to enhance oxygen removal (Fig. 12.2C and D). It is important to mention that the secondary HDO reactor will be operated at significantly milder conditions than standard bio-oil HDO, i.e., P(H2) ¼ 30 bar versus 100–300 bar (Marker et al., 2014). In hydropyrolysis, the choice of reactor configuration (Fig. 12.2) can significantly impact the operating conditions of the process, with each arrangement having specific advantages. For example, when no catalyst is used (Fig. 12.2A and C), the optimal reaction conditions of hydropyrolysis match those of fast pyrolysis, i.e., T  500°C, respectively. However, minimal HDO occurs (Zhang et al., 2011), making it crucial to minimize the residence time before the downstream HDO reactor to prevent secondary reactions (Hurt et al., 2013). On the other hand, in the presence of a catalyst (Fig. 12.2B and D), the hydropyrolysis reaction conditions must consider the HDO reaction, making the choice of the active material key. Indeed, most HDO catalysts are designed to operate at temperatures inferior to that of fast pyrolysis, i.e., 250°C < T < 400°C versus T  500°C, respectively, and are too reactive under such temperatures. Dayton et al. (2013) observed this overactive behavior when using a commercial hydrotreatment catalyst, which yielded only gas products before starting to deactivate after 20 h of time on stream.

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Fig. 12.2 Configurations for continuous fast hydropyrolysis systems: (A) non-catalytic fast hydropyrolysis; (B) catalytic fast hydropyrolysis; (C) non-catalytic fasthydropyrolysis with ex situ hydrotreating; (D) catalytic fast hydropyrolysis with ex situ hydrotreating. (Copy with permission from Resende, F.L.P., Recent advances on fast hydropyrolysis of biomass. Catal. Today 269 (2016), 148–155.)

The comparison between HDO performed on condensed bio-oil and hydropyrolysis is shown in Table 12.1. Even though all reactor configurations permit achieving a final product with low oxygen content, high energy content, and properties close to regular fuels, the HDO of condensed bio-oil faces multiple engineering drawbacks, such as bio-oil stability, reactor plugging, and catalyst deactivation. On the other hand, hydropyrolysis oil displays comparable properties to fixed-bed HDO. Much like fixed-bed HDO, hydropyrolysis has been proven at the pilot scale with hundreds of hours of operations (Marker et al., 2013). It is essential to mention that catalytic hydropyrolysis is not without drawbacks. By placing the catalyst directly in contact with biomass, deactivation by the deposition of inorganic elements, such as alkaline metals in biomass, can lead to severe and permanent catalyst deactivation, making this technology more sensitive to changes in biomass types.

Table 12.1 Comparison of fast-pyrolysis bio-oil properties after condensed HDO and catalytic fast hydropyrolysis. Catalytic fast hydropyrolysis Property HDO reactor

Carbon Hydrogen Nitrogen Oxygen Sulfur Viscosity (40–50°C) Density (15–40°C) HHV H/C O/C a

(wt%) (wt%) (wt%) (wt%) (ppm) (cSt) (kg/L) (MJ/kg) (molar) (molar)

Fast pyrolysis

HDO (from condensed bio-oil) Fixed bed

Single stagea Fluidized bed

Downstream HDOb Fluidized bed + fixed bed

44–58 5.5–7.2 0–0.2 35–50 300 psi would be required. Unfortunately, equilibrium conversions are lower at higher pressures, and because of the exothermic combustion step, process control is more difficult and can lead to potential temperature runaways.

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The direct route involves a mechanism of only a surface reaction on the catalyst: CH4 + 0:5O2 ! CO + 2H2 ðor 2CH4 + O2 ! 2CO + 4H2 Þ Compared with conventional syngas production methods, the direct route would drastically reduce the amount of catalyst used, making it possible to use compact reactors.

13.4.5 Membrane reactors Membrane technology combines air separation and natural gas reforming processes and has the potential to reduce the cost of syngas generation and hydrocarbon products (Carolan et al., 2002; Khassin, 2005). O2 transport membranes can combine five unit operations that are currently used in the industry: (i) O2 separation, (ii) O2 compression, (iii) POX, (iv) SMR and (v) heat exchange. The technology uses catalytic components along with the membrane to accelerate the reforming reactions. A patented two-step process for syngas generation was developed in 2000 (Nataraj et al., 2000) and can be utilized to generate syngas from several feedstocks, including natural gas, associated gas from crude oil production, light hydrocarbon gases from refineries and medium-weight hydrocarbon fractions such as naphtha. The first stage involves conventional steam reforming with partial conversion to syngas, followed by complete conversion in an ion transport ceramic membrane reactor. This combination resolves any issues associated with steam reforming feedstocks that contain hydrocarbons with a higher molecular weight than CH4, as such hydrocarbons tend to crack and degrade both the catalyst and membrane. By shifting the equilibrium in the steam reforming process by removing H2 from the reaction zone, membrane reactors can also be used to increase the equilibrium-limited CH4 conversion. By using palladium-silver alloy membrane reactors, close to 100% CH4 conversion can be achieved (Shu et al., 1995).

13.5 H2 production H2 is an important commodity in the chemical and refining industries (Table 13.2). While the gasification of residue and coke to produce H2 and/or power may become more frequent in refineries over the next two decades (Speight, 2011b), several other processes are available for the production of the additional H2 that involve the use of bio-feedstocks. This section will present a general description of these processes.

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Table 13.2 Summary of typical hydrogen production and applications in crude oil refinery. Hydrogen production

Steam-methane reforming: • Produces hydrogen for hydrotreating or hydrocracking Steam reforming of higher-molecular weight hydrocarbon derivatives: • Produces hydrogen from low-boiling hydrocarbons other than methane Gasification of crude oil residue and coke: • Produces synthesis gas from which hydrogen can be isolated Partial oxidation processes (analogous to gasification): • Produces synthesis gas from which hydrogen can be isolated Hydrogen applications

Hydrocracking: • Hydrogen is used to upgrade high-molecular weight crude oil fractions into low-boiling, valuable products Hydrodesulphurization: • Sulfur compounds are hydrogenated to hydrogen sulfide (H2S) as feed for Claus (sulfur-production) plants Hydroisomerization: • Normal (straight-chain) n-paraffin derivatives are converted into isoparaffin derivatives to improve product properties such as the octane number of gasoline constituents

13.5.1 Biomass Biomass encompasses a wide range of materials that produce a variety of products depending on the feedstock (Balat, 2011; Demirbas¸, 2011; Ramroop Singh, 2011). For example, typical biomass wastes include wood material (bark, chips, scraps and saw dust), pulp and paper industry residues, agricultural residues, organic municipal material, sewage, manure and food processing by-products. Agricultural residues such as straws, nut shells, fruit shells, fruit seeds, plant stalks and stover, green leaves and molasses are potential renewable energy resources. Many developing countries have a wide variety of agricultural residues in ample quantities, most of which are vastly underutilized. When agricultural residues are used as fuel through direct combustion, only a small percentage of their potential energy is made available due to the inefficiency of the burners used. Current disposal methods for these agricultural residues have given rise to widespread environmental concerns. For example, the disposal of rice and wheat straw by open-field

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burning causes air pollution. In addition, the heat content of the different types of biomass varies widely and must be taken into consideration when designing any conversion processes ( Jenkins and Ebeling, 1985). The choice of gasifier operation parameters (temperature, gasifying agent and catalysts) dictates the product gas composition and quality. Biomass decomposition occurs at relatively lower temperatures, and therefore, different types of reactors that are adaptable to such feedstock mixtures are required (Brar et al., 2012). Furthermore, the feedstock and gasifier type and operating parameters not only determine the product gas composition but also dictate the amount of impurities that need to be handled downstream. The downstream processes need to be modified if the biomass and/or solid waste are co-gasified with coal. Heavy metals and impurities such as sulfur and mercury in the feedstock can make syngas difficult to use and harmful for the environment. Alkalis present in biomass can also cause corrosion at high temperatures in the downstream pipes. An alternative option to downstream gas cleaning would be to remove mercury and sulfur prior to feeding into the gasifier. Before being used as feedstock, biomass needs to dried and reduced in size. Size reduction is required to obtain appropriate particle sizes, whereas drying is required to achieve the moisture content suitable for gasification operations. In addition, biomass densification can help prepare pellets and improve the density and material flow in the feeder. The recommended biomass moisture content is 50% v/v, the gas should first be considered for H2 recovery using a membrane or PSA unit (Br€ uschke, 1995, 2003; Speight, 2014). The tail or reject gas will still contain a substantial amount of H2, which can then be used as steam reformer feedstock. Generally, the feedstock purification process uses various methods to produce H2. For example, high-pressure hydrocracker purge gas is purified in a membrane unit that produces H2 at medium pressure and is combined with medium-pressure off-gas, which is first purified in a PSA unit. The lowpressure off-gas is compressed, mixed with reject gases from the membrane and PSA units and then used as steam reformer feed.

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Various processes can be used to purify the H2 stream, but as the product streams are available in a wide variety of compositions, flows and pressures, the most suitable method will depend on the requirement. Several factors that must also be taken into consideration when selecting a purification method: (i) H2 recovery, (ii) product purity, (iii) pressure profile, (iv) reliability and (v) cost. The last is an equally important parameter that is not considered here as the emphasis is on the technical aspects of the purification process. 13.6.2.1 Wet scrubbing Wet scrubbing systems, particularly amine or potassium carbonate (K2CO3) systems, are used for the removal of acid gases such as H2S or CO2 (Mokhatab et al., 2006). Most wet scrubbing systems depend on chemical reactions and can be designed to operate at a wide range of pressures and capacities. These systems were once widely used to remove CO2 from steam reforming plants, but have now been replaced by PSA units except in cases where CO needs to be recovered. Wet scrubbing is still used to remove H2S and CO2 in POX plants. Wet scrubbing systems remove only the acid gases or heavy hydrocarbons and not CH4 or other hydrocarbon gases. Therefore, they have little influence on the product purity. Wet scrubbing systems are thus most often used during pre-treatment or when H2-rich streams need to be desulfurized for use as fuel gas. 13.6.2.2 PSA units PSA units use beds of solid adsorbent to separate impurities from H2 streams, allowing the production of high-purity, high-pressure H2 (99.9% v/v purity compared to 98%.

13.7 The future The petrochemical industry is concerned with the production and trade of petrochemicals and has a direct relationship with the crude oil industry, especially with the downstream sector of the industry. Petrochemical industries are specialized in the production of petrochemicals that have various industrial applications and can be considered a sub-sector of the crude oil

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industry as the former cannot exist without the latter. Crude oil is the major prerequisite raw material for the production of petrochemicals both in terms of quality and quantity. In addition, the petrochemical industry is subject to the geopolitics of the crude oil industry, with each industry reliant upon the other for sustained survival. The growth and development of petrochemical industries depend on a number of factors and also vary across country based on the technical knowhow, marketability and applicability of petrochemicals for the manufacture of products through processes made feasible by the knowledge and application of petrochemistry. Petrochemistry is a branch of chemistry (chemistry being a branch of natural science concerned with the study of the composition and constitution of substances and the changes such substances undergo due to changes in the molecules that constitute said substances) that deals with crude oil, natural gas and their derivatives. However, not all of the petrochemical or chemical materials produced by the chemical industry are made in a single location. Groups of related materials are often made in adjacent manufacturing plants to foster industrial symbiosis, material and utility efficiency and other economies of scale (integrated manufacturing). Specialty and fine chemical companies are sometimes found in manufacturing locations adjacent to those of petrochemicals, but in most cases, they do not require the same scale of infrastructure (e.g., pipelines, storage, ports and power) and therefore can also be found in multi-sector business parks. These trends will continue to exist as long as the refining industry persists in its present form (Speight, 2011a, 2011b). The petrochemical industry is continuously affected by globalization and the integration of global economies. For example, large-scale petrochemical plants built during the past several years are substantially larger than those built over two decades ago. As a result, smaller, older and less efficient units are being shut down, expanded or, in some cases, retrofitted to produce different chemical products. In addition, crude oil prices were higher in the past decade and petrochemical markets are affected by sharp price fluctuations, creating a cloud of uncertainty in both upstream and downstream investments. Increasing concerns over fossil fuel supply and consumption with respect to their impact on health and the environment have led to global legislations that will affect chemical and energy production and processing for the foreseeable future. The recent shift from local markets to large global markets has led to an increase in the competitive pressures on petrochemical industries.

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Furthermore, fluctuations in product prices and the high price of feedstocks has reduced the economical attraction of petrochemical plants. The everincreasing cost of energy and increasingly stringent environmental regulations have also negatively impacted the operational costs. When cheap feedstocks are not available, the best method by which to make profits is to integrate petrochemical complexes with adjacent refineries and optimize them. This is valid for both installed plants and those under construction. Petrochemical-refinery integration is an important facilitator of the reduction of costs and increase in efficiency because it guarantees a regular supply of feedstock for petrochemical industries. Integrated schemes also harness the economy of scale and can produce more diverse products. Petrochemical-refinery integration therefore reduces the sale of crude oil, optimizes products, economizes costs and increases benefits. Basic chemicals and plastics are key building blocks for the manufacture of a wide variety of durable and nondurable consumer goods. The demand for chemicals and plastics is driven by global economic conditions, which are directly linked to the demand for consumer goods. In the future, recently introduced manufacturing processes will facilitate adaptation of the industry to new feedstocks, which will shift the distribution of products. This, in turn, will potentially lead to a supply/demand imbalance, particularly for smaller downstream petrochemical derivatives. In addition, growing environmental concerns and the variability of crude oil prices, which is usually upward, will expedite the development and commercialization of chemical products from sources other than natural gas and crude oil. As a result, feedstocks and technologies previously considered economically impractical will be developed to meet the increasing demand. Developments in homogeneous and heterogeneous catalysis have fostered effective approaches for the utilization of renewable sources. However, further advances are needed to realize technologies that can compete with established petrochemical processes. Catalysis will play a key role in this development, with new reactions, processes and concepts leveraging both traditional and emerging chemo- and bio-catalytic technologies. Thus, new knowledge and better technologies are required to improve chemical transformations that require mild oxidation conditions, selective reduction and dehydration, better control of bond cleavage and improvements in the direct polymerization of multifunctional monomers. For biological transformations, a better understanding of metabolic pathways and cell biology, lower downstream recovery costs, increased utility of mixed

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sugar streams and improved molecular thermal stability are necessary. Although it is possible to prepare a very large number of molecular structures from the fundamental building blocks, there is a scarcity of information on molecular product behaviors and industrial processing properties. A comprehensive database on biomolecular performance characteristics would prove extremely useful to both the public and private sectors. There is a significant market opportunity for the development of bio-based products from the four carbon building blocks. However, competition with petrochemical-derived products remains a significant technical challenge and should be undertaken with a long-term perspective. In summary, syngas and H2 offer many routes, either direct or indirect, to the production of industrial chemicals. The direct path involves methanation, Fischer-Tropsch chemistry and the synthesis of oxygenates. Direct conversion involves the straight hydrogenation of CO to paraffins, olefins and O2-containing products. The best known direct hydrogenation process of CO is Fischer-Tropsch synthesis, which mainly yields mixtures of linear alkanes and/or alkenes. Mechanistically, it can be described as a reductive oligomerization of CO that follows a geometric progression (Schulz-Flory distribution). A-values close to 1 yield broad product distributions, whereas small a-values predominantly yield CH4. The indirect path involves carbonylation, CH3OH and methyl formate chemistry.

References Aasberg-Petersen, K., Bak Hansen, J.-H., Christiansen, T.S., Dybkjær, I., Seier Christensen, P., Stub Nielsen, C., Winter Madsen, S.E.L., Rostrup-Nielsen, J.R., 2001. Technologies for large-scale gas conversion. Appl. Cat. A: General 221, 379–387. Aasberg-Petersen, K., Christensen, T.S., Stub Nielsen, C., Dybkjær, I., 2002. Recent developments in autothermal reforming and pre-reforming for synthesis gas production in GTL applications. Preprints. Div. Fuel Chem., Am. Chem. Soc. 47 (1), 96–97. Arena, U., 2012. Process and technological aspects of municipal solid waste gasification. A review. Waste Manag. 32, 625–639. Balat, M., 2011. Fuels from biomass—an overview. In: Speight, J.G. (Ed.), The Biofuels Handbook. Royal Society of Chemistry, London, United Kingdom. Part 1, Chapter 3. Balasubramanian, B., Ortiz, A.L., Kaytakoglu, S., Harrison, D.P., 1999. Hydrogen from methane in a single-step process. Chem. Eng. Sci. 54, 3543–3552. Basu, P., 2013. Biomass Gasification, Pyrolysis and Torrefaction, second ed. Practical Design and Theory, Academic Press, Inc., New York. Biermann, C.J., 1993. Essentials of Pulping and Papermaking. Academic Press Inc., New York. Brandmair, M., Find, J., Lercher, J.A., 2003. Combined autothermal reforming and hydrogenolysis of alkanes. In: Proceedings. DGMK Conference on Innovation in the Manufacture and Use of Hydrogen, Dresden, Germany, October 15–17, pp. 273–280.

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Brar, J.S., Singh, K., Wang, J., Kumar, S., 2012. Cogasification of coal and biomass: a review. Int. J. For. Res. 2012 (2012), 1–10. Brigwater, A.V. (Ed.), 2003. Pyrolysis and Gasification of Biomass and Waste. CPL Press, Newbury, Berkshire, United Kingdom. Br€ uschke, H., 1995. Industrial application of membrane separation processes. Pure Appl. Chem. 67 (6), 993–1002. Br€ uschke, H., 2003. Separation of hydrogen from dilute streams (e.g. using membranes). In: Proceedings. DGMK Conference on Innovation in the Manufacture and Use of Hydrogen, Dresden, Germany, October 15–17, p. 47. Carolan, M.F., Chen, C.M., Rynders, S.W., 2002. ITM syngas and ITM H2: engineering development of ceramic membrane reactor systems for converting natural gas to hydrogen and synthesis gas for liquid transportation fuels. In: Proceedings of the 2002 U.S. DOE Hydrogen Program Review NREL/CP-610-32405. National Renewable Energy Laboratory, Golden, CO. Chadeesingh, R., 2011. The Fischer-Tropsch process. In: Speight, J.G. (Ed.), The Biofuels Handbook. The Royal Society of Chemistry, London, United Kingdom, pp. 476–517. Part 3, Chapter 5. Demirbas¸, A., 2011. Production of fuels from crops. In: Speight, J.G. (Ed.), The Biofuels Handbook. Royal Society of Chemistry, London, United Kingdom. Part 2 (Chapter 1). Ehwald, H., K€ urschner, U., Smejkal, Q., Lieske, H., 2003. Investigation of different catalysts for autothermal reforming of i-octane. In: Proceedings. DGMK Conference on Innovation in the Manufacture and Use of Hydrogen, Dresden, Germany, October 15–17, p. 345. Enger, B.C., Lødeng, R., Holmen, A., 2008. A review of catalytic partial oxidation of methane to synthesis gas with emphasis on reaction mechanisms over transition metal catalysts. Appl. Catal. A Gen. 346 (1–2), 1–27. Gunardson, H.H., Abrardo, J.M., 1999. Produce CO-rich synthesis gas. Hydrocarb. Process., 87–93. Hagh, B.F., 2004. Comparison of autothermal reforming for hydrocarbon fuels. Preprints. Div. Fuel Chem., Am. Chem. Soc. 49 (1), 144–147. Hufton, J.R., Mayorga, S., Sircar, S., 1999. Sorption-enhanced reaction process for hydrogen production. AICHE J. 45, 248–256. Jenkins, B.M., Ebeling, J.M., 1985. Thermochemical properties of biomass fuels. Calif. Agric. (May-June), 14–18. Khassin, A.A., 2005. Catalytic membrane reactor for conversion of syngas to liquid hydrocarbons. Energeia 16 (6), 1–3. Lee, S., Shah, Y.T., 2013. Biofuels and Bioenergy. CRC Press, Taylor & Francis Group, Boca Raton, FL. Mastellone, M.L., Arena, U., 2007. Fluidized bed gasification of plastic waste: effect of bed material on process performance. In: Proceedings. 55th International Energy Agency— Fluidized Bed Conversion Meeting, Paris, France, October 30–31. McKendry, P., 2002. Energy production from biomass part 3: gasification technologies. Bioresour. Technol. 83 (1), 55–63. Mokhatab, S., Poe, W.A., Speight, J.G., 2006. Handbook of Natural Gas Transmission and Processing. Elsevier, Amsterdam, Netherlands. Nagaoka, K., Jentys, A., Lecher, J.A., 2003. Autothermal reforming of methane over monoand bi-metal catalysts prepared from hydrotalcite-like precursors. In: Proceedings. DGMK Conference on Innovation in the Manufacture and Use of Hydrogen, Dresden, Germany, October 15–17, p. 171. Nataraj, S., Moore, R.B., Russek, S.L., 2000. Production of Synthesis Gas by Mixed Conducting Membranes. United States Patent 6,048,472. April 11.

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Ramroop Singh, N., 2011. Biofuel. In: Speight, J.G. (Ed.), The Biofuels Handbook. Royal Society of Chemistry, London, United Kingdom. Part 1, Chapter 5. Ricketts, B., Hotchkiss, R., Livingston, W., Hall, M., 2002. Technology status review of waste/biomass co-gasification with coal. In: Proceedings. Inst. Chem. Eng. Fifth European Gasification Conference, Noordwijk, Netherlands, April 8–10. Rostrup-Nielsen, J.R., 1984. Sulfur-passivated nickel catalysts for carbon-free steam reforming of methane. J. Catal. 85, 31–43. Rostrup-Nielsen, J.R., Christiansen, L.J., Bak Hansen, J.-H., 1988. Activity of steam reforming catalysts: role and assessment. All. Cat. 43, 287–303. Rostrup-Nielsen, J.R., 1993. Production of synthesis gas. Catal. Today 19, 305–324. Rostrup-Nielsen, J.R., 2002. Syngas in perspective. Catal. Today 71, 243–247. Rostrup-Nielsen, J.R., Sehested, J., Norskov, J.K., 2002. Adv. Catal. 47, 65–139. Rostrup-Nielsen, J.R., Christiansen, L.J., 2011. Concepts in Syngas Manufacture. Catalytic Science Series, Volume 10. Imperial College Press, World Scientific Publishing (UK) Ltd, London, United Kingdom. Shu, J., Grandjean, B.P.A., Kaliaguine, S., 1995. Asymmetric Pd-Ag stainless steel catalytic membranes for methane steam reforming. Catal. Today 25, 327–332. Speight, J.G. (Ed.), 2011a. The Biofuels Handbook. Royal Society of Chemistry, London, United Kingdom. Speight, J.G., 2011b. The Refinery of the Future. Gulf Professional Publishing, Elsevier, Oxford, United Kingdom. Speight, J.G., 2013a. The Chemistry and Technology of Coal, third ed. CRC Press, Taylor & Francis Group, Boca Raton, FL. Speight, J.G., 2013b. Coal-Fired Power Generation Handbook. Scrivener Publishing, Salem, MA. Speight, J.G., 2014. The Chemistry and Technology of Petroleum, fifth ed. CRC Press, Taylor & Francis Group, Boca Raton, FL. V
elez, F.F., Chejne, F., Vald
es, C.F., Emery, E.J., London˜o, C.A., 2009. Cogasification of Colombian coal and biomass in a fluidized bed: an experimental study. Fuel 88 (3), 424–430. Vernon, P.D.F., Green, M.L.H., Cheetham, A.K., Ashcroft, A.T., 1990. Partial oxidation of methane to synthesis gas. Catal. Lett. 6 (2), 181–186. Wang, W., Stagg-Williams, S.M., Noronha, F.B., Mattos, L.V., Passos, F.B., 2004. Partial oxidation and combined reforming of methane on Ce-promoted catalysts. Preprints. Div. Fuel Chem., Am. Chem. Soc. 49 (1), 132–133. Zhu, Q., Zhao, X., Deng, Y., 2004. Advances in the partial oxidation of methane to synthesis gas. J. Nat. Gas Chem. 13, 191–203.

CHAPTER 14

Production of biofuels via Fischer-Tropsch synthesis: Biomass-to-liquids Hessam Jahangiria,b, Angelos A. Lappasc, Miloud Ouadib, and Elli Heracleousc,d a

Department of Engineering and Mathematics, Sheffield Hallam University, United Kingdom School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom c Chemical Process & Energy Resources Institute, Centre for Research and Technology Hellas, Thessaloniki, Greece d School of Science & Technology, International Hellenic University, Thessaloniki, Greece b

14.1 Introduction Growing environmental and supply-security concerns are the main drivers of changes in fuel products. European Union (EU) policies on local air quality, climate change, and sustainability, which are applied via Fuel Directives or Emission Directives, have strongly influenced research efforts and advances in conventional fossil, synthetic, and bio-origin fuels. According to a European Commission study, Europe was responsible for 10% of the total carbon dioxide (CO2) emissions, while India, the USA, and China contributed 7%, 14%, and 29%, respectively, in 2017–2018 (Bokde et al., 2021). The developing countries India and China have targeted to decrease CO2 emissions per gross domestic product by 25% and 40% of the 2005 levels by 2020, respectively. Furthermore, the Renewable Energy Directive I (well known as 20/20/20) has targeted to decrease CO2 emissions by 20% from the 1990 levels, increase the renewable energy share to 20%, and decrease energy consumption by 20% by 2020 (Bokde et al., 2021). According to the Eurostat data in 2019 (Eurostat, 2019), the renewable energy share was 19.7% of the energy consumed by the 27 member countries of the EU (in comparison with 9.6% in 2004), merely 0.3% short of the 2020 target of 20%. For individual EU member states, the renewable energy share was the highest in Sweden (56.4%), followed by Finland (43.1%), Latvia (41.0%), and Denmark (37.2%). According to the Renewable Energy Directive II, the minimum renewable energy share in the transport sector should increase to 14% by 2030, and a minimum of 3.5% of the transport energy must be produced from Handbook of Biofuels Production https://doi.org/10.1016/B978-0-323-91193-1.00013-5

Copyright © 2023 Elsevier Ltd. All rights reserved.

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advanced biofuels (Cadillo-Benalcazar et al., 2021), i.e., second- and thirdgeneration biofuels. Second-generation biofuels are produced from nonfood biomass (lignocellulosic material), such as sugarcane bagasse, oat hulls, and wheat husk (Santos et al., 2019), while third-generation biofuels are obtained from algae, sewage sludge, municipal solid waste, etc. (CadilloBenalcazar et al., 2021; Hornung et al., 2021, 2022). Gasification of second-generation feedstocks followed by Fischer-Tropsch (FT) synthesis is one of the most promising routes for producing sustainable fuels of top quality (Mahmoudi et al., 2020; Ouadi et al., 2019a). The production of fuels via FT synthesis involves the conversion of the feedstock to synthesis gas (syngas; carbon monoxide [CO] and hydrogen [H2]) and subsequent synthesis of hydrocarbons via the FT synthesis reaction: [i] CO + 2H2 ! d[CH2]d + H2O, where d[CH2]d represents a product consisting mainly of paraffinic hydrocarbons of variable chain length. In addition, side reactions such as those forming alcohols and undesired carbonaceous deposits may occur via Boudouard reaction ( Jahangiri et al., 2014). In general, the FT process is operated in the temperature range of 150–350°C to avoid the formation of methane (CH4)-rich by-products. Increased pressure leads to higher conversion rates and also favors the formation of desired long-chain alkanes. Typical FT pressures are in the range of 10–50 bar. The FT hydrogenation reaction is catalyzed mainly by Fe and Co catalysts, while the size and distribution of the hydrocarbon products of the reaction are generally governed by the Anderson-Schulz-Flory chain polymerization kinetics model ( Jahangiri et al., 2014; Zijlstra et al., 2020). One of the most important advantages of FT synthesis is its versatility in terms of both feedstock and products. The FT process can produce hydrocarbons of different lengths from syngas derived from any carbon-containing feedstock, such as coal, natural gas, and biomass. Depending on the feedstock, the process is referred to as coal-to-liquid (CTL), gas-to-liquid (GTL), or biomass-to-liquid (BTL). Moreover, synthetic fuels have distinct environmental advantages over conventional crude-refined fuels as they are virtually free of sulfur, nitrogen, and aromatics. In addition, they are largely compatible with current vehicles and fully blendable with conventional fuels and can thus be handled by the existing fuel infrastructure. However, the high energy demands and the considerable capital cost of FT plants both contribute to the high price of synthetic FT fuels, and as a consequence, the economic viability of the FT process largely depends on the price of crude oil (Teimouri et al., 2021).

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The FT process is not a new concept. It was first developed in Germany in the 1930s, as Germany was very poor in oil resources and needed to develop an independent source of transportation fuels based on its abundant coal resources during the Second World War (WWII) (Santos and Alencar, 2020). After WWII, the exploitation of the Middle East’s vast oil reserves made the FT process uneconomical, and thus, its interest decreased, except in South Africa. South Africa has vast coal deposits, and the high oil prices combined with the oil embargo during the 1970s led to the great development of the FT process by South African Synthetic Oil Limited (SASOL) (Overett et al., 2000). In the last few decades, the technical advances in the FT process combined with the depletion of crude reserves have increased worldwide interest in this route to transportation fuels. The FT process has already been commercialized on a large scale. SASOL Synfuels currently operates the Secunda CTL plant at Secunda in South Africa, processing 45 million tons (Mt) of coal per year and fulfilling approximately 28% of South Africa’s diesel and petrol needs (Dry, 2002). In China, the Shenhua Ningxia CTL plant (an FT plant) was commissioned in 2017 (Shenhua-Ningmei project). This plant is operational and focuses on processing low-grade coal (Li and Li, 2019; Xu et al., 2015). The ShenhuaNingmei project then launched a commercial CTL plant to produce 1.08 Mt of diesel oil, naphtha, and liquefied petroleum gas annually. An entrained flow gasifier (Siemens-GSP) with a 4 Mt/yr capacity is used in this project (Ra et al., 2021). Since 1993, Shell in Malaysia (Bintulu) and PetroSA in South Africa (Mossel Bay) have been operating industrial FT synthesis facilities, which produce liquid fuels from syngas derived from natural gas. In a joint venture with Qatar Petroleum, SASOL has been operating the Oryx GTL plant in Ras Laffan Industrial City, Qatar, since 2007. The facility is supplied with lean CH4-rich gas from the Qatar North gas field and can produce 34,000 barrels per day (bpd) of liquid products (24,000 barrels of GTL diesel, 9000 barrels of naphtha, and 1000 barrels of liquefied petroleum gas) (Qatar, 2021). In 2010, Shell constructed the Pearl GTL plant, also in Ras Laffan. Pearl GTL is now the world’s largest GTL plant, producing 140,000 bpd of GTL products (Shell, 2021). The plant also produces 120,000 bpd of natural gas liquids and ethane. Several other GTL plants are currently being planned or are in construction (SASOL, 2021). Escravos GTL (EGTL) is a collaboration between Chevron (Nigerian National Petroleum Corporation) and SASOL, which was developed to convert a waste product (methane flaring) into high-value-

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added products. Moreover, a joint venture between Uzbekneftegaz (Petronas) and SASOL is in the final stages of planning a facility to convert part of Uzbekistan’s gas reserves into transport fuel. In North America (the United States and Canada), the shale gas revolution and the reduction in natural gas prices have opened up new opportunities for GTL processes. SASOL is actively looking at the feasibility of establishing GTL project opportunities in Louisiana (the USA) and Alberta (Canada) (SASOL, 2021). A recent US project is the collaboration between Haldor Topsoe and SASOL. These two companies have long worked together on many GTL projects. SASOL’s FT technologies and Topsoe’s SynCOR™ technologies have been licensed into numerous world-scale GTL projects. The two global leaders in GTL technology signed an agreement to offer G2L™ single-point licensing of approved GTL solutions to produce diesel, naphtha, and kerosene from natural gas. Based on this collaboration agreement, these two companies will continue to offer these essential technologies, in addition to Topsoe’s hydroprocessing and hydrogen technologies. This offers potential customers access to a single-point licensing that covers the complete value chain from gas to liquid fuels (TOPSOE, 2019). The FT process is well known for processing coal- and natural gas-derived CO2-free syngas. The direct application of CO2 has not been explored significantly on a large scale. The injection of recycled CO2 back to the reforming section (for natural gas-based FT processing) or directly to the reactor have been studied by (Zhang et al., 2014). Due to the lack of experience with CO2-based gases and capitalizing on the early developments in catalysts for direct CO2 hydrogenation, power-to-FT fuel (PtFT-fuel) plants have been built, which focus on converting CO2 to CO through co-electrolysis or a reverse water-gas shift reactor (RWGS-reactor). Sunfire, Fraunhofer GmbH, and other partners developed the first pre-commercialization plant, named “Fuel 1,” which was commissioned in 2014 in Dresden, Germany. The “Fuel 1” plant can operate for more than 1500 h and produce 1 bpd of FT product. In this plant, CO2 is converted to CO via an RWGS-reactor, and the energy efficiency is approximately 60%. This process has been calculated to provide a CO2 mitigation potential of 85% in comparison with conventional fuels (Dieterich et al., 2020; Sunfire and Blue, 2021). The VTT Technical Research Centre of Finland and Lappeenranta University of Technology (LUT) started an initial pre-commercialization plant for 300 h in 2017 for the SOLETAIR project in Finland. In this plant, CO2 is captured by direct air capture (DAC), and H2 is formed by proton exchange membrane electrolysis. CO2 is transformed to CO in an

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RWGS-reactor before reaching the FT unit. In this process, the system produces 6.2 kg of combined wax and FT oil per day. The total conversion of CO and CO2 is around 60% and 40%, respectively, achieving carbon and energy efficiencies of 94% and 47%, respectively (Va´zquez et al., 2018). An FT-based power-to-liquid (PtL) plant started as part of the “Kopernikus P2X” initiative in 2019, which was funded by the German Federal Ministry of Education and Research. In this project, co-electrolysis, FT reactor, and hydrocracking units are joined with a DAC unit to produce liquid fuels. However, the present configuration can merely produce 10 L per day (Dieterich et al., 2020; Kopernikus, 2021). Finally, the first industrial PtL plant based on the FT process is planned to start by 2022 in the Heroya Industrial Park, Norway. This project will apply a 20-MW co-electrolysis unit of CO2 and H2O for syngas production to produce 8000 tons of FT crude liquid per year that can be treated further in the existing refineries. Hydropower plants will produce the necessary electrolysis power (Dieterich et al., 2020; NORDIC, 2021). Today, global warming and the universal efforts toward CO2 emission reduction have rekindled the interest in using FT technology with biomass to produce high-quality clean biofuels that are compatible with the existing infrastructure and vehicle technology. A wide variety of biomass resources can be used for the FT process. Materials foreseen to be used in the BTL process include woody biomass (lignocellulosic biomass) and by-products (bagasse, paper slurry, black liquor, etc.), which are the most common biomass feed (Bashir et al., 2021; Santos and Alencar, 2020). The use of renewable resources as feedstock, with all the associated environmental advantages, undoubtedly gives synthetic fuels a new dynamic. The production of synthetic fuels from biomass via the FT process comprises three basic steps: gasification of the feedstock (in this case biomass) to produce syngas (CO and H2) and gas cleaning/conditioning, FT synthesis to produce middle distillates, and upgrading of the FT liquids to high-quality fuel products. BTL via biomass gasification and the use of biomass-derived syngas for fuel production has limited commercial application compared with CTL and GTL. Gas cleaning between units poses the main problem when biomass gasification is integrated with FT synthesis. The generated syngas contains some contaminants (mainly tar, NH3 and H2S) that should be removed before entering the FT synthesis reactor (Ail and Dasappa, 2016). Therefore, research is actively ongoing on the steps of the FT process to improve the overall efficiency, focusing on the biomass gasification step and subsequent

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gas-conditioning prior to the FT reactor step to meet the strict FT gas purification (cleaning) requirements. Several gasification technologies (e.g., fixed-bed, circulating fluidized-bed, and entrained flow gasifiers) and operation modes have been considered and assessed and are discussed later in the chapter. In the following paragraphs, an overview of the essential topics and the current state of the art for biofuel production via FT synthesis is presented. The following section (Section 14.2) starts with a short discussion on biomass gasification, including gasifiers and gas-cleaning techniques. We then thoroughly describe the main types of reactors and catalytic materials currently used for FT synthesis, followed by a comprehensive discussion of the different processes and technologies used for upgrading the FT liquids to premium fuel products. In Section 14.3, we describe the final BTL fuel products and their properties. Finally, the most recent advances in the BTL process’s commercialization are presented, alongside a discussion of the advantages and limitations of the FT process and its outlook on the future fuel market.

14.2 Biomass-to-liquid process steps and technologies Notwithstanding the complexity of FT plants, all XTL (“anything”-toliquid) processes, where X ¼ C for coal, G for natural gas, or B for biomass processes, consist of the three main steps illustrated in Fig. 14.1: gasification to syngas and gas cleaning/conditioning, FT synthesis, and product upgrading. Variations in and different available options for biomass gasification (pressure, use of oxygen or air medium, etc.), types of FT reactor and catalyst, and target products lead to many possible process configurations to produce FT liquids from biomass (Martinelli et al., 2020). However, all concepts can be categorized into two main groups: (1) full-conversion

Off-gas

Cleaning Cleaning Biomass Biomass

Gasifier Gasifier Conditioning Conditioning

FischerFischer– Tropsch Tropsch reactor reactor

Electricity

Upgrading Upgrading

Fig. 14.1 Schematic line-up of the biomass-to-liquid process.

BTLBTLfuels fuels

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FT to maximize liquid production and (2) cofiring the FT off-gas with natural gas in a gas turbine for electricity production to maximize energy efficiency. Several studies have investigated the technical feasibility and economics of the different BTL-FT processes to identify the most promising system configurations (Gonzalez et al., 2011; Henrich et al., 2009; Martinelli et al., 2020). The outcomes of these studies are not conclusive, as there are significant uncertainties concerning technology status and economic values. Although both biomass gasification technologies and syngas conversion technologies are commercially available and have been demonstrated at a commercial scale, there is a very limited commercial experience in integrating biomass gasification with downstream processes to produce liquid transport fuels. There is a consensus that R&D efforts should focus on the following key issues: gasifier designs, syngas quality, product selectivity in chemical synthesis, and process integration and scale (Grim et al., 2019). The following paragraphs provide a description of the main processes, reactor types, and catalytic materials used in the three main steps of the BTL-FT process.

14.2.1 Biomass gasification to syngas 14.2.1.1 Gasifiers Gasification converts biomass into mixed syngas consisting of H2, CO, CH4, and CO2. Biomass gasification is a crucial stage for the application of the BTL process, as the establishment of BTL-FT technology has been hindered mainly by difficulties in syngas production from biomass and subsequent cleaning. Moreover, almost 75% of a BTL plant’s investment costs are accounted for by the pretreatment, gasification, and gas cleaning steps. Therefore, the gasification pressure, temperature, and catalyst significantly affect the economy of both the gasifier and downstream equipment ( Janajreh et al., 2021). Many technologies are available for syngas production, presented in Fig. 14.2 (Balat et al., 2009; Janajreh et al., 2021). Biomass gasifiers can be classified as: • Air-blown, oxygen-blown, or steam-blown; • Atmospheric or pressurized; • Slagging or nonslagging; • Fixed-bed updraft/downdraft, fluidized-bed, or entrained flow; and • Allothermal (indirect heating) or autothermal (direct heating by combustion of part of the feedstock).

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Fig. 14.2 Types of biomass gasifiers. (Reproduced from Balat, M., Balat, M., Kirtay, E., Balat, H., 2009. Main routes for the thermo-conversion of biomass into fuels and chemicals. Part 2: Gasification systems. Energy Convers. Manag. 50, 3158–3168).

A detailed description of the biomass gasification technology and the different types of gasifiers is provided in the chapter “Production of bio-alcohols via gasification,” which is dedicated to bio-syngas production via gasification. The present chapter focuses on the gasification technology suitable for integration in a BTL-FT plant to produce liquid fuels. Fixed-bed gasifiers have relatively low throughput and poor heat transfer and are therefore considered unsuitable for large-scale applications (Safarian et al., 2019). Based on throughput, complexity, cost, and efficiency issues, circulating fluidized-bed (CFB) and entrained flow gasifiers are more suitable for large-scale syngas production (Safarian et al., 2019). Some demonstration and commercial plants operate the biomass gasification process in fluidized-bed and entrained flow reactor modes. Such projects and technologies include the BIOFLOW and CHRISGAS projects, the CHOREN Carbo-V technology, the VTT pressurized circulating fluidized bed, and the RENUGAS technology. These technologies and projects investigate both the technical challenges and economic feasibility of high-pressure gasifiers (Motta et al., 2018). The CHOREN Carbo-V biomass gasification process is an excellent example of the application of entrained flow gasifiers in the BTL process (Higman and Van Der Burgt, 2008). The CHOREN Carbo-V patented

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Carbo-V gasifier Low-temperature gasifier (NTV) Oxygen Biomass

Pyrolysis gas Raw gas

Steam

M

Gas scrubber Syngas

BFW Oxygen Deduster Mill Char

Residual char/ash Vitrified slag

Waste water

Fig. 14.3 The CHOREN Carbo-V gasification process. (Reproduced from Heidenreich, S., Foscolo, P. U., 2015. New concepts in biomass gasification. Prog. Energy Combust. Sci. 46, 72–95).

gasification process consists of three stages: low temperature, high temperature, and endothermic entrained-bed gasification (Fig. 14.3) (Heidenreich and Foscolo, 2015). The biomass is continuously carbonized during the first stage through partial oxidation with oxygen at temperatures between 400°C and 500°C, i.e., it is broken down to a tar-containing gas (volatile parts) and solid carbon (char). The tar-containing gas is then fed into the hightemperature gasifier, where it is partially oxidized using oxygen as the gasification agent. The heat, which is released due to the oxidation process, warms up the carbonization gas to temperatures that exceed the ash melting point of the fuels used (1300–1500°C). At these temperatures, any unwanted longer-chain hydrocarbons, e.g., tar, and even CH4, are broken down. The gas that is produced primarily consists of CO, H2, CO2, and steam. The char from the low-temperature gasifier is cooled, ground to pulverized fuel, and then blown into the stream of hot gas from the combustion chamber in the entrained flow gasifier. A large amount of heat is absorbed by the char during gasification, which lowers the temperature of the gas to 800–900 °C in seconds. This “chemical quenching” process produces a tar-free gas with low CH4 content and high proportions of combustible CO and H2. The commercial status of the CHOREN project is discussed in Section 14.5. An example of the application of fluidized bed gasifiers in the BTL process is the G€ ussing Renewable Energy (GRE) multifuel gasification plant

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in G€ ussing, Austria. This plant is the world’s first fast internally circulating fluidized bed (FICFB) gasification plant. The plant’s gasification steps involve two interconnected fluidized-bed systems, as shown in Fig. 14.4 (Wilk et al., 2013). Biomass is ground into small pieces and fed into the reactor, where it is gasified under anaerobic conditions at 850°C in the presence of steam. The reactor’s bed material is olivine, which acts as a heat transfer medium and provides a stable temperature in the gasifier. After gasification, the syngas is purified and cooled. Syngas cleaning includes dedusting with a bag filter and washing to reduce the concentration of tar, ammonia (NH3), and acid gas components (Hongrapipat et al., 2020; Rauch et al., 2014). Special syngas technology makes it possible to redirect all side products back into the process. As a result, no waste or wastewater is created during gas purification. Moreover, the product gas is completely free from nitrogen. The use of steam as a gasification agent in the FICFB concept results in a product gas with high quality (low nitrogen, optimal H2/CO ratio, and high heating value) without the need for pure oxygen. It also enables different syngas applications to be realized at a smaller scale (10–100 MW fuel) (Rauch, 2014). A part of the remaining coke is directed to the burning section with the help of circulating channel material used as a carrier, where it is burnt (see Fig. 14.4). The heat produced from the combustion raises the temperature of the carrier and is transferred back to the gasifier to maintain the temperature of the gasification reaction at the desired level. The flue gas is discharged separately such that its heat content enters the district heating network.

Flue gas

Producer gas Heat

Gasification

Combustion

Biomass Circulation Steam

Air

Fig. 14.4 Concept of dual fast internally circulating fluidized-bed gasification. (Reproduced from Wilk, V., Schmid, J. C., Hofbauer, H., 2013. Influence of fuel feeding positions on gasification in dual fluidized bed gasifiers. Biomass Bioenergy 54, 46–58).

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The G€ ussing plant has been operating at the commercial demonstration scale of 8 MW thermal input, producing 2 MW of electricity and 4.5 MW of district heat for more than 10 years. Several new plants are being built or are already in operation utilizing the developed gasification technology, as shown in Table 14.1 (Ail and Dasappa, 2016). Besides the production of heat and power, an FT pilot plant has been built in the CHP plant to demonstrate the use of bio-derived syngas for fuel production. The specifics of the whole BTL process chain are discussed in Section 14.5, which presents the main advances toward the commercialization of the BTL process. Recently, supercritical water gasification (SCWG; hydrothermal gasification) has shown promising results and may be more beneficial than the traditional gasification processes. Traditional gasification requires pretreatment and drying of biomass, which adds additional costs to the economics of the process. However, SCWG converts biomass into H2-rich syngas without the need for pretreatment and drying. The SCWG technology uses water at high pressure (250–300 bar) and temperature (700°C) as the reaction medium and exploits the supercritical water properties that include low density and viscosity, as well as non-polar behavior, thus improving mixing and heat distribution (Boukis and Stoll, 2021). The VERENA pilot plant in the Karlsruhe Institute of Technology in Germany (Boukis and Stoll, 2021) has achieved considerable progress in SCWG technology. The VERENA pilot plant operates with 100 kg/h of wet biomass at 700°C and 350 bar. One of the plant’s features is the separation of solids and brine from the primary product stream, thus avoiding clogging and decreasing the fouling of the heat exchanger (Boukis and Stoll, 2021). The TU Delft/Gensos semi-pilot-scale plant has been recently established in the Netherlands. This pilot-scale plant applies SCWG in a fluidized-bed reactor with a capacity of 50 kg/h wet biomass at 240 bar and 500–600°C. The fluidized-bed reactor of this plant has solved reactor plugging problems due to char formation and salt deposition and thus improved the heat and mass transfer during the reaction (Yakaboylu et al., 2018). Although significant progress has been made in the SCWG technology, there is still no pre-commercial-scale plant utilizing this technology. One of the main challenges of this technology is controlling the reactor’s high pressure, as the vapor pressure of water is considerably higher than that of the produced gas under the reaction conditions (Okolie et al., 2019). This technology also has other challenges, such as corrosion, erosion, and mechanical connection stability (adaptors and welding) (Boukis and Stoll, 2021).

Table 14.1 Biomass-to-liquid technologies. Organization

Gasification

Scale

Year

Details

Velocys (G€ ussing, Austria)

Dual Fluidized-bed gasification

Pre-Commercial

2010

CHOREN, Sigma Plant (Freiberg, Germany)

Carbo-V gasification

Commercial

2010

Solena Fuels, Green Sky (Essex, UK)

Solena plasma gasification

Commercial

2015

• • • • • • • • • • •

Sierra Biofuels, Fulcrum Bio-energy (Nevada, USA)

Red Rock Biofuels (Oregon, USA)

Steam reforming gasification

Steam reforming gasification

Commercial

Commercial

2016

2017

• • • • • • • • •

150 t/d dry biomass 1 bpd FT products Co-based catalyst Microchannel reactor 3044 t/d dry biomass 5000 bpd liquid fuel Co-based catalyst Discontinued Municipal and commercial waste 1157 bpd jet fuel Velocys microchannel reactor Co-based catalyst Municipal solid waste 400 t/d municipal solid waste feed Velocys reactor Co-based catalyst Forest and saw mill waste 460 t/d biomass feed Velocys reactor Co-based catalyst

Reproduced from Ail, S. S., Dasappa, S., 2016. Biomass to liquid transportation fuel via Fischer Tropsch synthesis—Technology review and current scenario. Renew. Sustain. Energy Rev. 58, 267–286.

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14.2.1.2 Syngas cleaning and conditioning The syngas purification step is the most expensive part of the FT process, accounting for 60%–70% of the total cost in the case of the natural-gas-based FT process (the simplest option). This cost is increased by 50% in the coal-based FT process and by a further 50% in the biomass-based FT process (Zhang, 2010). Therefore, syngas cleaning is considered the greatest challenge to the commercialization of the BTL process. The presence of impurities in the syngas produced by the gasification step is inevitable. Syngas contains various types of contaminants, such as particulates, condensable tars, BTX (benzene, toluene, and xylenes), alkali compounds, hydrogen sulfide (H2S), hydrochloric acid (HCl), NH3, and hydrogen cyanide (HCN). The catalysts used in the FT reactor to synthesize the liquid fuels are notoriously sensitive to such impurities, especially sulfur and nitrogen compounds, which irreversibly poison the FT catalysts (G€ oransson et al., 2011; Van Der Drift et al., 2004). Alkali metals and tars deposit on the catalysts and contaminate the products, while particles cause fouling of the reactor. Therefore, extensive cleaning of the syngas is required prior to feeding it into the FT reactor. Indicative syngas specifications for FT synthesis are shown in Table 14.2 (G€ oransson et al., 2011; Gupta et al., 2021). The first step in all syngas cleaning configurations is removing BTX or larger hydrocarbons and tars. BTX should be removed upstream of the active carbon filters in the syngas cleaning train, as active carbon adsorbs BTX and would therefore require frequent regeneration, which would, in turn, reduce process reliability. Tars normally condensate under the typical FT reactor conditions and foul downstream equipment, coat surfaces, and enter pores in the filter and sorbents. Therefore, tars should be removed at a temperature below the condensation point at the FT reactor’s operating Table 14.2 Maximum allowable concentration of impurities in syngas. Impurity

Specification

H2S + COS + CS2 NH3 + HCN HCl + HBr + HF Alkali metals (Na + K) Particles (soot, ash) Hetero-organic components (including S, N, O) Organic compounds (tars)