Plant and Algae Biomass: Feasible Sources for Biofuel Production 303094073X, 9783030940737

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Rouf Ahmad Bhat Dig Vijay Singh Fernanda Maria Policarpo Tonelli Khalid Rehman Hakeem

Plant and Algae Biomass

Feasible Sources for Biofuel Production

Plant and Algae Biomass

Rouf Ahmad Bhat • Dig Vijay Singh  Fernanda Maria Policarpo Tonelli  Khalid Rehman Hakeem

Plant and Algae Biomass Feasible Sources for Biofuel Production

Rouf Ahmad Bhat Department of Environmental Sciences Cluster University Srinagar Srinagar, Jammu and Kashmir, India

Dig Vijay Singh Department of Environmental Science Babasaheb Bhimrao Ambedkar University Lucknow, India

Fernanda Maria Policarpo Tonelli Universidade Federal de Minas Gerais Belo Horizonte, Brazil

Khalid Rehman Hakeem Department of Biological Sciences Faculty of Science King Abdulaziz University Jeddah, Saudi Arabia Princess Dr Najla Bint Saud Al- Saud Center for Excellence Research in Biotechnology King Abdulaziz University Jeddah, Saudi Arabia

ISBN 978-3-030-94073-7    ISBN 978-3-030-94074-4 (eBook) https://doi.org/10.1007/978-3-030-94074-4 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Ecosystems worldwide are being impacted negatively by human actions, ignoring the sustainability principles. These actions, for example, the intensive production and use of fossil fuels to achieve rapid urbanization and industrialization, represent a serious risk to the survival of life on Earth. When it comes to the use of energy derived from petroleum, toxic pollutants are generated as a consequence of this kind of fuel combustion. To worsen the scenario, most of these pollutants are persistent contaminants capable of causing extensive damage to the environment for a long time. So, sustainable ways to produce green fuels must receive attention. In this context, biofuels emerge as an interesting option that can be produced from renewable organic material, which may be sourced indefinitely in contrast to fossil fuels. Plants and algae are vital organisms responsible for the majority of oxygen release on Earth. These organisms are photosynthetic in nature and evolve oxygen for animals and microbes settled in diverse kinds of habitats. Plant and algae metabolites have wide applications in several industries, such as food and cosmetics. Consequently, they have received wide recognition as a sustainable energy source. The utilization of plant and algae biomass aims to generate a large quantity of biofuel. Biofuel from plant and algae metabolites is a suitable alternative energy resource at the global level. Plant and algal biomass are considered viable green fuel sources. The exploitation of plant and algal biomass for energy production can alleviate energy scarcity and help in attaining energy goals. Furthermore, fuel from these sources is considered carbon neutral and can play an essential role in reducing the negative impact of high concentrations of greenhouse gases. In this book, some important plant and algal metabolites that can be used as a source for the production of biofuel have been highlighted. Mechanism and methods applied to extract these substances from these organisms have been elaborated in a versatile manner. Protocols dedicated to optimizing the obtainment processes (involving specific metabolic routes or other types of optimizations on steps involved in biofuel production) have also been given a valuable space. Aspects related to economic issues associated with biofuel and technical challenges related to biofuel obtainment have been discussed at length. This book is an v

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Preface

attempt to offer to the students of undergraduate, postgraduate, and researchers a comprehensive knowledge of biofuel production from plants and algae. We are extremely grateful to Springer for their generous cooperation and providing us the opportunity to publish this book.

Srinagar, Jammu and Kashmir, India  Rouf Ahmad Bhat  Lucknow, India  Dig Vijay Singh Belo Horizonte, Brazil  Fernanda Maria Policarpo Tonelli   Jeddah, Saudi Arabia  Khalid Rehman Hakeem  

About the Book

Environmental pollution is, nowadays, one of the most worrying problems worldwide, and fossil fuels are a relevant source of contamination. In this context, biofuel emerges as an eco-friendly alternative deserving attention. The book enters the aspects of environmental pollution caused by fossil fuels into an ideal of a pollution-free atmosphere. It is an attentive attempt in bringing forward the characteristics of plants and algae metabolites for the production of biofuel. The book contains 9 chapters, discussing different aspects of plant and algae as relevant sources of substances to biofuel obtainment. The introductory chapters of the book deal with the appreciative values of a pollution-free atmosphere, followed by the potentials of plants and algae as a liquid energy resource. Moreover, the subsequent chapters in the book deal with the mechanism and methods for the production of biofuel. A sizeable portion of the book has been dedicated to the metabolic routes to enhance the extraction of biofuel. The final section is dedicated to conclusions and also discusses future developments on biofuels. In general, the contents of this book are presented in logical order to assist the reader reflect upon the theme from a biotechnological point of view. We hope this book shall provide an up-to-date account of algae as a source of clean fuel for the future.

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Contents

1 Introduction: Values of Pollution-Free Atmosphere ����������������������������    1 1.1 Introduction��������������������������������������������������������������������������������������    1 1.2 Microalgae as a Biofuel Source��������������������������������������������������������    4 1.3 Macroalgae for Biofuel Production��������������������������������������������������    5 1.4 Benefits of Microalgal Biofuels��������������������������������������������������������    6 1.5 Challenges for the Commercialization of Algal Fuel ����������������������    8 1.5.1 Algal Growth������������������������������������������������������������������������    8 1.5.2 Bioprospecting: Utilizing Natural Diversity to Increase Productivity��������������������������������������������������������������������������    8 1.5.3 Diatoms ��������������������������������������������������������������������������������    9 1.5.4 Chlorophyceae and Trebouxiophyceae ��������������������������������   10 1.5.5 Cyanobacteria ����������������������������������������������������������������������   10 1.5.6 Breeding, Classical Genetics to Improve and Identify Traits����������������������������������������������������������������   11 1.5.7 Energy Security��������������������������������������������������������������������   11 References��������������������������������������������������������������������������������������������������   12 2 Plant and Algae Classes Recognition, Biomass Production and Potential Source of Biofuel��������������������������������������������������������������   15 2.1 Introduction��������������������������������������������������������������������������������������   15 2.2 Role of Algae in Biosphere ��������������������������������������������������������������   18 2.3 Crops and Plants as the Source of Biofuel����������������������������������������   19 2.3.1 Corn (Zea mays)��������������������������������������������������������������������   19 2.3.2 Sugar Cane����������������������������������������������������������������������������   19 2.3.3 Palm Oil��������������������������������������������������������������������������������   20 2.3.4 Jatropha ��������������������������������������������������������������������������������   20 2.3.5 Switchgrass ��������������������������������������������������������������������������   20 2.4 Different Types of Biofuels from Algae��������������������������������������������   21 2.4.1 Biodiesel ������������������������������������������������������������������������������   21 2.4.2 Bioethanol ����������������������������������������������������������������������������   22

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2.4.3 Biogas ����������������������������������������������������������������������������������   24 2.4.4 Bio-hydrogen������������������������������������������������������������������������   25 References��������������������������������������������������������������������������������������������������   27 3 Plant and Algae Metabolites Alternative and Clean Source of Energy��������������������������������������������������������������������������������������   33 3.1 Introduction��������������������������������������������������������������������������������������   33 3.2 Role of Plant and Algal Metabolites in Biofuel Production ������������   35 3.2.1 Lignin������������������������������������������������������������������������������������   35 3.2.2 Cellulosic and Hemicellulosic Biomass�������������������������������   37 3.2.3 Lipids������������������������������������������������������������������������������������   40 3.2.4 Fatty Acids����������������������������������������������������������������������������   42 References��������������������������������������������������������������������������������������������������   43 4 Mechanism and Methods of Extraction of Biofuels������������������������������   51 4.1 Introduction��������������������������������������������������������������������������������������   51 4.2 Energy Development and Environmental Welfare����������������������������   52 4.3 An Overview of Biofuels������������������������������������������������������������������   54 4.4 Classification of Biofuels������������������������������������������������������������������   55 4.4.1 First-Generation Biofuels�����������������������������������������������������   56 4.4.2 Second-Generation Biofuels ������������������������������������������������   60 4.4.3 Third-Generation Biofuels����������������������������������������������������   61 4.4.4 Fourth-Generation Biofuels��������������������������������������������������   62 4.5 Need of the Biofuel Production��������������������������������������������������������   64 4.6 Biofuel Production Techniques��������������������������������������������������������   66 4.6.1 Techniques for Production of Biofuels from Biomass����������   67 4.6.2 Biomass to Bioethanol Transformation��������������������������������   67 4.6.3 Structure of Biogas for Anaerobic Digesters������������������������   68 4.6.4 Preparing Biogas with Anaerobic Digestion������������������������   69 4.6.5 Biogas-Purifying Techniques������������������������������������������������   70 4.7 Cellulosic Biofuel Conversion Methods and Emerging Technologies ������������������������������������������������������������������������������������   71 4.8 Conclusion����������������������������������������������������������������������������������������   74 References��������������������������������������������������������������������������������������������������   74 5 Metabolic Routes to Biofuels Extraction ����������������������������������������������   87 5.1 Introduction��������������������������������������������������������������������������������������   87 5.2 First-Generation Biofuels������������������������������������������������������������������   90 5.3 Second-Generation Biofuels ������������������������������������������������������������   92 5.4 Third-Generation Biofuels����������������������������������������������������������������   94 5.5 Microfluidic Techniques for Enhancing Biofuel Based on Microalgae������������������������������������������������������������������������   95 5.5.1 Industrial Waste��������������������������������������������������������������������   96 5.5.2 Lignocellulosic Biomass������������������������������������������������������   96 5.5.3 Biohydrogen Production ������������������������������������������������������   98 5.5.4 Bioethanol and Biomethanol������������������������������������������������   99

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5.6 Challenges of Algal Fuel Commercialization ����������������������������������   99 5.7 Improved Oil Extraction ������������������������������������������������������������������  100 5.8 Future Perspectives ��������������������������������������������������������������������������  100 References��������������������������������������������������������������������������������������������������  101 6 Optimizations on Steps Involved on Biofuel Obtainment and their Validation ��������������������������������������������������������������������������������  107 6.1 Optimizing Raw Material for Biodiesel Production ������������������������  107 6.2 Optimizing Pretreatment for Biodiesel Production��������������������������  112 6.3 Optimizing Biofuel Manufacturing Process ������������������������������������  115 6.4 Modelling Optimizations: Validation������������������������������������������������  116 References��������������������������������������������������������������������������������������������������  119 7 Economic Consideration on Biofuel and Energy Security������������������  127 7.1 Introduction��������������������������������������������������������������������������������������  127 7.2 Economic Aspects of Biofuels����������������������������������������������������������  128 7.3 Biofuel Policies and Scenario ����������������������������������������������������������  130 7.4 Need to Focus on Biofuels����������������������������������������������������������������  131 7.5 Environmental Benefits��������������������������������������������������������������������  131 References��������������������������������������������������������������������������������������������������  132 8 Technical Challenges of Biofuel Obtainment����������������������������������������  135 8.1 Introduction��������������������������������������������������������������������������������������  135 8.2 Lignocellulosic Biomass-Derived Biofuel����������������������������������������  136 8.3 Algae-Derived Biofuel����������������������������������������������������������������������  139 8.4 Biodiesel from Oleaginous Microbes ����������������������������������������������  141 References��������������������������������������������������������������������������������������������������  143 9 Conclusion and Future Perspectives������������������������������������������������������  147 9.1 Main Conclusions ����������������������������������������������������������������������������  148 9.2 Future Perspectives ��������������������������������������������������������������������������  151 References��������������������������������������������������������������������������������������������������  153 Index������������������������������������������������������������������������������������������������������������������  157

About the Authors

Rouf Ahmad Bhat (PhD)  has pursued his doctorate at Sher-e-Kashmir University of Agricultural Sciences and Technology Kashmir (Division of Environmental Science) and presently working in the Department of School Education, Government of Jammu and Kashmir. Dr Bhat has been teaching graduate and postgraduate students of environmental sciences for the past 3 years. He is an author of more than 50 research articles (h-index 18; i-index 26; total citation >820) and 40 book chapters, and has published more than 28 books with international publishers (Springer, Elsevier, CRC Press Taylor and Francis, Apple Academic Press, John Wiley and IGI Global). He specializes in limnology, toxicology, phytochemistry and phytoremediation. Dr Bhat has presented and participated in numerous state, national and international conferences, seminars, workshops and symposium. Besides, he has worked as an associate environmental expert in World Bank-funded Flood Recovery Project and also as environmental support staff in the Asian Development Bank (ADB) funded development projects. He has received many awards, appreciations and recognition for his services to the science of water testing, air and noise analysis. He has served as editorial board member and reviewer of reputed international journals. Dr Bhat is still writing and experimenting with diverse capacities of plants for use in aquatic pollution remediation.

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About the Authors

Dig Vijay Singh  has obtained his master’s degrees from Sher-e-Kashmir University of Agricultural Sciences and Technology Kashmir (Division of Environmental Science) and is pursuing his doctoral studies in the School of Environmental Science, Babasaheb Bhimrao Ambedkar Central University, Lucknow. He has published 08 research articles in reputed journal (Springer and Elsevier) and 16 book chapters with international publishers (Springer, Elsevier, CRC Press Taylor& Francis, Apple Academic Press, John Wiley and IGI Global). His specialization is in wastewater treatment, phycoremediation, metal toxicity, stress physiology, antioxidants defence mechanisms and biofuel production. Singh has presented and participated in several state and national conferences, seminars and workshops. Fernanda  Maria  Policarpo  Tonelli  specializes in molecular biology and has been studying biotechnological topics like gene delivery approaches (using engineered viral particles and nanomaterials) aiming transgenesis. She has taught topics related to biochemistry and molecular biology to graduate students. Dr Tonelli has authored 12 scientific articles and more than 20 book chapters for international publishers and has reviewed various articles/book proposals. She has presented and participated in numerous national and international conferences and has also had the opportunity to contribute to the organization of various scientific events. Dr Tonelli has dedicated herself to the promotion of science and technology, co-­funding an NGO with this purpose, and has joined a scientific divulgation group composed only of women. Her efforts as a researcher have been recognized by various awards (including For Women in Science Brazil-L’Oreal/ UNESCO/ABC and Under30 Brazil – Forbes) and certificates of merit. Khalid  Rehman  Hakeem  (PhD, FRSB) is presently working as a professor at King Abdulaziz University, Jeddah, Saudi Arabia. After completed his PhD (botany; specialization in plant ecophysiology and molecular biology) from Jamia Hamdard, New Delhi, India in 2011, he has worked as an assistant professor at the University of Kashmir, Srinagar, for a short period. Later, he joined Universiti Putra Malaysia, Selangor, Malaysia, and worked there as a postdoctoral fellow in 2012 and fellow researcher (associate professor) from

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2013 to 2016, respectively. He joined King Abdulaziz University in August 2016 and was promoted to professorship recently. Dr Hakeem has more than 12 years of teaching and research experience in plant ecophysiology, biotechnology and molecular biology, medicinal plant research, and plant-microbe-soil interactions as well as in environmental studies. He is the recipient of several fellowships at both national and international levels. He has recently been elected as Fellow of the Royal Society of Biology, London. Dr Hakeem has served as a visiting scientist at Fatih Universiti, Istanbul, Turkey, and at Jinan University, Guangzhou, China. Currently, he is involved with a number of international research projects with different government organizations. So far, Dr Hakeem has authored and edited more than 70 books with international publishers, including Springer Nature, Academic Press (Elsevier) and CRC Press, among others. He has also to his credit 155 research publications in peerreviewed international journals and 65 book chapters in edited volumes with international publishers. At present, Dr Hakeem is serving as an editorial board member and reviewer of several high-impact international scientific journals by Elsevier, Springer Nature, Taylor, Cambridge and Francis and John Wiley Publishers.

Chapter 1

Introduction: Values of Pollution-Free Atmosphere

Abstract  Fossil hydrocarbons are needs of the global economy to function. Fossil hydrocarbons are required for producing light, heat and transportation. With the increase in our population and economy, there will be the increase in the use of fossil fuel. Additionally, atmospheric CO2 concentration was increased, and the potential for major greenhouse gas-mediated climate change now seems likely to affect the whole world. Electrical power and liquid fuels both are generated by the use of fossil fuels. Various renewable or low atmospheric technologies are there which are used to generate electrical power, such as solar, wind, hydroelectric, geothermal and nuclear. Nonetheless, renewable technologies to add or replace liquid fossil fuels are in their early developmental phases because for the time being, it is expected that future demand for energy is growing at a yearly growth rate of 5–7.9% for the upcoming 20 years. The demand for the total biofuel is expected to meet about 27% of the total transport fuel demand in 2050. It is thought that in the coming years, biofuels are the best resources to meet the increase in energy demand viewpoints. In 1970s, when the whole world faced a major oil crisis, the concept of biofuels was visualized. Most of the people in the developing countries rely on agriculture which improves the general well-being and also helps to elevate food security. Biofuels can help international development and poverty mitigation, due to the reason that a number of people in the developing countries take part in agriculture to elevate agricultural income, which strongly recover the general welfare and also elevated food security. Therefore, the present chapter aimed to discuss about the conclusion and future perspectives of biofuels produced from algae. Keywords  Algae · Biofuel · Fossil fuels · Biomass · Biofuel energy

1.1  Introduction Fossil hydrocarbons are needs of the global economy to function. Fossil hydrocarbons are required for producing light, heat and transportation. With the increase in our population and economy, there will be the increase in the use of fossil fuel. It © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4_1

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1  Introduction: Values of Pollution-Free Atmosphere

was suggested in the data that the use of fossil fuel will enhance, and competition will also increase for these limited resources because nations improve their gross domestic product per capita. Additionally, atmospheric concentration of carbon dioxide was increased, and the potential for major greenhouse gas-mediated climate change (Parry et  al., 2007) now seems likely to affect the whole world. At last, petroleum is a limited resource which is derived from earliest algae deposits and will finally come to an end or turn into too costly to improve (Schindler & Zittel, 2008; Dyni, 2006). Renewable energy resources are developed by these factors that may displace fossil fuels and authorize larger access to fuel resources for all countries and, at the same time, highly decrease carbon emission into the atmosphere. There are various technologies which have been examined as renewable energy resources and, though not a single strategy, are providing complete solution. It is possible that an employment of combined strategies will significantly reduce our reliance on fossil fuels. Renewable energy industries which function sustainably is becoming a challenge that remains, and it can be cost-competitive with existing energy resources. Electrical power and liquid fuels both are generated by the use of fossil fuels. Various renewable or low atmospheric technologies are there which are used to produce electrical power, such as solar, wind, hydroelectric, geothermal and nuclear. Nonetheless, renewable technologies to add or replace liquid fossil fuels are in their early developmental phases. It was expected by the “International Energy Agency” that 6% of the total fuel will be contributed by biofuels by 2030 but can increase considerably if undeveloped petroleum fields are not admitted or if extensive new fields are not recognized. The most capable sustainable substitutes are approximately wholly categorized under the “moniker biofuels”. A varied series of technologies come under this term which produces fuel with a minimum one part based on a biological system. The main technologies which are currently used for biofuels start with terrestrial plants and terminate with ethyl alcohol, whether it is corn starch to carbohydrates to ethyl alcohol. In Brazil, the success of these types of strategies is well reported, particularly the sugar cane to ethyl alcohol generation (Nass et al., 2007). The oil from some terrestrial plants, for instance, soy and palm, are used to generate biodiesel to a smaller extent. These strategies only work at small level; nonetheless, because their use has elevated, it is obvious that they are not sustainable, due to the huge amount of agricultural land that will be needed to add a considerable amount of petroleum by the use of this strategy (Fargione et  al., 2008; Searchinger et al., 2008). Presently, various hybrid schemes have been discussed or being installed. Some instances of these schemes are conversion of cellulose to sugars for fermentation into fuel and gasification of residual biomass into syngas then that can be used to generate liquid fuels (Huber et al., 2006). Though to generate fuels each of these strategies is being used, they are inadequate to provide the worldwide demand for liquid fuels. Nowadays, because fossil fuels are non-renewable energy resources and will diminish sooner or later in the coming time, energy safety is becoming a serious matter (Masjuki et  al., 2013). The greenhouse gas emission is caused due to the increase in the use of fossil fuel, which then causes adverse damage to the

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environment. The rehabilitated interests in the fuel’s production from renewable sources have come due to the instability of oil prices together with foremost concerns about the change in the climate, oil supply security and reducing raw materials (Dellomonaco et al., 2010a). After that, finding new fields and instigating energy from the future natural gases and non-conservative resources are a big challenge because for the time being, it is expected that future demand for energy is growing at a yearly growth rate of 6–8% for the upcoming 20 years. The demand for the total biofuel is expected to meet about 27% of the total transport fuel demand in 2050 (Müller-Langer et al., 2014). It is thought that in the coming years, biofuels are the best resources to fulfil the energy demand viewpoints. In 1970s, when the whole world faced a major oil crisis, the concept of biofuels was visualized. Most of the people in the developing countries rely on agriculture which improves the general well-being and also helps to elevate food security. Biofuels can help international development and poverty mitigation, due to the reason that a number of people in the developing countries take part in agriculture to elevate agricultural income, which strongly recovers the general welfare and also elevated food security (Dale et al., 2014). It has been reported that biofuels are easily available in locality; these are non-polluting, sustainable and reliable fuels and can be acquired from renewable sources (Demirbas, 2008). As compared to fossils and nuclear resources, the distribution of renewable resources is done uniformly. Biogas is another place of renewable resources; it has been derived by the treatment of manure and biomass material anaerobically. Nowadays, the volumes of biogas used for the purpose of transportation are comparatively very less (Naik et al., 2010). For the development and growth, biofuel sector requires both opportunities and threats (Bindraban et al., 2009). Currently, elevation in pressure on arable land is used for the production of food, and it can cause severe food scarcity, particularly in developing countries, where approximately 799 million people are facing the problem of hunger and malnutrition (Dragone et  al., 2010). Consequently, focussing towards the thorough research in biotechnology sector and plant agronomy sector and by using accuracy agricultural techniques, increase in the production rate of biofuels can be done within the available land (Masjuki et al., 2013). White biotechnology is the branch of biotechnology which holds the bio-generation of fuels and chemicals from renewable resources. Various primary factors which are responsible for the unstable development in division of biofuel and extensive interest for the technology are (i) chances to decrease dependence on fossil fuels through renewable energy and (ii) search for security and dependence on energy as rising economies like USA and (iii) as an ability to decrease the net emission of carbon dioxide into the atmosphere and to decrease global warming and (iv) to increase the prices of commodity, to enhance income and significantly to enhance the chances of rural employment (FAO, 2006). Modern advancements in synthetic biology may give novel tools for engineers to work on their plans and build new optimal biocatalysts for the generation of biomass sustainably (Dellomonaco et  al., 2010b). The sixth leading consumer of energy in the whole world is India. It has been estimated that by the year 2030, the demand for energy will grow eight times at the current growth rate of 4.9% per year. Annually, India is losing a considerable amount of foreign

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exchangers via importing crude fossil fuel, which is used about 72% of the total requisitions of the country (Kaushik et al., 2010). Up to now, in Asian countries, small research has particularly addressed biofuels. The main consumers of fossil fuels are the transportation and agricultural sectors, and these are the main contributors to environmental pollution, and it can be decreased by the replacement of fossil fuels by renewable energy resources of bio-origin (Agarwal, 2007). However, in the sector of transportation, fossil fuel is used in faster rate, and the drift emerges to be moving up considerably (Masjuki et al., 2013). Economic cost-benefit analysis is used to access the effectiveness of alternative policies of biofuels in attaining energy, environmental and agricultural policy goal (De Gorter & Just, 2010). It has been reported that various political concerns can motivate the policies of biofuels such as decreasing reliance on oil, improving the environment and enhancing the agricultural income (Rajagopal et al., 2007). Law makers should recognize from the future emergencies to make a short-medium and long-term policy taking into account all the observations, characteristics and options (Masjuki et  al., 2013). The major approaches should be (i) decrease in tax for biofuels and (ii) biofuel responsibility. Numerous concerns are elevated by these progresses without financial assistance because in various parts of the world, biomass has to compete price-wise with petroleum commodities (Doornbosch & Steenblik, 2008).

1.2  Microalgae as a Biofuel Source The single-celled and simply multicellular microorganisms are microalgae. These include prokaryotic microalgae such as cyanophyta or gram-negative bacteria and eukaryotic microalgae such as streptophyta or chlorophyta and diatoms (bacillariophyta). Microalgae are useful because these have the capability to produce whole year. Microalgae grow and reproduce in aqueous medium and therefore require a smaller amount of water as compared with terrestrial crops. Not like other biodiesel crops, microalgae also do not need pesticides. Various valuable products like protein and residual biomass after the extraction of oil are also generated by microalgae. These products may be used as feed or to make fertilizer, or fermentation can be done to generate ethyl alcohol or CH4 (Fig. 1.1). The considerable increase in the yield of oil can be done by changing the growth condition to adjust the biochemical composition of algal biomass (Rodolfi et al., 2009; Cantrell et  al., 2008). The choices of exact species for the generation and extraction of important co-products come under the algal biofuel technology. Algae are bioengineered for attaining complex photosynthetic effectiveness through uninterrupted growth of manufacturing system. There are various challenges including only “single species cultivation techniques” which are developed until now, and it has been recommended to follow these worldwide, but varied culture can give more algal oil as compared to single species culture. It has been assessed that generation

1.3  Macroalgae for Biofuel Production

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Fig. 1.1  Showing the use of macroalgae for the production of biodiesel and other co-products

of oil from algae costs less than extracting oil from other resources because various techniques are used for extracting oil from other resources such as water pumping, transmission of carbon dioxide, production and extraction (Ugwu et al., 2008; Ono & Cuello, 2006). The use of microalgae for the production of various products are depicted in Fig. 1.2.

1.3  Macroalgae for Biofuel Production The fastest growing algae are macroalgae. The size of macroalgae is 60 m in length. Macroalgae have growth rates go beyond those of terrestrial plants. For instance, the biomass of brown algae of the standard output for non-cultured algae was about 3.4–10.9 kg dry weight m−2 year−1 and for culture algae up to 12.9 kg dry weight m−2 more than 7 months as compared to 5.9–10.8 kg fresh weight m−2 year−1 for sugar cane, main fruitful terrestrial plant. They are accessible in the natural water basins during their favourable seasons. There is no need of arable land and fertilizer for the cultivation of macroalgae at sea and give a probable solution to the energy crisis. The biomass of macroalgae has high quantity of sugars, and these sugars might be used to produce ethanol fuel (Wi et al., 2009).

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Fig. 1.2  Showing the use of microalgae for the production of various products

1.4  Benefits of Microalgal Biofuels Microalgae are varied group of unicellular organisms. These have the capability to give numerous solutions for the requirements of liquid transportation fuel through various paths. For the growth and development of algal species, broad range of aquatic environment is required. Carbon dioxide is effectively used by algae and is accountable for over 41% of the worldwide carbon fixation, and the majority of this production comes from marine microalgae (Parker et  al., 2008; Falkowski et  al., 1998). Biomass can be made by algae very quickly, some species replicating as few as 6 h and most of the species showing two replications per day (Huesemann et al., 2009; Sheehan et al., 1998a). Algae that are having high energy are capable of producing oils, and numerous species are there which naturally accumulate high level of oil in total dry biomass (Rodolfi et al., 2009). For instance, identification of a number of Botryococcus spp. which have approximately 51% of their dry biomass stored as “long-chain hydrocarbons” have been done (Kojima & Zhang, 1999). The diversity of algae provides various options for researchers to identify the strains for its production and also provides basis for genetic information that might be useful for the improvement of the production strains. The different species of microalgae are examined as possible biofuel crops originating from that groups that have extensively diverse familial relationships than the most diverse terrestrial plants, giving very rich genetic diversity (Deschamps & Moreira, 2009; Reeb et al., 2009). The following groups are discussed under the microalgae such as chlorophyta, diatoms, phaeophyceae, prymnesiophytes, eustigmatophytes, and cyanobacteria, and the examination of members belonging to all these groups has a great potential to

1.4  Benefits of Microalgal Biofuels

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Fig. 1.3  Showing the various benefits of algal biofuel

produce a good quantity of biofuels. Nevertheless, it must be noted that cyanobacteria are not algae but a class of photosynthetic bacteria. There are various other advantages of microalgae more than terrestrial plants (Fig.  1.3). As these are unicellular organisms that replicate by division, various higher technologies might be used to fastly develop strains. These technologies might bring reduction in the processes which take years in crop plants down to some months in algae. The biomass terrestrial sources which are used to produce biofuels have high impact on the environment as compared to algae (Dismukes et al., 2008). Algae are capable of eliminating essential nutrients from the water and can be cultivated on land and will not be used for traditional agriculture. Therefore, not only would the manufacturing of algae biofuels reduce the land use as compared to biofuels generated from land plants, but in the culturing process of microalgae, waste tributaries can also be reformed. Municipal wastewater is coming under waste tributaries in which NO3− and SO42− are removed before the release and exhaust gas of coal or other power plants based on combustible material to detain SO42− and CO2 (Fierro et  al., 2008; Douskova et  al., 2009). The strains of algae generation are capable to be bioengineered, allowing development of particular traits (Rosenberg et al., 2008; Zaslavskaia et al., 2001) and manufacturing of important co-products which might permit biofuels of algae to compete efficiently with petroleum. All these important characters make algae a platform that has a high capability to generate cost-competitive biofuels.

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1.5  Challenges for the Commercialization of Algal Fuel The reasons which have been mentioned to spend important assets to change the algae into biofuels are higher growth rate, logical densities of growth and higher contents of oil. Nonetheless, for algae to mature as an economically practical platform to balance petroleum and, therefore, alleviate the discharge of carbon dioxide, there are numerous obstacles to defeat arrays from how and where to grow algae, for the improvement of oil extraction and the processing of fuel. The main challenges which come under the production of biofuels from algae are isolation of strain, use and sourcing of nutrient, construction management, harvesting, growth of co-­ products, extraction of fuel, refining and use of remaining biomass.

1.5.1  Algal Growth On the production of biofuel from algae, an important effect will be made by better engineering. These advancements comprise effective plans for the circulation of nutrients and exposure to light and have been checked somewhere else (Lehr & Posten, 2009). Briefly, for engineers, various important challenges are there to either design “photobioreactors” that are inexpensive for extensive use or for biologists or engineers to merge forces to develop those species which will grow effectively in economical systems (Borowitzka, 1997). Photobioreactors have various benefits above open systems that means photobioreactors can simply preserve axenic cultures and maintain controlled environments of growth which finally enhances the productivity (Chisti, 2007). Notwithstanding the advantages of reduced contamination and enhanced output, it is not clear whether photobioreactors will ever develop into cost-competitive with open pond systems. Despite the employment of the growth plan, considerable advancements in excess of technologies for the development, production and oil extraction from algae are required to be prepared.

1.5.2  B  ioprospecting: Utilizing Natural Diversity to Increase Productivity A number of identified species and probably hundreds of thousands of species come under the really varied group, i.e., algae. An extremely diverse group of algae gives a broad variety of preliminary strains for the generation of biofuel. It provides unbelievable chances but also gives an important challenge. It shows an unbelievable chance other than an important challenge too. There are requirements of large

1.5  Challenges for the Commercialization of Algal Fuel

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efforts for the application of characterizing species in the practices of industries. Following the recognition of species, considerable physiological, biochemical and genetic categorization must take place to move a species into a practical pipeline. Various established optimal conditions of growth (such as temperature, level of nutrients, salinity and pH), characterization of growth (such as growth rate and density of final culture) and examination of accumulation of metabolite (such as composition and accumulation of lipid) are included in this type of characterization. Separately, functional genomics such as “proteomics and metabolomics” may give insight into various pathways of metabolites there in the species and also provide a base for future metabolic engineering. The preliminary characterization of a small number of species is provided by the United States government-sponsored “Aquatic Species Program (ASP)” from the 1970s through to the mid-1990s (Sheehan et al. 1998b).

1.5.3  Diatoms Recently, the most productive major marine producers and the most diverse group are diatoms, having more than 100,000 species. In addition to this, it is thought that a number of oil dumps are made up of biomass (Armbrust, 2009). The differentiation of diatoms is done by their ornate bipartite shells, and these shells are made up mainly of polymerized silicates. It has been found that diatoms accumulate lipids in the silica-restricted environment. Nevertheless, growth is decreased (Roessler, 1988). Although this class of algae shows a comparatively unused pool of biodiversity for biofuels, there are very few studied about the mechanism of lipid accumulation by algae. Most of the work has been done on the generation of shells of diatoms. Additionally, various species of diatoms have been identified which produces various biomolecules, for instance, omega-3 fatty acids. These have a variety of commercial applications or show a number of benefits to human health (Petrie et al., 2009; Napier & Sayanova, 2005). The use of genetics for the better understanding of the basic biology of diatoms or breeding approaches has been disturbed because during the vegetative growth, these are diploid, not like numerous groups of algae, though diploid species such as Arabidopsis thaliana, Drosophila and mice have turned into introductory model organisms. Due to the poor understanding of the life cycle of diatoms, these are intractable to conventional forward exploration of genetics. Thalassiosira pseudonana and Phaeodactylum tricornutum are the two species for which whole-genome sequencing have been released, and the work has been done on these species showing nuclear conversion of these two species and some other diatoms (Poulsen & Kröger, 2005; Falciatore et al., 1999; Apt et al., 1996; Dunahay et al., 1995). Reverse genetic approaches, for example, small interfering RNAs, might be allowed by the sequenced genomes in combination with nuclear transformation to divide the molecular pathways which regulate the metabolism of diatom (De Riso et al., 2009).

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1.5.4  Chlorophyceae and Trebouxiophyceae The most deeply studied groups of algae are the group of Chlorophyceae and Trebouxiophyceae mainly because of the establishment of green algae, i.e., Chlorophyceae, and the algal model organism is Chlamydomonas reinhardtii. The reason behind is that Chlorophyceae or Trebouxiophyceae both share similar lineage with vascular plants. These two green algae have about 8000 species and may be present all over freshwater and marine water, and some species can live in elevated saline conditions. For generating phytochemicals, for instance, β-carotene and astaxanthin green algae have been used widely. The genome sequencing have been done of numerous species of green algae, and for the transformation of chloroplast and genome of Chlamydomonas reinhardtii nucleus, molecular tools are also present there. Lerche and Hallmann (2009) studied the transformation of nucleus in Chlorella and volvox species of green algae (Lerche & Hallmann, 2009). It has been identified that different members of Scenedesmus genus are capable of producing oil with fast growth and comparatively elevated content of lipid. Additionally, several species are there which generate various metabolites, for instance, Botryococcus species. This species contains a fuel molecule, i.e., triterpenoid botryoccenes, that needs minimum refining (Metzger & Largeau, 2005).

1.5.5  Cyanobacteria In the beginning, the examination of cyanobacteria was done for the production of biomass, and it was found that cyanobacteria accumulate comparatively low lipids level (Sheehan et al., 1998b) though nowadays, cyanobacteria has got new interests because it is probably a biofuel resource and has the capability to grow in extreme environmental conditions. Though as compared to the eukaryotic algae, cyanobacteria cannot produce that much of lipid as the genome of cyanobacteria has the ability to simply manipulate and may produce other precursor molecules of biomass. The main advantage of cyanobacteria is that it is capable in fixing nitrogen. Though the development of species of more than two as in association have not been circumspectly modelled so far, it is likely that a synergistic relationship may also be formed due to the culturing of cyanobacteria with algae rich in lipids. It allows the total biofuel density to enlarge over recent approximates whilst reducing the rate of adding nitrates to the system. Actually, the characterization of a diatom species has been done, and a cyanobacterium is endosymbiosed by this diatom species signifying that these types of associations are really helpful to as a minimum one of the two species (Carpenter & Janson, 2000).

1.5  Challenges for the Commercialization of Algal Fuel

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1.5.6  B  reeding, Classical Genetics to Improve and Identify Traits From the beginning of agriculture, artificial selection of required trait in agricultural plants has possibly been taking place. The recognition of desired traits from the variety of naturally occurring plant species and the selective combination of these traits through interbreeding are the two conventional strategies. The variety of cultivars of Brassica oleracea from broccoli to cabbage is the obvious accomplishment of this strategy. There are a few species of microalgae in which direct and successful breeding is possible, but the sexual life cycle of enormous species is little known. This is somewhat due to the vast variety of algal group, from the diatoms having diploid nature to the Chlorophyceae having haploid nature, such that the occurrence of various processes in one species of algae is not appropriate to other species of algae. Nevertheless, due to the small size and watery life cycle of numerous algae, the knowledge about their whole life cycle is limited. The tools are developed to improve the algae for the production of biomass and for selective breeding. Further, these tools are used to move desired traits that affect the biomass between directly linked species or to increase particular strains of one species. The benefit of these breeding strategies is that polygenic traits may be moved among strains. Nevertheless, at present for the improvement of the strain of algae, mutagenesis and molecular genetics are at the front position.

1.5.7  Energy Security There are additional benefits of energy produced by energy crops which are being produced domestically. The energy security could be enhanced due to the utilization of energy crops for the production of biofuels by reducing the dependency on foreign oil. Earlier, 51% of the oil was imported by USA which was used for transportation, and it was estimated by the Department of Expenditure (DOE) that by 2010, the increase will be 75%. Considerable economic and social costs are due to the dependency on foreign imports. It was estimated that per year, $10–23 billion dollars is the total cost of defending foreign oil. For the maintenance of “Strategic Petroleum Reserve”, it costs almost 590 million barrels of oil. According to present consumption rate, if all overseas imports were withholding, the reserve would last nearly 75 days. Buhroo et al. (2018) stated that the total cost for the maintenance of such reservoir is more than 200 million dollar per year.

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Rajagopal, D., Sexton, S.  E., Roland-Holst, D., & Zilberman, D. (2007). Challenge of biofuel: Filling the tank without emptying the stomach? Environmental Research Letters, 2, 044004. Reeb, V. C., Peglar, M. T., Yoon, H. S., Bai, J. R., Wu, M., Shiu, P., Grafenberg, J. L., Reyes-Prieto, A., Rümmele, S. E., Gross, J., & Bhattacharya, D. (2009). Interrelationships of chromalveolates within a broadly sampled tree of photosynthetic protists. Molecular Phylogenetics and Evolution, 53, 202–211. Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., & Tredici, M. R. (2009). Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnology and Bioengineering, 102, 100–112. Roessler, P. G. (1988). Effects of silicon deficiency on lipid composition and metabolism in the diatom Cyclotella cryptica 1. Journal of Phycology, 24, 394–400. Rosenberg, J. N., Oyler, G. A., Wilkinson, L., & Betenbaugh, M. J. (2008). A green light for engineered algae: Redirecting metabolism to fuel a biotechnology revolution. Current Opinion in Biotechnology, 19, 430–436. Schindler, J., & Zittel, W. (2008). Crude oil–The supply outlook. Energy Watch Group. Searchinger, T., Heimlich, R., Houghton, R.  A., Dong, F., Elobeid, A., Fabiosa, J., Tokgoz, S., Hayes, D., & Yu, T. H. (2008). Use of US croplands for biofuels increases greenhouse gases through emissions from land-use change. Science, 319, 1238–1240. Sheehan, J., Dunahay, T., Benemann, J., & Roessler, P. (1998a). A look back at the US Department of Energy’s aquatic species program: Biodiesel from algae (Vol. 328, pp.  1–294). National Renewable Energy Program. Sheehan, J., Dunahay, T., Benemann, J., & Roessler, P. (1998b). Look back at the US Department of Energy’s aquatic species program: Biodiesel from algae; close-out report. (No. NREL/ TP-580-24190). National Renewable Energy Laboratory. Ugwu, C. U., Aoyagi, H., & Uchiyama, H. (2008). Photobioreactors for mass cultivation of algae. Bioresource Technology, 99, 4021–4028. Wi, S. G., Kim, H. J., Mahadevan, S. A., Yang, D. J., & Bae, H. J. (2009). The potential value of the seaweed Ceylon moss (Gelidium amansii) as an alternative bioenergy resource. Bioresource Technology, 100, 6658–6660. Zaslavskaia, L. A., Lippmeier, J. C., Shih, C., Ehrhardt, D., Grossman, A. R., & Apt, K. E. (2001). Trophic conversion of an obligate photoautotrophic organism through metabolic engineering. Science, 292, 2073–2075.

Chapter 2

Plant and Algae Classes Recognition, Biomass Production and Potential Source of Biofuel

Abstract  Energy scarcity is a worrying problem for the global population. The limited fuel reserve has pressed for the utilization of renewable fuel sources, mainly biomass. Biomass is considered as the potential substrate for renewable fuel, and several crops and plants have been utilized as the source of renewable fuel. Owing to certain constraints, the biomass from crops and plants is considered as reasonable alternative for biofuel production. Due to comparatively higher solar conversion efficiency and lipid-producing potential as compared to plants and crops, the biomass from algae is considered as the promising and futuristic source of renewable energy. Algal biomass acts as the foremost source of different types of fuels such as bioethanol, biodiesel, biogas and bio-hydrogen. The conversion of biomass to different types of biofuels is doable by thermochemical and enzymatic process. Furthermore, cheap nutrient requirements, short life cycle, carbon sequestration and extraction of different valuable products from algal biomass make the process of biofuel production more economical, eco-friendly and sustainable. Keywords  Energy · Plants · Algae · Biomass · Biofuel · Bio-hydrogen

2.1  Introduction Globally, energy demands are increasing at unprecedented rate, and total dependence on fossil fuels is responsible for the releases of different types of pollutants into the environment (Upadhyay et al., 2021). The entire world is relying on the fossil fuels as the source of energy. The crisis of energy is one of the biggest problems the global community is facing, and the escalating demands can lead to fuel depletion very soon (Droege, 2002). Fossil fuel as energy source is an unsustainable approach as the combustion of the fuel dissipates toxic pollutants responsible for degradation of environmental health (Singh et  al., 2020b). Escalating energy demands and fuel prices are majorly caused by industrialization, urbanization and population growth (Shahzad et al., 2021). The major portion of the energy demands is fulfilled by fossil fuels, and rapid use may lead to fuel exhaustion in the middle of © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4_2

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2  Plant and Algae Classes Recognition, Biomass Production and Potential Source…

this century (Shafiee & Topal, 2009). In addition, several environmental problems such as pollution and global warming are accelerated by use of fossil fuels. The limited energy reserves, rapid use of fossil fuels, rising fuel price, growing global population, climate change and environmental deterioration have provided impetus for exploitation of alternative and sustainable energy sources (Alam et  al., 2012; Singh et al., 2020b). For environment stability and sustainability, the production of environment friendly fuel is necessary (Singh et al., 2020b). The different renewable energy source such as solar, wind, water and biomass can be very helpful in sustaining the energy demands (Nematollahi et al., 2016). Among different renewable energy source, biomass can assist in developing alternative energy for the growing population. The fuel produced from living organisms such as algae, plants and animals is called biofuel. Biofuel can minimize the impact of pollution and is a sustainable approach towards proper management of the resource (Upadhyay et al., 2021). The countries such as Brazil and the USA are the largest producer of biofuel (Balat, 2007). Plants and algae are gaining importance globally as the cheap source of biofuel. Crops such as corn, wheat, sugar cane (Groom et al., 2008), palm and jatropha (Sarin et al., 2007) known as energy crops can be used as the substrate for biofuel. Utilization of food crops as biomass for biofuel production can become the cause of conflict as the food shortage can occur (Demirbas, 2007). The decline in arable lands, deteriorating soil health and increasing food demands have raised several questions on the use of food crops for biofuel production (Singh et al., 2020b). Thus, the use of different crops and plants can never be the feasible option for biofuel production. The necessity is to exploit the doable alternative which neither depends upon arable land nor compete with food crops. The best alternative seems to be algae which, because of multitude of benefits, can act as the sustainable and alternative option for biofuel production (Fig. 2.1) (Trivedi et al., 2015). Biomass

Sources of Biofuel First generaon

Second generaon

Crops such as Corn, sunflower, coconut, soyb ean, sugarcane, potato, wheat Sustainable fuel produc on

Jatropha, grass, solid waste, sludge, wood, waste and residue Advantages Economical and sustainable fuel produc on

Disadvantages Use of arable land, economically and environmentally not feasible approach for biofuel produc on

Third generaon Algae and microbes

Use of arable land and complex technologies

Economical, sustainable, grow under adverse condi ons, less land and technology requirement, mi gate green house gases and high lipid produc on

Fig. 2.1  Different sources of biomass and biofuel for energy production

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2.1 Introduction

from algae is viable feedstock for renewable energy production in economical and eco-friendly manner (Upadhyay et al., 2021). The biomass production rate and lipid content in algae are several times higher as compared to land plants; thus, exploiting algae for biomass production can be the energy-efficient and economical approach for sustainable biofuel production (Aratboni et  al., 2019; Singh et  al., 2020b). Biofuel from algae is considered as the feasible alternative energy source which can accomplish the future energy demands in economical and sustainable manner (Singh et al., 2020b). Both macro- and microalgae have potential to grow in short time and can also be cultivating on large scale (Bošnjaković and Sinagav, 2020). Algae is rapidly gaining attention as feedstock for third-generation biofuel due to its capability to tolerate extreme conditions, rapid growth, simple cultivation, short lifespan, carbon dioxide fixation and high lipid production (Singh et al., 2020a). The lipid content in algal cells is much higher (Lam & Lee, 2012) than that of soybeans as well as palm oil (Kligerman & Bouwer, 2015). In microalgae, the lipid content of the cell varies from 30% to 40%, and in some species such as Botryococcus braunii, the lipid content can reach 85% (Mirza et al., 2008). Globally, algae are now considered as the promising source of biofuel. Methane, bioethanol, biodiesel and bio-hydrogen are the different types of biofuel produced from microalgae (Fig. 2.2). Moreover, algae can also grow efficiently on different wastewaters recovering nutrient resource for growth, removing pollutants and producing biomass which can be used for biofuel production (Singh et al., 2020b). Cultivation of algae on wastewater can minimize the dependence on nutrients and water, which can increase wide-scale adoption and cultivation of algal-based systems (Upadhyay et al., 2021). Thus, multitude of benefits from algal biomass compared to crop plants has provided huge impetus to the scientific community to exploit microalgae on wide scale and optimize the different conditions responsible for enhanced biofuel production (Singh et al., 2021).

Ethanol Hydrolysis + Fermentaon Butanol

Anaerobic digeson

Carbohydrate enriched algal biomass

Fermentaon

Methane

Hydrogen Methane

Fig. 2.2  Utilization of different processes for biofuel production from algal biomass

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2.2  Role of Algae in Biosphere Macroalgae are diverse, eukaryotic, multicellular macroscopic organisms that belong to Chlorophyta, Rhodophyta, Phaeophyceae and Xanthophyceae (Graham et al., 2009). On the basis of the photosynthetic pigment, macroalgae are divided as green, red and brown (Jard et al., 2013). The primary producer in oceans is macroalgae/seaweeds which lack the composite structures as that of plants. Macroalgae are gaining ample consideration due to its high growth, biomass, carbohydrate (Sudhakar et al., 2018), biofuel production potential and ability to grow efficiently on non-arable land (Kim et al., 2016). Sugar kelp, the potential macroalgal species if produced and utilized in sustainable manner, can become the viable substrate for biofuel production (Yazdani et al., 2015). Environmental conditions as well as light quality and quantity are the major factors which affect the distribution of macroalgae. Macroalgae are low in lipid content (1–5%), while carbohydrate and proteins are present in high amount (Sudhakar et al., 2013). The few species of macroalgae contain lignin which is easily digested by microbes during biofuel production (Vergara-Fernández et al., 2008). Some species of macroalgae have very high productivity as compared to plant and tropical rainforest (Gao & McKinley, 1994). The biomass from macroalgae is enriched with certain chemical compounds which have several applications in food and fodder industry (Bruhn et al., 2011). In the USA, the offshore cultivation of macroalgae rose in 1970 (Huesemann et al., 2012), but the plans of biofuel production from macroalgae ceased for decade due to finding of natural gas (Roesijadi et al., 2010). The interest for alternative fuel in the USA and Europe has increased with focus back on macroalgae as the alternative, cheap and sustainable source of biofuel (Rothe et al., 2012). Macroalgae as source of biofuel is still in its infancy stage, and results are mostly based on laboratory scale studies (Cherad et al., 2014). Microalgae are dominant, photoautotrophic and oxygen-evolving microorganisms that appeared on biosphere 4–5 billion years ago (Hanada, 2016). Microalgae are unicellular that exists individually or in the form of colonies and involve green algae, diatoms as well as dinoflagellates. Microalgae can be found in diverse habitats (Alam et al., 2012) and are more efficient converters of solar energy as compared to plants (Scott et al., 2010; Singh et al., 2021). Several microalgae species can have oil content of 80% on dry weight basis and can double their biomass within 1 day which makes biomass from microalgae as feasible feedstock for biofuel production. The high oil content in algae can be easily converted into different types of biofuels. Microalgae can produce 15–300 times more biofuel as compared to plants on area basis (Dragone et al., 2010). Microalgae’s ability to grow in different wastewater, saline water and non-arable land and its no competence with food crops have increased the interest of scientific community to utilize them as promising source of biofuel (Singh et al., 2021). Thus, algal biomass as the source of biodiesel, bioethanol, biogas and bio-hydrogen can certainly reduce the use of conventional fuel, and large-scale production can stimulate the production of

2.3  Crops and Plants as the Source of Biofuel

19

industrially important compounds. Therefore, extensive research and large-scale exploitation of biomass can strengthen the renewable source and amplify the energy production by utilizing algal biomass.

2.3  Crops and Plants as the Source of Biofuel 2.3.1  Corn (Zea mays) Corn is produced in huge quantity and is an important crop for production of bioethanol (Veljković et al., 2018). It is considered as the king of ethanol-based biofuels. The carbohydrate-enriched corns are converted into ethanol in presence of yeast. Kernels of corn are only used for biofuel production compared to the main body due to its high cellulose which is very difficult to degrade. The kernel is composed of root, pericarp, germ and endosperm, and each part varies in its composition. The root and pericarp contain mainly cellulose, the germ is rich in oil and carbohydrate, while the endosperm contains mainly starch, proteins and fats (82%) (Gulati et al., 1996). Approximately 80% of oil is in germ and only 5% of oil in endosperm to total oil in kernel. The oil content of >6% in the corn is considered as high-oil corn (Rajendran et al., 2012). In kernel, the germ is the most important part containing the majority of total oil which can be used as biofuel (Veljković et al., 2018).

2.3.2  Sugar Cane Brazil is working tirelessly to use biofuel widely as fuel in vehicles and has invested huge financial resource to enhance biofuel production from sugar cane. Utilizing sugar cane for biofuel production is cheaper than that of corn. Due to the enhancement in the agricultural production, the increment in waste production has also increased which can be used as the low-cost material for biofuel production (Chandel et al., 2011). Bagasse is the by-product obtained after extraction of juice from sugar cane. The other important waste from sugar cane is sugar cane trash which releases harmful pollutants after burning trash in the fields. Both sugar cane leaves and bagasse contain cellulose as well as hemicelluloses which can be converted into monomers by chemical and enzymatic mechanisms (Krishnan et  al., 2010). The monomers can be converted into biofuels and other industrially important products that have potential to increase revenue (Soccol et al., 2011). Thus, utilizing sugar cane bagasse and leaves can be a cheap and eco-friendly approach for production of bio-based products such as biofuel (Abraham et al., 2020).

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2.3.3  Palm Oil Palm oil is a perennial crop, and cultivation is carried throughout the year (Mukherjee & Sovacool, 2014). Palm oil is a rapidly growing industry, and the huge waste generated during oil extraction can be used for production of biofuel (Kurnia et  al., 2016). The necessity of sustainable fuel has been driven to develop technologies responsible for processing palm oil and its waste into biofuel. Palm oil is considered as one of the energy-efficient biodiesel fuel. Globally, the high production efficiency has led to a wide-scale plantation of palm plants (Kurnia et al., 2016). Among different oilseeds, palm oil is the most efficient and has higher per hectare (4000 kg/ ha) yield in the world (Salim et al., 2012). Palm oil contains good amount of both palmitic and oleic acids which are suitable fatty acid for biofuel production. Malaysia and Indonesia are the major palm oil producers and suppliers and has developed several techniques to produce biofuel from palm oil and its waste (Vijay et al., 2016). In Malaysia, biodiesel from palm industry is an attractive option and can lead to decline in the release of greenhouse gases by 50–70% (Hassan et al., 2011). The modification in diesel engine is not required if palm oil is used as fuel in vehicles (Ndayishimiye & Tazerout, 2011).

2.3.4  Jatropha Biomass from different crops have been utilized for energy production, but Jatropha has been regarded as promising source of green energy (Mkoma & Mabiki, 2011). Biodiesel industry revolves around jatropha as the poisonous weed is produced in India in huge quantity. Jatropha is a bushy weed which requires very less amount of water for its growth. Jatropha, because of its easy production and eco-friendly nature, has attained attention as renewable fuel source (Moniruzzaman et al., 2017). The oil content in seeds of jatropha has been around 40–60% and is extracted by crushing the seed, and oil is used for biofuel production (Koh & Ghazi, 2011). Jatropha oil contains proteins, fat and fatty acids. Saturated fatty acids such as palmitic, stearic, oleic and linoleic are present in jatropha oil in abundant amount. The oil from jatropha plant has energy value similar to that of crude oil but higher than that of anthracite coal (Sotolongo et al., 2007).

2.3.5  Switchgrass The dependence on non-renewable fuels can be minimized by exploiting the green fuel. Switchgrass is found mainly in North America and has gained huge attention due to its adaptability and productivity (David & Ragauskas, 2010). The cellulose in switchgrass requires less energy to convert it into biofuel. Ethanol from

2.4  Different Types of Biofuels from Algae

21

switchgrass contains more energy as compared to that obtained from corn. The large-level plantation is lacking, and work to exploit this crop as biofuel source is still in its initial phase (Schmer et al., 2008). Furthermore, exploiting the environment friendly and sustainable resource can provide assistance in elevating the socio-­ economic status and employment and improve environmental health.

2.4  Different Types of Biofuels from Algae 2.4.1  Biodiesel The content of lipid is highly dependent upon the species of algae (Nelson et al., 2002). Neutral lipids, glycolipids and phospholipids are the three types of lipids in algae (MacDougall et al., 2011). Glycolipids are abundant in thylakoid membrane of chloroplast, and phospholipids are vital component of cell membrane (Khotimchenko, 2006). Dictyota, a brown macroalgae, is a lipid rich with variation in lipid content of 71–202 mg g−1 of dry weight. In benthic red algae, the glycolipids are major lipid constituents representing 50.3–75.1% of the total lipids while 47.2–83.1% in brown algae (Khotimchenko, 2006). Several microalgae species such Scenedesmus, Chlorella, Nannochloropsis and Botryococcus are rich in lipid and can be used for the biodiesel production (Vadivel et al., 2020; Arvindnarayan et al., 2017). Some species of microalgae can have lipid content even up to 80% of dry cell weight (Arvindnarayan et al., 2017). The lipid content in microalgae can be also enhanced by culturing them under different stress conditions. The stress conditions such as nutrients, salinity, light, temperature, pH and metals can improve the lipid yield of microalgae (Jaiswal et al., 2020; Cheng & He, 2014). The fatty acid content in microalgae involves both saturated and unsaturated fatty (C12–C24), and the composition is very similar to vegetable oil. Biodiesel is produced by extracting oil from algal biomasss followed by the process of transesterification (Fig.  2.3) (Maceiras et  al., 2011). Transesterification is the process used to convert triacylglycerols (TAGs) and storage lipid to fatty acid methyl ester such as diesel (Daroch et al., 2013). TAGs, due to abundance of fatty acid and high rate of conversion (99%), are preferred for biofuel production (Williams & Laurens, 2010). The extraction process such as solvent extraction or hydrocracking improves the biodiesel stability and can be used directly in the vehicle engine (Sarpal et al., 2016). The presence of phosphorus, sulphur and nitrogen in glycolipids and phospholipids can be problematic for biofuel production (MacDougall et  al., 2011). Phosphate is known to inhibit the process of transesterification, and it has been reported that only 70% of biodiesel has been extracted from phospholipids. Thus, for biodiesel production, the content of TAG is important to elevate the biofuel production potential of alga. The quality of biodiesel from algae depends upon the fatty acid profile which is essential to determine the cetane number, stability and melting point. Cetane number and melting point of the biofuel from algae increase with

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2  Plant and Algae Classes Recognition, Biomass Production and Potential Source… Cultured under laboratory or open environmental condions In presence sunlight, CO2 H2O

of and

Algae

DRYING OF BIOMASS Mechanical and chemical cell disrupon OIL EXTRACTION

Filtraon, Sedi mentaon, floc culaon

Biomass

Under sunlight or thermal treatment

BIOMASS HARVESTING

Transesterficaon BIOFUEL PRODUCTION

Fig. 2.3  Process of biofuel extraction from microalgal biomass

degree of saturation and chain length of fatty acids (Suutari et al., 2015). The high cetane number of biofuel is often linked with short ignition and very low emission of NOx (Suutari et al., 2015). Different techniques have potential to modify the fatty acid profile, but it is economical to utilize the good-quality fatty acid-rich feedstock for biofuel production (Sajjadi et  al., 2018). The biodiesel from microalgae and gasoline blending (85% blend) can be used without any modification in the engine (Hossain et al., 2019).

2.4.2  Bioethanol Biofuel encompasses different types of fuels obtained from algal biomass after necessary treatment. Carbohydrate as the source of biofuel from algal biomass is still developing (Jung et al., 2013). The main constituent of the algae is carbohydrate which involves both reserve and structural carbohydrate (Yoza & Masutani, 2013). Carbohydrate is a vital compound for the production of bioethanol and biogas from alga (Adams et al., 2009). Since, the composition of carbohydrate in algae varies within different classes leading to variation in bioethanol production. The bioethanol is extracted from algal biomass by hydrolysis of polysaccharides which is followed by the fermentation of monomeric carbohydrates (Lee et al., 2015). Bioethanol is produced from algal biomass by fermentation process in the presence of yeasts and bacteria (Daroch et al., 2013). Glucan, a polysaccharide which can be easily converted into ethanol, is present in algae in very low amount (Schultz-Jensen et al., 2013). The recovery of ethanol from algal biomass not only requires glucan but also mannitol, agar and carrageenan (Wargacki et al., 2012). Mannitol, due to no requirement of pretreatment phase and easily dissolvable, could be used to improve ethanol production yield (Wang et al., 2013). Furthermore, the favourable redox potential of mannitol may lead to production of higher amount of ethanol. It has also been

2.4  Different Types of Biofuels from Algae

23

suggested to supplement nicotinamide adenine dinucleotide externally and modify metabolic pathway of bacteria to increase the ethanol production (Wang et  al., 2013). The entire conversion of algal sugars to ethanol in brown algal biomass has not been attained fully as the microbes used were not capable to metabolize alginate. In order to convert all sugars in brown algae into ethanol, the modified strain of Escherichia coli was used, and excellent results were obtained (Wargacki et al., 2012). The genetically modified Saccharomyces cerevisiae was able to degrade alginate and produce theoretical ethanol production of 83% (Enquist-Newman et  al., 2014). Escherichia coli and Saccharomyces cerevisiae have shown better results by converting different types of sugar from algal biomass (Wargacki et al., 2012). The pretreatment such as temperature, pH and enzymes has been used to increase the availability of carbohydrate for fermentation (Lee et  al., 2015). The algal biomass should not contain salt as presence of salt may cause cellulose hydrolysis (Schultz-Jensen et al., 2013). The ethanol yield from algae is not only dependent upon the carbohydrate content but also on microorganisms and inhibitory substances which hinder the activity of microbes and ultimately bioethanol production (Lee & Lee, 2012). The inhibitory compounds formation during the process of hydrolysis depends upon the algal species and on the prevailing conditions during the bioethanol production (Park et al., 2012). The formation of inhibitory compound can be diminished by using enzymes or by maintenance of mild process conditions. The continuous hydrolysis with dilute acid has proved to enhance ethanol production from algal biomass (Park et al., 2012). Ethanol production from algae can be economically feasible by utilizing the algal biomass for other industrially important compounds (Horn et al., 2000). The integrated agar and ethanol production are considered economically viable but still require extensive research in order to make it environmentally sustainable (Kumari & Pramanik, 2013). Biomass from alga is converted into biofuel through microbiological (Jung et al., 2011) and thermochemical processes (Ferrera-Lorenzo et al., 2014) such as pyrolysis, gasification and liquefaction. Pyrolysis, gasification and liquefaction are considered as the better processes for the treatment of algal biomass (Fig.  2.3) (Bruhn et al., 2011). The oil produced through different thermal processes is processed into biodiesel or refined through chemical transesterification (Elliott et al., 2014). The oil extracted from the pyrolyzed biomass contains lighter oil and char which can be used as source of energy. In pyrolysis, the oil and char production from algal biomass depends upon temperature and heating rate during the process (Bae et  al., 2011). The oil may pose problems due to presence of high nitrogen and sulphur content in the algal biomass. The high sulphur content in the oil from biomass of Saccharina spp. through liquefaction process has been reported by Elliott et  al. (2014). Through pyrolysis process, the amount of ash formed can be decreased through pretreatment of algal biomass with acid (Ross et al., 2009). Aresta et al. (2005) reported that the liquefaction process of oil extraction from algal biomass is more efficient as more oil is produced from algal biomass. The species of algae to be used for biofuel production depends upon lipid content, TAG content and degree of unsaturation of fatty acids. Sargassum myriocystum, a brown algae, have low lipid content, but better-quality lipid composition is responsible for biofuel production (Renita & Amarnath, 2011).

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2.4.3  Biogas The biomass from alga can also be used for biogas production with the assistance of methanogenic bacteria under anaerobic conditions (Fig.  2.4) (Vergara-Fernández et al., 2008). Both bioethanol and biogas production from algal biomass varies with carbohydrate content, C/N ratio and lignin content of biomass (Hughes et al., 2012). The low content of lignin in algal biomass is very advantageous for both bioethanol and biogas production (Yoza & Masutani, 2013). The carbohydrate content from algal biomass through different thermochemical processes can be converted into biofuel (Fan et al., 2020). Among different thermochemical processes, hydrothermal gasification is considered good processing method for biofuel production from carbohydrate-enriched biomass (Cherad et al., 2014). Apart from methane and carbon dioxide, the biogas also contains hydrogen sulphide (H2S) and ammonia (NH3) in small amount (Vergara-Fernández et al., 2008). The sulphur content in algal biomass can be high and leads to higher content of H2S in biogas (Bruhn et al., 2011). The biomass after conversion in biogas may contain high ammonium content due to presence of nitrogen in higher amount in algal biomass (Sialve et al., 2009). Bird and Ryther (1990) reported that macroalgae biomass can be excellent feedstock for biogas production due to its good efficiency and conversion rate. Langlois et al. (2012) reported that the biogas from algal biomass can become an efficient alternative in place of fossil fuels. The multitude of studies has been carried out to evaluate the biogas production of different types of algae (Morand et al., 1990). The biogas yield from algal biomass can be comparable to that from cattle manure (Jard et al., 2013).

Gasificaon Ethanol Methane Hydrogen

Pyrolysis

Liquefacon

Combuson

Heat, power

Algal biomass

Fig. 2.4  Thermal processes utilized for the production of different types of biofuel

2.4  Different Types of Biofuels from Algae

25

2.4.4  Bio-hydrogen Hydrogen is the most abundant element (Zohuri, 2019) and is considered as the renewable fuel and appropriate substitute with energy density very higher compared to other fuels (Edwards et al., 2008). The commercial production of hydrogen is still at infancy stage; however, the laboratory scale production of hydrogen has been observed with lower yield (Kumar et al., 2013). Thus, to maximize the hydrogen production, modification and optimization of designs and maintaining of optimal growth conditions can be very helpful (Moreno-Garrido, 2008). The major source of hydrogen is fossil fuel which is responsible for production of more than 95% hydrogen (Wang and Wan, 2009). Relying on fossil fuel for hydrogen production to accomplish future demands is undeniably an unsustainable approach. It is necessary to identify and exploit alternative and eco-friendly sources (Rozendal et al., 2006). Water is the cleanest source of hydrogen, but huge electricity required during electrolysis of water has increased the operation cost for hydrogen production. Moreover, water can become the cheap source of hydrogen in countries with cheap electricity (Saifuddin and Priatharsini, 2016). Several microorganisms are involved in hydrogen production, but algae, the third-generation resource, is considered efficient due to multilevel benefits as compared to other plants (Moreno-­Garrido, 2008). Several pretreatment methods have been employed to produce hydrogen from biomass. In order to rapture the cell and release fermentable sugar, diverse physico-chemical and biological methods are used (Behera et al., 2010). Several algal species are able to produce hydrogen under certain environmental conditions (Li & Fang, 2007). But certain barriers such as cheap method of biomass harvesting, continuous large-scale biomass production, competition with invasive species and less light penetration can be the major limitation in sufficient biofuel production (Radakovits et al., 2010). Utilizing algae for hydrogen production can reduce the environmental damage by using solar energy to breakdown water into H2 and O2 (Eroglu & Melis, 2011). In algae, hydrogen metabolism was initially explored by Gaffron and Rubin (1942) and in bacteria by Gest and Kamen (1949). The inherent capability of these organisms to grow efficiently in aerobic as well as anaerobic conditions and ability to shift to different cultivation modes initiates hydrogen production (Melis, 2007). Chlamydomonas reinhardtii and Anabaena cylindrica is the algal species responsible for hydrogen production under sunlight. These organisms’ direct protons and electrons are produced after water photolysis to hydrogen production with the help of chloroplast hydrogenases (Kruse and Hankamer, 2010). Kruse and Hankamer, (2010) reported that certain modification in respiratory metabolism can increase the bio-hydrogen production (4 mL/h) in C. reinhardtii. Fermentation and photolysis are the main mechanism responsible for production of hydrogen from algae (Hallenbeck & Benemann, 2002) (Fig. 2.5), but still these methods require intensive research to make the hydrogen production frugal and less energy intensive (Mathews & Wang, 2009). In photolysis, direct and indirect biophotolysis are the mechanisms adopted for hydrogen production (Nikolaidis & Poullikkas, 2017). Direct photolysis is the cheap method as it involves only water and sunlight for hydrogen production (Kim & Kim, 2011). Algae, after absorption of light enhance the water

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2  Plant and Algae Classes Recognition, Biomass Production and Potential Source…

Direct photolysis

Bio-hydrogen

Carbohydrate

Algal biomass

Biomass as substrate for bacteria Carbon dioxide

Bacteria

Fig. 2.5  Mechanism of bio-hydrogen production from algal biomass

oxidation, and with the help of photosystem I, electron and proton are transported to the chloroplast ferredoxin (Melis, 2007) which make possible the reduction of proton to hydrogen (Eroglu & Melis, 2011). Hydrogen production through indirect photolysis occurs in two stages. In first stage, algae allowed to grow under normal condition leads to the synthesis of carbohydrate, and in second stage, the synthesized carbohydrate is used by nitrogenase enzymes to produce hydrogen. The solar light to hydrogen conversion efficiency is very low (16.3%) in indirect photolysis which can be due to involvement of multiple steps and use of energy by enzymes (Pilon et al., 2011). Cynobacteria mainly filamentous one due to the presence of heterocyst is considered as efficient organism for hydrogen production through indirect process (Eroglu and Melis, 2011). Both direct and indirect photolysis has not been carried out in large scale for economical and sustainable hydrogen production (Nikolaidis and Poullikkas, 2017). In contrast to photolysis, fermentation, especially dark fermentation, is considered superior mechanism for hydrogen production. In dark fermentation, the process is totally dependent upon the bacteria rather than on algae (Brentner et  al., 2010). Yoshida et al. (2005) reported that E. coli SR 13 by dark fermentation produces 300 L H2/L/h, while only 0.012 L H2/L/h was obtained from Chlamydomonas reinhardtii through direct photolysis (Laurinavichene et al., 2008). Hydrogen production through bacteria is not economical as the substrate as energy source are expensive which prevents the commercial implementation of such processes for hydrogen production (Kapdan & Kargi, 2006). The integration of direct photolysis and dark fermentation would be beneficial in enhancing hydrogen production and overcoming the limitation posed by individual photolysis and fermentation process (Akhlaghi & Najafpour-Darzi, 2020). Algae through direct photolysis produces hydrogen and is grown till the production process is stopped. The algal biomass through dark fermentation is then used by bacteria as substrate for hydrogen production. During dark fermentation, carbon dioxide along with hydrogen is produced, and if gas separation is possible, then carbon dioxide can be utilized during microalgae cultivation for enhanced biomass production. Thus, integrating both methods can be the efficient approach to enhance biomass and hydrogen production in cost-effective and sustainable manner (Lam et al., 2019).

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Chapter 3

Plant and Algae Metabolites Alternative and Clean Source of Energy

Abstract  Plants and algal metabolites are believed to be the potential source of biofuel. High cellulose and lignin content in plant biomass is the major obstruction towards the sustainable fuel production. Owing to expensive pretreatment methods for lignin and cellulose degradation have raised several questions on the use of plants biomass for energy production. However, biotechnological approaches to promote inherent enzyme production for cellulose degradation and modify process responsible for inhibition of lignin synthesis can overcome the pretreatment cost and enhance the profitability of plants biomass for biofuel production. On the contrary, algal biomass due to very low cellulose content can act as the cheap source of carbohydrate, lipid and fatty acids. Thus, employing biotechnological approaches and exploiting algal biomass can be the potential source of different metabolites and eventually biofuel. Keywords  Lignin · Cellulose · Lipids · PUFA · Metabolites · Biofuel

3.1  Introduction Global warming is the major environmental issue accelerated by anthropogenic activities, and based on data and prediction models, International Panel on Climate Change (IPCC) has warned the people about the repercussions of global warming. IPCC is working tirelessly by motivating and promoting involvement of different world societies to take concrete steps towards CO2 emissions (Canadell & Schulze, 2014). Global warming changes the local climate, and rapid melting of glaciers poses severe risks to the survival of organisms on earth (McMichael & Lindgren, 2011). The balance of carbon cycle is disturbed through continuous emission of carbon dioxide by various human activities such as fossil fuel combustion and land use change (Churkina, 2008). From 1750 to 2012, anthropogenic activities lead to huge emission of carbon dioxide (570 petagrams), and almost 70% was due to the use of fossil fuels (Canadell & Schulze, 2014). The global energy consumption is increasing at swift rate, and it is expected that fossil fuel will get exhausted in the © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4_3

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coming three decades. The swiftly expanding global population along with industrial growth has increased the per capita energy consumption. The rising global energy demand is satisfied mainly by conventional fuels. The usage of energy at such rate is estimated to increase by >50% by 2030. The consumption rate of fossil fuels is 105 times higher than replenishment rate, creating a huge gap between production and consumption which may become the major cause of fuel exhaustion (Shuba & Kife, 2018). The total dependence of world on fossil fuel has been the originator of several severe environmental problems, and the paradigm is shifting towards exploitation and utilization of sustainable and green fuels. In order to meet the future energy demands and reduce reliance on fossil fuel, it is high time to exploit reliable, economical and carbon-neutral fuel (Singh et al., 2020). Different counties of the world are experiencing the changes in climate year after year, and increasing severity has forced to utilize the eco-friendly alternatives (Akanwa & Joe-Ikechebelu, 2019). The yearly global consumption of energy is 15 terawatts, and renewable energy contributes a meagre amount (7.8%) to the total consumption (Jones & Mayfield, 2012). It has been estimated that 5500 × 1021 J of energy reaches on the earth and plants, and algae plays a crucial role in converting solar energy to chemical energy (Smil, 2005). Terrestrial plants sequester huge amount of carbon (121.7 × 109 metric tons) from atmosphere and utilize it as the carbon source for synthesis of different types of chemical compounds (Beer et al., 2010). Exploiting photosynthetic organisms for biofuel production is an attractive alternative to expedite the fuel availability and reduce the concentration of gases responsible for enhanced global warming (Sarkar & Shimizu, 2015). Photosynthetic organisms are considered as green and sustainable factories for production of carbohydrate, biofuel and vital bio-compounds having immense value in food and feed industry (Singh et al., 2020). Utilizing plants can maintain the atmospheric balance by fixing carbon dioxide which is the main gas responsible for 60% of global warming (Salih, 2011). Agro-waste is produced in enormous quantity (Singh, Bhat, & Geelani, 2021), and utilizing it as the feedstock for biofuel production can certainly reduce the burden on first-­generation sources (Mahro & Timm, 2007). Thus, the second-generation feedstock’s main residue of different crops is usually considered superior for biofuel production as compared to first-generation sources (Valentine et al., 2012). The first-generation biofuel produced from corn and sugar cane can become the cause of food and energy issue, while the second-generation biofuels from lignocellulosic biomass being energy intensive have limited the exploitation for biofuel production (Shimizu, 2014). One of the factors that affect the price of biofuel is the type of substrate used for biofuel production (Qureshi & Blaschek, 2000). Wheat straw is the most commonly available material globally, but unsustainable method of disposal by burning can cause serious human health hazard and global warming (Zhu et al., 2006). Thus, utilizing the crop residue as substrate for biofuel could be the sustainable approach towards waste management and biofuel production. Moreover, rice straw (Nimcevic & Gapes, 2000) and corn (Kadam & McMillam, 2003) can be used as the ideal substrate for fuel and chemicals production. Still certain shortcoming in the crop and plant biomass such as high lignin, cellulose and

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hemicelluloses content and expensive methods of their degradation has limited utilization of plant biomass for biofuel production. After second-generation source of biofuel, algae are considered as the potential source of third-generation biofuel and can revolutionize the fuel industry due to its renewability and sustainability (Upadhyay et al., 2021). Microalgae are eukaryotic, photosynthetic organisms with size of 1–100 μm, and cyanobacteria is prokaryotic with size of 1–10 μm (Larkum et al., 2012). Microalgae have higher solar to chemical energy conversion efficiency (10–50 times) (Khan et al., 2009), cell growth and smaller land footprint for energy production than that of terrestrial plants (Chisti, 2007). Microalgae are the most common photosynthetic organism utilized for biofuel and bio-compounds production (Singh, Upadhyay, et  al., 2021). The higher efficiency of converting CO2 into lipids is only few steps far from biofuel. Microalgae are composed of more than 50% of carbon, and approximately 1.8  kg of CO2 is biologically fixed for production of 1 kg of biomass (Chisti, 2007). Photosynthetic microorganisms have several benefits such as high solar conversion efficiency, ability to grow on non-arable land, wastewater and saline water (Singh et al., 2020). Algae as third-generation biofuel have attained global attention due to multitude of benefits (Sheehan, 2009). Utilizing photosynthetic organism for biofuel, biochemical compounds (Singh, Bhat, & Geelani, 2021) and CO2 fixation are considered as the efficient approach for protection of environmental health (Savakis & Hellingwerf, 2015; Singh et al., 2020). Microalgae strains such as Chlorella sp., Scenedesmus sp. and Botryococcus braunii have shown good potential of CO2 fixation (200–1300 mg/L/day) (Sydney et al., 2010). The biomass can act as nutrient supplement in food and feed industry and source of antioxidants and fatty acids (Rosenberg et al., 2008). The microalgal biomass acts as potential substrate for biodiesel production, carbohydrate for bioethanol (Chng et al., 2017) and PUFA for food and pharmaceutical industry (Singh, Upadhyay, et al., 2021). Their ability to grow under different conditions such as wastewater (Upadhyay et al., 2021), saline water and contaminated ecosystems has increased the interest of global community towards microalgae (Sarkar & Shimizu, 2015). Furthermore, employing different stress condition and genetic engineering can be the important tools to accelerate the production of different valuable compounds from algae (Sarkar & Shimizu, 2015).

3.2  R  ole of Plant and Algal Metabolites in Biofuel Production 3.2.1  Lignin Lignin is a high molecular weight secondary compound and important constituent of cell wall (Terrett & Dupree, 2019). It is considered as the second most important polymers of biological origin on biosphere which accounts for about 3/10th of the carbon content (Ralph et  al., 2004). Lignin is essential for plant growth and

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development and imparts resistance towards several biotic and abiotic stresses conditions (Liu et  al., 2018). As the complex compound, lignin in plant cell wall enhances rigidity and mineral transport (Schuetz et al., 2014) and also acts as the barrier against pathogens (Ithal et  al., 2007). Plant lignin is the economical and renewable resource generally used for synthesis of different materials or source of energy (Espinoza-Acosta et al., 2018). The structure of lignin in plants is strongly influenced by monomers composition (Constant et al., 2016) which plays vital role in determining the degradability as well as workability of the plant biomass (Lupoi et al., 2015). The intense research is undergoing on development and commercialization of non-food crops (Sticklen, 2008) as the energy source as the generation of green and carbon-neutral fuel is necessary to minimize the damage caused by global warming (Sims et al., 2010). Presently, utilizing lignocellulosic biomass for biofuel production is very expensive due to complexity of cell walls (Somerville et  al., 2004). The continuous research in conversion technologies can make possible to convert lignocellulosic biomass into biofuel (Naik et al., 2010), and development of desired biomass can increase the conversion efficiency in cost-effective manner (Mansfield, 2009). Lignin is the most important cell wall component that prevents the biomass to be used for biofuel production. Lignin being very resistant to degradation through both physical and chemical treatments can eventually affect the biofuel production potential of the plant biomass. Treatment with the help of organic solvents (Hallac et al., 2010), ionic liquids (Singh et al., 2009) and kraft pulping (Olsson et al., 2006) is considered as efficient approach for lignin breakdown, but being expensive can increase the cost of biofuel production. Dilute acid (Taherdanak et al., 2016), ammonia fibre expansion (Shao & Zhao, 2016) and hydrothermolysis (Hashmi et al., 2017) are the most common pretreatment methods used for lignin degradation in plant biomass but require very high temperature to remove lignin. The other important approach can be the use of feedstock in which the lignin is easily dissolvable at low temperature. The research on lignin synthesis in plant has grown tremendously over the few decades, and utilization of genomics and proteomics has enabled to explore technologies responsible for modification in lignin content in order to improve the degradability and reduce the pretreatment cost of lignin degradation (Simmons et  al., 2010). Several researchers have utilized the genetic engineering to alter the lignin content in plants (Vanholme et al., 2012). The main approaches considered to reduce cell wall reluctance are to alter the pathways responsible for lignin synthesis and convert the lignin biomass into valuable aromatic compounds (Simmons et al., 2010). Plants and crops such as Populus trichocarpa (Jansson & Douglas, 2007) and switchgrass (Kim et  al., 2020) such as Arabidopsis (Kumar et al., 2009) are important systems in terms of biofuel production. Utilizing these plants and genomic tools can increase the biofuel production and provide new avenues by modifying the lignin synthesis in plants (Vogt, 2010). Genetic manipulation mediated through gene-gun or Agrobacterium tumefaciens is considered as mature practice to reduce lignin content. One of the feasible approaches to reduce the lignin content in lignocellulosic biomass is to modify the activity of enzymes responsible for lignin biosynthesis (Simmons et al., 2010). In plant cell, the presence of lignin can hamper the extraction from biomass through

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enzymatic degradation (Zhao et al., 2020). In order to degrade lignin, the biomass is pretreated with hydrolysis under acidic or basic condition before enzymatic degradation. The presence of lignin in plant biomass needs to be reduced for enhanced biofuel production, and substantial steps through metabolic engineering have been adopted to reduce the lignin content in plant biomass (Brar et al., 2020; Ghosh & Das, 2020). A systematic approach to improve the saccharification yield of lignin was performed on Arabidopsis thaliana, and the author successfully restricted the lignin biosynthesis to vessels with increase in thickening of cell wall and enhancement of carbohydrate upon saccharification (Yang et al., 2013). Crops such as sugar cane (Mason et al., 2020), sorghum (Stamenković et al., 2020), eucalyptus (Thoresen et al., 2021), and pine (Bay et al., 2020) as substrate can easily be genetically tailored to increase the biofuel production.

3.2.2  Cellulosic and Hemicellulosic Biomass Biofuel can provide the potential alternative to avert problem of global instability and environmental pollution caused by non-renewable fuels (Upadhyay et  al., 2021). Biofuel is generated mainly from carbohydrates but can meet only the small fraction of energy requirements (Ma et al., 2020). The cell wall of plants mainly consists of cellulose, hemicelluloses and lignin (Chundawat et al., 2011). Cellulose which is present in abundant amount is also the promising alternative for biofuel production. The use of cellulosic biomass for biofuel production is advantageous in the countries having climate unsuitable for growth of energy crops. Several steps have been taken by different countries to accelerate biofuel production by using cellulosic biomass as feedstock for bioethanol (Spyridon & Willem Euverink, 2016). In order to accelerate the biofuel extraction, it is necessary to break cellulose into smaller sugar molecules (Dodd & Cann, 2009). In cell wall, the cellulose digestibility by cellulase is one of the most imperative aspects for biomass conversion into glucose (Abramson et al., 2010). The amorphous portion is susceptible to degradation by cellulase, but the important aspect is to reduce the crystallinity which could be helpful in cellulose degradation (Demura & Ye, 2010). The major drawback is the incorporation of cellulase enzymes and pretreatment methods which are expensive and have limited the biofuel production from plant biomass (Dey et al., 2020). The utilization of pretreatment processes increases the price of biofuel production; thus, finding economical ways to enhance cellulose degradation can certainly increase the biofuel potential of plant biomass. However, the biofuel production from cellulosic and hemicellulosic biomass can be made feasible by genetically introducing the synthesis of cellulase and hemicellulases which can be the very frugal approach for biofuel production. The enhancement in carbohydrate content and plant biomass can increase the bioethanol production from plants (Sticklen, 2008). Bioethanol is the type of biofuel used mostly throughout the world. Ethanol is produced from plant biomass as well as plant-based material such as carbohydrates, cow dung and oil crops. Bio-alcohol has been produced through traditional

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methods as alternative fuel source. The plant material mostly from sugar cane and grain crop has been utilized due to abundance of starch and sugar. Bio-alcohol, mainly ethanol, is produced through fermentation of carbohydrate-rich plant biomass (Shah & Sen, 2011). Several agricultural crops and their waste can be used as feedstock for energy production. Sugar cane, sugar beet, corn and wheat are utilized for the production of bioethanol (Dias et al., 2009). Bioethanol from sugar cane is produced in higher amount when the bagasse is used as substrate as huge quantity of bagasse waste is generated per ton of sugar that can be easily used for biofuel and electricity generation (Buddadee et al., 2008). Similarly, the starch-rich corn is used for bioethanol production by applying the process of fermentation and distillation. Utilizing sugar and starch for ethanol production can compete with the food production. It is important to mention that use of non-food crops as well as waste is very feasible approach to enhance biofuel production. Recently, the focus has also shifted towards switchgrass, miscanthus (Rodionova et  al., 2017) and hybrid poplar (Demura & Ye, 2010) as the substrate for biofuel production. Switchgrass and miscanthus are perennial crop and have no competition with food crops (El Akkari et al., 2020). The biomass of these plants requires the fermentation and distillation before converting into sugars (Rodionova et al., 2017). Bioethanol is low in toxicity and produce fewer pollutants compared to that of petroleum fuel. Plant biomass reduces the greenhouse gases concentration by sequestering carbon dioxide from atmosphere. The ethanol derived from carbohydrates sustains the small but valuable contribution in renewable energy supply to the world especially Brazil that is known to produce ethanol from sugar cane in cost-effective manner and is responsible for supplying 1/4th of the ground transportation demands, while in the USA, the bioethanol is produced mainly from corn grain, and it is estimated that the quantity of corn grain produced if converted into biofuel can meet the 15% of transportation demand. Meeting the energy demands of the USA, the corn grain must be directed towards biofuel production than utilizing it as feed (Schlamadinger et al., 1997). The future bioethanol production from plant biomass along with grain ethanol can be helpful in reducing the use of fossil fuel (Bordetsky et  al., 2005). Bioethanol production from cellulosic biomass is very advantageous due to its huge abundance as compared to grain and sugar (Sarkar et al., 2012). In 2006, the government of the USA has made the goal to reduce dependence on non-renewable fuels by 30% till 2030 and accelerates the use of crop biomass for biofuel production. Utilizing conventional crops for biofuel generation can have certain impacts on food industry while miscanthus, switchgrass and hybrid poplar are considered as the cheap feedstock for production of biofuel (Lawrence & Walbot, 2007). In the USA, it is anticipated that 1.3 billion tons of dry plant biomass can produce 130 billion gallons of bioethanol annually having theoretical yield of 100 gallons/ton of biomass (Carroll & Somerville, 2009). In the USA, the department of energy put forward the plans to establish six cellulosic bioethanol refineries which, after being operational, can produce 130 million gallons annually (DOE, 2007). The continuous research and funding across the globe from different sources can certainly increase the biofuel

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Pre-treatment of cellulose and lignin

Plant/crop Lignocellulosic biomass

Carbohydrate

Energy crops Biofuel Lipid and Carbohydrate extracon Algal biomass

Fig. 3.1  Biofuel production from plant and algal biomass

production from cellulosic biomass. Still, several problems are associated with commercial ethanol reduction from cellulosic biomass. The huge cost involved in cellulase production and pretreatment methods needs to be minimized in order to produce biofuel in economical manner. The pretreatment process degrades the lignin and exposes the cellulose to the cellulase. Both cellulase and pretreatment methods increase the cost of ethanol from cellulose by two- to threefolds. In order to decrease the cost of biofuel production from cellulosic biomass, it is important to introduce the cellulose degrading enzymes (cellulases and hemicellulases) within the crop which can lead to less or no production of such enzymes in bioreactors. Genetic engineering can be helpful in modifying the lignin content of plant biomass and thus reduces the need of pretreatment methods (Sticklen, 2008). Absence of lignin, presence of low cellulose content (Chen et  al., 2013) and abundance of carbohydrates, lipids and proteins in microalgae as compared to plants have increased the utilization of algal biomass as feedstock for bioethanol production (Fig. 3.1) (Heilmann et al., 2010). Bioethanol production from microalgae has been improved by utilizing different strains such as Porphyridium cruentum (Kim et al., 2017), Tetraselmis suecica (Reyimu & Ozçimen, 2017) and Desmodesmus sp. (Rizza et al., 2017). Synechocystis sp. PCC 6803, the microalgal species, was capable of producing bioethanol (0.69 g/g) by converting CO2 under photoautotrophic mode (Dexter & Fu, 2009). Recently, biobutanol production has been also observed from carbohydrate-rich biomass of Neochloris aquatica CL-M1 (Wang et al., 2017), Chlorella vulgaris JSC-6 (Wang et  al., 2016) and biomass residue (Cheng et al., 2015).

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3.2.3  Lipids Lipids are crucial for proper growth, development and survival of organisms. Lipids are vital component of cell membrane and helps in energy storage as well as cell signalling (Napier et al., 2014). In cell, lipids are stored as energy molecules to carry out different cellular functions under unfavourable conditions (Hess et al., 2018). Plant biomass is mainly due to vegetative growth, and increment in growth increases biomass generation in plants. Several metabolites from plants have been extracted from plant biomass, and the production can be improved by upregulating pathways responsible for increased production of these compounds (Tatsis & O’Connor, 2016). Different products from plant biomass have been utilized by human from time immemorial. The by-products from plant biomass have myriad of uses in paper, textile industry and nutraceutical industry (Crini et al., 2020). Recently, biomass from several plants is considered as an efficient alternative for bioenergy production (Alhazmi & Loy, 2021) (Fig. 3.1). Plant seed plays important role by acting as the prominent source of food and fuel (Napier et al., 2014). Storage molecules accumulated in the seed act as the energy source during seed germination (Napier et al., 2014). Neutral lipids are the prominent storage oil which has numerous industrial value, and substantial efforts from time to time have been focused on improving the oil composition and yield (Napier et al., 2014). The storage lipid in plant seeds is triacylglycerol (Murphy, 2020). Essential fatty acid such as linoleic acid and α-linolenic acid are present in plant oil in abundant amount but are devoid of some important unsaturated fatty acids. The properties of fatty acids are strongly affected by the position of double bond in fatty acid chain. TAGs, the storage lipids, are abundant, and energy-rich compound in plants can be easily converted into biofuels. The most important strategy to improve biofuel production is by increasing the TAG content in plants (Tatsis & O’Connor, 2016). Genetic modification can assist in improving the TAG content in plants (Huang et  al., 2020). It has been observed that silencing enzymes responsible for starch synthesis can divert carbon towards TAG biosynthesis. Promoting the expression of certain gene (WRINKLED1) and silencing some genes (AGPase) resulted in TAG accumulation, and increase of 95 and 43 folds was observed in leaves and stem of transgenic sugar cane plant (Tatsis & O’Connor, 2016). Microalgae are considered as microplants, and the diverse by-products from algal biomass is similar to that originated from terrestrial plants (Singh, Upadhyay, et al., 2021). Broadly, microalgae are divided as green, blue green, red, brown and diatoms (El Gamal, 2010). Like plants, microalgae are also the source of vital biomolecules such as carbohydrate, proteins and lipids (Singh et al., 2020). As compared to plant biomass, microalgae have low lignin content and require mild pretreatment for lignin degradation (Passos et al., 2014). After comparing with different plants, microalgae seem to be the most feasible, economical and environment friendly substrate for production of different biofuels. Microalgae considered as the important component of third-generation biofuel can overcome the limitation due to the use of other feedstocks for biofuel production (Nigam & Singh, 2011). Lipid

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content varies in microalgae (1–70%) and is also directly dependent upon the species, culture and environmental conditions (Kumar et al., 2019). The lipids in microalgae are categorized as neutral and polar lipids. Neutral lipid known as TAGs is composed of saturated and monosaturated fatty acids (Shen et al., 2016). Microalgae produce biofuel several times more efficiently (200 times) as the biomass can be harvested numerous times as compared to plants (Schenk et  al., 2008). Different microalgal species such as Chlamydomonas reinhardtii (Scranton et  al., 2015), Chlorella protothecoides (Xu et al., 2006), Nannochloropsis (Vieler et al., 2012), Botryococcus braunii (Ge et al., 2011), Chlorella pyrenoidosa (Shen et al., 2016) and Chlorella minutissima (Li et al., 2011) are known to have high lipid and desired fatty acid content and thus can be used for large-scale biofuel production. The process of biofuel extraction from microalgae biomass is a complex process as different steps such as drying, cell disruption, lipid extraction and transesterification are involved in biofuel production (Alam et al., 2012). Biodiesel extracted from microalgal biomass have properties such as viscosity and density similar to fossil fuels (diesel) (Schenk et  al., 2008). Microalgae as the third-generation biofuel can be made economical by utilizing the wastewater as nutrient media for growth (Upadhyay et al., 2021). The other way to reduce the cost of biofuel is the biorefinery approach which relies on use of algal biomass for production of industrially valuable compounds (Cuellar-Bermudez et  al., 2015; Singh, Upadhyay, et  al., 2021). The production of the desired products in algae can be increased by employing different biotic and abiotic stresses (Singh, Upadhyay, et al., 2021). As per the literature, majority of the studies on microalgae has been carried on biodiesel production due to abundance of lipid in microalgae (Alvira et al., 2010). Additionally, Chlorella, Scenedesmus and Chlamydomonas are the few microalgal strains grown under specific conditions that have carbohydrate content more than 50% as per dry cell basis and are important stains in terms of ethanol production (Chen et al., 2013). Exploiting carbohydrate-enriched biomass for bioethanol production is a suitable paradigm as microalgae have high growth and carbon sequestration potential as compared to terrestrial plants. It has also been observed that mild treatment such as dilute acid can help sugar extraction from microalgae (Ho et  al., 2013). Several microalgae species have potential to produce TAG comparable to vegetable oils (Draaisma et al., 2013), and some of them even acts as the potent source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Mühlroth et  al., 2013; Singh, Upadhyay, et al., 2021). The TAG content in microalgae varies with different microalgal strains as well as the stress conditions (Breuer et al., 2012). Microalgae are diverse in nature, and still several species remain unexplored, but proper screening may lead to identification of TAG abundant strains. The other importance of different microalgae aspects under adverse condition strains is to sustain the photosynthetic rate for proper growth. Under stress conditions, it has been observed that the decline in photosynthesis is species specific and also have significant effect on the TAG synthesis (Klok et  al., 2014). Nutrients starvation especially nitrogen is considered as the most feasible approach to amplify the TAG accumulation in microalgae (Yang et al., 2017). In Neochloris oleoabundans, it has been observed that under nitrogen limited conditions, the majority of the energy is dissipated as

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heat or used for catabolic processes, and only 8.6% of generated electrons in the microalgae ended up in TAG (Klok et al., 2013). For TAG synthesis, the first step involves acetyl-CoA carboxylase which leads to the formation of malonyl-CoA. The second step involves the formation of malonyl-ACP from malonyl group of malonyl-CoA which is mediated by malonyl-CoA:Acyl Carrier Protein transacylase. Acyl Carrier Protein is a substrate for fatty acid elongation and is catalysed by ketoacyl-ACP synthases (Brown et  al., 2009). The substantial increase in acetylCoA, malonyl-CoA and CoA has been observed in different species of microalgae such as Chlorella desiccata, Chlamydomonas reinhardtii, etc. cultured under unfavourable conditions (Avidan et al., 2015). After elongation, the fatty acid transferred to cytoplasm acts as substrate for acyltransferases which are responsible for TAG synthesis in the cell. TAG is the important energy storage molecule in the cell due to the presence of acyl chains. Several authors have reported that under different stress condition such as nutrient starvation, temperature and light, the TAG synthesis in microalgae can be amplified (Hu et al., 2008; Martín et al., 2016).

3.2.4  Fatty Acids The natural source of polyunsaturated fatty acids (PUFA) is fish, but due to the increasing aquatic pollution, the fish can be the major source of contamination for humans (Yaakob et al., 2014). The possible accumulation of metals, pesticides and recalcitrant pollutants in fish has raise question marks on the use of fish as source of PUFA. Additionally, the poor taste, smell and stability have restricted the use of fish for PUFA production (Spolaore et al., 2006). However, the other sources such as walnuts, sunflower oil, soybean oil and flaxseed are cultivated on arable land, and production is totally decided by several other agricultural and environmental conditions. The presence of already limited arable land can be the limiting factor for exploitation of such resources for PUFA production. Thus, exploiting microalgae can beat several hurdles and elevate the production of important bio-compounds. Algae as the promising substrate for the production of valuable compounds have been started in the recent past (Villarruel-López et  al., 2017; Singh, Upadhyay, et al., 2021). The diverse products from algal biomass include proteins, lipids, vitamins, antioxidants and PUFA which have multiple industrial benefits. PUFA are gaining consideration due to their presence in limited organism, and human body also lacks the pathway responsible for their synthesis (Singh, Upadhyay, et  al., 2021). Microalgae are considered as the viable and potent source of these compounds, and large-scale exploitation can enhance their availability in cosmetic, nutraceuticals and pharmaceutical industry (Sun et  al., 2019; Singh, Upadhyay, et al., 2021). Fatty acids having 12 or more carbon in the chain are the main constituents of lipids (Hess et al., 2018). In microalgae, fatty acids ranging from C14 to C20 are important in terms of biofuel production (Sun et al., 2019), while fatty acids with more than 20 carbon atoms in the chain are PUFA.  Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the main PUFA which are essential for

References

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growth and development of humans (Singh, Upadhyay, et al., 2021). Both DHA and EPA are used as dietary supplement and provide protection against several diseases (Chandra et al., 2019). Two pathways (aerobic and anaerobic) have been identified in microalgae responsible for PUFA biosynthesis. In aerobic pathway, fatty acids in presence of oxygen reacts with enzymes such as desaturase and elongase leading to the synthesis of PUFAs, while in anaerobic pathways, PUFA synthesis in microalgae occurs by the synthesis of polyketide synthase (Sun et al., 2019). Nannochloropsis sp. (Zittelli et al., 2004), Phaeodactylum tricornutum, Nitzschia laevis (Hemaiswarya et al., 2011), Isochrysis galbana (Zamora & Richmond, 2004) and Porphyridium cruentum (Arad & Richmond, 2004) are some of the algal species important in terms of different types of PUFA. Several reports on the use of PUFA as dietary supplement have proved very helpful in protecting human from different chronic diseases (Singh, Upadhyay, et al., 2021). Apart from being the promising source of futuristic biofuel, and predominant source of diverse industrial compounds, microalgae are green, economical, reliable and sustainable bio-resource for present as well as future generations. Thus, exploiting algal farming and involving farmers, societies, industries and governments can certainly enhance the production of diverse compounds in sustainable manner and can represent the potential sector responsible for increasing income of the farmers.

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Chapter 4

Mechanism and Methods of Extraction of Biofuels

Abstract  The utilization of non-renewable sources of energy has directly affected the economies of developing countries. So far, man has succeeded to utilize nature’s renewable forms of energy to a large extent. However, deriving energies from such kind of sources involves the huge amount of costs. In the recent times, energy production from bio-sources has gained a huge focus. This chapter discusses the various methods and mechanism involved in the production of biofuels from organic wastes and biomass. Furthermore, the chapter also stresses the involvement of microalgae and other microbes for the sustainable recovery and generation of biofuels. Keywords  Biofuels · Fermentation · Biotransformation · Carbon cut · Biomass · Wood waste

4.1  Introduction Over the last several generations, the world has experienced an increase in energy demand and resource plunder by emerging regions, particularly India and Africa, in order to meet rising norms (Peng et al., 2019). Starting with a nation’s desire, alterations in the environmental scenario and conflict among nations over fuel usage, the drives are onerous and must be addressed. Rapid urbanization gobbling up fossil fuels, historic high gasoline production, increasing reliance on Middle Eastern oil sources, detrimental effect of natural fuels on greenhouse emissions exerting strain on civilization and elevated levels of “nitrogen oxides”, “sulphur dioxide” and metal particles in the atmosphere due to the use of modern fuels are some of the biggest concerns associated with conventional energy insecurity (Curtin et al., 2019; Piotr et  al., 2019). The society’s never-ending search for a fuel that can solve today’s challenges indicates a finite number of sources. Within a quarter, rising countries with a high desire in economic expansion have consumed two-thirds of the energy depleted in prior years. Without a source of energy, the world’s energy hunt used to be tedious. However, with the help of wise decisions in the direction of biofuels, it © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4_4

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is now very close to looping in the twentieth century. The International Energy Agency (IEA) and the Organization for Economic Cooperation and Development (OECD) have described the worldwide energy demand that will be confronted in the near future. According to the late economist Angus Maddison, developing countries will overtake developed countries in economic weight by 2030. India and China have all of the important resources for improving a poor country’s economy, including investment and trade (Ting & Boqiang, 2019). The development of “SouthSouth linkages” is critical. Despite the fact that the global economy has strengthened, there has been a huge loss in reserves. Saudi Arabia reigns supreme in the market, with 260 billion barrels of oil and 115 billion barrels of natural gas (Lippman, 2019). In opposition to the major energy-seeking countries, the global oil demand is constrained by a small number of countries.

4.2  Energy Development and Environmental Welfare The energy need is getting increased day by day from the last few years because of the economic growth and growth of world’s population. Nonetheless, at present, the main part of this energy utilized is the fossil sources of energy. Fossil fuels are non-­ renewable sources, and this is the main problem because these are limited for supply, and one day, there will be shortage of these fossil fuels. Due to this main problem, researchers have found their interest in other renewable fuels. To reduce the requirement of fossil fuels; decrease the emission of greenhouse gases, particularly in the transport area; and enhance the safety of fuel supply, there is a production of biofuels from renewable sources (Passoth, 2014; Valentine et  al., 2012). Nevertheless, nowadays, the production of biofuels is done mainly by the use of first-generation substrates, for example, sugar cane, wheat, or vegetable oil. Human beings can also use these products as their food. The utilization of these crops can lead to various changes in land use and also causes loss of natural ecosystem. That is why their use is being criticized (Kim & Dale, 2004; Kluts et al., 2017). As a result, the generation of biofuel has been conducted by the use of second-generation biomass such as lignocelluloses which include straw or wood remains (Gnansounou, 2010). However, these straw or wood residue crops can also do competition with the generation of food due to the requirement of land for the generation of these plants (Kluts et al., 2017). Generally, the manufacturing yield of keen energy crops is poor due to the production on poor-yielding land; therefore, most likely, the production rates are higher and the profits low. Various aspects are there which determine whether the production of food or biofuel is cost-effective. If the generation of biofuel is cost-effective, then production can be done of those crops in place of food plants which give energy (Glithero et al., 2015; Shortall, 2013; Sims et al., 2010). On the contrary, the supreme source for the generation of biofuel may be cereal straw because it is a co-product of the synthesis of food, and therefore, the generation of this resource does not do competition with food production (Townsend et al., 2017), and increase in the level of grain generation did not show any adverse

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consequences on the use of cereal straw as raw material for the generation of biofuel (Jørgensen et al., 2019). Biodiesel is an environmental friendly, non-hazardous and most importantly free from sulphur and aromatics. It is best for the replacement of fossil diesel because biodiesel has lesser emissions as compared to fossil diesel (Staat & Vallet, 1994). Nonetheless, by using biodiesel as a fuel, there is an increase in NO (Dorado et al., 2003). Earlier, vegetable oil and animal fats have been utilized as raw products for the generation of biodiesel (Singh & Singh, 2010). The “transesterification” reactions of “fat or oil triacylglycerols” with methanol or ethyl alcohol is generally used for the production of biodiesel in the presence of alkaline or acid catalyst (Marchetti et al., 2007; Sharma & Singh, 2008; Vicente et al., 2004). Therefore, the carbons that will be highly prone to the instigation of peroxidation are allylic to two olefinic groups at the same time. For the methyl ester, the oxidation relative rates of oleic acid are 18:2, linoleic acid 18:3 and α-linolenic acids 18:3 1:12 as reported in a study. There are various studies which were conducted on the storage and oxidative potency of biodiesel produced from vegetable oil and frying oils such as sunflower, soybean, rapeseed, etc. (Bondioli et al., 2002; Bouaid et al., 2007; Bouaid et al., 2009). Though information regarding the storage stability of biodiesel which are generated from corn oil does not exist, diverse states of storage like increased temperature and contact to light, air, water and other harmful substance are there under which the studies of stability were carried out. The different methods of oxidation such as “pressurized differential scanning calorimetry” and “EN 14112” also called as “Rancimat methods” are there under which biodiesel was subjected in some other studies (Dunn, 2002; Moser, 2009). The process of raw material generation is not only the process on which oxidation stability depends by; it also depends on the pressing and refining process of oil (Chahine & Macneill, 1974). Globally, 79% of the total requirements of energy are because of the use of crude oil, natural gas and coal and cause the release of CO2 which further leads to the change in global climate (BP, 2017; Olivier et al., 2016). This condition can be alleviated by better use of biomass for active functions (Shuit et  al., 2009). Distinct approaches have been industrialized by the European countries to use more and more renewable sources which are present in every locality (Papadaki, 2012). In the European Union countries from the last decade, the generation of energy by the use of renewable resources has increased gradually and arrived at 16% of the total energy requirement in 2016  in order to meet the demand of future targets (IEA, 2017; Papadaki, 2012). Different sources such as “wastes and biomass, hydropower, wind energy, solar energy and geothermal energy” are the different resources of sustainable generation of energy (REN21, 2016), while 68% of the total renewable energy is the share of conventional biomass, biofuels and wastes amounts (Alzate et al., 2018; Eurostat, 2017). The use of biomass as a source of energy and food is in conflict (Koizumi, 2015; Solarte-Toro et  al., 2019) with the use of remaining biomass from agricultural and industrial activities to energy utilization, and these conflicts can be avoided. There are some examples of these types of energy transporters such as “wood pellets, biogas, bioethanol, biodiesel and syngas” which are acquired from stand-alone processes (Claassen et al., 1999; Surendra et al., 2014).

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In European Union nations, the generation of bioethanol has developed which is primarily based on 42% corn, 33% wheat, 18% sugar beet, and 7% other cereals, which supplied 6.9% of the world’s bioethanol production. There is an increase in the share of wheat and sugar beet (Eurostat, 2017), while less than 0.2% of bioethanol production is dependent upon lignocellulosic biomass, for instance, Miscanthus sinensis and Panicum virgatum. Broad studies have been done on the generation potential of lignocellulosic feedstocks, but only a small number of services are available in Italy (ePURE, 2015). Bioethanol production from lignin cellulose needs pretreatment. Bioethanol can be pretreated by dilute acids (Del Campo et al., 2006). Subsequently, there is a requirement of a “saccharification stage” for the conversion of cellulose moiety of biomass into fermented sugars (Humbird et al., 2011). These treatments are very costly and require an increase in asset expenses (Hernández et al., 2014). The charges of these enzymes may be approximately 19–20% of the total generation cost (Macrelli et al., 2012). The enduring goal of the research is the optimization of the process (Martínez-Patiño et  al., 2017). Further, the methods based on biorefinery will be successful as compared to the stand-alone methods because biorefinery-based processes generate and commercialize additional value-­ added products besides ethyl alcohol (Moncada & Aristizábal, 2016).

4.3  An Overview of Biofuels Biofuels are a type of energy fuel made from organic substances (often referred to as biomass) produced by plants and living organisms that can be cultivated and collected again. Biofuels include “biodiesel” (made from vegetable oils, reclaimed wax or animal fats), “bioethanol” (alcohol made from fermenting sugar and starch crops like corn) and “biogas”. One of the world’s most essential energy sources is petroleum oil. Because of the rapid increase in petroleum use, it is predicted that the world would run out of petroleum oil by 2070–2080 (Ting & Boqiang, 2019). More than 70% of all petroleum fuel is used in the transportation industry. Due to greenhouse gas emissions (GHG), which include CO2 and other harmful gases including “methane”, “carbon monoxide” and “chlorofluorocarbons”, its overuse has raised worries about health and global warming. By 2040, greenhouse gas emissions are expected to reach over 43 billion metric tons. Complementary power sources that are “conveniently accessible”, “sustainable” and “economical” are thus required. Agricultural and critical harvesting, woods and residual streams are the primary sources of biofuels utilized to substitute non-renewable energy fuels (Curtin et al., 2019; Peng et al., 2019; Piotr et al., 2019). Biofuels are being developed as a replacement for petroleum since they are “harmless”, “sulphur-free” and “biodegradable”, and they come from renewable sources. These remarkable biofuels are derived from renewable resources, as demonstrated by a Swiss study. The amount of ozone-­ depleting substances released by biofuels is lower than that of petroleum oils

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(Shahid et al., 2019). The main problem is that such biofuels require a great deal of harvesting as raw resources, which means that a huge area is being used to grow crops for power rather than sustenance. According to a frequently recognized estimate, it takes a certain amount of grain to produce enough biofuel to fill a UK family’s vehicle once as it does to feed a child for nearly seven months (Mathimani et  al., 2019; Satputaley et  al., 2018). Furthermore, because it is progressively beneficial for ranchers to sell their harvests to biofuel organizations rather than to food producers, this “green fuel” raises grain prices (Vasudev et al., 2019). In small biofuel-producing countries, such as Brazil, it is common to clear vast areas of rainforest to make room for farms dedicated to producing ethanol (Kumar et al., 2019; Vasudev et al., 2019). Despite the controversy surrounding biofuels, they only account for about 2% of the global fluid fuel supply, but we shouldn’t dismiss them. Scientists estimate that second-gen biofuels, for which the European Parliament approved a 2.5 percent priority, might provide up to 30% of the world’s transportation energy (Yanik et al., 2013). As a result, the most pertinent issue concerning biofuels is whether they are the safest alternative. Stakeholders will agree that a biofuels guideline, i.e., a limitless green fuel that is considered necessary, is indispensable (Vargas e Silva & Monteggia, 2015). Existing fuels can be substituted with biofuels, but there are several obstacles to overcome, the most notable of which is that biofuels have a reduced energy density ratio than conventional fuels. This is a problem since producing energy equivalent to petroleum fuel requires a larger quantity of biofuel crops. Petroleum is a fossil fuel that harms the environment, and this is possibly the most compelling argument for us to pursue biofuel development. It’s also worth noting that in order to meet the increased demand for biofuel crops, we’re destroying forests, rainforests and grasslands. This is a significant problem in countries like “Brazil”, “the USA”, “Argentina” and “Indonesia” (Adamczyk & Sajdak, 2017; Li et al., 2020; Trinh et al., 2012). Biodiesel can be made from a variety of raw materials, including “algae oil”, “animal fats”, “vegan oils” and “microbial oil resources”, albeit the content and quality of biodiesel made from different sources might vary (Li et al., 2017).

4.4  Classification of Biofuels Biofuels are classified into four kinds based on the feedstock: first-, second-, thirdand fourth-generation biofuels (Ullah et al., 2009).

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4.4.1  First-Generation Biofuels The dispute over nourishment and fuel is aided by the first generation of biofuels (Jin et al., 2015; Li et al., 2017). The advancement of genetically modified yields has steadily increased since their introduction in 1996–1997 (Areal et al., 2013). The first generation of biofuels are made from “oil-based plants”, “sugar” and “starch” yields (Fig. 4.1). By using fermentation process, sugars inside various raw materials can be used to attain alcohol, and the metabolic breakdown can be done by utilizing microorganisms such as “Kluyveromyces, Zymomonas, Zygosaccharomyces, and Saccharomyces”. Raw Materials with Available Fermentable Sugars Fermentable sugars are mainly occurring in sugar beets, sweet sorghum and sugar cane. Only milling, fermentation, distillation and denaturalization processes are used upon these raw materials (therefore, the sugar is not apt for consumption of human). Furthermore, it should be dried out for their use as mixtures in gasoline (Solomon et al., 2007), and it is obligatory to transfer its azeotropic (constant boiling mixtures) point. There are several methods of dehydration such as

Fig. 4.1  It shows the different processes for the generation of biofuels in first generation, second generation and third generation

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pervaporation, vacuum distillation, extractive distillation and adsorption with molecular meshes, along with solvents or salts or with both at the same time. Hence, by combining two or more of the above processes or hybrid processes, ethanol can also be dehydrated (Uyazán et al., 2004). Subsequently, for over 50 years of practice with first-generation ethanol, advancement was concerned with further expansion of the fundamental utilization of unprocessed materials and the by-products of the methods, incorporation of processes of first generation with processes of 2G, examination of the special effects of the variabilities of the same unprocessed material, crop situations, harvesting periods and procedures and storing time of the unprocessed material on ethanol harvests (dos Passos Bernardes et al., 2016; Gumienna et al., 2016). Another significant phase is programmed sugar cane harvest, which proliferates the occurrence of inorganic composites including copper, calcium, silica, potassium and iron as compared to the old process of manual cutting and burning. Furthermore, it has been reported that mechanical harvesting increases 7.6% silica, 13% calcium and 32% magnesium in the sugar cane juice when compared to old processes of burning and cutting (Thai et al., 2012). The process of fermentation is greatly affected by these minerals as magnesium stimulates yield of ethanol while excess copper decreases the production (Costa et al., 2015). Attention has increased for ethanol produced by utilizing sweet sorghum and other type of sorghum. This crop could be grown with less water as compared to other grains and is greatly proficient at photosynthesis for converting CO2 into energy. In addition, aggregate sugar tranquil of the sweet sorghum stem juice is analogous with the juice of sugar cane (Chuck-Hernandez et al., 2012). The technology of sugar cane processing has been modified to perform sweet sorghum harvesting and milling. Various researchers have proposed the crafty apparatus specifically for stems of sorghum to increase indexes of juice extraction (Peralta-­ Contreras et al., 2013). Sorghum stems has a number of abilities but has great moisture content, i.e., 70%, and the less density bound the spaces inside which transferring it later garner is cost-effective (Chuck-Hernandez et al., 2012; Zegada-Lizarazu & Monti, 2012). Furthermore, preservation of the sugary substances inside the stems is challenging due to the native crop bacteria, including Leuconostoc, damaging a great fraction of sugars through passage and stowage. Blocking of growth of Leuconostoc through transportation, by using SO2 (sulphur dioxide) gas, has been confirmed though packing the gas in enclosed tanks at the sites of sorghum collection, along with its eradication when it reaches at the dispensation unit, marks it an affluent structure (Lingle et al., 2012). For 40 years, first-generation technology has been scientifically functional. That’s why in addition to the studies stated, there are various researches linked to scrutiny of ecological harms of this technology. Among all these, the major problem is the production of biomass known as vinasse (residue from the sugar or ethanol industry) which is formed on ratio of 12 L vinasse L-1 ethanol (da Silva et al., 2007). Vinasse is a liquid organic matter with suspended constituent part, high COD (chemical oxygen demand) and lower pH. About 40 years long ago, the by-product (vinasse) was an extremely polluting fluid that brings about ever ecological harms

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in the surface water, the place where it was disposed. The final disposal of this liquid was a problem due to less technology and its high rates. But advancement in technology has changed the whole scenario as it comes up with cost-effective methods of treatment. Various methods including physicochemical, chemical, alcoholic fermentation and biological methods were tested to manage different kind of by-­ products, and the most appropriate method of treatment is the biological methods because of the presence of huge number of biological and ecological mixtures in their arrangements (Sheehan & Greenfield, 1980). A number of substitutes were projected for the management of vinasse such as production of biogas (Cruz-­ Salomón et  al., 2017), fertigation (fertilizer application) (Mijangos-cortes et  al., 2014) and feeding chickens (Hidalgo et al., 2009). Furthermore, uniform approaches were created to life cycles analysis to define entry and departures of energy, to check by-products production in the whole supply chain and to reduce effects from institution of crop to ethanol fuel consumption (Cavalett et al., 2012; Gallejones et al., 2015; Miret et al., 2016). Raw Materials with High Starch Content Raw materials that have high content of starch are cereals, rhizomes and tubers which are accustomed to attain ethanol and come under first-generation sources. For ethanol production, following methods are followed by these raw materials including amalgamation, saccharification, fermentation, milling, distillation and dehydration. In between 2012 and 2015, countries including China, the USA and India have more published studies on ethanol derived from starch with 187, 181 and 74 printed papers individually. The beginning of the process occurs with dry milling of starch and then association with proteins and lipids. Afterwards, in amalgamation and saccharification, these macromolecules reduce activities of enzymes due to their inhibiting properties in the ongoing process (Srichuwong et  al., 2009). Therefore, investigation of the interactions between non-starch and starch components and their impacts amid hydrolysis is needed. Wang et al. (2016) have tested the use of pigmented sorghum to comprehend whether pigments inhibited the process or not and resolute that the raised amount of anthocyanins had no impact upon yield of ethanol. On the industrialized platform, there are many inventive techniques for removing fermentable sugars and amorphous carbohydrates with diffusion process that utilized biomass from sorghum-chopped sweet grains and stems (Appiah-­ Nkansah et  al., 2016). A number of researchers have studied the interaction of enzymes with the starch during the process of milling and elimination of physical barriers. According to Chuck-Hernandez et  al. (2012), production of ethanol increases during the decortication of the sorghum grains. In the studies revealed, cereal management skills and ethanol production are matched; energy expenses decline and yields of ethanol proliferate. Nonetheless, quantifiable examination of expenses and evaluation of altered practice plans must be considered. It is also essential to assess balances in water and profitable investigation of the method before commencing industrialized scale.

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Li et al. (2015) has highlighted a practical method for the detection of starch and ethanol content by the use of spectrophotometer FT-NIR. Due to this method, the production of ethanol can be systematically controlled on industrial scale. The generation of ethanol from bananas in Costa Rica and Ecuador has been analysed previously (Graefe et al., 2011). Banana shows a benefit above tubers due to their ripening and maturation, and starch is naturally hydrolysed, and therefore, there is no need of enzyme treatment to reach the fermentable sugars (ASTEDU, J., 1987; Bugaud et al., 2009). It has been showing that there is a high yield of ethanol generation from green bananas at various phases of maturity instantly. The yield of the generation of ethanol from overmature bananas was 23%. It was less than that of immature bananas. The metabolic changes during the maturity of bananas cause the reduction in a dry-base matter (Hammond et al., 1996). It has been recommended that because of the high yield of green bananas, only green bananas should be used to generate ethanol though there are some technological problems also like peeling the banana. The Production of Biodiesel from Vegetable Oil The production of vegetable oil can be done on a large scale because these are capable feedstock for the generation of biodiesel. Vegetable oil can vary according to the climate conditions or availability, and approximately 94% of the generation of diesel comes from feedstock of vegetable oil. The main crop which is used for the production of biodiesel in the USA is soybean oil; on the other hand, in Europe and other countries, rapeseed oil and palm oil are most commonly used. Nonetheless, the rate of biodiesel is generally influenced by the cost of natural resources usually in the 70–80% proportion (Shi & Bao, 2008). For the production of biodiesel from waste edible or non-edible oil such as “Mahua, Jatropha or Karanja”, various studies are done by the researchers (Ghadge & Raheman, 2006; Tiwari et al., 2007). When looking to other manufacturing sites, various substitutes come into mind. One of them is the ethyl alcohol whose main feedstock is corn. A hydrocarbon-based source of renewable carbon is the integrated biorefineries that are provided by the ethyl alcohol acquired from corn plants for the generation of fuel and chemicals. The formation of ethanol takes place when starch undergoes hydrolysis followed by the fermentation of glucose. All through the process, there is also the formation of other by-products such as corn gluten meal, gluten feed and oil of corn. As a result, new technology can be used to extract corn oil as a remaining product which leads to the production of ethanol more effectively. The conversion of corn oil can be done into biodiesel. The commercialization of biodiesel is done as an alternative or amalgamation stock of regular diesel though as compared to distinctive fossil fuel, oxidation of biodiesel is less resistant, and thus the stability of fuel will be influenced by the doping of biodiesel in regular diesel considerably (Dunn & Knothe, 2003). Therefore, due to the generation and requirement of biodiesel rising quickly, the improvement of methods to check the quality of biodiesel industry and regularity develops into an important topic for the introduction of biodiesel in the market. The biodiesel produced from corn plants is a chemical mixture of “long-chain fatty

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acid methyl esters”. Fatty acid methyl esters (FAMEs) are highly prone to autoxidation, and therefore, it is highly reactive as compared to fossil diesel. The volatility depends on the number and place of double bond present in the fatty acid methyl esters that are interrupted by methylene.

4.4.2  Second-Generation Biofuels Second-generation biofuels are non-food outputs derived primarily from agricultural and woody leftovers (Aguilar et al., 2018; Andree et al., 2017; Leu & Boussiba, 2014). Biofuel made from second-generation sustainable lignocellulosic biomass reduces food safety risks. “Forest residuals”, “agriculture detritus”, “yields” and processing unit scrap make up the biomass of lignocellulose. Lignocellulosic biofuels are aiding to alleviate environmental and nutritional concerns. Lignin (26–31 percent), hemicellulose (25–32 percent) and cellulose (41–46 percent) are commonly used in the synthesis of lignocellulosic biomass. Different techniques are used by various scientists for the generation of biodiesel from first-generation and second-generation feedstocks (Martínez et  al., 2014; Razack & Duraiarasan, 2016; Saydut et al., 2016). In a previous study, single-step transesterification with methanol was used for the production of biodiesel from filbert and sunflower oil, and potassium hydroxide was used as a catalyst (Saydut et al., 2016). In a previous study, the biodiesel was produced by the use of waste cooking oil using response surface methodology, and encapsulated mixed enzyme was used as a catalyst (Razack & Duraiarasan, 2016). In another study, “Jatropha curcas and Ceiba pentandra” oils were used for the production of biodiesel using “response surface methodology” (Dharma et al., 2016). The same methodology was used by another researcher for the generation of biodiesel from “Calophyllum inophyllum” (Ong et al., 2014). There are several studies which have produced biodiesel from stone fruit oil, but not any of them used any statistical representation. For example, alkali transesterification with methanol was used to produce stone fruit oil methyl ester, and potassium hydroxide was used as a catalyst (Gumus & Kasifoglu, 2010). In a study, stone fruit oil biodiesel was generated through alkali transesterification with catalyst of 0.75% of KOH, and methanol ratio was 6:1 (Fadhil, 2017). It was also reported that the production of biodiesel by the use of “Prunus armeniaca L.” oil using single-step transesterification process through utilizing sodium hydroxide as a catalyst is at a ratio of methanol:oil of 6:1, and yield was 93% (Ullah et al., 2009). Transesterification single-step process was used, and 1% KOH as a catalyst was used for the production of biodiesel, and the yield was 96.5% at a 55°C for 60 minutes with an invariable stirring at 400 rotation per minute (Yadav et al., 2018). Therefore, it has been found that there are various parameters such as temperature during reaction time, concentration of catalyst and type of catalyst, alcohol type, molar ratio between oil and methanol, reaction time and speed of stirring, etc., which influence transesterification process (Atabani et  al., 2012; Atadashi et al., 2012; Hamze et al., 2015).

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4.4.3  Third-Generation Biofuels Algae-based third-generation biofuels can be produced on a large scale, absorb CO2 and are relatively simple to refine and accruing a lot of attention. There is a fast growth in interest with the increase in the search for raw materials for the production of ethanol with high energy crops. Persistent grasses, algae and bacteria especially nitrogen-fixing bacteria, all these crops are coming under third generation of biofuels. For the ethanol generation, algae are not raw material, but these produce hydrogen for thermal and chemicals conversion and act as a substrate (Brennan & Owende, 2010). The conversion of biomass from macroalgae leads to the production of starches. Then these starches undergo hydrolysis and fermentation and generate bioethanol (Adams et al., 2009; Khambhaty et al., 2013; Scholz et al., 2013; Sudhakar et al., 2016) and biobutanol. Biobutanol shows high energy density and high compatibility with gasoline as compared to bioethanol (Dürre, 2008). Konda et al. (2015) also reported the economic viability of microalgae as raw material for bio-refineries. The possible benefits for the production of wide range of chemical products from microalgae to give the economic feasibility to industrial groups-denominated biorefineries were represented in this study. Isolated algae strains can be handled in the laboratory conditions as compared to natural conditions because eutrophication process takes place in natural conditions which leads to the proliferation of algae due to the presence of more nutrients inside the water body. Therefore, to improve quality of water, algae were proposed for the making of ethanol. Chen et al. (2017) patented a technique (pre-technique) using electrocoagulations and acid saccharification of algae. They extract about 156 mg glucose g-1 of algae that can transform the ethanol. Seaweed is the mostly used macroalgae (pluricellular organism) which can convert nutrients in the water bodies and carbon dioxide in the biomass. Mohammed (2013) stated that out of 9200 species of seaweeds, only 221 are efficiently important. Macroalgae serves as a basic material for the preparation of bioethanol as they do not contain high amount of lipids. However, macroalgae have a large amount of sugar and more carbohydrates which can be fermented easily. The production of carbohydrates is influenced by different growth variable including light, nutrients, temperature, salinity and pH. George et al. (2014) reaved that the intensity of photosynthetic light of 60 μmol m-1s-1 and 12:12 cycle attained a biomass output of 7.9 mg L-1 d-1 with “Ankistrodesmus falcatus” with reference to effect of light. The knowledge about the difficulties between the pretreatment technologies like liquefaction, saccharification of the macroalgae and the second-generation raw material is very important because polysaccharides are also present there that are hydrolysed into fermented sugars. Lipids and carbohydrates are present in very high amount in microalgae. Therefore, for the integrated generation of bioethanol and biodiesel, Scenedesmus species was planned to be used as a raw material (Sivaramakrishnan & Incharoensakdi, 2018).

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4.4.4  Fourth-Generation Biofuels Engineered cyanobacterial growth is used in the fourth generation of biofuels, which is a novel and fast expanding field (Adeniyi et al., 2018; Sharma et al., 2020). The pretreatment stage is critical for enzymatic hydrolysis, as well as fermentation to enhance the volume of the target product. To achieve the desired result, pretreatment biomass could be used in conjunction with enzymatic saccharification and microbial fermentation. Synthetic, biological and physical pretreatment methods are the three categories of pretreatment methods (Abdullah et al., 2019; Kumari & Singh, 2018). The use of a comprehensive biomass transformation method to specialize microalgae growth and production within industrial unit facilities utilizing wastewater-rich exhaust gas and sewage water as nutrient repositories is one way to create ample biomass to fulfil the expanding requirements of energy consumption. Both emission regulation and sewage water treatment units will profit more (Sivaramakrishnan & Incharoensakdi, 2018). Oleaginous organisms use the fermentation technique to convert carbohydrates to fatty acids or oil through a range of metabolic pathways (Shields-Menard et al., 2018). Microalgae have a simple “cell structure” and require “light”, “water”, “carbon dioxide” and “nutrients” (phosphorus and nitrogen) for growth (Behera et  al., 2019; Sivaramakrishnan & Incharoensakdi, 2018). Microalgae are eukaryotes related to the Protista family, i.e., membrane formation all around nucleus, whereas Cyanobacteria are prokaryotes, which are imperfect membrane-binding organelles of the domain of bacteria family categorized in the specimen as algae (Chiaramonti et al., 2017; Daroch et al., 2013). Nitrogen and phosphorus, which make approximately 10–20 percent of the biomass in microalgae, are the most important additions. Algae have a diverse set of strategies for allocation, food organization, photosynthetic coloration and rejuvenation. Increased light assimilation into microalgae by restricting the size of certain chlorophyll antenna and pigment administration are two fundamental approaches for genetically engineering microalgae (b; Achinas & Euverink, 2016; Nguyen et al., 2017; Srivastava et al., 2020a; Van et al., 2019) (Fig. 4.2). Furthermore, microalgal metabolic technology has the potential to significantly boost lipid or carbohydrate content. Microalgae feedstock, a raw material rich in “carbohydrates”, “lipids” and “enzymes” can be converted into “bio-hydrogen”, “biodiesel”, “bio-oil”, “biomethane”, “biocrude oil” and other products (Anto et al., 2020; Saini et al., 2020). The most acceptable criterion for it is to maximize lipids and carbohydrates, which improves the performance of microalgae biomass yields (Ryckebosch et al., 2011; Sampath et  al., 2020). To successfully manufacture bioenergy, fourth-­generation biofuels incorporate genetic engineering feedstock, genomically produced microorganisms such as cyanobacteria and microalgae. They were created using non-arable lands, similar to third-generation biofuels. General view of biofuel production from algal culture, involvement of different processes and microbes is depicted in Fig. 4.3.

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Fig. 4.2  Showing the use of biomass for the biofuel generation in first-, second-, third- and fourth-­ generation biofuels

Fig. 4.3  Various techniques applied for the processing of microalgal biofuels

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Various advancements in biological engineering have directed towards the fourth biofuel generation, in which “genetically modified organisms” are used that detain more carbon dioxide, for example, genetically modified sugar cane that contains higher lipid content for concurrent generation of bioethanol and biodiesel (Huang et al., 2016). As well, “genetically modified Escherichia coli” (a bacterium) is used for the fermentation and leads to the production of triglycerides from juices of sugar cane and afterwards altered into biodiesel. Various companies in the USA such as “Amyris, LS9 and Sapphire Energy Solazyme” and in Terrebonne, Canada, have studied the methods to extend the process to industrial levels (Steen et al., 2010; Westfall & Gardner, 2011). The utilization of genetically modified organisms showed higher yield and effectiveness. “Scheffersomyces shehatae JCM 18690” is a genetically modified yeast which has the capability for the simultaneous hydrolyzation and fermentation of starch, and after 10 days, it gives the production rate up to 0.92 gram L-1 d-1, as compared with the high production rate of Saccharomyces cerevisiae from maize crop (Tanimura et al., 2015). The yield of the conversion of glucose or xylose into ethanol has increased approximately 28% by the use of genetically modified Escherichia coli strain, i.e., KO11 (Huerta et al., 2005). The higher production cost of biofuels is related to the raw material (Neto et al., 2016). Biorefineries are the integrating processes that widen the range of products of a generation plant and, thus, commercialization possibilities. But the difficulty range is very high, posing challenges in the vital utilization of raw material and asking for more study due to the involvement of number variables. Earlier, a study was there which concerned about the generation of ethanol from paper, wood and manure. The remains are dried in the plant where ethyl alcohol is generated. Storage was done for the short period of time and then set up into the gasifier along with carbon for the generation of CO, H2 and C. The collection of raw material showed a significant effect on the cost of end product in this process, and the C which is used for the improvement of economic effectiveness raises expenses. The production of ethanol with the combination of wood and carbon has increased the economic viability. It was demonstrated by Gwak et al. (2018) that the use of carbon in this combination might be a better option for obtaining energy with utilizing household-produced organic waste (Gwak et al., 2018). The carbon dioxide produced by the combustion of ethyl alcohol is less than that produced by fossil fuels, and photosynthesis can fix it, and therefore, its emission into the atmosphere is less and releasing less greenhouse gases (Quintella et al., 2011).

4.5  Need of the Biofuel Production The rapid surge and decline in fossil fuel costs, as well as a complete change towards green energy, have contributed to an increase in the use of biomass conversion processes. The development of appropriate conversion processes permits the chemical transition to energy and even fuels, and the increased available biomass capacity indicates a best ability in the role of biomass in the global energy blend. Techniques

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for changing biomass make it easier to convert unprocessed biomass into a variety of solid, gaseous or liquid fuels, as well as generate electricity and heat simultaneously, allowing for the provision of energy services. Even though, in addition to the low conversion efficiency technologies, the current high investment costs hinder the rapid development of such technologies, preventing the use of biomass for direct burning in order to preserve a favourable process (Sampath et al., 2020). On the flipside, the limitations associated with effectiveness during the chemical transition of biomass may be accounted for through rapid reformulating new formulation of the revisioning, using a number of subprocesses that ultimately improve productivity. A large range of potential alternatives generate multiple transformation chains in all manufacturing processes between raw materials and finished products, varying in conversion effectiveness and integrated process costs. The systematic detection, creation and assessment of relevant pathways wherein biomass is reliably turned into various prospective anaerobic digestion-based products can be learned via literature searches. This dynamical challenge is examined in a wider context that uses a technique edifice route to evaluate acceptable conversion paths and their integration from a set of new innovations that serve as important aspects. A well-defined biomass garbage collection and repository system is essential for developing a large-scale biofuel system for managing organic trash. Advanced biofuel generation technologies, on the other hand, are critical for reducing non-­renewable fuel use in global economies and achieving renewable energy stability in the future. As a result, it is critical to make rapid progress in developing biofuel resources to meet the world’s energy needs. As little more than a result, a novel method is required to address the ongoing waste management concerns, which include increased area and water use (De et al., 2015). The best structure of evolving conversion frameworks is determined using the MILP maximization formulation, which can further strive to minimize the gross capital plus processing costs. Moreover, process aggregation factors are used to verify that the energy requirements of each plant are decreased and that the processes in economics are improved by the effective achievement of a supersystem of heat exchanger (Jamil et al., 2020). Emerging technologies can be efficiently estimated for prospective economic productivity, allowing for the selection of the most efficient conversion method based on the type of raw biomass required and the anticipated outcome (Ni et al., 2020). Traditional biofuels will be referred to as first-generation biofuels, whereas second- and third-generation biofuels will be referred to as contemporary biofuels, depending on feedstock types and manufacturing processes. Biodiesel is made from oil yields, and bioethanol is made from starch and sugar yields in traditional fluid biofuels. These are currently produced in a cost-effective manner. Biodiesel is the most renewable energy source that may be used as a superior transportation fuel (Carsanba et al., 2018; Ledesma-Amaro & Nicaud, 2016; Matsakas et al., 2017). Biodiesel will reduce brown haze by 40% while also lowering CO levels significantly. The burning of biodiesel with mineral diesel minimizes particulate matter, CO and hydrocarbon emissions to a considerable extent, with only a tiny loss of power. The higher price of raw material is the biggest hurdle to developing a

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profitable business (Lucia & Grisolia, 2018) because using biodiesel is extremely efficient and enticing. The integration of undesired cooking oil into the system of persistent transesterification, which includes the recovery of high-quality glycerol as a biodiesel by-product, is critical for lowering biodiesel prices. The potential of bio-oil being replaced because of its increased viscosity, raised ash and water content, limited heating value, volatility and maximal corrosiveness, substituting bio-­ oil with fossil, requiring chemical feedstock, is negated (Aziz, 2015). Engineered science entails the creation of new organic components and gadgets, as well as the redesigning of existent potential arrangements. Enhanced biofuels require a photosynthetic or non-photosynthetic structure, either naturally occurring or created (Srivastava et al., 2020a, b).

4.6  Biofuel Production Techniques Presently, biofuels are highly safe for the environment than traditional ones and can reduce pollution levels to a great extent, plummeting CO2 discharges. Innovative inexhaustible “energy sources” can be manufactured from “agricultural and woodland lignocellulosic biomass” as well as from “algal feedstock” (Stephen & Periyasamy, 2018). The liquid biofuels engendered increasingly are “human-made hydrocarbons” degradation of carbohydrates by different biochemical processes (Lucia et al., 2016; Matsakas et al., 2017). Key features of the manufactured biofuels obtained from “organic wastes” are better yield and abridged capability to contaminate, while most particular “biofuel techniques” are still under investigations (Aziz, 2015). The chief standard procedures include fermentation of organic matter especially starch towards ethyl alcohol with “transesterification of components of waste to produce biodiesel”. First-generation biofuels are generated principally from “barley, wheat, maize, potatoes, sugar cane and oilseed, while biodiesel is synthesized from “sunflower and soybean”. Starch breakdown microorganisms such as “Saccharomyces cerevisiae and Rhizopus sp” are applied to produce ethyl alcohol by the “fermentation of corn” (Ullah et  al., 2009). The main “enzymatic hydrolysis process” at the manufacturing comprises transformation of carbohydrates to “bioethanol” (Saini et al., 2020). Biofuels of the “second generation” are characterized as “bioethanol” improvement from wood-based wastes and accessible crops, including dead leaves (Anto et al., 2020). Third-generation biofuels depend on the progress of biodiesel through algae and “cellulolytic bacterial species metabolism” and microorganisms due to the enhanced progression pace and even the competence of carbon sequestration. Numerous strategies are applied to produce biofuels of the “first, second, third and fourth generations” (Agarwal et al., 2018).

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4.6.1  Techniques for Production of Biofuels from Biomass A complete variety of organic waste may be utilized during resourceful biofuel yield which offers enormous visions to the “methods and biofuel potentialities”. Biomass “feedstocks are used to produce assured biofuels, and they can substitute ancient fuel sources and reduce carbon emanations. Most of the “biomass utilized today is from three primary categories: forestry, cultivation, and trash. This includes traditional cutting of trees into natural wood, sawmill deposits of wood, and some timber handling businesses, rural vitality crops, rural build-ups, and waste” (Fonseca et al., 2011; Wyman, 2002). Biofuels prepared from “fats and oils” are alike to “diesel in their chain length and other fuel characteristics and are termed biodiesel” (Luo et  al., 2012). Convinced pioneers to the production of “biodiesel involve microalgae, edible and non-edible oil” (Bet-Moushoul et al., 2016). Biodiesel, one of several biofuel sources, is produced through green crops as a replacement to petroleum diesel (Karmakar et al., 2010; Morshed et al., 2011; Takase et al., 2014). Bioenergy conversion through “microalgal and lignocellulosic biomass” has gained momentous appreciations in recent years. Furthermore, numerous “lignocellulosic/ bioenergy” materials offer the utmost cost-effective biomass to bioenergy. The foremost hindrance confronted during the transformation of biomass is the composite organization and features of the “cell walls components” in the biomass and should be treated before the biotransformation. The “pretreatment process” is accompanied to enrich for the “broad spectrum of applications”.

4.6.2  Biomass to Bioethanol Transformation Biomass leads to bioethanol generation in two distinct ways, namely, thermochemical and biochemical transformations (Chen et  al., 2011). Such conversion procedures are distinguishable. According to Mu et al. (2010), feedstock gasifying at 800 ° C and catalytic development are utilized in the “thermochemical pathway”. This kind of performance required a vast expanse of heat energy, contributing to the synthetic process to produce gases like H2, CO and small amounts of CO2 so that syngas can be transformed into a mixture of alcohol by the chemical process at 300 °C (Gamage et al., 2010; Guo et al., 2016). Bioconversion usually contains five progressions, i.e., “fermentation, hydrolysis, distillation, dehydration and pretreatment” (Younesi et  al., 2005). The microbes Clostridium ljungdahlii, Zymomonas mobilis and Saccharomyces cerevisiae can be used for ethanol production by “syngas processing” (Mishra et al., 2019; Pradhan et  al., 2018). Degradation of “hemicellulose and cellulose” into simple carbohydrates involves certain enzymes such as cellulose or acids in the “hydrolysis” (Pradhan et al., 2018). Sugars are produced by the hydrolysis of polysaccharides and transformed into bioethanol consecutively (Chisti, 2007; Martinopoulos et al., 2018). Enzymatic hydrolysis has benefits over acids, including low energy

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consumption due to mild process conditions, increased sugar yields and no undesirable waste. Substrate characteristics including porosity influence enzymatic cellulose hydrolysis, degree of polymerization, the crystallinity of the cellulose fibre, hemicellulose, lignin content, optimum blending, the concentration of the substrate, end product, the activity of the enzyme and reaction parameters like temperature and pH (Ayala-Parra et al., 2017; Pradhan et al., 2018). During fermentation, yeast or bacteria convert sugars to ethanol in a liquid or solid state. The economy is greatly improved if sugars of both C5 and C6 are used. However, fermentation efficiency of C5 sugars is poor. Besides, the yeasts do not tolerate low pH, high concentrations of ethanol and the by-products. Isolation of clear ethanol is generated through the distillation process (Martinopoulos et al., 2018). Thermophilic framework based on amount of raw material and volume of digester produces biogas at high speed as compared to mesophilic frameworks (Adarme et  al., 2017; Harun et  al., 2010). The degree of humidity in anaerobic digestion is one of the fundamental elements for biogas production. Due to less wastewater generation in dry modification operation for AD, it will be economical as it lowers the working cost but at the same time increases the maintenance costs of the system (Mussgnug et al., 2007; Yang et al., 2018). Contrary, wet model anaerobic digestion is known to lower the setup cost of the system. In solitary, the microorganism in each digester degrades substrate, but each one of them has certain limitations with the type of digester which eventually affects the biogas production. The biogas digester for conversion of waste into biogas can be used in groups to make the process economical and efficient. Therefore, utilizing multistage digester can certainly increase the gas yield per unit of substrate and overcome the limitations posed by the single digester (Singh & Singh, 2010; García et al., 2017). The biogas generated after anaerobic digestion contains 55–75% of methane and 30–45% of carbon dioxides along with trace amount of gases such as water, hydrogen sulphide, oxygen, nitrogen and hydrocarbons (Ward et al., 2014). Apart from generation of different gases during digestion of waste, the nutrient-enriched digestate is also produced which after continuous processing can be used as the renewable fertilizer in the agriculture fields. The nutrient composition of digestate is highly dependent upon the feedstock used for anaerobic digestion, and sometimes, the potentially toxic pollutants such as heavy metals and organic compounds could make the process expensive and hamper their subsequent utilization in the field (Bonturi et al., 2017; Piotrowska et al., 2013).

4.6.3  Structure of Biogas for Anaerobic Digesters The composition of biogas differs greatly with feedstock and types of system used for digestion of feedstock. The biogas obtained from sewage treatment plants contains several trace amounts of gases such as siloxanes, ammonia and particulate matter (Liska et al., 2014). Due to varying feedstock composition and operational

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conditions of digester, the biogas structure can vary significantly from feedstock of different plant. The different constituents of biogas such as moisture, siloxanes and ammonia are pretreated during processing of biogas. The anaerobic digestion of feedstock produces the significant amount of hazardous gases such as hydrogen sulphide and sulphur-containing compounds. During the ignition of biogas, the hydrogen sulphide reacts with oxygen resulting in the production of sulphurous acid and ultimately sulphuric acid. Several techniques such as adsorption and scrubbing by using the biotrickling filters have been utilized for the elimination of hydrogen sulphide from biogas. The iron sponge adsorption is the most widely used adsorption system for the elimination of hydrogen sulphide from biogas. In iron sponge adsorption, the alternate adsorbent materials such as Sulfa-Bind, Sulfa-Treat, and Sulfur-Rite are used to eliminate the hydrogen sulphide from biogas (García et al., 2017; Liska et al., 2014; Sakuragi et al., 2016; Singh & Singh, 2010). Similarly, the siloxanes in anaerobic digester can be volatized or processed into silicon dioxide. For silicon dioxide production, the siloxanes are ignited at high temperature in the incorporated system in anaerobic digester such as IC machine and turbine (Awe et al., 2017; Lee & Park, 2016; Zhang et al., 2020). The activated carbon adsorption is considered as the major techniques to remove the siloxanes from biogas. In kitchen waste, the siloxanes are either in small amount or absent, but co-digestion with wastewater increases their concentration in the biogas. The efficient adsorption of siloxanes by granulated activated carbon and the upstream elimination of moisture and hydrogen sulphide are responsible for increasing the efficiency of granulated activated carbon absorbers. Silica gel as compared to granulated activated carbon have high siloxanes removal rate and thus can be used as the efficient alternative absorber (Littlejohns et al., 2018; Ojeda et al., 2011).

4.6.4  Preparing Biogas with Anaerobic Digestion The biogas obtained after digestion in anaerobic digester is majorly methane; thus, biogas can be called as biomethane (Liska et  al., 2014). The other toxic compounds present in biogas are dependent upon the end use of the fuel (Salman et al., 2020). Before using as the fuel in vehicle, the moisture, carbon dioxide and hydrogen sulphide should be removed from the biogas (Nguyen et  al., 2017; Singh & Olsen, 2011). The biogas generated must meet the regulation standards established by regional government before introducing the biogas in gas grid (Sakuragi et al., 2016). The handling and transportation of biogas should be done carefully because it contains high content of moisture, hydrogen sulphide and microorganisms. In addition, to remove such toxic gases, several purification procedures are utilized leaving up to 98% of methane per gas stream per unit volume (Joshi et al., 2009).

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4.6.5  Biogas-Purifying Techniques Water vapour evacuation: The water vapours from biogas must be removed to comply with designs of vehicles. The different techniques such as physical partition and adsorption drying are utilized to remove the water vapours from biogas (Awe et al., 2017). Physical Partition Refrigeration is considered as the easiest method to remove the water vapours from biogas. In physical partition, either demister is used to isolate the microscopic pores or cyclone separators by using centrifugal forces to remove the water vapours (Lee & Park, 2016). Adsorption Drying In adsorption drying, the pressure of the gas is increased followed by feeding it to column containing silica adsorbent. This method is cyclic method in which the bed is thermally regenerated, and the water vapours after every few hours are converted into vapours (Littlejohns et al., 2018). Removal of H2S The removal of hydrogen sulphide is necessary to avoid the biogas consumption during channelling and stockpiling of biogas in tanks. Hydrogen sulphide is very responsive, and under high temperature, and pressure, the reactivity of hydrogen sulphide is increased. To reduce or eliminate the hydrogen sulphide, it reacts with oxide or hydroxide of iron which leads to the formation of iron sulphide (Sakdasri et al., 2019; Soltanian et al., 2020). Removal of CO2 The emission of carbon dioxide is important to examine the biogas production potential. The removal of CO2 decreases the fuel weight but increased the fuel value of the gas. The following methods are believed to be efficient for carbon dioxide removal from biogas (Awe et al., 2017).

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Absorption of CO2 by Physical Method The carbon dioxide is removed by scrubbing the biogas using water. In this method, the raw gas under pressure of 1000–2000 kPa is transferred to the bottom of column, and water supplied from top allows the mass transfer between liquid and the raw gas. The concentration of the carbon dioxide in the column reduces as the gas moves upward and become enriched with methane (Soltanian et  al., 2020). The methane-rich biogas then exits from the top of column after depressurization of water. Finally, desorption column is used to revive water and discharged after mixing with air to release the carbon dioxide from water (Ryckebosch et al., 2011). Chemical Absorption Several chemical reactions have been used to remove carbon dioxide from biogas (Fonseca et al., 2011). In chemical absorption, amine under negligible pressure is utilized to trap the carbon dioxide from biogas (Fonseca et al., 2011). The process is very similar to water or glycol scrubbing (Persson et al., 2006). Before chemical scrubbing, it is important to remove the hydrogen sulphide to prevent amine intoxication (Andersson et al., 2020). The amine is regenerated using heat or steam, and it has been reported that 93% of pure carbon dioxide obtained is isolated and recuperated (Bauer et al., 2013). Cryogenic Separation In cryogenic isolation of carbon dioxide, it is well established that the carbon dioxide forms bubbles at 78 °C while methane has melting point of 161 °C which reflects that carbon dioxide can be extracted as fluid on cooling of gas by increasing pressure (de Baan et al., 2013). Methane from the biogas can be extracted in liquid or vapour form depending upon the design of the device.

4.7  C  ellulosic Biofuel Conversion Methods and Emerging Technologies Advancements in technical knowledge development associate the amount of energy of biomass, and a number of new skills to make it available for assorted uses are now emerging. In such case, thermochemical methods appear predominantly capable to astound the prevailing complications correlated to biochemical transformation, including elongated response periods, little proficiency of transformation by microorganisms and enzymes and elevated manufacturing rates (Raheem et  al., 2015; Sims et  al., 2010). Furthermore, thermochemical procedures permit direct

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conversion of biomass into liquefied petroleum, therefore aggregating the process effectiveness and later will enable easier conduct of the energy density of the product formed and storage and distribution of the biofuel produced by consuming current setup. The capability for cognate bio-oils to be assimilated into prevailing organization customary for petroleum-established oil has given rise to the word “drop-in” biofuels. There are three thermochemical translation processes which are customarily utilized on the basis of the amount of oxygen in the procedure. These three processes are pyrolysis (thermal degradation without oxygen), gasification (partial oxidation) and combustion (complete oxidation) (Silva et al., 2016). Bio-oil or crude-like oil production from thermal decomposition processes including pyrolysis and hydrothermal distillation possibly will remove the compulsion to bifurcate biomass of plant because these techniques are premeditated to be feedstock idiomorphic, so far as they are responsive to the usage of numerous raw materials with exceedingly inconstant configurations. Pyrolysis of biomass is at temperature of 200oC–750oC without oxygen and produces three main by-products such as sustainable bio-oil, carbonized char and gases/smokes (Raheem et  al., 2015). Long-last pyrolysis process is mainly implemented for fabricating charcoal, whereas “fast pyrolysis process” is used to increase production of bio-oil (Bridgwater, 1999; Bridgwater, 2010; Bridgwater et al., 1999; Peacocke & Bridgwater, 2000). This oil can also be used directly to produce electricity in a generator or, more sophisticated, may use as a transportation fuel. Nonetheless, the utilization of the technical knowledge has been restricted by its need for biomass having low moisture and by complications practiced in uncontrollable problems associated to the increased acidity, increased reactivity and increased oxygen amounts of biomass obtained from pyrolysis oils. Therefore, pyrolysis oils that are derived from biomass are difficult to separate due to the presence of water in it, and gross calorific value gets limited, i.e., up to 17 MJ/kg (Bridgwater, 2010). The principles of various technologies like fast pyrolysis and others including “fluid beds, rotating cone and vacuum pyrolysis ablative and twin-screw pyrolysis” were reviewed and assessed by Venderbosch and Prins (2010). “Hydrothermal liquefaction (HTL)” is a re-emerged technology that is strong and can provide various resources of biomass. This technology shows higher benefit above various methods like pyrolysis. Hydrothermal liquefaction technology is able to use wet biomass resources with no requirement of expensive drying steps (Akhtar & Amin, 2011). It is a thermal and chemical process. This technique utilizes 150–180 bar pressure, and temperature ranges from 300 to 350°C (Behrendt et al., 2008; Goudriaan & Peferoen, 1990; Toor et al., 2014) in which water reaches at its significant point and then it becomes an extremely reactive medium that go through solid biomass and imitate geological methods. In this technology, there is no need of pretreatments. During hydrothermal liquefaction technique, there is a release of various small components which are unstable and highly reactive leads to form hydrocarbons. Decarboxylation and dehydration processes convert carbon dioxide from O2 in the biomass (Goudriaan & Peferoen, 1990; Toor et al., 2014). After the completion of the reaction, the development of high pressure triggers the phase separation and the comparatively simple separation of the product, which is

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usually called bio-oil or bio-crude. It has comparatively higher density of “33.8–36.9 MJ/kg” and has low content of O2 as compared to pyrolytic bio-oil, therefore permitting the option of combining with conventional hydrocarbon fuels without emulsification. In the hydrothermal liquefaction technique, comparison of the values of the energy density of hydrothermal liquefaction and the value for the energy density of ethanol is estimated at 26.4 MJ/kg (Fersi et al., 2012), while present fossil fuel is estimated at “44.4 MJ/kg” (Chattanathan et al., 2012). The chemical characteristics of bio-oil are greatly dependent upon the composition of biomass because every compound such as polysaccharides, lignin, proteins and lipids generates a different variety. The reduction of non-cellulosic polysaccharides is taken place into saturated hydrocarbons, and lignin remains there in the residual part (Akhtar & Amin, 2011). Hydrothermal liquefaction generally requires costly reactors as compared to the pyrolysis process because of the high-pressure requirement. Nevertheless, the main part of the hydrothermal liquefaction is the reactor, and generally, the expenses of HTL and fast pyrolysis might be same (Nabi et al., 2015). The design of “continuous flow system” would be the most significant development in the field of technology that prevents the repetitive need for application of batch-wise temperature. The “continuous-flow processing system” has been re-evaluated for microalgae and lignocellulosic biomass, in addition to the downstream processing of hydrothermal liquefaction products (Elliott et  al., 2015). The expenses for the energy balance and theoretical process have been intended, and the business potential of hydrothermal liquefaction technique has also been assessed (Elliott et al., 2015). It has been estimated in a thermal and chemical processing of biomass of microalgae that pyrolysis oils extracted from microalgae are more stable and having less oxygen as compared to pyrolysis oil extracted from lignocellulosic biomass. It has been estimated that the thermal liquefaction is a very effective pathway to high amount of bio-oils, along with calorific values closer to that of petroleum oil (Raheem et al., 2015). For the alteration of sunflower oil to higher purity biodiesel, various techniques have been developed such as “continuous flow vortex” and “fluidic production” with no requirement for “saponification”, solvents or composite catalysts (Britton & Raston, 2014). However, there are various challenges before the consideration of bio-oils as commercially feasible “drop-in fuels”. There is also the requirement of alterations to present storage and shifting facilities. Both HTL and fast pyrolysis oil techniques may experience from increased acidic and iodine level. Various efforts are under progress for the improvement of hydrothermal liquefaction of bio-oil into fossil fuel terms through hydrogenation of solvent, cracking of catalyst, esterification reaction and hybrid procedures (Ramirez et  al., 2015). Various concerns might arise from the emission of derivatives from the thermal and chemical changes into the environment. The growth of various techniques like “catalytic hydrothermal gasification (CHG)” to clean the organic remains was found as water derivatives in hydrothermal liquefaction (Elliott et  al., 2014; Elliott et  al., 2015). The conversion of gas can be done into heat and electricity and is composed of CO2 or hydrogen and methane with less quantity of contaminants like CO or HC (Elliott, 2008). Moreover, the application of hydrothermal liquefaction was done in

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a broad variety of biomass and organic remains chemicals such as biomass from wood (Zhu et  al., 2014), “mixed culture algae” (Ou et  al., 2015; Chen, Zhang, Zhang, Schideman, et al., 2014a), manure (Chen, Zhang, Zhang, Yu, et al., 2014b), residues from food and agricultural industry (Pavlovic et al., 2013) and “municipal waste” (Minowa et  al., 1995). The compounds of renewed bio-crude from HTL techniques of lignin and cellulosic biomass can be used again, unprocessed apart from distillation, and as a biofuel for present diesel engines when combined with automotive diesel fuel (Nabi et al., 2015).

4.8  Conclusion The requirement of energy is increasing day by day from the last few years because of the economic growth and growth of world’s population. Nonetheless, at present, the main part of this energy utilized is the fossil sources of energy. Nowadays, the production of biofuels is done mainly by the use of first-generation substrates, for example, sugar cane, wheat or vegetable oil. Human beings can also use these products as their food. The utilization of these crops can lead to various changes in land use and also causes loss of natural ecosystem. That is why their use is being criticized. Generally, the production yield of keen energy crops is poor due to the production on poor-yielding land; therefore, most likely, the production rates are higher and the profits low. Various aspects are there which determine whether the production of food or biofuel is cost-effective. The supreme source for the generation of biofuel may be cereal straw because it is a co-product of the synthesis of food, and therefore, the generation of this resource does not do competition with food production, and increased level of generation of grain did not have any adverse effect on the use of the cereal straw as raw material for the generation of biofuel. The present chapter aimed to discuss about the effective steps which are used for the generation of biofuels.

References Abdullah, B., Syed Muhammad, S. A. F., Shokravi, Z., Ismail, S., Kassim, K. A., Mahmood, A. N., et al. (2019). Fourth generation biofuel: a review on risks and mitigation strategies. Renewable and Sustainable Energy Reviews, 107, 37–50. https://doi.org/10.1016/j.rser.2019.02.018 Achinas, S., & Euverink, G. J. W. (2016). Consolidated briefing of biochemical ethanol production from lignocellulosic biomass. Electronic Journal of Biotechnology, 23, 44–53. https://doi. org/10.1016/j.ejbt.2016.07.006 Adamczyk, M., & Sajdak, M. (2017). Pyrolysis behaviours of microalgae nannochloropsis gaditana. Waste and Biomass Valorization, 1–15. Adams, J.  M., Gallagher, J.  A., & Donnison, I.  S. (2009). Fermentation study on Saccharina latissima for bioethanol production considering variable pre-treatments. Journal of Applied Phycology, 21(5), 569–574.

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Chapter 5

Metabolic Routes to Biofuels Extraction

Abstract  Biofuel is a chemical generated by biological processes. Mainly living organism biomasses (microalgae, pants and bacteria) are the source of it. An increase of population needs more energy for the betterment of life, and to fulfil their needs, biofuel is the best example of source globally. Since many years ago, the fossil fuels are the major source of energy. They were utilized having no sustainability that further affect environment as related towards combustion of fossil fuel. The biofuel production is well developed in recent approaches. There is a possible strategy for microalgal cultivation in which energy is conserved in direct way for the biofuel production. For biofuels, plant biomass is the one of the best source known since few decades. However, there is an increase in the evidences that shows that “algal biomass” is one of the best sources for its production. The production of biomethanol, bioethanol, biohydrogen, carbohydrates, proteins and different compounds depends upon species and method utilized for microalgae cultivation that are further utilized in different pharmaceutical industries. In algae, for the production of biofuels, only three components are required, i.e. carbon dioxide, water and sunlight that help to generate an eco-friendly and renewable product. Bacterial co-­culture is also an important part for the biofuel production. The present chapter aimed to discuss about various technical challenges that are required for the production of biofuels. Keywords  Biofuel · Fossil fuel · Microalgae · Bacteria · Algal biomass

5.1  Introduction Biofuel is a chemical generated by biological processes. Mainly living organism biomasses (microalgae, pants and bacteria) are the source of it. An increase of population needs more energy for the betterment of life, and to fulfil their needs, biofuel is the best example of source globally. Since many years ago, the fossil fuels are the major source of energy. They were utilized having no sustainability that further affect environment as related towards combustion of fossil fuel (Voloshin et  al., 2015; Razzak, Hossain, Lucky, Bassi, & De Lasa, 2013; Allakhverdiev et al., 2009). © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4_5

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To avoid environment effect due to this, it can be utilized as a substitute through renewable energy source which is eco-friendly in nature (Dragone, Fernandes, Vicente, & Teixeira, 2010). Heimann (2016) reported that the biofuel production is well developed in recent approaches. There is a possible strategy for microalgal cultivation in which energy is conserved in direct way for the biofuel production. The cultivation of cyanobacteria biofilm is an example and good pathways to produce biomass and also utilized for “biofuel processing pathways”. Demirbas & Demirbas (2011) reported that the biofilm cultivation is a good production source of biofuel through cyanobacteria or microalgae. For biofuels, plant biomass is the one of the best source known since few decades. However, there is an increase in the evidences that shows that “algal biomass” is one of the best sources for its production (Dragone et  al., 2010). In plants and algae, photosynthesis occurs by which they differ from other sources. Carbon dioxide shows carbohydrate formation with the help of sun energy, and the process is known as photosynthesis (Voloshin et al., 2015). It is known for carbon fixation in plant and green algae. It shows that it is an exclusive process that is responsible for plant and algal biomass growth which is a raw material for the production of biofuel. Sugar is known to be a basic substrate by which formation of bioethanol and biomethanol occurs (Dias et al., 2009). The use of photosynthetic organisms is a very environment-friendly and inexpensive source (in which carbon dioxide and solar light are used as carbon source and as energy source, respectively. Voloshin et al. (2015) reported two stages in which the photosynthesis process occurred: one is light dependent and second is light independent. They also studied that light is responsible for electron activity (transfer) by which it gets activated. It is also accomplished by electron carriers in large membrane complexes (coupled with plastoquinone, cytochrome c and plastocyanin). The main reason of the reduction of NADP+ molecule into NADPH is water electron and ferridox in NADP oxidoreductase. In “oxygenic photosynthesis” the electron source is water. The water decomposition forms due to protons and molecular oxygen. Not all the time, but in few situations, the protons come beyond hydrogenase and not over ferredoxin: “NADP oxidoreductase”. Molecular hydrogen is also a biofuel that is highly reached in chemicals formed by biological processes and arrived from living organism biomass (microalgae, plants and bacteria). Globally, population increases day by day, and to fulfil energy requirement is on high demand for betterment of life for which biofuel is one of the best sources. Various researchers reported that fossil fuels are also one of the best sources of energy, but they are utilized unsustainably and lead to various environmental issues due to combustion of them (Voloshin et al., 2015; Razzak et al., 2013; Allakhverdiev et al., 2009). The fossil fuels are utilized as substitute through source of renewable energy (eco-friendly biofuels) (Dragone et al., 2010). Recently, it was reported that the production of microbial biofuel is well-known, and also there were recommendations of some strategies for the cultivation of microalgae through direct energy conversion to form biofuels. Slade and Bauen (2013) reported that for the production of biofuels, microalgae are utilized as attractive source or feedstock. The production of biomethanol, bioethanol, biohydrogen, carbohydrates, proteins and different compounds depends upon species and method utilized for microalgae cultivation that are further utilized in different

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pharmaceutical industries (Carlsson et  al., 2007). In algae, for the production of biofuels, only three components are required, i.e. carbon dioxide, water and sunlight that help to generate an eco-friendly and renewable product. In algal biofuel, about 100 times increase than that of higher plants. Fermentation occurs through microorganisms to form biomass into biofuel. However, biofuel production can be performed in various ways; (i) cyanobacteria genetic engineering (to increase the H+ production) (Lindberg, Park, & Melis, 2010); (ii) hydrogen production optimization and metabolic engineering (for the production of biofuel in bacteria) (Cha, Chung, Elkins, Guss, & Westpheling, 2013; Carere et al., 2012); (iii) the conversion of carbohydrates to biohydrogen through dark fermentation; (iv) the production of biohydrogen from microalgae by photobiological methods (Poudyal et al., 2015); (v) in high concentration of alcohol, to increase the production of ethanol, genetic engineering of yeast is formed; (vi) by performing microorganisms genetic engineering helps in the carbohydrate fermentation for bioethanol and butanol production increases; (vii) some microalgal screening for the production of biodiesel; and (viii) the plant cell wall carbohydrates fermented through microorganisms or yeast for the biofuel production. Different algal species have ability by which an alternative energy source can be produced. There are some biofuel sources formed, i.e. alcohols, triglycerides, fatty acids, biodiesel, lipids, cellulose, living organism biomass and carbohydrates. However, several attempts were considered for the identification of algae strains broadly. Out of all algae species, majority of them are utilized as significant source due to their huge production of biomass as carbohydrates, proteins and lipids, for example, in the composition of Spirulina maxima having about 60–71% proteins, in Porphyridium cruentum about 40–57% w/w carbohydrates and in Schizochytrium species about 50–77% w/w lipids. Razaghifard (2013) reported that in some specific conditions, various microalgae are used as a biomass source. Various workers studied that microalgae produce more biodiesel than that of cotton plant and palm plants (Singh, Nigam, & Murphy, 2011). Atsumi, Higashide, and Liao (2009) gave an example; to improve the isobutyraldehyde and butanol production, keto acid decarboxylase gene (in cyanobacteria Synechococcus) elongates PCC 7942. There are some algae species (Botryococcus braunii and Chlorella protothecoides) having terpenoid hydrocarbons in huge amount and glyceryl lipids that lead to crude oil (shorter hydrocarbons). They are also utilized as bioethanol, isobutyraldehyde, triterpenic hydrocarbons and isobutanol (as petroleum fuels). However, in some species of bacteria (Escherichia coli and Bacillus subtilis), genetic engineering is having potential to produce bio-alcohol, fatty acids and isoprenoids in huge quantity. Some species such as Clostridium acetobutylicum and Clostridium beijerinckii are utilized for biofuel production through fermentation of acetonebutanol-ethanol (Gronenberg, Marcheschi, & Liao, 2013). Bacillus and E. coli are the bacteria species known for the production of lactic acid and glutamic acids (as chemical source) (Hasunuma et al., 2013). However, different bacterial species are known for the production of ethanol. In bacteria, biofuel production depends upon the species of bacteria. Caldicellulosiruptor, Pyrococcus, Thermotoga and Thermococcus species which produce huge quantity of hydrogen and decreased amount of ethanol have been showed in genetic studies. All these evidences pointed

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out the biochemical pathway of bacteria cell and significant relationship in between biofuel production and all these pathways. Nowadays, bacterial co-culture is also an important part for the biofuel production. Verbeke et  al. (2013) reported that Thermoanaerobacter species co-culture plays a significant role in the production of ethanol (along cellulolytic organisms). Saccharomyces cerevisiae is a model organism. It is utilized as to produce ethanol and lipids through the process of fermentation (Tai & Stephanopoulos, 2013). They are categorized in primary and secondary biofuels, in which primary are those having direct production from different sources (plants, forest, crop residue, etc.); however secondary biofuels are produced directly from plants as well as microorganisms. They are further divided into three generations. In first generation biofuels are produced from starch-rich food crops (wheat, corn, potato, barley) and soybean, sunflower and animal fat responsible for biodiesel production. In second generation the generation of biofuels occurs to form bioethanol and biodiesel from jatropha, straw, grass, cassava and wood. However, in third biofuel generation, it leads to formation of biodiesel from microorganisms and microalgae (Abdelaziz, Leite, & Hallenbeck, 2013; Dragone et al., 2010).

5.2  First-Generation Biofuels This generation produces ethanol and biodiesel and has relation towards biomass. Saccharomyces cerevisiae is responsible for the generation of ethanol from glucose mostly through “GMO yeast strains”. Most of the time, corn and sugarcane are utilized to produce bioethanol of first generation. In Brazil, sugarcane utilization is on first number. It is the most common feedstock for the generation of biofuel and simplest process to produce ethanol. Sucrose is removed by crushing sugarcane in water and the end product is either raw sugar or ethanol. The high rates of sugar are one of the major concerns for bioethanol business. In 2012, the prices of raw sugar and ethanol were closer to “US$0.20/P and US$0.68/L”, respectively. The cost of 1 litre of ethanol production from raw sugar is around “US$0.30 to US$0.35”. Due to this there is a demand of raw sugar either than ethanol. The great source for the generation of ethanol is corn due to richness of carbohydrates. Also, both corn and sugarcane required initial hydrolysis of starch to separate sugar and form ethanol through fermentation. Mc Aloon et al. (2000) reported that an enzyme α-amylase is commonly utilized for starch hydrolysis (US$0.04/gallon of ethanol production). In August 2012 the market price of corn is about US$339/t that produces about 401–460 litre ethanol (depends on efficiency of process). The production of bioethanol and biodiesel from yeast and algal biomass through fermentation and transesterification are depicted in Fig. 5.1. Biodiesel is one of the most utilized fuels at industrial scale. In this oily plants and seed are utilized, but it is mainly used for oil extraction and conversion into biodiesel by bond breaking among the long chain of fatty acid to glycerol and replacement of methanol through transesterification. It is a simple form of lipids that are utilized in biodiesel production. Globally, the market prices of oil depend upon the source of

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Fig. 5.1  Showing the production of bioethanol and biodiesel from yeast and algal biomass through fermentation and transesterification

vegetables. There are various examples that showed the variation among the market price of oil such as in 2012, the market values of soybean oil and palm oil were “US$1,230/t and US$931/t”, respectively. The market value of canola oil was US$1180/t, which is one of the common feedstocks for biodiesel production. About 10 tons of feedstock produced about 1000–1200 litre biodiesels having US$0.85/L market price. Methanol is also required for biodiesel generation having market value US$0.35/L. However, this market price is a major factor that affects production of biodiesel. So due to this reason, various alternative sources were recommended which are not costly, and they are at high demand (used oil, non-edible plant oil) having the price rate in between “US$350 and US$500/ton”. The main source of lipids is algal biomass for biodiesel formation and relates with third-­ generation biofuels. As discussed, corn and sugarcane are the best source of biodiesel from edible plant oils, and they depend upon the price rate decided by international market. Instead of these, the used cooking oil and jatropha have the lowest prices and may be an alternative source of corn and sugarcane. These oils were found less than that of soil bean oil. But the used oil requires more work for its purification. Jatropha has minimum production and also low market values so it could not be recommended for cultivation at good land.

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5.3  Second-Generation Biofuels Second-generation biofuels can be defined as fuels produced from a broad accumulation, exclusively comprising of different feedstocks but not limited to non-eatable lignocellulosic biomass. For fabrication of second-generation biofuels, biomass utilized is generally divided into three leading classes: (a) homogeneous, for example, white wood chips having a price value of “US$100–US$120/t”; (b) quasi-­ homogeneous, for instance, forest and agronomic deposits set a price concerning “US$60–US$80/t” and (c) non-homogeneous, containing feedstock with low value by way of MSWs (municipal solid wastes) and ranges between “US$0 and US$60/ ton” (Lavoie, Beauchet, Berberi, & Chornet, 2011). The amount for this biomass is considerably fewer than the rate for sugarcane, corn and vegetable oil. Alternatively, such biomass is commonly extra composite to transform, and their construction relies on new and different expertise. Product flea market is associated with the production of biofuel; accordingly, the asking price of translating the novel raw material into the concluding/final manufactured article should be possibly insignificant to retain cost-effectiveness. In contrast, numerous biomasses, for example, corn with the ethanol forage dichotomy, permit the chance to produce a multiplicity of by-products through similar raw material, therefore stick to the conception of a biorefinery. For construction of second-generation biofuels, the transformation technique is frequently prepared conferring to binary altered methods, commonly bringing up “thermo” or “bio” passageways. The “thermo” method enfolds detailed methods wherever biomass is warmed up with a slight quantity of reacting mediator. Altogether, developments in that kind bring about transformation of biomass into three divisions: (a) one solid form identified as biochar, (b) one liquid form recognized as pyrolytic bio-oil and (c) and a gas identified as syngas, which is generally formed of CO (carbon monoxide), short-chain alkanes, hydrogen and CO2 (carbon dioxide). While treated at low temperature between 250 °C and 350 °C devoid of oxygen, biomasses go through a method, namely, torrefaction, and the biochar is the main translation product. The heating of an organic material, such as biomass, in the absence of oxygen at greater temperatures, i.e. 550  °C–750  °C, is recognized as pyrolysis (whichever firm or sluggish provisional on the rate heat exchange with the biomass), and the main manufactured article is bio-oil. Gasification process takes place at higher temperatures, i.e. 750  °C–1200  °C, and with restricted oxygen induction, generating customarily biogas along with biochar and bio-oils as end products. Warm air procedures are assured abundant concerning energy due to the energy requisite to high temperature of the biomass capable of the bidden heats that may be delivered through the incomplete or complete carbon oxidation from the biomass that are usually very exothermic reactions. Biochar, deliberated as a compact biofuel, acquires countless consideration in the trade of pelletizing, particularly in portions of the sphere where biomass lignocellulosic is relatively reasonably priced (Clarke & Preto, 2011). On the other hand, for fuel passage, construction of syngas sorbent or pyrolytic oil is generally deliberated as additional auspicious mediators. Pyrolytic oil is a fluid intermediate which relatively overlooks analogous

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to petroleum fuel; however, it is chemically different. Consequently, to yield transferable fuel from this intermediate pyrolytic oil, an additional conversion needs to be made, which is a relatively challenging chore due to the presence of extra water along with the harsh kind of bio-oil. Many researchers have studied the four potential methods for the transformation: (a) hydro-deoxygenation (decreasing the quantity of oxygen generates an assortment of alkanes analogous to petroleum fuel), (b) steam reforming, (c) catalytic cracking and (d) the manufacture of an emulsion using diesel (Zhang, Chang, Wang, & Xu, 2007). Gasification (conversion of carbon-­based raw material such as coal into fuel gas), as compared to pyrolysis (thermal decomposition of materials at elevated temperatures), generated syngas, generally consisting of solitary carbon and hydrogen composites. However, even though manufacturing of transportation fuels is attainable from syngas, it depends upon the compound catalysts used to bring the construction of C-C bonds. The Fischer-Tropsch process is a characteristic example of such technique (Jun, Roh, Kim, Ryu, & Lee, 2004). The production of methanol is a specific modest method for the industrialized formation of biofuels out of syngas. Methanol can be obtained from hydrogen and carbon monoxide straight under the action of a reducing catalyst. In recent reports it has been mentioned that degrees of manufacture fluctuate from 500 to 560 litres per ton of biomass by means of municipal solid wastes, a non-homogeneous biomass (Lavoie et al., 2012). Methanol is a final product but it cannot be utilized as chemical addition in fuel at this stage. For that reason, requirement of additional conversion is there. Depending upon methanol, many end products have been formed such as alkanes by way of “methanol-to-gasoline” method and ethanol by process of carbonylation (Lavoie et al., 2012). It has been studied that methanol is also utilized for the production of a new generation of fuels including Bio-DME, manufactured via cation chemistry of two molecules of methanol. Methanol has unique advantage; it can be produced under the action of an acid catalyst (Yoo et  al., 2007) and also reported as a stabilizer to diesel (Ribeiro et  al., 2007). However, Bio-DME has particular activities that incline to bound its usage in the transference fuel market, especially due to its low viscosity rates in contrast to diesel fuel initiating extreme array in systems of fuel injection (Ribeiro et al., 2007). The distinctive amount of produced ethanol/t of biomass is 360 litres, having the ethanol worth of US$0.68 per litre and manufacture cost adjacent to US$0.30 per litre. Consequently, the method greatly relies on the expense of raw material due to the transformation from biomass to biogas, the syngas purification and the catalytic production of ethanol which embody important technical tasks. Thus, the most expensive and homogeneous biomasses could not be adequate contenders for such expertise. Biomasses including non-homogeneous or quasi-homogeneous would be much appropriate (Marie-Rose, Lemieux-Périnet, Lavoie, & Bernardes, 2011). And subsequent passageway is bio-path in contrast to a squashing practice because cellulose is initially sequestered from the lignocellulosic biomass. Numerous methods have been deliberated, such as “classical pulping processes” (Jin, Jameel, Chang, & Phillips, 2010), “steam explosion” (Lavoie, Capek-Menard, Gauvin, & Chornet, 2010) and “organosolv processes” (Brosse, Sannigrahi, & Ragauskas, 2009).

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The technical challenge is the separation of cellulose due to the production of high purity of cellulose to eradicate most of the inhibitors without losing more energy or more chemicals. There are two techniques such as enzymes or chemical hydrolysis by the use of acids which are used for saccharification of cellulose once it is purified (Chornet, Chornet, & Lavoie, 2010; Sun & Cheng, 2002). These techniques have some limitation, mainly from expensive point of view due to the enzyme prices especially predicted to increase “US$0.12 to US$0.20/L” of the production of ethanol in 2015 (Mielenz, 2001). Alternatively, the conduction of chemical processes depends upon cheap chemicals (e.g. sulphuric acid). In a forest of North America, the weight of biomass is about 46% glucans (Lavoie et al., 2012), which cause the probably high generation of 323 L of ethyl alcohol per ton of raw biomass. The high ramified polymers of carbohydrates having five carbon and six carbon sugars are hemicelluloses and produce 16–24% of the “lignocellulosic biomass”. Typically, in hemicelluloses the xylans and glucans ratio is differing from 49% to 74% of the total carbohydrate content. The main problem with hemicellulose is presence of C5 and C5 sugars does not undergo fermentation with classical strains of yeast and needs “genetically modified organisms” for the ethanol generation (Matsushika, Inoue, Kodaki, & Sawayama, 2009). In addition, the fermentation process inhibited by acetic and formic acids needs extra operation for detoxification. Chemical pathways can be used for the valorization of C5 (Fuente-Hernández, Corcos, Beauchet, & Lavoie, 2013). According to Lavoie et al. (2011), the second main natural polymer which is present abundantly is lignin; it is present at 24–36% in “lignocellulosic biomass” (Lavoie et al., 2011), in which mostly phenyl propane units are present. Paper industry used lignin for the generation of biofuel (Dayton & Frederick, 1996), and it is a highly energetic macromolecule (Dickinson, Verrill, & Kitto, 1998). About 10–20% of weight of lignin can be converted into value-added components like “guaiacol”, “catechol” and “phenol” (Beauchet, Monteil-Rivera, & Lavoie, 2012). It has also been showed that a part of it can also be converted into transportation fuel, for example, jet fuel (Shabtai, Zmierczak, & Chornet, 1999).

5.4  Third-Generation Biofuels The third-generation biofuels are generated from algal biomass. The algal biomass has a very different kind of yield as compared with mass of lignocellulose (Brennan & Owende, 2010). The generation of biofuels by using algae mainly depends on the lipid content of the microorganism. Commonly, Chlorella species is widely used due to the presence of high amount of lipids (Liang, Sarkany, & Cui, 2009) and high production rate (Chen, Yeh, Aisyah, Lee, & Chang, 2011). A number of technical and geographical challenges are there which are related to the biomass of algae. Usually, in ultimate growth condition, the biomass production from algae is very high (Chen et al., 2011). For this process, the water is required in higher volumes for industrial scale; this becomes a major problem of other countries like Canada where the temperature is less than 0° during a considerable year part. When the extraction

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of lipids is done from the biomass of algae, high water content becomes a problem which needs dewatering, through the process of centrifugation or filtration before the separation of lipids. The process of transesterification is used to process lipids or can be hydrogenolysed for the production of kerosene grade alkane fit for use as “drop-in aviation fuels” (Tran et al., 2010). The relation of third-generation biofuels is mostly with algae. Thus, the key difference between second- and third-generation biofuels is the feedstock. Algae do not need any land surface or freshwater bodies for its production as compared with “lignocellulosic biomass” (Lee & Lavoie, 2013).

5.5  M  icrofluidic Techniques for Enhancing Biofuel Based on Microalgae The well-thought-out bio-based cell factories are microalgae. These are capable to quickly inhabit a liquid medium and generate a wide range of chemicals that were produced from their medium (Michalak & Chojnacka, 2015). Following the practices of fractionation and purification, a number of chemicals can be assessed such as intracellular lipids, starch, chlorophyll, carotenoids or protein pigments (Suganya, Varman, Masjuki, & Renganathan, 2016). To enhance the production of these products, several efforts have been performed such as finding of productive strains, increasing the generation of biomass and changing metabolic pathways (Markou & Nerantzis, 2013). A number of techniques such as bioreactor design, techniques of harvesting of microalgae, various methods for the extraction of metabolic components (Ranjith Kumar, Hanumantha Rao, & Arumugam, 2015) and downstream physicochemical treatments (Khanra et  al., 2018) were thoroughly studied to decrease the cost of generation. However, the various confront of higher cost of production and decreased yield are still there because of the commercial generation of most of the products of microalgae due to the use of laboratory methods for the product optimization. There are various microfluidic processes which demonstrated that they have higher throughput and low value of several microbial applications, for instance, assessment and small microbial cells of fuel (Wang, Bernarda, Huang, Lee, & Chang, 2011). For the cultivation of microalgae, there are a number of microfluidic screening platforms that have been designed (Juang & Chang, 2016). Moreover, various conditions for the culturing can be mastered concerning various fluidic circumstances, supply of nutrients and light diffusion. The investigation of growth kinetics and dissimilarity of single cells, optimization of pigment and lipids production from a number of cell strains with higher throughputs were done by using microfluidic methods. Nevertheless, still there is a need of novel miniaturized detection expertise for the in situ analysis of many microalgae metabolites (Kim, Devarenne, & Han, 2018). The main point of future perspectives is the viability of the use of microfluidic techniques for the optimization of large-scale cultivation of microalgae and goods production. In view of microalgae valorization, the additional method which is needed to isolate, disinfect and change the products during the

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culturing of microalgae is the biorefinery method (Laurens et  al., 2017; Trivedi, Aila, Bangwal, Kaul, & Garg, 2015).

5.5.1  Industrial Waste Industrial waste particularly waste from food industry can also be used for the generation of biofuels. An inexpensive feedstock assortment is really most essential for the generation of biofuels. Gradually, there is an increase in the production of food waste. In literature, loss of food and food waste is defined to as recognition of substances that are projected for human use which can be polluted, despoiled, released or lost. The definition for food loss was given by the “Food and Agricultural Organisation” of the USA that it is any alteration in the accessibility, edibility, cleanliness or quality of edible food that avoid this food consumption by people (Girotto, Alibardi, & Cossu, 2015). During the processing of food, food waste and food losses are generated, and these are also generated in the manufacturing industry during the whole phase of generation. Because of various conditions like unsuitable transport system, systems for the waste storage and improper packaging, the production of waste can be done. At last, food waste and food losses can also be generated from retail system and retail markets due to the inappropriate conservation or treatment and lack of cooling (Parfitt, Barthel, & Macnaughton, 2010). The environment can also be affected due to food waste and food loss and contributes various greenhouse gases such as methane during the disposal of food waste into landfills. These also cause other impacts on the environment including degradation of natural sources and disruption of biogenic cycle (Girotto et al., 2015). Therefore, the food waste can also act as a capable approach for the biofuel production since their treatment and processing is very expensive. Moreover, the composition of food processing waste is it includes “cellulose, hemicelluloses, lignin, lipids, organic acids, proteins and starch”, and these can act as carbon source for the generation of biofuels (Zhang et al., 2018).

5.5.2  Lignocellulosic Biomass The pretreatment of biomass from lignocellulose can be done by using various methods such as physicochemical, physical and chemical, biological or combined pretreatment (Fig. 5.2). Techniques like “chipping, milling, grinding, freezing and radiation” are used in the physical pretreatment. The decrease in the size of element is caused by these techniques, and at the same time, they enhance the surface area of lignocellulosic substances (Kumari & Singh, 2018). Acid treatment such as sulphuric acid and hydrochloric acid can be given in chemical pretreatment that enhances the enzyme hydrolysis of biomass to liberate fermentable carbohydrates (Kumar, Barrett, Delwiche, & Stroeve, 2009) or reactive substances like H2O2 or

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Fig. 5.2  Showing the various pretreatment methods such as physical and chemical pretreatment, physicochemical pretreatment, biological pretreatment or combined pretreatment of lignocellulosic biomass

peracetic acid. In lignocellulosic substances lignin is present which is capable to form soluble fragments with peracetic acid, and pretreatment with hydrogen peroxide eventually causes improvement in enzyme digestibility (Sheikh et  al., 2015). Alkaline substances are also utilized for the treatment of lignocellulosic biomass such as NH3, Ca(OH)2, KOH and NaOH. Polysaccharides are solubilized due to the treatment with alkaline substances, and porosity is improved due to this solubilization. Ozonolysis is also a pretreatment method used to treat lignocellulosic biomass that comes under chemical pretreatment method in which ozone is used for the treatment. Due to this method, the lignin content in lignocellulosic wastes is really decreased (Kumar et  al., 2009). Another chemical pretreatment method is “ionic liquid pretreatment” in which carbohydrates and lignin are dissolved at the same time. When the lignocellulosic waste is treated with organic solvent, it causes delignification of lignocellulosic wastes. Various methods such as “ammonia fibre explosion”, carbon dioxide explosion, “liquid hot water pretreatment”, “steam explosion”, “ultrasonication” and “wet oxidation pretreatment” come under physicochemical pretreatment (Kumari & Singh, 2018). The treatment with microorganism and enzymes comes under biological pretreatment. The methods which come under combined treatment are as follows: (i) Alkali and electron beam irradiation pretreatment method. (ii) Combined alkali and ionic liquid pretreatment method.

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(iii) Combined alkali and photocatalysis pretreatment method. (iv) Combined biological and steam explosion pretreatment method. (v) Combined dilute acid and microwave pretreatment methods. (vi) Combined dilute acid and steam explosion pretreatment method. (vii) Combined enzyme hydrolysis and superfine grinding with steam pretreatment method. (viii) Combined SO2 and steam explosion pretreatment method. (ix) Microwave-assisted alkali pretreatment (Kumari & Singh, 2018). By the use of agrochemical waste, there can be the production of various kinds of biofuels.

5.5.3  Biohydrogen Production In the future one of the safe, clean and non-hazardous biofuels is biohydrogen. Water is the only derivative that is used as a fuel and is free of carbon and does not act as a contaminant (Show, Lee, & Zhang, 2011). Currently, hydrogen is approximately generated by physical and chemical processes which split fossil fuels. Therefore, under various conditions like high temperature and high pressure, the production of hydrogen takes place from contaminated and restricted sources and releases greenhouse gases (Yun et al., 2018; Ewan & Allen, 2005). As a result, the use of other resources becomes significant in order that hydrogen can be attained in a renewable, sustainable and environmental way. The generation of hydrogen can be done from biological processes that are eco-friendly and consume a lesser amount of energy in contrast to physical and chemical ones. The biological processes are “dark fermentation, direct and indirect biophotolysis, and photo-fermentation” (Yun et al., 2018). Dark fermentation process is most widely used and practically applicable for the generation of hydrogen because it is capable in degrading organic waste with higher rate of hydrogen generation. Diverse bacterial cultures, for example, Clostridium species, Enterobacter species, Lactobacillus species, Megasphaera species, Prevotella species and Selenomonas species, are used to carry out dark pigmentation (Lopez-Hidalgo, Alvarado-Cuevas, & De Leon-Rodriguez, 2018; Palomo-Briones, Razo-Flores, Bernet, & Trably, 2017; Cheng, Yu, & Zhu, 2014; Cheng & Zhu, 2013). Moreover, lignocellulosic wastes like “husk of beans, corn stalk and cob, rice straw, wheat straw, vegetable waste” are utilized for the production of biohydrogen (Lopez-Hidalgo et  al., 2017; Sen, Chou, Wu, & Liu, 2016; Zhang et al., 2016b; Bansal, Sreekrishnan, & Singh, 2013; Sekoai & Kana, 2013). The most positive source that has been found for the production of biohydrogen is the heating of lignocellulosic waste with sulphuric acid and sodium hydroxide pretreatment.

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5.5.4  Bioethanol and Biomethanol The most interesting chemical is ethyl alcohol because it shows various characteristics including “antifreeze, beverage, depressant, fuel, germicide, and solvent” (Braide, Kanu, Oranusi, & Adeleye, 2016); ethanol is getting important day by day because of the environmental problems like global warming and climatic change. Greater attention is paying towards bioethanol at various levels such as globally, nationally and regionally. Actually, the market for bioethanol has come into a stage of quick and intermediary growth globally. Renewable sources are getting much attention by a number of nations for controlled production (Sarkar, Ghosh, Bannerjee, & Aikat, 2012). Yearly, the ethanol generation enhanced up to 86.6 billion litres in 2010 all over the world (Kumari & Singh, 2018). The most appropriate approach for the bioethanol generation is the utilization of the wastes that came from the agriculture sector. The waste that came from the agriculture sector is vegetable remains, bagasse, stock of corns, cobs of corns, husk from corn and rice, leaves, rapeseed waste and sugarcane bark wood feedstock. This agricultural waste has the potential for the generation of “bioethanol” (Kumari & Singh, 2018; Braide et al., 2016; Zhang, Wang, Su, Qi, & He, 2010). And the most effective biofuel for the power production is “biomethanol” (Suntana, Vogt, Turnblom, & Upadhye, 2009). Biomethanol shows various applications in vehicles having fuel cell power; additionally, biomethanol is the simple organic liquid carrier used to store hydrogen compound. Biomethanol has various physicochemical properties that is why it is used as an attractive automotive fuel. Additionally, bioethanol is better to fossil fuel because it burns at low temperature (Shamsul, Kamarudin, Rahman, & Kofli, 2014). Methanol generation by the use of lignocellulosic waste is thought to be the most significant because of its financial and environmental advantages (Chandra, Vijay, Subbarao, & Khura, 2011). Various agricultural remains such as rice bran, vegetable leaves, banana peel and biomass are utilized for the generation of biomethanol (Arteaga-Perez, Gomez-Capiro, Karelovic, & Jimenez, 2016; Anitha, Kamarudin, Shamsul, & Kofli, 2015; Nakagawa et al., 2007).

5.6  Challenges of Algal Fuel Commercialization There are various factors such as increased growth rates, economical growing densities and increased oil filling that have been considered as cause of investment of capital to make biofuel from algae. However, there are various difficulties such as how and where to cultivate algae, oil extraction and fuel processing improvement and reduction of CO2 that are required to overcome and which ultimately make algae as economically feasible platform. The production of algae as biofuel becomes the major challenge that includes isolation of strain, utilization and sourcing of nutrients, management of production, reaping, development of co-product, mining and refining of fuel and utilization of remaining biomass. If the manufacturing

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practice will be improved, this may ultimately make an impact on the production of algae production. The amendments include resourceful strategies aimed at nutrient movement and light exposure (Lehr & Posten, 2009; Christ, 2007). Concisely, either to design photobioreactors or to develop species which can grow significantly in affordable cost in open environment is a major challenge for engineers as well as biologist. Christ (2007) stated that photobioreactors have benefits over open environment where axenic culture and growth in controlled environment can be maintained effortlessly that ultimately provides high productivity. However, implied systems are confronted by productivities in gas exchange and needed for additional cooling. This is still unclear that photobioreactors may prove cost competitive with open environment despite having recompenses of decreased contagion and high productivity.

5.7  Improved Oil Extraction Another challenge that also depends upon the engineers is oil extraction. Krichnavaruk, Shotipruk, Goto, and Pavasant (2008) revealed that oil press, extraction of hexane and removal of CO2 fluid are the main tactics of oil extraction from algae. These practices are very expensive; however, it has been successfully demonstrated. Providentially, all are needed to improve by engineers. However, once the extraction was done, there is requirement of conversion of algal oil in the usable liquid fuel as the crude oil is chemically comparable to that of crude fossil fuel oil. However there is basic need of catalyst that improves the gasoline production from bio-oil (Maher & Bressler, 2007). Due to similarities, it is practically assuming that the associations among algae-producing companies and oil companies are alike, as these firms have wide capability that ultimately expands the downstream processing productivities.

5.8  Future Perspectives Nowadays there are various challenges, for example, energy security, rates of oil, resource exhaustion and changes in climatic conditions, in front of present world, and all these challenges are directly or circuitously causing various damages to the environment. Notable progress has been aggravated due to all these challenges, and there is also the production of energy and fuel that has been derived from biomass. Consequently, it was expected that biofuels are the most expensive components and ameliorate various problems in a viable way. The reasonable alternatives for the decrease of CO2 release are the biofuels in the transport area. Moreover, locally available plants can be used for the production of biofuels. Nowadays, much attention has been given to biofuels, and it has been considered that biofuels are capable to defeat the energy crisis worldwide. The use of algae for the production of biofuels

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gives a major benefit that is it gives probably higher yield and no land or freshwater resources are required to grow algae (Daroch, Geng, & Wang, 2013). Recently, many researchers are engaged globally for recovering the biofuel generation. Certainly, the fast-developing and fast-moving industry is the biofuel industry, and for the generation of biofuel, there have main research processes in technology being made, and a higher understanding about the generation of biofuels has been attained too. But the complete replacement of biofuels with fossil fuels cannot be done, and several steps of engineering and biology are still needed for the effective generation of biofuels at industrial range. Moreover, knowledge about the generation of biofuels that how biofuels will affect the future changes in climate is very important in order that a sustainable biofuel financial system can be attained. Therefore, in the future biofuels will surely replace fossil fuels and will be a most important provider of energy in a sustainable manner with the ability to enhance the supply safety, and the quantity of release of vehicles will definitely decrease by biofuels (Daroch et al., 2013).

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Chapter 6

Optimizations on Steps Involved on Biofuel Obtainment and their Validation

Abstract  The biodiesel obtainment through transesterification of fat and oils from animal or vegetable sources can be optimized to allow quality and quantity enhancements, important to attend the increasing demand for this eco-friendly fuel. World population is expected to rise up to nine billion people by 2050 increasing energy demand preferentially in a way that avoids also environmental damage. Bio-based solutions to reduce greenhouse gas emission, fossil fuel dependency and waste production and to improve security regarding energy are necessary, being biofuel a strategic alternative. When it comes to biofuel obtainment, however, a higher energy return of investment is aimed once they still cannot compete economically with fossil fuels. However, various studies have been developed to try to change this scenario in the future through improvements on production efficiency. These improvements can be performed before (optimizations on raw material) or during manufacturing process of converting oil/fat into biodiesel. This chapter will be dedicated to discuss the optimization of biofuel obtainment and also discuss validation methods for improvements on economically feasible biorefinery and extraction strategies. Keywords  Biofuel · Optimization · Environmental friendly · Production efficiency · Extraction

6.1  Optimizing Raw Material for Biodiesel Production A large array of raw material for biodiesel production is available, such as edible plant material (e.g. wood to synthesize first-generation biofuel), non-food vegetable like feed stocks (e.g. sugarcane to generate second-generation biofuel), macroalgae, microalgae, bacteria, yeasts (employed on third-generation biofuel’s synthesis) and CO2 directly used through phototrophic mode mainly through genetically modified organisms (source of fourth-generation biofuel) (Fig. 6.1) (Gray et al., 2006; Hahn-­ Hägerdal et al., 2006; Jouzani et al., 2018; Ganesan et al., 2020).

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4_6

107

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6  Optimizations on Steps Involved on Biofuel Obtainment and their Validation

Fig. 6.1  Biofuel and its generations

So, raw material for biodiesel production can be optimized (Table 6.1) in order to offer better rate on fuel obtainment, for example, through culture conditions optimization and/or genetic engineering. Researchers have been trying, for example, to obtain higher biomass and energy yields by enhancing the efficiency of photo-­ conversion (Ketzer et al., 2018). Third-generation biofuel generated from algae, for example, can have its production optimized by adjustments on illumination, temperature, pH, availability of dissolved nutrients and CO2. The stress-tolerant microalga Parachlorella sp. BX1.5, for example, can generate improved quality oil for biodiesel production when cultivated at higher CO2 concentration (2%) when compared to a 0.04% of CO2 condition. At optimized carbon dioxide supply, cells presented different triacylglycerol (TAG) production depending on growth medium composition. BG11 medium was assayed, as same as its version without NaNO3 (nitrogen-deficient condition) and its version without K2HPO4 (phosphorus-deficient condition); TAG accumulation was obtained in higher concentration at a phosphorus-deficient condition, followed by nitrogen-deficient condition. BG11 medium offered the lowest intercellular oil production. The same order was observed for amount of fatty acyl methyl esters (FAMEs) in dry-cell weight (Sasaki et  al., 2020). To evaluate light intensity and NaNO3 concentration influence on biodiesel production from raw material derived from Amazonian cyanobacteria strains Synechocystis sp. CACIAM05, Microcystis aeruginosa CACIAM08, Pantanalinema rosaneae CACIAM18 and Limnothrix sp. CACIAM25, factorial planning (22) with central points was applied considering the variables independent. Low level of NaNO3 optimized all strains production parameters. When it comes to luminous intensity, CACIAM05 and CACIAM25 produced better fatty acid raw material at low luminous intensity, whereas CACIAM08 and CACIAM18 worked better at high luminous intensity (Aboim et al., 2019).

6.1  Optimizing Raw Material for Biodiesel Production

109

Table 6.1  Examples mentioned in this chapter of raw material optimization for biofuel obtainment Species involved Microalga Parachlorella sp. BX1.5

Optimization Increase in CO2 concentration to 2% and use of BG11 medium in a phosphorus-­ deficient formulation Low level of NaNO3 at high luminous intensity

(Aboim et al., 2019)

Low level of NaNO3 at low luminous intensity

(Aboim et al., 2019) (Wang et al., 2016)

Jatropha curcas

CRISPR/Cas-induced frameshift mutation on nitrate reductase gene to induce nitrogen deficiency Metabolic engineering for terpene synthase overexpression combined with a miRNA-based repression of competing pathways Introduction of synthetic metabolic pathways to increase hydrocarbon accumulation Introduction of synthetic metabolic pathways to increase hydrocarbon accumulation Genetic engineering involving OsSUS3 gene Genetic manipulation involving the gene of the cellulolytic enzyme endo-β-1,4-­ glucanase from Trichoderma reesei Metabolic engineering using the genes from diacylglycerol acyltransferase 1 and also WRI1 and OLEOSIN Genetic manipulation using the genes of diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase Genetic manipulation using WRI1 gene or hemoglobin gene Genetic manipulation using gene for auxin response factor 19 from J. curcas Genetic manipulation using maize’s leafy cotyledon1 gene β-Ketoacyl-ACP synthase II’s gene silencing Casbene synthase gene silencing

Brassica napus

Fatty acid desaturase 2 gene silencing

Camelina sativa

Induced mutation on fatty acid elongase 1’s gene

Cyanobacteria strains Microcystis aeruginosa CACIAM08 and Pantanalinema rosaneae CACIAM18 Cyanobacteria strains Synechocystis sp. CACIAM05 and Limnothrix sp. CACIAM25 Microalgae Nannochloropsis oceanica Microalga Chlamydomonas reinhardtii

Eukaryotic algae Chlamydomonas reinhardtii Prokaryotic cyanobacteria Synechocystis sp. PCC 6803 Oryza sativa Oryza sativa

Solanum tuberosum

Camelina sativa

Lepidium campestre Arabidopsis thaliana and Jatropha curcas Arabidopsis thaliana and Camelina sativa Camelina sativa

Reference (Sasaki et al., 2020)

(Wichmann et al., 2018)

(Yunus et al., 2018) (Yunus et al., 2018) (Fan et al., 2020) (Li et al., 2019) (Liu et al., 2017) (Chhikara et al., 2017) (Ivarson et al., 2017) (Sun et al., 2017) (Zhu et al., 2018) (Kim et al., 2015) (Li et al., 2016) (Okuzaki et al., 2018) (Ozseyhan et al., 2018)

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Biofuel precursors’ production can also be enhanced by genetic engineering. Nannochloropsis spp. can accumulate large amounts of TAG (a raw material for biofuel production) under nitrogen deficiency condition (You et  al., 2020). So, through CRISPR/Cas methodology, scientists have developed a methodology to induce a frameshift mutation on nitrate reductase gene from the microalgae Nannochloropsis oceanica to make it like permanently in a nitrogen deficiency situation, failing to grow under NaNO3 and proving to be an interesting option for scalable oil production (Wang et al., 2016). Protein and metabolic engineering are also important strategies to aim to improve biodiesel production. Metabolic engineering approach involving serial enzyme loading for terpene synthase overexpression, combined with a miRNA-based repression of competing pathways, allowed the enhanced production of sesquiterpene biodiesel precursor (E)-α-bisabolene by engineered green microalga Chlamydomonas reinhardtii (Wichmann et al., 2018). Introduction of synthetic metabolic pathways increased hydrocarbon accumulation at eightfold in Chlamydomonas reinhardtii (eukaryotic algae) and at 19-fold in Synechocystis sp. PCC 6803 (prokaryotic cyanobacteria) (Yunus et al., 2018). The prokaryotic cyanobacteria could also significantly improve isobutanol production (2.4 times) due to protein engineering of α-ketoisovalerate decarboxylase (by combining the modifications V461I/S286T) (Miao et al., 2018). It is also possible to improve biofuel obtainment by favouring bio-based raw material survival and biomass production; for example, if it is plant-based, improving its resistance against pests, drought and pollutants enhances the amount of plant available to be used to produce fuel (Kraic et al., 2018). However, more efficient strategies aim to facilitate pretreatment step (that can be physical, chemical, physio-­ chemical or biological (Rocha-Meneses et al., 2020)) especially when the raw material is plant-based and is necessary to deal with cellulose disruption on cell wall. When a pretreatment is necessary, it is not only a crucial step to determine the success of the process, but it also largely influences the process’ costs (Haghighi Mood et al., 2013). Strategies to remove hemicellulose and lignin, improve hydrolysis and diminish cellulose crystallinity are welcome (Barsanti & Gualtieri, 2018; Ketzer et al., 2018). An interesting alternative to pretreatment and to optimize the process is to genetically engineer plant species to reduce the recalcitrance lignocellulose offer in biofuel generation, trying to eliminate or reduce at maximum rate the need of pretreatment (Rocha-Meneses et al., 2020). This can be done through transgenes or induced mutations (Vermerris & Abril, 2015). However, a special attention must be dedicated to these strategies in order to avoid reducing also the vegetal’s response to stress. A successful strategy involved OsSUS3 gene (coding for sucrose synthase) to produce transgenic rice with improved biotic stress response and enhanced biomass saccharification, consequently offering increased bioethanol production (Fan et al., 2020). The overexpressing of fungal (Trichoderma reesei) cellulolytic enzyme endo-β-1,4-glucanase to be deposited into plant cell walls resulted in increased biomass porosity in transgenic rice plants being also an option to optimize biofuel generation (Li et al., 2019).

6.1  Optimizing Raw Material for Biodiesel Production

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Influencing on the expression of enzymes related to lipid metabolism is also a viable strategy. There are strategies, for example, that successfully improved oil content. Diacylglycerol acyltransferase 1 (that promotes the conversion of diacylglycerol into triacylglycerol) gene as same as WRI1 and OLEOSIN was used to promote metabolic engineering in Potato, increasing at 100-fold TAG generation (Liu et al., 2017). Combined transgenesis involving diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase also induced an increase in oil production on engineered Camelina sativa (Chhikara et al., 2017). Oil content was successfully increased in Lepidium campestre overexpressing Arabidopsis thaliana WRI1 or hemoglobin gene from A. thaliana or Beta vulgaris (Ivarson et al., 2017). There are also strategies to obtain more oil from plant seeds through transgenesis aiming to increase seed’s size or influence on embryo’s development. By promoting overexpression of Jatropha curcas’ gene for auxin response factor 19 on Arabidopsis thaliana and J. curcas, seed size could be significantly enhanced (Sun et al., 2017). Oil content could be enhanced in Arabidopsis thaliana and Camelina sativa seeds as a consequence of maize leafy cotyledon1 gene insertion; the gene is responsible for not only enhancing the expression of genes related to fatty acid biosynthesis but also influencing on embryo’s development (Zhu et al., 2018). It is also interesting to address strategies to optimize the obtainment of biofuel by promoting gene suppression such as RNA interference (RNAi) and CRISPR/Cas. RNA interference technology uses designed interference RNA to promote the gene silencing phenomenon (Qi et al., 2019). Camelina sativa’s oil could be successfully increased in jet fuel-type fatty acids’ content due to β-ketoacyl-ACP synthase II’s gene silencing through RNAi (Kim et al., 2015). By using an iRNA to silence casbene synthase genes from Jatropha curcas L., it was possible to optimize the seed oil reducing phorbol esters content (Li et al., 2016). CRISPR (clustered regularly interspaced short palindromic repeats)/Cas technology can also serve this purpose. The system involves a nuclease, commonly Cas9, guided by a gRNA (designed to pair to the target DNA sequence) to the place in which genome edition is desired. The enzyme is able to cause a double-strand break in DNA that is repaired by non-­ homologous end-joining: an error-prone mechanism that results in small insertions or deletions (indels) near to cleavage site. This may cause gene suppression by inducing premature termination codons or frameshift mutations (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Sander & Joung, 2014; Yin et al., 2017; Basharat et al., 2018). The silencing of the fatty acid desaturase 2 gene from Brassica napus cv. Westar was promoted by using two guide RNAs; this strategy increased the content of oleic acid in plant’s seeds (Okuzaki et al., 2018). Changes in fatty acid content could also be performed in Camelina sativa through CRISPR/Cas technology. By inducing mutation on fatty acid elongase 1’s gene, it was possible to reduce the amount of C20–C24 very long-chain fatty acids on the seeds increasing the desirable generation of C18 unsaturated fatty acids (Ozseyhan et al., 2018).

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6.2  Optimizing Pretreatment for Biodiesel Production There are three main routes to obtain biofuel: thermochemical, hydrothermal and biochemical ones, being the last one more feasible, eco-friendly and cost-effective – consequently receiving more attention on this chapter. The direct thermochemical route converts biomass into heat and electricity, and the indirect allows the generation of bio-oils and syngas (that can be converted into biofuels). Hydrothermal uses supercritical water in the process, and biochemical commonly includes steps of pretreatment, hydrolysis and fermentation (Zabed et al., 2019). Focusing on biochemical route, pretreatment optimization (Table  6.2) can involve, for example, physical approaches such as milling to reduce particle size. Attrition mill made possible facile and cost-effective generation of biofuel production from corn stover particles enhancing enzymatic hydrolysis and turning Table 6.2  Examples mentioned in this chapter of pretreatment optimization for biofuel obtainment Species involved Zea mays Zea mays

Optimization Attrition mill Vibro-ball milling

Salix gracilistyla Helianthus annuus Algae from Scenedesmus genre Chlorella sp. ABC-001 Organisms from genre Saccharum Dunaliella salina

Extrusion Microwave Microwave digester-assisted solvent extraction method Acid hydrolysis Alkaline pretreatment

Elaeis guineensis Zea mays Triticum aestivum Pinus sylvestris Arachis hypogaea Salix viminalis Oryza sativa Zea mays Organisms from the genre Picea Organisms from the genres Betula and Picea Hordeum vulgare

Ozone-rich microbubbles

Reference (Gu et al., 2019) (Monlau et al., 2019) (Han et al., 2020) (Athar et al., 2020) (Mamo & Mekonnen, 2020) (Seon et al., 2020) (Zhang et al., 2019)

(Kamaroddin et al., 2020) Ozonolysis (Omar & Amin, 2016) Superacid SO4H-functionalized ionic liquid (Hui et al., 2019) Acidic 1-ethyl-3-methylimidazolium (Lopes et al., 2018) hydrogen sulphate ionic liquid IonoSolv pretreatment with protic (Gschwend et al., hydrogen sulphate ionic liquids 2019) Hydrothermal pretreatment using (Ge et al., 2020) high-pressure CO2 Steam explosion (Devin et al., 2019) Steam explosion produced (Steinbach et al., 2019) Steam explosion (Wang et al., 2020) Two-stage steam explosion with (Seidel et al., 2019) 2-naphtholin Organosolv-steam explosion fractionation (Matsakas et al., 2020) Nitrogen explosion (Raud et al., 2019)

6.2  Optimizing Pretreatment for Biodiesel Production

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unnecessary steps of washing, detoxification and solvent recovery (Gu et al., 2019). Vibro-ball milling could significantly increase dry solid digestate cellulose and hemicelluloses hydrolysis from a mixture composed of 42  wt% of corn silage, 5 wt% of cow manure and 53 wt% of cow sewage, optimizing bioethanol and methane production (Monlau et  al., 2019). Extrusion can also offer enhancements on biofuel obtainment: Salix gracilistyla Miq. that underwent extrusion at 160 °C and 5 rpm using 1-ethyl-3-methylimidazolium acetate under solid loading of 15% (Han et al., 2020). Pretreating biofuel’s raw material using microwave is also an interesting strategy. It allows the reduction on reaction time of acid-catalysed transesterification of refined sunflower oil, allowing the achievement of conversion rate next to 100% at 76  °C in only 30  min (Athar et  al., 2020). Algal oil from Scenedesmus could be used to efficiently generate biofuel (yield of 92%) through microwave digester-assisted solvent extraction method at 60 °C (Mamo & Mekonnen, 2020). Chemical pretreatment approaches are also options to improve biofuel production. The acid hydrolysis using H2SO4, for example, offered better results than the alkaline hydrolysis (using NaOH) offering a higher ethanol/g sugars rate when Chlorella sp. ABC-001 was used as raw material for biofuel obtainment (Seon et al., 2020). However, sometimes alkaline pretreatment is more suitable to favour enzymatic digestion; for sugarcane bagasse an alkaline 6.25% hydrogen peroxide pretreatment at 160 °C for 60 min in the presence of Tween 80 proved to be an efficient strategy (Zhang et al., 2019). Ozonolysis is also an option to optimize energy usage and consequently costs when obtaining biofuel. Ozone-rich microbubbles offered an increased lipid extraction from Dunaliella salina slurry at 60 ̊C requiring less energy than conventional methods (Kamaroddin et al., 2020). When it comes to producing second-generation biofuel, this technique is also useful; ozonolysis promotes oil palm frond’s lignin-efficient degradation and increases total reducing sugar recovery (Omar & Amin, 2016). Ionic liquids can also be prepared to be used in pretreatment step to enhance hydrolysis process, consequently contributing to biofuel production. A superacid SO4H-functionalized ionic liquid proved to be able to enhance xylose dehydration and hydrolysis presenting positive correlation between this capacity and the acid strength when acting over corn cob (Hui et al., 2019). Hemicellulose fraction of wheat straw could be efficiently hydrolysed by an aqueous solution of the acidic 1-ethyl-3-methylimidazolium hydrogen sulphate ionic liquid, also overcoming challenges related to the reuse and recycling of these liquids, recovering a high rate of pentoses (Lopes et al., 2018). To deal with cellulose from plant raw material, highly efficient solvents could be developed by coupling diallylimidazolium methoxyacetate with dimethyl sulfoxide, N,N-­dimethylformamide and N,N-dimethylacetamide (there are polar aprotic solvents) (Xu et al., 2019). IonoSolv pretreatment with protic hydrogen sulphate ionic liquids (such as N,N-dimethylbutylammonium hydrogen sulphate) proved to be a low-cost, inexpensive pretreatment of Pinus sylvestris allowing glucose release in an amount which equated to a projected glucose release (Gschwend et al., 2019). There are also physicochemical pretreatment approaches to improve biofuel production. Hydrothermal pretreatment technology, for example, does not require toxic chemical inputs; it uses liquid water or steam and heat to contribute to

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second-­generation biofuel production dealing well with lignocellulose biomass facilitating its processing (Ruiz et  al., 2020). Hydrothermal pretreatment using high-pressure CO2 (190 °C at 60 bar) offered an acid environment and increased porosity of raw material which made possible the enhancement of enzymatic hydrolysis of peanut shells’ biomass by reducing hemicellulose content facilitating enzyme accessibility to the cellulose (Ge et al., 2020). Steam explosion is also a viable pretreatment option that consists in heating biomass using pressurized steam followed by sudden pressure release. It could not only favour obtainment of bioethanol from woody biomass (at 220 °C after a 2% sulphuric acid presoaking) but also the decontamination from trace elements such as Zn and Mn (Devin et  al., 2019). Rice straw that has suffered pretreatment through steam explosion produced an increased amount of biomethane by anaerobic digestion (Steinbach et al., 2019). This eco-friendly approach has also favoured hydrolysis of corn lignocellulosic biomass (Wang et al., 2020). Adaptations on standard protocols can be made in order to enhance success rate. When it comes to softwood, it is necessary to prevent lignin repolymerization, and addition of 2-naphtholin in a two-stage steam explosion pretreatment revealed to be a good alternative to optimize biofuel obtainment (Seidel et al., 2019). A novel method of hybrid organosolv-steam explosion fractionation of birch and spruce woodchips allowed high methane production rates to be obtained (Matsakas et al., 2020). Industrial vinegar residue from solid-state fermentation is also a viable raw material to biofuel production, and steam explosion pretreatment could increase by 13-fold enzymatic hydrolysis rate (Xia et al., 2020). Pretreatment based on explosion can also be performed with nitrogen; nitrogen explosion, although was not able to surpass steam explosion in removing hemicelluloses from biomass from second-­generation biofuel’s production, proved to be of similar efficiency in bioethanol generation and more effective when the biofuel production process involves anaerobic digestion (Raud et al., 2019). Supercritical carbon dioxide (scCO2) is an abundant and renewable solvent but is lethal to a lot of microbes; however tolerant strains such as Bacillus megaterium can efficiently produce biofuel in its presence, and to optimize the process, genetic modification can allow the generation of organisms in which scCO2 induces the expression of genes involved in isobutanol production from 2-ketoisovalerate (Boock et al., 2019). Biological strategies of pretreatment also deserve to receive attention specially when it comes to obtaining second- and third-generation biofuel. It can be performed using pure cultures of fungi, bacteria, only their enzymes, consortia (mix of microorganisms capable of, synergistically, promoting biomass’ pretreatment) or ensiling (addition of mainly lactic acid bacteria to biomass ensilages to promote acidification favouring polysaccharides processing) approaches being the use of fungi the most time-consuming strategy among them. When pretreating lignocellulosic biomass, the increase in efficiency of biofuel generation was optimized in a highest way (more than 400%) through enzymatic pretreatment (Zabed et al., 2019). Not only optimization of pretreatment step can be performed, especially when raw material involves lignocellulosic biomass. To produce bioethanol, for example, steps from manufacturing process performed after pretreatment, such as hydrolysis (converting the polymer into glucose monomers), fermentation and distillation, are also possible to be modified to obtain better results (Rocha-Meneses et al., 2020).

6.3  Optimizing Biofuel Manufacturing Process

115

6.3  Optimizing Biofuel Manufacturing Process The optimization of biodiesel manufacturing process can involve choosing the best catalyst (to perform an acidic, basic or enzymatic reaction) and its better concentration, process’ temperature, agitation rate, alcohol/oil molar ratio and time of reaction, among other parameters (Murthy et al., 2020). The presence of non-ionic surfactants, for example, can favour the process of enzymatic conversion of lignocelluloses into fermentable sugars when raw material for biofuel production is lignocellulosic biomass. It may reduce enzyme loading making the process more cost-effective in an eco-friendly and relatively nontoxic way (Al-Azkawi et al., 2020). Ionic liquids are useful not only as a pretreatment, but can also be used during biofuel production step. Especially non-volatile hydrophobic ionic liquids (ILs), for example, are an interesting option as solvents to biofuel synthesis being easier to recycle and safer to handle (Taher et al., 2019). The use of ILs in biofuel generation is already a reality; transesterification of soybean oil can be performed to obtain fatty acid methyl esters (soybean-oil-to-methanol ratio – 1:2 v/v) using ionic liquid as catalyst (8.0%w/v) at constant reflux for 4 h at 300 rpm (Panchal et al., 2020). Levulinic acid production from bamboo biomass could be performed at a satisfactory rate by using dicationic ionic liquids, containing 1,4-bis(3methylimidazolium-­1-yl) butane ([C4(Mim)2]) cation with counter anions [(2HSO4) (H2SO4)4] at 110  °C for 60  min (Khan et  al., 2018). Phosphomolybdenum-based sulphonated ionic liquid-­ functionalized MIL-100(Fe) metal-organic framework could promote in an efficient way the transesterification of soybean oil and esterification of free fatty acids with long-term catalytic durability at methanol/oil molar ratio of 30:1 and 120 °C (Xie & Wan, 2019). It is also possible to enhance biofuel production by using genetically modified organisms to present enhanced performance during fermentation step. Escherichia coli, for example, can produce ethanol but in a low yield as the fermentation process converts more efficiently sugars to organic acids (Ingram et al., 1998). In order to optimize ethanol production, E. coli was engineered to overexpress alcohol dehydrogenase and pyruvate decarboxylate from Zymomonas mobilis (Wang et al., 2008; Tomás-Pejó et al., 2008). Clostridium acetobutylicum ATCC 824 (a butyrate minus mutant strain) after receiving plasmids containing synthetic isopropanol operons could at optimized pH offer a productivity of a biofuel composed of an isopropanol/ butanol/ethanol mixture never reached before (Dusséaux et al., 2013). 2,3-Butanediol could efficiently be produced by genetically engineered cyanobacteria Synechococcus elongatus PCC7942 from CO2 (Oliver et al., 2013). Clostridial n-butanol pathway was inserted through genetic manipulation into Thermoanaerobacterium saccharolyticum JW/SL-YS485 to allow n-butanol production from xylose offering more than 20% of the theoretical maximum yield (Bhandiwad et al., 2014). Engineered Klebsiella pneumoniae could produce 1-butanol by expressing the genes ter-bdhB-­ bdhA and kivd and suffering suppression of native fermentation pathways by antisense RNA strategy (Wang et al., 2014). The hyperthermophile Pyrococcus furiosus

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could be successfully engineered by using the genes thl, hbd, crt, ter, bad and bdh to allow butanol production reaching 70 mg/L after 48 h (Keller et al., 2015). In order to reduce acetone production by Clostridium acetobutylicum and enhance alcohol generation, the introduction of a secondary alcohol dehydrogenase was an interesting strategy; the gene for the enzyme was obtained from Clostridium beijerinckii NRRL B593 and made the target species capable to convert 50% of acetone generated into isopropanol (Bankar et al., 2015). Fungi species can also be engineered to biofuel-efficient production. Cellobiose, for example, is a sugar present in plant biomass that is naturally poorly fermented by microbes; however, the cellulolytic thermophilic filamentous fungus Myceliophthora thermophila could be metabolically engineered to ferment this sugar into ethanol. The insertion of alcohol dehydrogenase’s gene from Saccharomyces cerevisiae and overexpression of the glucose transporter GLT-1 from Neurospora crassa converted the fungus into a promising platform for bioethanol production (Li et al., 2020). Beta-glucosidase is an important enzyme when it comes to fungi and biofuel production specially because filamentous fungi are known to be a good producer of it. The genes coding this enzyme are interesting in order to genetically modify fungi species to offer useful organism with cellulolytic potential (Singhania et al., 2017): promising alternative to the use of Trichoderma reesei. Aiming to reduce the cost for biomass-based biofuel, Penicillium oxalicum was engineered to overexpress β-glucosidases, enhancing organisms’ cellulolytic ability (Yao et al., 2016). Algae genetic modification is also possible to improve biofuel production. In the USA, the first field study using genetically engineered algae approved by the Environmental Protection Agency was already performed. Acutodesmus dimorphus is modified to contain DNA sequence to enhance fatty acid biosynthesis. Besides being optimized to biofuel production, genetically modified organism has not disrupted ecosystem’s balance as it has not outcompeted native strains (Szyjka et al., 2017). Scenedesmus obliquus (microalgal species) that can naturally be used to generate biofuels could be metabolic engineered to enhance this capacity. Overexpression of type 2 diacylglycerol acyltransferase gene from Chlamydomonas reinhardtii enhanced lipid content nearly twofold and offered 29% more biomass (Chen et al., 2016). Although metabolic engineering of algae is a popular strategy to make them even more suitable to biofuel production, strategies to enlarge the absorbing spectrum range in photosynthesis are also interesting (Abdullaha et al., 2019).

6.4  Modelling Optimizations: Validation Modelling the fatty acid ethyl ester yield of biodiesel manufacturing process is crucial to allow the understanding on how changes in variables impact the process final result. However it is not an easy task once biological systems are unsteady and non-­ linear. It is complex to set formula to efficiently describe and predict the physical performance related to the bioprocess (Franco-Lara et al., 2006). Strategies such as response surface methodology (RSM) are widely used to perform this role; however

6.4  Modelling Optimizations: Validation

117

it is also possible to obtain success through modelling strategies based on artificial intelligence such as artificial neural network (ANN). These methodologies have the intention to provide insight on input parameters on the target output analysing not only individual inputs but also their interactive effect. Modelling optimizations not based on artificial intelligence tend to offer more limited resources for optimization. One-variable-at-a-time approach (OVAT), for example, does not consider interactive effect of input parameters and may drastically fail to set optimized conditions. However there are studies that apply this method to set conditions for a target input parameter. For example, to generate biodiesel from waste cooking oil through microwave-assisted intensification, researchers at first estimated best conditions to each input parameter (reaction temperature, catalyst loading and methanol-to-waste cooking oil molar ratio) using OFAT, and then RSM was applied to optimize process variables (Gupta & Rathod, 2018). Factorial design of experiment (DOE) can also be applied to model and optimize biofuel obtainment; however, it is a time-consuming approach specially if there is a high number of input factors to be considered. To pretreat lignocellulose from elephant grass leaves to ethanol production, 2ν5–1 fractional factorial design was applied allowing optimization of temperature, time, number of treatments, concentration and pH (Rezende et al., 2018). RSM, on its turn, is useful to provide analysis and validation only in the range data was collected. It is not suitable for interpolation or extrapolation (Murthy et  al., 2020). RSM involves the use of a set of statistical methods generally applied to optimize situations in which there are several factors influencing a performance characteristic/response of interest or lead to the meeting of a given set of specifications (Myers & Montgomery, 1995). However, inside the interval of collected data, there are research groups that successfully apply this method, while others consider that it may disregard parameters considered “less important” that could possess interactive effects on bioprocess’ output (Desai et al., 2008). A full two-level factorial design 22 and amplified to RSM was applied to study optimization in the transesterification of Jatropha oil to generate the biofuel n-butanol. The best condition ended up being set as 75 °C at 10 minutes and 1.2 wt% of catalyst percent (Sánchez et al., 2015). Transesterification of olive and palm oils by using a mixture of immobilized lipases was optimized by simplex-centroid design (triangular surface analyses): for olive oil 95% conversion efficiency was achieved in 18 h of reaction using 29.0% of Lipozyme TL IM, 12.5% of Lipozyme RM IM and 58.5% of Novozym 435 and for palm oil 80% conversion efficiency in the same time using 52.5% of Lipozyme TL IM and 47.5% of Lipozyme RM IM (Poppe et al., 2015). Palm oil transesterification catalysed by mesoporous K/SBA 15 was optimized through RSM combined to central composite design (CCD) and at 70 °C, for 5 h, 11.6 mol/mol of methanol/oil ratio and 3.91 wt.% for the catalyst to successfully achieve 93% of biodiesel yield (Abdullah et  al., 2009). Transesterification of palm oil using as catalyst sulphated zirconia alumina could be optimized by RSM/CCD; a product yield of 83,3% was obtained by methanol-to-­ palm oil ratio of 8, 6 wt.% of catalyst for 3 hours at 127 °C (Yee et al., 2010). The transesterification of oil from Jatropha curcas catalysed by CaO–MgO was successfully optimized through RSM/CCD to allow 93.55% biodiesel yield (Lee et  al.,

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2011). RSM also allowed optimization of biohydrogen production by Rhodobacter capsulatus allowing the obtainment of a yield 85% higher than the one previously achieved (Ghosh et al., 2012). However, recently analysis methods based on artificial neural network are more commonly applied due to the superiority they are able to offer in the optimization process when it comes to predictive capability. As examples of tools based on artificial intelligence, it is possible to highlight ant algorithm, fuzzy logic, particle swarm optimization, artificial neural network and genetic algorithm (Garlapati & Banerjee, 2010; Sewsynker-Sukai et al., 2017). There are studies that use artificial neural network and other different methodology not based on artificial intelligence such as RSM, for example, the one from Sivamani and coworkers that set conditions to use Simarouba glauca as raw material for biofuel production by applying oil-to-­ alcohol ratio of 1:6.22 at 67.25 °C for 20 hours (Sivamani et al., 2017). Adaptive neuro-fuzzy inference system (ANFIS), for example, proved to be superior to RSM to optimize biodiesel production from Thevetia peruviana seed oil. It offered smaller: standard error of prediction and mean absolute percentage deviation (Ighose et al., 2017). To generate biodiesel from Sesamum indicum L. oil, the catalyst barium hydroxide (1.79 wt%) was ultrasonicated at 31.92 °C for 40.30 min, using methanol-to-oil molar ratio 6.69:1, to obtain 98.6% of biofuel yield. To elucidate this optimal condition, artificial neural network (ANN) proved to possess better prediction capability than RSM once the first presented lower root mean square error, standard error of prediction and relative percent deviation and higher correlation coefficient (Sarve et al., 2015). The optimization of biofuel production from sunflower oil using calcium oxide as a catalyst was performed by applying RSM and ANN. ANN also in this situation proved to be the better approach, and experimental value for biofuel yield was similar to the theoretical value (Avramovic et al., 2015). ANN associated with particle swarm algorithm could more precisely optimize parameters for biodiesel production from sesame oil than RSM, although both methods offered results in good agreement with actual results (Soltani et al., 2020). ANN is recently the preferable method once it can capture non-linear relationships among parameters from biological processes such as microbial fermentation allowing high-quality analysis (Sewsynker-Sukai et al., 2017). ANN proved to be powerful to optimize the production of bioethanol: from sugarcane using Saccharomyces cerevisiae (Ahmadian-Moghadam et al., 2013); from intermediates and by-products of sugar beet processing using the same yeast (Grahovac et  al., 2016); and from different types of biomasses considering composition regarding elemental analysis, proximate analysis and operating parameters (Safarian et  al., 2021). When it comes to biodiesel, the optimization of its obtainment could be performed using a neuro-fuzzy estimator to the process involving the use of Synechococcus nidulans (Furlong et  al., 2013); ANN surpassed RSM and could offer more precise optimization of this biofuel’s production (60 °C for 55 minutes and oil-to-methanol ratio 16 (v/v)) using Nannochloropsis salina and calcium oxide as a solid nanocatalyst (3% (w/v)) (Raj et al., 2021). Biogas generation using microbial fuel cell could be optimized by ANN; processing just small amounts of current, the overall performance of the bioreactor could be predicted regarding methane

References

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production (Harper et al., 2013). In order to develop more effective strategies for biogas production, integrated biogas-wastewater treatment plant was modelled through ANN successfully optimizing seven plant parameters (Sakiewicza et  al., 2020). Input variables such as substrate and inoculum type, pH, temperature and substrate concentration were optimized regarding cumulative volume of biohydrogen generated per gram substrate in a successful way using ANN; the method was capable of considering the non-linear relationship between the inputs and biohydrogen yield (Sewsynker & Kana, 2016). Biohydrogen’s production by microbial electrolysis cell after being modelled by ANN could be optimized in a more sensitive way than by ANFIS, and it was possible to understand that cathodic H2 recovery (rcat) is the most effective factor for H2 production rate optimization (Hosseinzadeh et al., 2020). It is also possible to associate methods. For example, RSM and ANN were combined to analyse in a precise and cost-effective way hydrogen production performance. ANN allowed to generate data matrix for statistical analysis through RSM.  The ANNs-RSM model was robust to be applied to non-linear noisy processes such as dark hydrogen fermentation (Wang et al., 2021).

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Wang, Z., Chen, M., Xu, Y., Li, S., Lu, W., Ping, S., Zhang, W., & Lin, M. (2008). An ethanoltolerant recombinant Escherichia coli expressing Zymomonas mobilis pdc and adhB genes for enhanced ethanol production from xylose. Biotechnology Letters, 30, 657–663. Wichmann, J., Baier, T., Wentnagel, E., Lauersen, K.  J., & Kruse, O. (2018). Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metabolic Engineering, 45, 211–222. Xia, M., Peng, M., Xue, D., Cheng, Y., Li, C., Wang, D., Lu, K., Zheng, Y., Xia, T., Song, J., & Wang, M. (2020). Development of optimal steam explosion pretreatment and highly effective cell factory for bioconversion of grain vinegar residue to butanol. Biotechnology for Biofuels, 13, 111. Xie, W., & Wan, F. (2019). Biodiesel production from acidic oils using polyoxometalate-based sulfonated ionic liquids functionalized metal–organic frameworks. Catalysis Letters, 149, 2916–2929. Xu, A., Chen, L., Wang, Y., Liu, R., & Niu, W. (2019). Development of Diallylimidazolium Methoxyacetate/DMSO (DMF/DMA) solvents for improving cellulose dissolution and fabricating porous material. Polymers, 11, 845. Yao, G., Wu, R., Qinbiao Kan, Q., Gao, L., Liu, M., Yang, P., Du, J., Li, Z., & Qu, Y. (2016). Production of a high- efficiency cellulase complex via β- glucosidase engineering in Penicillium oxalicum. Biotechnology for Biofuels, 9, 78. Yee, K. F., Kansedo, J., & Lee, K. T. (2010). Biodiesel production from palm oil via heterogeneous transesterification: Optimization study. Chemical Engineering Communications, 197(12), 1597–1611. Yin, K., Gao, C., & Qiu, J.  L. (2017). Progress and prospects in plant genome editing. Nature Plants, 3, 17107. You, W., Wei, L., Gong, Y., El Hajjami, M., Xu, J., & Poetsch, A. (2020). Integration of proteome and transcriptome refines key molecular processes underlying oil production in Nannochloropsis oceanic. Biotechnology for Biofuels, 13, 109. Yunus, I. S., Wichmann, J., Wördenweber, R., Lauersen, K. J., Kruse, O., & Jones, P. R. (2018). Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel. Metabolic Engineering, 49, 201–211. Zabed, H. M., Akter, S., Yun, J., Zhang, G., Awad, F. N., Qi, X., & Sahu, J. N. (2019). Recent advances in biological pretreatment of microalgae and lignocellulosic biomass for biofuel production. Renewable and Sustainable Energy Reviews, 105, 105–128. Zhang, H., Huang, S., Wei, W., Zhang, J., & Xie, J. (2019). Investigation of alkaline hydrogen peroxide pretreatment and Tween 80 to enhance enzymatic hydrolysis of sugarcane bagasse. Biotechnology for Biofuels, 12, 107. Zhu, Y., Xie, L., Chen, G.  Q., Lee, M.  Y., Loque, D., & Scheller, H.  V. (2018). A transgene design for enhancing oil content in Arabidopsis and Camelina seeds. Biotechnology for Biofuels, 11, 46.

Chapter 7

Economic Consideration on Biofuel and Energy Security

Abstract  The economy of algae biomass is probably affected due to the extraction and auction of natural co-products and algal oil. There can be the addition of proteins or many other purification products or an increase in these facilities if the infrastructure for the generation of algal biofuel is in place. Keywords  Economic benefits · Energy security · Algal biofuel · Proteins · Eco-friendly products

7.1  Introduction After the extraction of oil from algae, the remaining residue mainly contains proteins and carbohydrates. The use of these remaining products traditionally may comprise of anaerobic digestion for the generation of methane gas (Sialve et al., 2009), incineration for the generation of energy or, maybe utilization as feed for animals, though currently algae are not sold as feed for the animals outside the industry of aquaculture. Most of the microalgae are most appropriate for the nutrition of human beings and animals due to the high content of protein and amino acids. Spirulina is a cyanobacterium that contains 60–70% of the dry weight of total protein. It is extensively used as a food supplement for human beings, domestic animals, poultry farms, aquarium fishes and horses. In the industry of aquaculture, biofuel of algae is also an important nutrient for fishes, molluscs and shrimps. The mainly admired genera of algae are “Tetraselmis, Nannochloropsis, Isochrysis, Pavlova, Navicula, Nitzschia, Chaetoceros, Skeletonema, Phaeodactylum and Thalassiosira” (Borowitzka et al., 1997). Another most important source of nutrient is Chlorella; it causes the production of a high costly molecule, i.e. “β-1,3-glucan”. β-1,3-Glucan is a polysaccharide and documented as an immune system booster, ability to act as a free radical scavenger, and can also decrease blood lipids (Iwamoto, 2003). Due to the varied nature of microalgae, these have the ability to generate a broad range of nutrients and natural products which are useful for both humans and for animals. The most important or useful co-products are “carotenoids” and © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4_7

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“long-­chain polyunsaturated fatty acids (LCPUFAs)”. Carotenoids include many other important metabolites such as “lutein, zeaxanthin, lycopene, bixin, β-carotene and astaxanthin”. Nonetheless, mainly β-carotene and astaxanthin are commercially produced (Vilchez et al., 1997). The halophile green microalga Dunaliella salina is capable of producing β-carotene, and under stress conditions, this pigment can accumulate up to 15% of its dry weight. It is an orange colour pigment, broadly used as a natural food colour. It has strong antioxidant ability and also acts as precursor of vitamin A (Ben-Amotz, 2004). The freshwater algae, Haematococcus pluvialis, mainly produce astaxanthin. Haematococcus pluvialis can accumulate up to 5% of its dry weight as this pigment. This pigment is reddish in colour and primarily used in feed additives for colouring fishes, carps, red seabream, shrimps and chickens. Humans in their food supplements also used this pigment because it is an amazing antioxidant (Cysewski and Lorenz, 2004; Guerin et al., 2003). “Long-chain polyunsaturated fatty acids” such as “omega-3” and “omega-6” can also be produced by microalgae. These compounds are very important for human beings as well as for animals, but these are present in a very limited variety of foods (Ratledge, 2004). Crypthecodinium and Schizochytrium are the two organisms which commercially produce “docosahexaenoic acid (DHA)”. It is an “omega-3 fatty acid” produced for infant formulas and feeds for aquaculture. “Long-chain polyunsaturated fatty acids” can also be effectively synthesized by algae, but presently these are not the primary commercial source of these fatty acids. “Eicosapentaenoic acid” which is synthesized by “Nannochloropsis, Phaeodactylum and Nitzschia”, arachidonic acid which is synthesized by Porphyridium and γ-linoleic acid which is synthesized by Arthrospira all come under long-chain polyunsaturated fatty acids (Spolaore et al., 2006). Supplementary products which are also synthesized by microalgae are “phycobiliproteins” (utilized in food and dyes), extracts for cosmetics (Nannochloropsis and Dunaliella) and various other stable isotopes biomolecules useful in research (Phaeodactylum and Arthrospira) (Stolz and Obermayer, 2005; Acien Fernandez et  al., 2005). Various distinctive molecules can also be produced by microalgae which are also having commercial potential such as vitamins, toxins, antibiotics, halogenated components and polyketides. In some examples, biomolecules that increase crop protection might have pharmacognostical potential (Kothari et  al., 2012; Cardozo et al., 2007; Hallmann, 2007; Pulz and Gross, 2004). A bridge is provided by these natural products, while there is an improvement in the economics of algal biomass. Additionally, co-extraction of most of these co-products will be done with that of lipids by the use of present strategies, reducing their value as a co-product.

7.2  Economic Aspects of Biofuels The beginning of biofuel production of algae started with oil surprises in the 1970s, but recently the fast increase or growth of industries of biomass has been mainly driven by consents, financial supports, concerns about the climate change,

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emission objectives and energy security. Generally speaking, presently the rates of biofuels are higher than fossil fuels. The generation rates of ethanol fuel increased by 25% and biodiesel increased by 173% from 2004 to 2006. In 2004, the 11% corn crop generated 3.5  billion of gallons of ethyl alcohol fuel. It is expected that the demand of ethyl alcohol will be more than double in the upcoming 10  years. The newer technologies must come out of the laboratories to the commercial certainty to fulfil this requirement (Bothast, 2005). Globally, the generation of ethyl alcohol from sugar crop is approximately 61%. Presently, consumption of biodiesel for transport is less than 0.3% as compared with diesel. Duncan (2003) assessed that biodiesel is approximately US$0.28 per litre more costly than normal diesel. The feedstock which is approximately 76–81% of the total cost of operation is the main economic factor believed for input costs of the production of biodiesel. On the basis of energy, in all countries of the world except Brazil, presently ethyl alcohol is more costly than gasoline. In the USA and Europe, ethyl alcohol synthesized from corns and grains costs more than from sugarcane in Brazil. Normally, biofuels cost higher than fossil fuels, and these are used by customers only then if the cost is waged by the government or if pressure is put on the customers to use biofuels. At present, for the economic feasibility, various requirements are needed for biofuels such as financial support, tax, fuel consents and other government supports. Therefore, both government and customers are giving a considerable premium to add the usual profit of biofuels. Numerous policies such as consumption of financial assistance, consent for least amount of consumption and generate financial support such as feedstock, introducing barrier and standard of sustainability are in use for the biofuels of the USA (Gardner and Tyner, 2007). The generation of biofuels needs supplementary use of land, water and fertilizers in Asia. Supplementary fertilizers are needed to considerably enhance the production of biofuel crop. In India, Jatropha crop plantation has been endorsed for the generation of biodiesel; supplementary 15 mt of organic manure and 2.7 mt of fertilizer will be needed per year for its plantation to fulfil the target of production. For the synthesis of biodiesel and bioethanol, there is a requirement of advanced technology for the extraction of oil, transesterification and processes of fermentation to fulfil the biofuel demands. Furthermore, to achieve the biofuel production, there is a requirement of kinetic models development that includes exact parameters of regulatory network to assist the recognition of enzyme bottlenecks in metabolic pathways may be attached (Dellomonaco et al., 2010a). Because of the fastest increase in population and urbanization, energy demand is also enhanced day by day (Demibas, 2008). Various laws have already been passed in some countries that authorize biofuel production to fulfil future biofuel demands. The composition of total cost of biofuels is the cost of production of biofuels, transportation of biofuels, conversion of biofuels and labour. The decrease in the demand for petroleum products can decrease its cost too and will produce economic profit for customers (Dave et al., 2013).

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7.3  Biofuel Policies and Scenario Sorda et al. (2010) stated that the biofuels which can be obtained commercially are substantially produced from various foodstuffs such as sugarcane, corn, oil seeds and sugar beet. So, the policies supporting biofuel production have impact on the stock associated with biofuel generation. The primary reasons of the green energy production are to overcome the dependency on fossil fuels or energy security, to decrease the climate change migration (GHG emission) and to enhance the need of some agricultural commodities which suffer from production oversupply. However, these all reasons have been disapproved. For instance, energy security can be succeeded not only by supporting the utilization of biofuels but also by using alternative form of natively generated renewable energy including wind and solar energy. Also, the involvement of biofuels in the reduction of greenhouse gases has been disputed. In the present time, agricultural commodities are used to generate biofuels like ethanol are produced from corn, whereas biodiesel are prepared from palm oil. By putting more land under cultivation and by higher the commodity prices, farmers can be encouraged more intensively. This phenomenon may cause rise in CO2 level from agronomy which can ultimately balance the greenhouse gas emission reduction attained from rise in biofuel utilization. Hence, the income of farmers can be increased by enhancing the demand for food as well as non-food farming stuffs. Policies related to biofuels are increasing the pressure on agriculture charges. These policies can also weaken the environment as they promote the augmentation of agricultural regions at the rate of rainforest and wildness. Therefore, it becomes important to comprehend the policies allotted by the chief biofuel manufacturer and consumer countries. This is because their verdict can have extensive effect on global markets of bioenergy as well as agriculture stuffs. The second-generation biodiesel which is made from Jatropha has become centre point of biofuel production in India. However, obligatory blends are presently E5, there are deliberations to increase the basic to E10 and finally E20 blends as biodiesel (from Jatropha) becomes more economical. Various regions of India have implemented different strategies which supports the rising of Jatropha and exploration into biofuel generation. Andhra Pradesh, which is one of the Indian states, constituted a partnership along with the firm Reliance Industry, providing the firm about 200 ac of the land for Jatropha farming for biodiesel custom. Also, other states like Chhattisgarh, Karnataka and Rajasthan are supporting the cultivation of Jatropha plants. The state of Chhattisgarh became self-contained on energy in the year 2015 utilizing biodiesel and vending Jatropha seeds for yield. Singh and Thakur (2016) have reported that the state of Tamil Nadu has eliminated the purchase tax over the Jatropha to encourage the Jatropha cultivation and use.

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7.4  Need to Focus on Biofuels The prices and huge importing bills of the petroleum products are increasing with rise in demand of these products for the energy in different sectors such as transport, industry, agriculture and others, and the repository of the fossil fuel is also declining. The enormous utilization of fossil fuel is responsible for hazardous pollution of the environment. Therefore, there is much need of an alternative source of energy to accomplish the needs that are being considered all over the world. Use of energy crops has various possible benefits. Increased rural cost-effective development, energy protection and ecological benefits are three main advantages discussed in this section. The use of energy crops also serves as additional benefit for the habitat of wildlife. It was stated by a scientist from the “National Audubon Society” that these energy crops might be helpful for the protection of natural forest that provides a substitutional source of wood. These crops may be grown on farm or grazing land which is no more appropriate for row crops. Buhroo et al. (2018) stated that utilization of the crop may generate additional market and another source of income for peasants, take underused land to use, make environment and energy protection advantages and give employment avenue.

7.5  Environmental Benefits Use of energy crop provides various environmental benefits including improvement in water quality, reduction in emission at generation facilities and improvement in wildlife habitat on conventional crops. Energy crops serves as filter system as it helps to remove pesticides and surplus chemical fertilizers from the shallow water before polluting the streamline water due to which soil and water quality also improve. Due to filtering abilities of energy crops, these are supposed as additional crop to be planted with convention crop in order to control pollution. An article of “Oak Ridge National Laboratory (ORNL)” on the utilization of energy crop reported that a neutral zone of grass or tree just 22 yards extensive can conserve coastal area and water from erosion, deposition and chemical flow. And these trees still can be reaped for energy. In comparison to conventional crop, most energy crops require less fertilizer. With the reduction in the use of chemical fertilizers, the water pollution and other environmental problems are overcome due to zero source pollution. It was concluded by Oak Ridge National Laboratory (ORNL) that groundwater contamination and surface water decrease with any alterations from annual to perennial herbaceous or woody crops. It was reported that in comparison to conventional crops, energy crops have high soil stability, reduced surface water overflow and reduced passage of nutrition and high soil water content. “UCS or Union of Concerned Scientist” has concluded that if the corn farm will be converted into grass field, then it may save 66 truckloads of soil from erosion every year. In some areas of Michigan, soil erosion benefits of energy crops have become of particular

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interest. It has been listed by “USDA National Resource Inventory” that there are about 557,300 acre areas in the state that have much potential for soil erosion. Decrease in the emission is one more advantage from the utilization of the energy crop against fossil fuel. Plants used for energy production also absorb some quantity of carbon dioxide which gets liberated during their ignition. Hence, no carbon dioxide is produced by the use of biofuel for the production of energy as the released carbon dioxide has been previously utilized by plants during their growth periods. In some plants like poplar, the quantity of carbon production is considerably less as compared with that of natural gas, petroleum and coal. In 1994, about 20% of carbon dioxide worldwide was produced by the USA. “Oak Ridge National Laboratory (ORNL)” revealed that 73% of SO2, 36% of CO2 and 33% of NO were emitted in the USA during power plant utilization. Major contributor of these emissions is coal power plants which supply about 74% of the electricity in the midwestern USA. This is due to the fact that most of the midwestern coal plants were made before 1970 when there was no modern pollution control system. “Oak Ridge National Laboratory (ORNL)” also reported that replacing fossil fuel for coal as energy source may help in the reduction of emission of such pollutants. However, growing energy plants in approximately 35 million acres would help in elimination of 6% of annual carbon dioxide release in the USA. Gases emitted from power plant have many ecological and health issues. Carbon dioxide gas is the primary cause of global warming. Sodium dioxide and nitrogen oxide gas cause acid rain. NOx emission contributed to the ground-level ozone. Acidification of the lake has been caused by acid rain that kills various aquatic animals including fishes. Acid rain also affects the forest, automobiles, buildings and many more things. It also causes various health diseases including asthma. Buhroo et al. (2018) reported that total healthcare cost related with air pollution estimated by “American Lung Association” about billion dollars per year.

References Ben-Amotz, A. (2004). Industrial production of microalgal cell-mass and secondary products-­ major industrial species. In Handbook of microalgal culture: Biotechnology and applied phycology, 273. Blackwell Science. Borowitzka, M. A. (1997). Microalgae for aquaculture: Opportunities and constraints. Journal of Applied Phycology, 9, 393–401. Bothast, R. J. (2005). New technologies in biofuel production (No. 1449-2016-119610). Buhroo, Z. I., Bhat, M. A., Malik, M. A., Kamili, A. S., Ganai, N. A., & Khan, I. L. (2018). Trends in development and utilization of sericulture resources for diversification and value addition. International Journal of Entomological Research, 6, 27–47. Cardozo, K. H., Guaratini, T., Barros, M. P., Falcão, V. R., Tonon, A. P., Lopes, N. P., Campos, S., Torres, M.  A., Souza, A.  O., Colepicolo, P., & Pinto, E. (2007). Metabolites from algae with economical impact. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 146, 60–78.

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Cysewski, G. R., & Lorenz, R. T. (2004). Industrial production of microalgal cell-mass and secondary products-species of high potential. In Handbook of microalgal culture: Biotechnology and applied phycology, 281. Blackwell Science. Dave, A., Huang, Y., Rezvani, S., McIlveen-Wright, D., Novaes, M., & Hewitt, N. (2013). Techno-­ economic assessment of biofuel development by anaerobic digestion of European marine cold-­ water seaweeds. Bioresource Technology, 135, 120–127. Dellomonaco, C., Fava, F., & Gonzalez, R. (2010a). The path to next generation biofuels: Successes and challenges in the era of synthetic biology. Microbial Cell Factories, 9, 1–15. Dellomonaco, C., Rivera, C., Campbell, P., & Gonzalez, R. (2010b). Engineered respiro-­ fermentative metabolism for the production of biofuels and biochemicals from fatty acid-rich feedstocks. Applied and Environmental Microbiology, 76, 5067–5078. Demirbas, A. (2008). Biofuels sources, biofuel policy, biofuel economy and global biofuel projections. Energy Conversion and Management, 49, 2106–2116. Duncan, R. (2003). The dawning era of polymer therapeutics. Nature Reviews Drug Discovery, 2, 347–360. Fernández, F. A., Sevilla, J. F., Egorova-Zachernyuk, T. A., & Grima, E. M. (2005). Cost-effective production of 13C, 15N stable isotope-labelled biomass from phototrophic microalgae for various biotechnological applications. Biomolecular Engineering, 22, 193–200. Gardner, B., & Tyner, W. (2007). Explorations in biofuels economics, policy, and history: Introduction to the special issue. Journal of Agricultural & Food Industrial Organization, 5, 1210. Guerin, M., Huntley, M. E., & Olaizola, M. (2003). Haematococcus astaxanthin: Applications for human health and nutrition. Trends in Biotechnology, 21, 210–216. Hallmann, A. (2007). Algal transgenics and biotechnology. Transgenic Plant Journal, 1, 81–98. Iwamoto, T., Sonobe, T., & Hayashi, K. (2003). Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. Journal of Clinical Microbiology, 41, 2616–2622. Kothari, R., Pathak, V. V., Kumar, V., & Singh, D. P. (2012). Experimental study for growth potential of unicellular alga Chlorella pyrenoidosa on dairy waste water: An integrated approach for treatment and biofuel production. Bioresource Technology, 116, 466–470. Pulz, O., & Gross, W. (2004). Valuable products from biotechnology of microalgae. Applied Microbiology and Biotechnology, 65, 635–648. Ratledge, C. (2004). Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie, 86, 807–815. Sialve, B., Bernet, N., & Bernard, O. (2009). Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnology Advances, 27, 409–416. Singh, R. S., & Thakur, S. (2016). Global demands of biofuels. In Biofuels: Production and future perspectives, CRC Press, Taylor & Francis Group, Boca Raton, London, New York (p. 41). Sorda, G., Banse, M., & Kemfert, C. (2010). An overview of biofuel policies across the world. Energy Policy, 38, 6977–6988. Spolaore, P., Joannis-Cassan, C., Duran, E., & Isambert, A. (2006). Commercial applications of microalgae. Journal of Bioscience and Bioengineering, 101, 87–96. Stolz, P., & Obermayer, B. (2005). Manufacturing microalgae for skin care. Cosmetics & Toiletries. Science Applied, 120, 99–106. Vilchez, C., Garbayo, I., Lobato, M. V., & Vega, J. (1997). Microalgae-mediated chemicals production and wastes removal. Enzyme and Microbial Technology, 20, 562–572.

Chapter 8

Technical Challenges of Biofuel Obtainment

Abstract  There is also an increasing concern regarding sustainability of biofuel generation/use that will influence on raw material preferences and consequently influence on biofuel’s obtainment chosen protocol. This chapter will explore main challenges related to biofuel obtainment presenting also interesting strategies to surpass some of these difficulties here addressed. Consortium-based bioconversion technologies, strategies involving synthetic biology/metabolic engineering and microfluidic platforms are examples of attempts to reduce cost and make the process of generating biofuel more efficient. Keywords  Algal consortium · Challenges · Biofuel · Bioconversion technology · Bioengineering

8.1  Introduction The process to obtain biofuel possesses some challenges related to technical aspects that may impact on its success. They may result, for example, in expensive, wasteful and energy-consuming pretreatment steps, high enzyme costs, difficulties to deal with moisture on extraction steps and fractionation techniques that may not offer high efficiency (Gaurav et al., 2017). When using terrestrial plants as raw material, for example, the biomass recalcitrance is challenging, and when using algae high biochemical diversity can make it difficult to choose the correct pretreatment, limiting biofuel’s obtainment efficiency (McCann & Carpita, 2015; Ward et al., 2014). Processes may also require a large amount of resource inputs which altogether may impair scale-up from a technological and economical point of view as all these challenges can make it difficult to achieve high rate of conversion and high yield of biofuel (Table 8.1).

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Table 8.1  Main challenges associated with biofuel obtainment Lignocellulosic raw material Choosing the right pretreatment method Avoiding carbohydrate loss and guaranteeing great biofuel yield Maintaining optimum growth conditions when using living organisms in fermentation step Adjusting protocols to avoid spending a lot of time on the process Difficulties associated with scaling-up Elevated costs

Algal raw material Choosing the adequate harvesting protocol Difficulties on large-scale cultivation Dealing with large biodiversity

Adjusting procedures to biomass composition variation due to different stages in microalgal development Choosing the right extraction method Costs can be elevated especially if it is necessary to dry biomass

Oleaginous microbes as raw material Avoiding culture contamination Dealing with native isolates Separating biomasses when dealing with co-cultures Choosing the adequate culture protocol

Maintaining optimized condition during cultivation Adjusting protocols to achieve the necessary yield

Fig. 8.1  Common steps performed to obtain bioethanol from lignocellulosic biomass

8.2  Lignocellulosic Biomass-Derived Biofuel Pretreatment is commonly applied when dealing with lignocellulosic raw material. For example, to obtain bioethanol from lignocellulosic biomass pretreatment, hydrolysis, fermentation and distillation are common steps (Rocha-Meneses et al., 2020) (Fig. 8.1). So, challenges related to this step are of special concern in this type of biosynthetic strategy. When treating biomass with bacteria to try to enhance process’ efficiency, anaerobic digestion can allow improvements on methane production. Anaerobic and facultative bacteria under thermophilic condition, for example, could

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improve in a significant way methane yield generated from corn straw (Fu et al., 2015). However, maintaining the optimum growth conditions for these microorganisms is challenging. The time consumed during pretreatment and the yield of biofuel obtained can also turn a methodology of pretreatment economically unfeasible. Fungal pretreatment of lignocellulosic biomass is an interesting example. Although temperature is not a problem to be maintained in optimal level, a long time of incubation is required. It has also been observed that lower yields of biofuel can sometimes be obtained when compared to traditional pretreatment methods. So, the production cost is estimated to be at least 4–15 times the one estimated for conventional pretreatment technologies, limiting large-scale use of this type of methodology (Vasco-Correa & Shah, 2019). Changing fungi to bacteria in pretreatment, the time of the procedure can be largely reduced; however, without any previous enzymatic digestion, it can be not useful once the yield of biofuel production is low if lignocellulose breakdown is not performed before the microorganism action. Physicochemical methods to pre-digest the raw material or facilitate the pre-digestion can enhance the process’ efficiency (Zhuo et al., 2018), but the additional cost related to that should be analysed. For example, rice straw submitted to dilute acid pretreatment contains lignin droplets generated during the pretreatment; the bacterium Cupriavidus basilensis B-8 can then dig out these droplets, and a porous structure is generated facilitating enzyme to access inner cellulose. Consequently enzymatic digestibility is increased up to 244% when compared to untreated rice straw (Yan et al., 2017). Pretreatment can also have it time reduced and the efficiency enhanced favouring biofuel obtainment by applying microbial consortia; co-cultivation systems can broaden the substrate utilization spectra when compared to pure cultures of microorganisms, and synthetic microbial consortia can be specially designed to suit processes necessary to efficiently produce biofuels (Jiang et al., 2020). For example, although it is possible to generate in an efficient way biofuel, biobutanol, using Clostridium sp. such as new strains like the WST, isolated from mangrove sediments. However, these microorganisms due to the inexpression of polysaccharide-­ degrading enzymes (Jiang et  al., 2018) cannot directly utilize polysaccharides to generate the fuel. The referred new strain, for example, is highly efficient but when using glucose or galactose as sugar source (Shanmugam et al., 2018). So, the most efficient and low-cost way to optimize the biofuel obtainment would be mixing species with different behaviour regarding fermentation process (e.g. mixing solventogenic species with cellulolytic ones) (Xin et  al., 2018). A mesophilic microbial consortium N3, for example, involved Clostridium ramosum, Clostridium celevecrescens, Proteiniphilum acetatigenes, Desulfovibrio africanus, Aminobacterium colombiense and some uncultured bacteria and could generate a high concentration of reducing sugars from cellulose. The addition of Clostridium acetobutylicum butanol production was highly stimulated (Xin & He, 2013; Wang et  al., 2015). Consortia in biofuel production involving algae and bacteria are also considered a very relevant strategy offering the possibility of cultivation on large-scale wastewater ponds and removal of pollutants such as heavy metal from contaminated water

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improving its quality, and substances like fatty acids produced by algae can be used by other organisms to grow (Naghavi & Sameipour, 2019). In fact, it is also important to highlight that microorganisms’ capacity to promote bioremediation and generating biomass for biodiesel production simultaneously is an important contribution to sustainability. Wastewater containing trace elements proved to be able to assist the upsurge of microorganisms’ biomass; lipids produced presented capacity to generate substances with efficient fuel combustion properties, being relevant for production of biodiesel (Leong et  al., 2020). However, there are also challenges related to the use of microbial consortia; for example, a large number of interaction mechanisms among microorganisms (that may be crucial to optimize the process) are still uncharacterized; there are technical limitations on studying interactions among cells and metabolites exchange specially for a large number of different species mixed, maintaining a stable coexistence of these different species and finding optimum fermentation conditions (Jiang et al., 2019). Pretreatment can also be performed using isolated enzymes aiming to optimize biofuel obtainment, but associated with this process are two main challenges: high cost and high time consumption (especially when optimal conditions are not provided for enzymatic work) (Castellini et al., 2021). These challenges can be faced performing, for example, assays to determine optimized conditions for enzyme’s activity (De La Torre et al., 2017). Using more than one enzyme is also interesting; however, the synergic effect of the enzymes working together should be previously analysed since certain enzymes working together can offer no gain in product generation and elevate total costs; laccases from Streptomyces ipomoeae and from Trametes villosa used together caused no enhancement on degradation products such as acetic acid, 5-HMF and furfural obtained from wheat straw steam explosion pretreatment (De La Torre et al., 2017). Enzyme recycling is also an alternative to reduce costs, reusing the proteins (Østby et al., 2020). Other important challenge faced during pretreatment (biological, physicochemical or chemical) is loss of carbohydrates (Satari et  al., 2019). Before applying a pretreatment, it is prudent to make sure that the whole biofuel production process is able to generate approximately or more than 400 g total carbohydrates per kg of lignocellulosic biomass (Marks et al., 2020). Attempts to enhance biofuel yield sometimes involve genetic modification/metabolic engineering. When it is the case, various challenges obstacles the progress such as the unavailability of genetic information for some species, optimizing DNA modification protocols, optimization of the metabolic pathway to achieve the expected results (Adegboye et  al., 2021). Thermoanaerobacterium saccharolyticum, for example, naturally possesses the ability to ferment hemicelluloses (but not cellulose) to generate ethanol at high titre and high yield. In this process the genes adhE, nfnA, nfnB and adhA present important roles. Clostridium thermocellum, on its turn, can ferment cellulose. However, after introducing these genes from T. saccharolyticum into C. thermocellum, ethanol productivity was significantly improved (Hon et al., 2017).

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8.3  Algae-Derived Biofuel Life cycle assessment approach studies, for example, have indicated that greenhouse gas emissions are fewer when the biofuel burned in some activity comes from algae raw material instead of coming from corn biomass, for example (Dutta et al., 2014). In fact, biofuel based in algae material presents advantages also when discussing technical aspects as it allows a sustainable fuel generation, does not require arable land, consumes less energy, maximizes biomass utilization, makes it more feasible the recycling of nutrients, can be grown in diverse environs (such as wastewater or the ones with high salt level), generates significantly more biomass than terrestrial plants, possesses a low operational cost and offers versatility in the form of a large number of species to explore (Chaudry et al., 2018; Kumar et al., 2016; Ishika et al., 2017; Ishika et al., 2018; Phwan et al., 2018; Saratale et al., 2018; Alam et al., 2019; Lu et al., 2019). However an important feature associated with using microalgae in large-scale procedures, for example, is technological challenges of harvesting and large-scale cultivation. Each species and strain possesses its specific cultivation technique to optimize the organism functions, and when it comes to harvesting, a similar situation occurs. A large variety of methods are available (such as sedimentation, flotation and centrifugation), but no all-purpose harvesting technique that suits all types of microalgae suspensions is available (Koller et  al., 2014; Hattab, 2015; Wang et  al., 2020). Bacteria with high flocculation activity are suggested as tools to improve microalgae biorefineries through optimization in the harvesting process (Gerardo et al., 2015). Bacillus sp., for example, offers 95% efficiency in aggregating Nannochloropsis oceanica in 30 s (Powell & Hill, 2013). Bacterium strain HW001 could aggregate several microalgae species (flocculating activity reaching up to 94%) such as Nannochloropsis oceanica and Chlorella vulgaris (Lee et al., 2013). As seasonal variations in temperature and insolation occur year-round, microalgal biomass productivity also varies and not only temperature and light intensity are relevant aspects to be observed; cell metabolism can also be affected by culture conditions such as pH and nutrients available, for example (Yew et al., 2019). These are some reasons why developing a large-scale cultivation that can optimize algae development and biofuel obtainment is considered challenging (Khanra et al., 2018). So, cultivation of algae with intension of studying biodiesel generation or large-­ scale production is generally not performed in uncontrolled environment. Bioreactors are commonly used, and research groups worldwide dedicate to optimize the technology associated with them, aiming also optimization of biofuel obtainment and cost reduction. Microfluidic photobioreactors are an interesting option that can be used for algae single-cell cultivation, offering low cost (Yang & Wang, 2016). It is also challenging the fact that algal biofuel production is largely related to biomass composition that can change significantly according to different stages in

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microalgal cultures growth cycle. In fact, compositional characteristics of biomass also impact on the susceptibility to pretreatment. Observing Chlorella vulgaris, Scenedesmus acutus and Nannochloropsis granulate behaviour, it was possible to conclude that although acid pretreatment may allow the obtainment of high energy yields, the optimum condition varies according to the algae strain involved and the composition of the biomass they are able to offer (Dong et al., 2016). The incongruous nature of biomass and lipid accumulation as same as slow-growing rate can convert, for example, oleaginous algae (that are well-known as biodiesel producers) into not so interesting option (Li et  al., 2011; Singh & Olsen, 2011; Sharma et al., 2012). Therefore, it is not an easy task selecting a suitable robust algae strain presenting a combination of favouring factors to biofuels’ production such as high growth rate, being capable of offering quality raw material to optimized biofuel synthesis and also being capable of surviving well in culture: resisting to other microbes’ invasion and providing conditions to a low-cost and efficient culture (Halim et al., 2012). Other important challenge is final cost of the biofuel when the protocol applied involves drying the biomass (Sathish & Sims, 2012). Working with wet biomass to perform lipid extraction significantly reduces costs once it requires less energy. However, from wet biomass lipid recovery is generally too low (high moisture content impairs lipid extraction  – it provides mass transfer limitation.) to allow this procedure to be competitive at large-scale industrial application (Howlader & French, 2020). That is why a pretreatment on wet biomass is normally performed prior to the solvent extraction; lipid recovery can be increased through physical, chemical, physical-chemical and enzymatic treatment, for example (Howlader et al., 2018). More than one strategy can be performed together to offer synergistic effect; microwave (energy dissipation of 140 W) and ultrasound irradiations (also at 140 W), for example, increased biodiesel production from Nannochloropsis sp. biomass (Martinez-Guerra et al., 2018). Algal consortium containing different species to try to surpass difficulties related to cultivation and to optimize the raw material for biofuel production can also be performed involving microalgae, for example. Using Chlorella sorokiniana (capable to produce higher biodiesel amounts than Botryococcus braunii) and Botryococcus braunii (that offered more biomass than Chlorella sorokiniana) allowed a combined algal processing obtaining 29 g/L ethanol with a recovery of lipids from 84 to 89%; the high level of polyunsaturated fatty acids favours the reduction in cost of biofuel generated (Singh et  al., 2020). However, challenges such as incompatibilities leading to competition for growth resources and different growth rates resulting in the outgrowth of one species over the other after cultivation under uniform growth condition are some of the possible obstacles to be faced (Jiang et al., 2019). Oil extraction process from algae also presents important challenges. Microwave-­ assisted extraction, for example, is a rapid and efficient extraction, but is related to high maintenance cost particularly on a commercial scale. Organic solvent-based methods, although being largely used, are not suitable for all algal strains, consume long time, involve a lot of labour and commonly generate toxic residues. Mechanical

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approaches may cause damage to the end products and possess a high cost (Kumar et al., 2015). The efficiency of extraction may be affected not only by the method chosen. Biomass size, for example, can affect the process’ efficiency. Varying algal biomass’ size and maintaining the total mass and volume of solvent, it was possible to observe that a reduced size probably offers a larger contact surface with solvents oil extracted efficiency. Increasing the amount of solvent not always represents a significant gain in extraction; it may result in a very slight increase in efficiency that is not a good option considering associated costs related to more solvent used (Baig et al., 2018). To favour biofuel production, some research groups have dedicated to metabolic engineering and synthetic biology genetically modifying algae’s DNA.  Although recent research indicated that population in Europe would accept well biodiesel produced using engineered algae (Villarreal et al., 2020), challenges are also present in this pathway. Important examples of these challenges are slow workflow, difficulties and technical limitation to reduce off-target integration, intensive labour and regulatory issues associated with genetically modified organisms (Kumar et al., 2020). Other challenge important to be discussed regards sustainability of algae-based biorefinery. However there are already strategies related to circular bioeconomy that aim to reuse wastes in biotechnological processes. Brewery effluent, for example, could be used to the growth of Scenedesmus obliquus; the microalga could use nutrients such as N and P to generate a biomass capable of providing biohydrogen by dark fermentation and bio-oil and biogas by pyrolysis (Ferreira et al., 2019). A consortium using Chlorella sp. and Scenedesmus sp. proved to be relevant to promote wastewater treatment, serve as a biofertilizer and also produce biodiesel in an effective way (Silambarasan et al., 2020).

8.4  Biodiesel from Oleaginous Microbes Although it is not the focus of this book, biodiesel can also be produced using microorganisms such as yeasts and bacteria. In this topic the main aspects and challenges related to this type of raw material will be addressed. In fact a large-scale application of this kind of biofuel production is less complex nowadays to be performed when compared to microalgae’s cultivation (Howlader & French, 2020). Certain species of microbes such as bacterium Rhodococcus opacus can produce a large amount of oil being an excellent material for biodiesel production (Shields-­ Menard et al., 2018). R. opacus PD630, for example, is considered an oleaginous organism as it can present a high percentage of TAGs in relation to dry cell weight: 76% when grown in gluconate, 38% when grown in hexadecane and 87% when grown in olive oil (Alvarez et al., 1996). In fact R. opacus PD630 presented the most significant yields (converting, after 48 h, 6.2% of organic content and accumulating 42.1% in oils based on cell dry weight) when compared to R. rhodochrous and R. jostii using corn stover waste (Le et al., 2017).

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Yeasts are also suitable for large-scale biodiesel production due to their fast growth rate, higher productivity that can be reached and their ability to use a wide range of substrates to survive (Adrio, 2017). It is possible that the chosen yeast does not offer high productivity at first; but this challenge can be easily solved with adjustments on the process. Rhodotorula glutinis, for example, is an oleaginous yeast that can offer a high oil production through microbial fermentation. The amount of oil content, as same as cell density, can be optimized by continual feeding instead of pulsed feedings supplying nutrients at rates superior to 0.8 g/L/h; the rates should be different to each of this two stages: a cell proliferation stage and a lipid accumulation stage – resulting in a high oil yield of 16.28 g/L (Karamerou et al., 2017). The oleaginous yeast Cryptococcus curvatus could deal with waste office paper as raw material to generate lipids offering biomass of 6.32 g/L, lipid yield of 1.39 g/L, lipid content of 22% and lipid coefficient of 99.9 mg/g sugar with the productivity of 0.02 g/L/h. However, after enzymatic pretreatment it could offer biomass of 15.20 g/L, lipid yield of 5.75 g/L, lipid content of 37.8% and lipid coefficient of 234.6 mg/g sugar with the productivity of 0.08 g/L/h (Annamalai et al., 2018). However, problems such as culture contamination still exist when using yeasts cultures. It was necessary, for example, to combine NaCl and high glucose concentrations to suppress bacterial contamination of Rhodosporidium toruloides cultures. Fed-batch bioreactor cultures could allow production of 37.2 g/L of biomass and 64.5% w/w of lipid yield (mainly composed of oleic acid and also containing palmitic and stearic acids) to generate second-generation biodiesel (Tchakouteu et al., 2017). There are also species of fungi interesting to be used in biodiesel production due to their high capacity of lipid synthesis and accumulation; the genre Mordellistena, for example, offers species that can reach over 40% lipid accumulation (Papanikolaou et al., 2017). An enormous challenge faced on biofuel’s production using microbes is when native isolates are chosen to be used. The unavailability of information regarding the organisms can impair the process. For these organisms lack information regarding behaviour, genetic engineering platforms and metabolic pathways, making it difficult optimization and cost reduction associated with the protocol (Adegboye et  al., 2021). However, engineering of biosynthesis pathways could already be performed in native strains, such as Clostridium thermocellum (that had its electron metabolism engineered) increasing bioethanol formation (Lo et al., 2017). To improve performance and reduce costs, co-cultures involving microbes is also an interesting option. Organisms can grow better in the symbiotic environment; the products produced by a species and trace elements released in culture medium, for example, may be used to enhance other species growth and metabolism (Yen et al., 2015). Cost reduction in the process of biodiesel generation is expected to be achieved using the oleaginous obligate heterotrophic fungus Aspergillus awamori co-cultured with photoautotrophic green algae Chlorella minutissima. The association made it possible fatty acids generation from pure glycerol instead of glucose in a system that presented 3.9-fold increase in biomass and 5.1-fold increase in total lipid yields (being C16:0, 35.02%, and C18:1, 24.21%  – the major composites) when compared to the axenic cultures (Dash & Banerjee, 2017). A challenge

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associated with this kind of strategy is separating the microalgal and bacterial biomasses when necessary; that is why bacterial exudates or volatiles are sometimes preferred (González-González & de Bashan, 2021).

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Chapter 9

Conclusion and Future Perspectives

Rapid industrialization and urbanization the world has experienced caused several consequences that negatively impact life on Earth. Environmental pollution is a severe consequence that threatens the survival of several species and disrupts the balance of ecosystems. Soil, water and air containing organic (e.g. hydrocarbons) and inorganic pollutants (e.g. heavy metals) are dangerous consequences from activities such as industrial ones, mining and farming, among others (Ali et  al., 2019; Kurwadkar, 2019; Peuke & Rennenberg, 2005; Tonelli & Tonelli, 2020). When it comes to the use of fossil fuel, global warming is a very dangerous consequence. This kind of fuel, by being burned, generates some gases that are the main cause of global warming (Ramakrishnan, 2015). From 1860 to 1914, for example, occurred the Second Industrial Revolution that brought a large array of technological benefits, specially to the USA, such as electricity and internal combustion engine. However, pollution sources increased in number and pollution itself in amount and diversity of contaminants. It is necessary to highlight here the role of fossil fuels enhancing greenhouse gas emissions that still persists worldwide (Mohajan, 2013; Mohajan, 2020). Industries are responsible for emitting a large amount of these gases, especially in developing countries such as China and India (Liu et al., 2020). So, to deal with this problem aiming to reduce the negative impact it causes on living beings, greener fuels generated from renewable raw material must be produced to substitute the ones derived from petroleum. In the situation the world is nowadays, the energy demand increases constantly, and it is also necessary to not only diminish pollution related to fuels but also to disrupt the dependence on fossil fuels that are not renewable and from which main oil sources are found concentrated on the Middle East (Curtin et al., 2019; Peng et al., 2019; Piotr et al., 2019). Environmental-friendly processes need to be developed to provide these fuels in a cost-effective way and in scale capable of supplying the amount needed to maintain activities in which they are required. For example, during the COVID-19 pandemic, lockdown led to a reduction in air pollution capable of inducing a cooling of © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4_9

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around 0.01 ± 0.005 °C by 2030. However, if a reduction in fossil fuel investments occurs associated with the development of green options such as biofuel, it is expected to be possible to avoid future warming of 0.3 °C by 2050 (Forster et al., 2020). This chapter is dedicated to summarize the main topics discussed and to present conclusion and future perspectives on plant and algae potential as raw material to provide ingredients for biofuel production and also the challenges, economic aspects and aspects related to sustainability associated with their production/use.

9.1  Main Conclusions Environmental pollution is one of the most worrying problems worldwide nowadays. Its main cause is reckless anthropic actions, ignoring sustainability principles. As a result, pollutants from different chemical nature (organic and inorganic substances) contaminate air, soil and water causing severe consequences to life forms on Earth (Brevik et al., 2020; Manisalidis et al., 2020; Yin, Brauer, et al., 2020, b; Quinete & Hauser-Davis, 2021). The introductory chapter revisited negative consequences related to environmental pollution caused by fossil fuel. As population grows on Earth, the use of fuel derived from petroleum increases and consequently the necessity of producing them. However, the generation and the burn of this kind of fuel represent a risk to the living beings’ survival (Franta, 2021). Toxic pollutants are generated as a consequence of this kind of fuel combustion, and most of them are persistent contaminants capable of causing extensive damage on ecosystems (Raimi, 2020). Not only greenhouse gases are consequences of fossil fuels but also particulate matter and petroleum and organic molecules derived from it that can reach the environment especially when accidents occur on extraction platforms or while dealing with the fuel. Airborne fine particulate matter (PM2.5) is a product of burning fossil fuels responsible for approximately 10.2 million premature deaths, each year in the world which is a good reason to incentive a shift to clean sources of energy (Vohra et al., 2021). That is why an increasing number of researchers have been dedicating themselves to propose protocols to remediate environmental pollution, restoring environments reducing the negative impact contaminants may cause to ecosystems (Wang et al., 2017). It reflects how important and how urgent are efforts aiming to achieve a world free of pollution, not only caused by fossil fuels (Bespalov & Gurova, 2021; Bhandari et al., 2021; Ganie et al., 2021; Sajjadi et al., 2021). Addressing specially fuels, the desire is to achieve an atmosphere free of contaminants through sustainable, efficient and low-cost technologies to generate energy from renewable green sources (Panoutsou et al., 2021; Shote et al., 2018). Biofuels are fuels produced from renewable organic material which may be provided indefinitely in contrast to fossil fuels, which are exhaustible resources and that are already causing extensive damage to the environment being responsible for

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climatic hitches posed by the escalated emissions of greenhouse gases (Hill et al., 2016). However, it is not an easy goal to achieve and it will demand a lot of time and dedication. For example, there are studies that indicate that several types of biofuels could yield lower life cycle greenhouse gas emissions than gasoline, but over a 30-year time horizon (Khanna et al., 2011). Anthropogenic activities, especially the ones related to fuel production and transportation, are increasing the concentration of greenhouse gases in the atmosphere, enhancing greenhouse effect and negatively impacting life on Earth. Besides being a natural phenomenon, greenhouse effect can also turn into a deleterious event if an increase in concentration of greenhouse gases occurs, causing, for example, global warming (Manabe, 2019). Through years, different climate models have predicted for global climate change and allowed phenomenon analysis. Greenhouse gases, such as CO2, nitrous oxide and chlorofluorocarbons (CFCs), can increase infrared opacity of air enhancing the absorption of longwave radiation, consequently increasing temperature: which is very harmful to ecosystems (Manabe, 2019). In order to try to obtain an atmosphere free of pollution, the demand for green fuel is increasing. Plant and algae (both micro and macro), besides being responsible for producing the majority of the oxygen available on atmosphere and being the base of food chain, are viable sources of biomass to produce this kind of fuel. They can be applied as doable option for producing, for example, bioethanol, biomethanol, biodiesel, biogas or other biofuels (Ullmann & Grimm, 2021; Valdivia et al., 2016). When it comes to biofuel generations, it is important to highlight that there are difficulties associated with all types of classes. For example, the first-generation biofuels that derive from crops, such as soy and palm (produced from edible oil), are advantageous when it comes to reducing CO2 emissions, but possess important disadvantages associated with them, being the main ones competing with food production (e.g. increasing its price) in arable land, nutrients and water supply and inducing deforestation (Sayre, 2010; Singh et al., 2020). However, it is still the main kind of biofuel produced; it is estimated that more than 90% of biofuels produced are generated from edible biomass (Oh et al., 2018). The second-generation biofuels (produced from oils of non-edible plants) do not compete with food production; the species may grow with minimal requirements for fertility and humidity, present the possibility to produce useful by-products and stability over different climatic conditions, but may cause consequences on land-use change, impair energy security, present technical difficulties to deal with lignocelluloses and may generate a low oil yield; and cost-ineffectiveness is commonly reported (Adenle et al., 2013; Bankovic-­ Ilic et al., 2012; Ganesan et al., 2020). Biofuels from third generation can be produced from used cooking oil, waste animal fats and mainly algae material. When it comes to algae, it can offer a difficulty in setting process’ optimized condition (once they may present necessity of a high amount of nitrogen and phosphorus and some species may produce harmful toxins) and scale-up due to the large source biodiversity existent; however, it is considered to be the raw material capable to provide the most sustainable procedure (Kadir et al., 2018; Merlo et al., 2021; Miranda et al., 2016). Algae can offer high rate of biomass with high lipid content in a fast way and do not require fertile soil (Coh et al., 2019; Yin, Brauer, et al., 2020, b).

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Regarding used cooking oil and waste animal fats, it is interesting because it offers a utilization to this material that would be discharged, presenting economic, environmental and food security advantages. However, dealing with impurities related to these raw materials and difficulties related to collecting them are relevant disadvantages (Foteinis et al., 2020). There are species of organisms naturally more suitable to generate each type of biofuel that can be generated from different metabolites obtained through algae and plants. Polyunsaturated fatty acids from algae and cellulose from plants’ biomass are versatile examples (Ganesan et al., 2020). Feedstock selection is not a task so simple to be performed; there are various parameters related to technical, environmental, economic and social issues that can impact the performance and cost of fuel production (Anwar et al., 2019). Consequently, there are different manners/methods/protocols that can be used to obtain biofuel from plant and algae, and new ones are continuously been proposed by research groups worldwide. When it comes to protocols to obtain biofuel from algae, for example, the main source of diversity lies on different oil extraction methods (Ellison et al., 2019; Onumaegbu et al., 2019; Sovova & Stateva, 2019; Yusuff, 2019). Extraction methodology influences on final cost and whole processes efficiency, and the chosen protocol involving mechanical extraction, solvent extraction, enzymatic extraction or a combination of these methods depends mainly on the feedstock source (Atabani et al., 2012; Li et al., 2019; Taher et al., 2019). In plants, a larger variation is found as the process generally requires a pretreatment step to deal with lignocelluloses, for example, Aftab et al. (2019). Each strategy possesses advantages and disadvantages that should be analysed before being chosen. It is also possible to notice that the protocols are possible to be optimized aiming to obtain a desirable result or to promote cost reduction, for example. There are various relevant limitations in the process of obtaining biofuels: related to the nature of the raw material, cost-effectiveness, time necessary to conclude the process and final yield generated, among others (Pikula et al., 2020). As it was presented on this book, most of these challenges are related to technical aspects that may impact on its success. They may result, for example, in expensive, wasteful and energy-consuming pretreatment steps, difficulties to deal with moisture on extraction steps and fractionation techniques that may not offer high efficiency. However, researches also present interesting strategies to surpass these barriers (Malode et al., 2021). It is also necessary to reinforce that the challenges related to biofuel production may impact on an important aspect that should be highlighted: the possibility/viability to scale-up to obtain amounts large enough to attend human necessities (Panoutsou et  al., 2021). When it comes to biofuel generated using microalgal material, for example, specialists are emphatic to assure that the ideal process should be environmentally sustainable, economically feasible and replicable (Kumar et al., 2020). However, the advancements on recombinant DNA technology have made possible genetic and metabolic engineering aiming to enhance processes efficiency, to generate the biofuel known as fourth generation (Alalwan et  al., 2019). In algae, for example, fatty acid, isoprenoid and alcohol pathways could be engineered to successfully enhance the obtainment of biofuel making the process more economically feasible. Ethical and regulatory aspects are debatable,

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and discussions on aspects related to these approaches are receiving increasing attention (Kanjilal and Saha, 2019; Villarreal et al., 2020). As most of countries on the world are capitalist, economic aspects should also be considered. From an economical point of view, for example, it may increase the income that farmers obtain, but also may negatively impact the environment if not managed properly (the land, water and other resources it requires during producing should be used rationally and in a sustainable way) (Chia et al., 2018; Hartley et al., 2019; Subramaniam & Masron, 2021). For being generated by renewable sources, biofuels may be sustained indefinitely; studies using different economic models have concluded that biofuels can lead to reductions in life cycle greenhouse gas emissions and also offer the possibility to reduce imports (reducing also the impacts of supply disruptions and petroleum price). There are, for example, economic incentives (grants, income tax credits, subsidies and loans) to promote biofuel research and future large use worldwide. However, there are also economic models showing that biofuel use can result in higher crop prices (Chen et al., 2021; Jeswani et al., 2020; Mizik & Gyarmati, 2021; Navarro-Pineda et al., 2017).

9.2  Future Perspectives It is expected that strategical fields related to biodiesel production continue to develop, in special fields related to fourth-generation biofuel; molecular biology tools to promote site-directed genetic engineering, more rapid and reliable genome sequencing platforms and multi-omics datasets are interesting examples of technologies from which improvements directly would positively impact optimized biodiesel production. These advancements would offer the opportunity to promote efficient and fast improvements on biofuel obtainment through robust bioengineered strains for high productivity. Making these strains commercially available, process economics would also be possible to be optimized. However, it is necessary to develop also genetic strain design principles considering also aspects related to the safety not only to human beings but also to the environment as a whole. Genetically modified organisms require strict regulations and monitoring to be used in a safe way, especially in outdoor cultivation (Kumar et al., 2020). Economic competitiveness of biofuel would also be enhanced by more efficient cultivation protocols and extraction and/or fractionation methods, for example. Optimizations in these steps would benefit the field significantly (Abdelhamid et al., 2019; Figueroa-Torres et  al., 2021; Gifuni et  al., 2019; Kings et  al., 2017; Solis et  al., 2021). There are relevant limitations in the process of obtaining biofuels related to the nature of the raw material used for their generation, for example. An efficient and economically viable removal of cell walls on a large scale would make the process easier and more advantageous. Improvements on microalgal cell disruption optimizing lipid recovery (Quesada-Salas et  al., 2021) or in lignocellulosic biofuel production (Lee et al., 2021), for example, would be interesting and desirable. Bioethanol, for example, is already used as biofuel for the transportation

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sector; however, recalcitrant structure of lignocellulosic biomass is still a challenge that genetic manipulation of cereal plants is trying to help to efficiently deal with. Grain and straw of modified plants can be useful material to a process less dependent on pretreatment and with larger amount of available cellulose in biomass (Rocha-­Meneses et al., 2020). When it comes to microalgae, in order to optimize nutrients supply also optimizing energy consumption, bioremediation of wastewater is suggested to be associated with cultivation step. However, further research needs to address these protocols to verify how secure and interesting this strategy is (Merlo et al., 2021). Optimized biofuel formulations are also desirable, taking into consideration ignition delay and consumption, for example. There are compositions that add water to biofuels, generating hydrofuels that offer reduced nitrogen oxide emissions (Brock et al., 2020). It is also expected that methodologies to allow efficient modelling aiming optimization of biofuel production surpass their obstacles. Artificial neural networks, for example, still possess several limitations specially to modelling bioprocesses with very small data sizes. Scientists need to be able to perform the determination of factors that influence the model development more efficiently. Flexible learning algorithms and enhanced capacity to deal with non-­ linear processes, for example, can contribute to significant improvements (Sewsynker-Sukai et al., 2017). When it comes to social impact of the process, it is desirable that strategies that do not threaten biodiversity and food production are preferred (Hill et al., 2016). Food security of future generations should be considered, avoiding contributing to make cropland insufficient to food production. Agricultural systems capable of conjugating in an efficient way food production and generation of material to generate biofuel would be a great contribution: allowing, for example, reduction in greenhouse gas emissions and optimizing nutrient use (Rahmann & Grimm, 2020; Ullmann & Grimm, 2021). Reporting to the population the benefits of biofuels is also necessary and expected to continue to happen in the future. Acceptance by final consumers is very relevant, and evidence of benefits and full comprehension of potential risks help to reach that. Issues that impair public acceptability should receive attention (Villarreal et al., 2020). Governments support to research programmes related to biofuels is also essential, motivating sustainable production and pollution reduction: aiming a zero-­ carbon future (Reid et al., 2020). It is also crucial to perform several field testing projects to access real risks to the environment and the possibility of scale-up success. When the amount of residues generated is relevant, disposal of these residues should be considered as same as waste reduction strategies (Abdullah et al., 2019). To improve investors’ confidence and improve biofuel industry technical and financial performance, tailored policy interventions targeting challenges should be integrated along the biofuels value chain: to achieve all governance levels guiding the sector integration and alignment with Sustainable Development Goals (Panoutsou et al., 2021). However, the most important aspect that will define success of strategies aiming this goal is the ability to efficiently balance profit and environmental impact. If prioritization is dedicated to profit, environmental negative impacts increase, and if only environment is a concern, cost of process can be excessively elevated to the point of not being able to cover production expenses (Solis et al., 2021). Profitability and sustainability should be considered at once.

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Index

A Acid saccharification, 61 Acid treatment, 96 Activated carbon adsorption, 69 Adaptive neuro-fuzzy inference system (ANFIS), 118 Adsorbent materials, 69 Adsorption drying, 70 Advanced biofuel generation technologies, 65 Agricultural and critical harvesting, 54 Agricultural and woodland lignocellulosic biomass, 66 Agricultural plants, 11 Agro-waste, 34 Alcoholic fermentation, 58 Algae, 17 Algae-based biorefinery, 141 Algae-based third-generation biofuels biobutanol, 61 economic feasibility, 61 electrocoagulations, 61 eutrophication process, 61 lipids and carbohydrates, 61 macroalgae, 61 nitrogen-fixing bacteria, 61 pretreatment technologies, 61 seaweed, 61 Algae deposits, 2 Algae-derived biofuel acid pretreatment, 140 advantages, 139 algae strain, 140 biodiesel generation, 139 biomass composition, 139 biorefinery, 141

challenges, 141 dark fermentation, 141 ethanol, 140 extraction efficiency, 141 large-scale cultivation, 139 lipid accumulation, 140 lipid extraction, 140 mechanical approaches, 140–141 metabolic engineering, 141 microalgae, 139, 140 microalgae suspensions, 139 oil extraction, 140 pretreatment, wet biomass, 140 synergistic effect, 140 temperature and light intensity, 139 terrestrial plants, 139 Algae genera, 127 Algal biomass, 88 Algal consortium, 140 Algal farming, 43 Algal feedstock, 66 Algal fuel biofuels, 8 commercialization, 99 functional genomics, 9 oil extraction, 8 production, 8 species, 8 Alkaline pretreatment, 113 Alkali transesterification with methanol, 60 Amalgamation, 58 American Lung Association, 132 Anaerobic digester, 69 Anaerobic digestion, 65, 68, 69, 127 Ankistrodesmus falcatus, 61

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 R. A. Bhat et al., Plant and Algae Biomass, https://doi.org/10.1007/978-3-030-94074-4

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158 ANNs-RSM model, 119 Anthropogenic activities, 149 Arabidopsis thaliana WRI1/hemoglobin gene, 111 Artificial neural network (ANN), 117, 118, 152 Aspergillus awamori, 142 Astaxanthin, 128 Attrition mill, 112 B Bagasse, 19 Banana, 59 Beta-carotene, 128 β-1,3-Glucan, 127 Beta-glucosidase, 116 Bio-alcohol, 37 Biobutanol, 61 Bio-crude, 73 Biodiesel, 21, 22, 53, 54 bioethanol, 65 oleaginous microbes, 141–143 reduced emissions, 65 renewable energy source, 65 transesterification, 66 Biodiesel manufacturing process optimization alcohol dehydrogenase, 116 algae genetic modification, 116 algae metabolic engineering, 116 beta-glucosidase, 116 2,3-Butanediol, 115 catalyst selection, 115 clostridial n-butanol pathway, 115 fungi species, 116 genetically modified organisms, 115, 116 hyperthermophile, 115 ILs, 115 levulinic acid production, 115 non-ionic surfactants, 115 Biodiesel production, 151 cost reduction, 142 Bioenergy conversion, 67 Bioethanol, 22, 23, 38, 39, 54, 66 Biofuel, 16, 21–26, 35, 37, 53, 87, 148, 149 amorphous portion, 37 biodiesel, 54 bioethanol, 37, 54 biogas, 54 cellulose, 37 challenges, 55, 135 classification, 55 commercialization, 59

Index crops, 55 definition, 54 development, 54, 55 extraction, 22 generations, 52, 149 global fluid fuel supply, 55 issues, 55 lipid, 21 obtainment challenges, 136 obtainment efficiency, 135 organizations, 55 precursors’ production, 110 production, 17, 38, 39, 89 raw materials, 55 sources, 54 starch-rich corn, 38 sugar and starch, 38 Biofuel policies and scenarios agricultural commodities, 130 agricultural regions augmentation, 130 biofuel production, 130 energy security, 130 firm Reliance Industry, 130 GHG emission reduction, 130 green energy production, 130 purchase tax, 130 Biofuel production aggregation factors, 65 biogas preparation, anaerobic digestion, 69 biogas-purifying techniques, 70–71 biogas structure, anaerobic digesters, 68, 69 from biomass, 67 biomass garbage collection and repository system, 65 biomass to bioethanol transformation, 67–68 conversion frameworks, 65 enzymatic hydrolysis process, 66 photosynthetic/non-photosynthetic structure, 66 potential alternatives, 65 technologies, 65 transesterification, 66 Biogas, 3, 24 Biogas-purifying techniques adsorption drying, 70 chemical absorption, 71 CO2 absorption, physical method, 71 CO2 removal, 70 cryogenic isolation, 71 H2S removal, 70 physical partition, 70

Index Bio-hydrogen production, 25, 26, 119 Biomass, 2, 3, 16, 20, 24, 34, 53, 149 conversion processes, 64 and hydrogen, 25, 26 source, 16 Biomass to bioethanol transformation anaerobic digestion, 68 conversion procedures, 67 enzymatic hydrolysis, 67 fermentation, 68 hemicellulose and cellulose degradation, 67 nutrient-enriched digestate, 68 progressions, 67 thermochemical pathway, 67 Biomass transformation method, 62 Biomethane, 69 Biomethanol, 99 Biomolecules, 128 Bioreactors, 139 Biorefinery, 41, 54, 92 Biorefinery-based processes, 54 Biotransformation, 67 Biotrickling filters, 69 2,3-Butanediol, 115 C C20–C24 very long-chain fatty acids, 111 Calophyllum inophyllum, 60 Camelina sativa, 111 Carbohydrate-enriched biomass, 41 Carbohydrate fermentation, 89 Carbon dioxide, 132 Carbon fixation, 88 Carbons, 53 Carotenoids, 127 Catalyst sulphated zirconia alumina, 117 Catalytic hydrothermal gasification (CHG), 73 Cellulolytic bacterial species metabolism, 66 Cellulolytic thermophilic filamentous fungus, 116 Cellulosic biofuel conversion methods CHG, 73, 74 continuous flow system, 73 decarboxylation and dehydration, 72 direct biomass conversion, 72 fluid beds, 72 HTL, 72, 73 microalgae biomass, 73 pyrolysis, 72 thermal decomposition, 72 thermal liquefaction, 73

159 thermochemical methods, 71 thermochemical translation, 72 Cellulosic biomass, 39 Central composite design (CCD), 117 Cereal straw, 74 Chemical absorption, 71 Chemical fertilizers, 131 Chemical pathways, 94 Chemical pretreatment approaches, 113 Chlamydomonas reinhardtii, 110, 116 Chlorella minutissima, 142 Chlorella vulgaris, 139 Chlorofluorocarbons (CFCs), 149 Chlorophyceae, 10 Classical pulping processes, 93 Clostridium acetobutylicum, 89, 115, 116, 137 Clostridium beijerinckii, 116 Clostridium thermocellum, 138, 142 Clustered regularly interspaced short palindromic repeats (CRISPR), 111 Co-cultures, 142 Co-extraction, 128 Combustion, 72 Complementary power sources, 54 Contemporary biofuels, 65 Continuous-flow processing system, 73 Continuous flow vortex, 73 Conversion efficiency, 117 Corn, 19 carbohydrate-enriched, 19 ethanol-based biofuels, 19 COVID-19 pandemic, 147 CRISPR/Cas, 111 Cryogenic isolation, 71 Cupriavidus basilensis B-8, 137 Cyanobacteria, 10, 62 Cynobacteria, 26 D Dehydration, 56 Diallylimidazolium methoxyacetate, 113 Diatom, 9, 10 Docosahexaenoic acid (DHA), 128 Drop-in fuels, 73 Dry milling, 58 E Eco-friendly approach, 114 Economic aspects biodiesel transport, 129 biofuel production, 128

160 Economic aspects (cont.) cost, 129 ethyl alcohol, 129 financial assistance, 129 petroleum products, 129 regulatory network, 129 Economic competitiveness, 151 Eicosapentaenoic acid, 128 Electrical power, 2 Energy, 15, 16 biodiesel, 53 bioethanol, 54 biofuel, 52 crops, 52, 131 economic growth, 52 European Union countries, 53 fossil fuels, 52 requirements, 53, 74 security, 11, 129, 130 transporters, 53 Engineered Klebsiella pneumoniae, 115 Environment and energy protection, 131 Environmental benefits, 131 Environmental-friendly processes, 147 Environmental pollution, 148 Environmental scenario and conflict, 51 Enzymatic hydrolysis, 66, 67, 114 Enzymatic pretreatment, 142 Enzyme recycling, 138 Ethanol formation, 59 Ethanol generation, 59 Ethanol production, 22, 23, 58 Eutrophication process, 61 Extraction methodology, 150 Extrusion, 113 F Factorial design of experiment (DOE), 117 Fast pyrolysis process, 72 Fat/oil triacylglycerols, 53 Fatty acid desaturase 2 gene, 111 Fatty acid methyl esters (FAMEs), 60 Fatty acyl methyl esters (FAMEs), 108 Fed-batch bioreactor cultures, 142 Fermentable sugars azeotropic, 56 biological methods, 58 dehydration, 56 ecological harms, 57 Leuconostoc, 57 magnesium, 57 programmed sugar cane harvest, 57 as raw materials, 56

Index sugary substances preservations, 57 sweet sorghum stem juice, 57 vinasse, 58 Fermentation, 89 First-generation biofuels corn and sugarcane, 90 ethanol and biodiesel, 90 fermentable sugars, 56–58 fermentation process, 56 processes, 56 raw materials, high starch content, 58–59 sources, 56 soybean oil and palm oil, 91 sugar and ethanol, 90 traditional biofuels, 65 vegetable oil, 59–60 First-generation ethanol, 57 Fischer-Tropsch process, 93 Fluidic production, 73 Food crops, 16, 18 Fossil fuels, 2, 34, 52, 88 Fossil hydrocarbons, 1 Fourth-generation biofuels biological engineering, 64 biorefineries, 64 emission regulation, 62 engineered cyanobacterial growth, 62 enzymatic hydrolysis, 62 enzymatic saccharification, 62 ethanol production, 64 genetically modified organisms, 64 genetic engineering feedstock, 62 light assimilation, 62 microalgae, 62 microbes, 62 photosynthesis, 64 storage, 64 Fractionation techniques, 135 Fungal (Trichoderma reesei) cellulolytic enzyme endo-β-1,4-glucanase, 110 G Gasification, 72, 92 Gene suppression, 111 Genetically engineered cyanobacteria, 115 Genetically modified Escherichia coli, 64 Genetic engineering platforms, 142 Genre Mordellistena, 142 Global energy consumption, 33 Global warming, 33, 36 Glycolipids, 21 Green fuel, 55 Greenhouse gas emissions (GHG), 2, 52, 54

Index H Haematococcus pluvialis, 128 Halophile green microalga Dunaliella salina, 128 Harmful gases, 54 Human-made hydrocarbons, 66 Hydrogen, 25, 26 commercial production, 25 pretreatment methods, 25 respiratory metabolism, 25 source, 25 Hydrolyzation, 64 Hydrothermal distillation, 72 Hydrothermal liquefaction (HTL), 72, 73 Hydrothermal pretreatment technology, 113 I Industrialized platform, 58 Industrial waste, 96 Inhibitory compounds formation, 23 Innovative inexhaustible “energy sources”, 66 The International Energy Agency (IEA), 52 International Panel on Climate Change (IPCC), 33 Ionic liquids, 115 IonoSolv pretreatment, 113 J Jatropha, 20, 91 Jatropha crop plantation, 129 “Jatropha curcas and Ceiba pentandra” oils, 60 Jatropha curcas L., 111 K Keen energy crops, 74 L Large-scale biodiesel production, 142 Leuconostoc, 57 Levulinic acid production, 115 Life cycle analysis, 58 Life cycle assessment approach studies, 139 Lignin, 35, 36 biosynthesis, 36, 37 degradation, 36 genetic engineering, 36 plants and genomic tools, 36 Lignocelluloses, 52, 96, 115 Lignocellulosic/bioenergy materials, 67 Lignocellulosic biofuels, 60

161 Lignocellulosic biomass, 94, 97 Lignocellulosic biomass-derived biofuel biodiesel production, 138 bioethanol, 136 biosynthetic strategy, 136 challenges, 138 Clostridium sp., 137 consortia, 137 enzymatic digestibility, 137 enzymatic digestion, 137 ethanol productivity, 138 fungal pretreatment, 137 genetic modification/metabolic engineering, 138 5-HMF and furfural, 138 interaction mechanisms, 138 microbial consortia, 137 optimum fermentation, 138 physicochemical methods, 137 production cost, 137 time consumption, 137 Lignocellulosic feedstocks, 54 Lipids, 40 accumulation, 142 energy molecules, 40 extraction, 140 metabolites, 40 in microalgae, 41 in plant seeds, 40 source, 91 TAG, 40, 41 Long-chain fatty acid methyl esters, 59–60 Long-chain polyunsaturated fatty acids (LCPUFAs), 128 Long-last pyrolysis process, 72 M Macroalgae, 18, 61 advantages, 7 agriculture, 7 benefits, 7 biofuel, 3, 4 biomass, 4 biotechnology, 3 carbon dioxide, 2 cellulose, 2 cultivation, 18 diversity, 6 fossil fuels, 4 growth and development, 6 multicellular microorganisms, 4 technologies, 2 use, 5, 6

Index

162 Mesophilic microbial consortium N3, 137 Metabolic engineering approach, 110 Metabolic pathways, 142 Metabolites, 128 Metal particles, 51 Methane gas, 127 Methanol, 91 Microalgae, 17, 18, 35, 40–42, 62 Microalgae’s cultivation, 141 Microalgal and bacterial biomasses, 143 Microalgal and lignocellulosic biomass, 67 Microalgal biofuels processing techniques, 63 Microalgal biomass productivity, 139 Microalgal cultivation, 88 Microalgal metabolic technology, 62 Microfluidic photobioreactors, 139 Microfluidic techniques bioethanol generation, 99 biohydrogen, 98 chemicals, 95 industrial waste, 96 lignocellulosic substances, 97 microalgae, 95 oil extraction, 100 Microorganisms, 56, 66 Microwave, 113 Microwave-assisted extraction, 140 Mixed culture algae, 74 Modelling optimizations AI, 117 ANFIS, 118 ANN-associated particle swarm algorithm, 118 ANN-based analysis methods, 118 biological processes, 118 calcium oxide, 118 CCD, 117 conversion efficiency, 117 DOE, 117 hydrogen production, 119 integrated biogas-wastewater treatment plant, 119 microbial electrolysis, 119 OVAT, 117 physical performance, 116 proximate analysis, 118 RSM, 116–118 transesterification, 117 two-level factorial design, 117 variables, 116 Molecular hydrogen, 88 Municipal waste, 74

N NADP oxidoreductase, 88 Nannochloropsis oceanica, 110, 139 Nannochloropsis salina, 118 Nannochloropsis sp. biomass, 140 Nannochloropsis spp., 110 National Audubon Society, 131 Native crop bacteria, 57 Nitrogen oxides, 51 Non-cellulosic polysaccharides, 73 Non-linear noisy processes, 119 Non-starch and starch components, 58 Non-volatile hydrophobic ILs, 115 NOx emission, 132 O Oak Ridge National Laboratory (ORNL), 131, 132 Oil extraction, 100 Oleaginous organisms, 62 One-variable-at-a-time approach (OVAT), 117 Organic solvent-based methods, 140 Organic wastes, 66, 67 Organization for Economic Cooperation and Development (OECD), 52 Oxidation methods, 53 Ozone-rich microbubbles, 113 Ozonolysis, 113 P Palm oil, 20 Penicillium oxalicum, 116 Peroxidation, 53 Petroleum, 55 Phosphomolybdenum-based sulphonated ionic liquid-functionalized MIL-100(Fe) metal-organic framework, 115 Photobioreactors, 100 Photo-conversion efficiency, 108 Photolysis, 25, 26 Photosynthetic microorganisms, 35 Photosynthetic organisms, 35, 88 Phycobiliproteins, 128 Physical partition, 70 Plant biomass, 88 Plants and algae, 16 Pollutants, 148 Pollution, 131 Polysaccharide, 22, 97 Polysaccharide-degrading enzymes, 137

Index Polyunsaturated fatty acids (PUFA), 42, 43, 150 Pretreatment optimization, biodiesel production acid hydrolysis, 113 alkaline pretreatment, 113 attrition mill, 112 biochemical routes, 112 biological strategies, 114 environmental friendly, 112 extrusion, 113 hemicellulose fraction, 113 hydrothermal routes, 112 ionic liquids, 113 IonoSolv pretreatment, 113 lignocellulosic biomass, 114 microwave, 113 ozonolysis, 113 physicochemical pretreatment approaches, 113 pure cultures, 114 scCO2, 114 steam explosion, 114 thermochemical routes, 112 vibro-ball milling, 113 Pretreatment process, 67 Product flea market, 92 Production efficiency, 114 Prokaryotic cyanobacteria, 110 Proteins, 127 “Prunus armeniaca L.” oil, 60 Pyrococcus furiosus, 116 Pyrolysis, 23, 72 R Rancimat methods, 53 Raw materials optimization, biodiesel production aim, 108, 109 bio-based raw material, 110 CO2 concentration, 108 combined transgenesis, 111 CRISPR/Cas technology, 111 edible plant material, 107 gene suppression, 111 genetically modified organisms, 107 genetic engineering, 110 light intensity and NaNO3 concentration, 108 lipid metabolism, 111 OsSUS3 gene, 110 pretreatment alternatives, 110

163 pretreatment process, 110 protein and metabolic engineering, 110 synthetic metabolic pathways, 110 TAG accumulation, 108 transgenesis, 111 Refrigeration, 70 Renewable energy industries, 2 Renewable energy resources, 2 Response surface methodology (RSM), 60, 116 Rhodococcus opacus, 141 Rhodosporidium toruloides cultures, 142 Rhodotorula glutinis, 142 RNA interference (RNAi), 111 Rural cost-effective development, 131 S Saccharification, 54, 58 Saccharomyces cerevisiae, 118 Saponification, 73 Scenedesmus obliquus (microalgal species), 116 Scenedesmus species, 61 Second-gen biofuels, 55 Second-generation biofuels agricultural and woody wastes, 60 alkali transesterification, 60 biochar, 92 bio-path, 93 cellulose, 94 chemical processes, 94 definition, 92 fabrication, 92 generation parameters, 60 generation techniques, 60 lignocellulosic, 60 methanol, 93 Second-generation biomass, 52 Second Industrial Revolution, 147 Sesamum indicum L. oil, 118 Sesquiterpene biodiesel precursor (E)-α-­ bisabolene, 110 Silica gel, 69 Siloxanes, 69 Single-step transesterification, 60 Sorghum-chopped sweet grains, 58 Sorghum grains, 58 South-South linkages, 52 Spectrophotometer FT-NIR, 59 Spirulina, 127 Steam explosion, 114 Straw/wood residue crops, 52

Index

164 Substrate characteristics, 68 Sugar, 88 Sugar cane Brazil, 19 production, 19 Sulphur dioxide, 51 Superacid SO4H-functionalized ionic liquid, 113 Supercritical carbon dioxide (scCO2), 114 Supplementary fertilizers, 129 Supplementary products, 128 Sustainability, 129 Sustainable Development Goals, 152 Switchgrass, 20 Synechococcus nidulans, 118 T Terpenoid hydrocarbons, 89 Terrestrial plants, 34 Thermal decomposition processes, 72 Thermo method, 92 Thermoanaerobacterium saccharolyticum, 115, 138 Thermoanaerobacter species, 90 Thermochemical methods, 71 Thermochemical translation processes, 72 Thermophilic framework, 68 Third-generation biofuels, 91, 94, 95 Toxic pollutants, 68, 148 Transesterification, 21, 53

Trebouxiophyceae, 10 Triacylglycerol (TAG), 108 Trichoderma reesei, 116 U Union of Concerned Scientist (UCS), 131 Urbanization, 51 USDA National Resource Inventory”, 132 V Vegetable oils by-products, 59 feedstock, 59 hydrocarbon-based source, 59 soybean oil, 59 waste edibles, 59 Vibro-ball milling, 113 Vinasse, 57 W Wastewater-rich exhaust gas, 62 Wastewater treatment, 141 Water vapour evacuation, 70 Z Zymomonas mobilis, 115