Algal Biofuel: Sustainable Solution 9781032425450, 9781032425467, 9781003363231

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Algal Biofuel

SUSTAINABLE SOLUTION

Algal Biofuel SUSTAINABLE SOLUTION

Editors

Richa Kothari • Vinayak V Pathak • V VTyagi

THE ENERGY AND RESOURCES INSTITUTE

Creating Innovative Solutions for a Sustainable Future

First published 2023 by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN and by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 © The Energy and Resources Institute CRC Press is an imprint of Informa UK Limited The right of the contributors to be identified as the author(s) of this work has been asserted in accordance with sections 77 and 78 of the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying and recording, or in any information storage or retrieval system, without permission in writing from the publishers. For permission to photocopy or use material electronically from this work, access www. copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Print edition not for sale in South Asia (India, Sri Lanka, Nepal, Bangladesh, Pakistan or Bhutan). British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 9781032425450 (hbk) ISBN: 9781032425467 (pbk) ISBN: 9781003363231 (ebk) DOI: 10.4324/9781003363231 Typeset in Times New Roman by TERI Press, New Delhi – 110 003

Foreword

Application of microalgal biomass as a renewable source of energy has gained the attention of researchers around the world. Biomass-based fuel generation became more important after the 1990 oil crisis. Deterioration of air quality and climate change appeared as a result of rapid consumption of fossil fuel. Hence, it is extremely important to develop sources of energy that can not only replace the conventional fuel but also have a positive impact on the environment. Algal Biofuel: Sustainable Solution primarily focuses on the different aspects of bioenergy production using algal biomass as microalgae are considered the optimum feedstock for bioenergy production. The major aim is to thoroughly review the available bioenergy options, challenges in bioenergy production, availability of bioenergy feedstock, and biomass to bioenergy conversion process. This book also highlights the feasibility of lignocellulosic biomass, crop residues, and non-edible oil seeds for generation of different bioenergy products. It will be helpful for researchers and other stakeholders working in the area of bioenergy production for development of innovative concepts in emerging areas of bioenergy. It is not possible for a single book to convey all dimensions of the bioenergy technologies, so application of algal biomass for other value-added products has not been included in this book. Algal Biofuel: Sustainable Solution begins with an introduction of a biorefinery approach and its importance in promoting bioeconomy (Chapter 1). With a biorefinery approach, we can implement bioenergy in less economically advanced countries and create new job opportunities in the bioenergy sector. Microalgae are considered as potential bioenergy feedstock and this source of energy is constantly being researched, so the recent advancements happening with microalgae are presented in Chapter 2. In Chapter 3, potential of biogas as a bioenergy option is discussed with support of recent updated trends in the fields. Chapter 4 provides an overview on the potential of algal-based biohydrogen as afuture bioenergy option. Crop residues, which are mainly composed of lignocellulosic biomass, can be used for bioenergy production as a part of waste management process. In this context, Chapters 5 and 6 review the comparative

vi

Foreword

potential of lignocellulosic biomass and algae for bioethanol production and the potential of crop residues for bioenergy production. The main downstream process in algal biofuel production involves harvesting. Chapter 7 provides a detailed analysis on magnetic harvesting of algal biomass for cost effective biofuel production. The potential of non-edible oil for biodiesel production is explained in Chapter 8. Chapter 9 not only reflects on the bioenergy application of algal biomass but also its key role in climate change mitigation through carbon sequestration. Environmental aspects of biomass-based bioenergy generation is equally important to ensure the sustainability of bioenergy. Thus, Chapter 10 describes the role of modern bioenergy applications for control of particulate emissions. In Chapter 11, applicability of immobilized algal cells for wastewater treatment is reviewed, which is extremely important to achieve a cost-effective biofuel production process. The role of a biotechnological approach for generation of various bioenergy products is explained in Chapter 12, while methods for enhancement of lipid content, which is one of the key attractions for researchers, is reviewed in Chapter 13. By providing this information in an easily accessible format, my hope is that newer workers in this field will be able to produce more reliable research, which will be significant to understanding newer dimensions of bioenergy technologies based on sustainable feedstock.

Professor John Korstad

Oral Roberts University

Preface

Energy sustainability has become a very important concern among researchers and policymakers. Present energy situation around the globe is unsustainable due to unequal distribution of natural resources as well as different environmental, geopolitical, and economical concerns. Rise in population with accelerated increase in industrial sector has led to rapid increase in the consumption of energy sources, which will make them extinct soon. Major developing countries such as India are heavily dependent on fossil fuels (coal, oil, natural gas) to meet their energy demand of the transportation and industrial sector. Further, to combat environmental pollution and mitigate the effects of greenhouse gases, it has become imperative that an alternative energy source is found which is sustainable and renewable. Biofuel is one such renewable, sustainable, and affordable energy source that has the potential to replace conventional energy sources. Biomass has been directly combusted for a long time; however, it generates severe incontinence cost and pollution cost. Therefore, modern applications that use advance conversion processes can make biomass more efficient for energy production. By employing specific conversion processes, bioenergy can be derived in solid, liquid or gaseous forms. Liquid biofuels such as biodiesel and bio-alcohol can replace fossil fuels. They can also be used in combination with fossil fuels so that the amount of fossil fuel consumption can be reduced. Despite such promising potential, biomass-based energy generation requires intensive research and development to become sustainable and affordable. Algal Biofuel: Sustainable Solution provides focused information on progressive trends in research and development with conversion strategies under the impactful stress conditions in bioenergy sector. The most-effective ways to valorize the available biomass resources with the concept of biorefinery, where each part of this biomass is utilized with generation of zero or near to zerowaste, have also been included in this book. Governments of many countries have constituted policy frameworks that ensure the optimum consumption of fossil fuel and develop renewable energy

viii Preface sources. The policy initiatives in various countries have set a target to blend diesel with bio-alcohol and biodiesel so that dependency on fossil fuels can be reduced. The book points out the governance of biofuel at global and national level and explores the potential of biofuel in meeting the rising energy demand. Conventional feedstock of first-generation biofuel is associated with foodfeed and biofuel trilemma; hence, it has less impact as a fossil fuel alternative. Similarly, second-generation biofuels are also involved with high water input and fertilizer support for feedstock development. Researchers have explored the potential of microalgae as a cleaner and greener alternative for fossil fuel replacement. Various algal strains of marine and freshwater habitat have been successfully investigated for high lipid accumulation, which is considered as precursor of biodiesel. The availability of algal biomass can be ensured for biofuel production process by employing effective cultivation and harvesting techniques, which is also termed as upstream processing for biofuel production. Algal biomass can be grown in open as well as close system and in this book authors have investigated the optimum culture conditions for enhancement of biomass. Researchers have found that culture conditions influence the lipid synthesis in algal biomass to a greater extent. Nutrient starvation and other stress conditions have been studied for the lipid induction in algal biomass. The practicality of these investigations for large-scale biodiesel production is still unexplored and plays crucial role in developing algal-based biofuel sector. The other technical step towards biodiesel production involves the conversion of fatty acid into fatty acid methyl esters. Conventional ways of transesterification have been found with limited yield efficiency and direct environmental impact. The approach of green transesterification process and other innovative conversion process have been found with higher efficiency than the conventional process. The feasibility of algal-based biofuel production depends on its yield efficiency and should be compatible in demand and supply chain. The chapters in this book focus towards the strategies to ensure the availability of algal biomass, effective cultivation, and harvesting techniques. The book explores a wide spectrum of bioenergy sources, including their applications. It also covers the latest information in the field of bioenergy technologies and their future prospect in relation to algal biomass. Bioenergy scenario provides waste to energy generation potential of various energy wastes including agriculture and solid waste. The book also gives impactful solutions to practical challenges with bioenergy technologies such as microalgal lipid for biodiesel extraction processes, different biotechnological approaches for bioenergy production, biodiesel from non-edible seeds among others. This book will be beneficial to both students and researches in the field of bioenergy generation.

Contents

Foreword Preface

v

vii

1. Biorefinery: A Future Approach for a Sustainable Bioeconomy 1

Introduction 1

Role of Government, Public, and Private Stakeholders 13

Conclusion 17

2. Algal Biomass Harvesting for Biofuel Production Introduction Harvesting Process Future Prospects and Conclusions

23

23

26

48

3. Biogas as Bioenergy Option: Advances and Challenges Introduction Biochemical Processes of Anaerobic Digestion Feedstock Materials Microbial Community Important Parameters Properties of Biogas Upgradation of Biogas Types of Digesters

Applications of Biogas Challenges/bottlenecks Conclusion

67

67

69

72

74

74

78

79

81 81 85 87

4. Application of Algal Biomass as a Feedstock for Fermentative Biohydrogen Production Introduction Microalgae

93 93 95

x

Contents

Advantages and Limitations of Biohydrogen from Microalgae Conclusion

101

103

5. Bioethanol Production from Lignocellulosic/Algal Biomass: Potential Sustainable Approach

Introduction Bioethanol from Lignocellulosic Material Conclusion

107

6. Crop Residues as a Potential Substrate for Bioenergy Production: An Overview

Introduction Agricultural Residues for Bioenergy Production Biomass-to-bioenergy Conversion Pathways Conclusion

121

7. Magnetic Harvesting of Microalgae Biomass for Cost-effective Algal Biofuel Production

Introduction Magnetic Materials for Microalgae Harvesting Factors Influencing Magnetic Harvesting Process Recovery of Magnetic Materials and Biomass Detachment Biocompatibility of Magnetic Nanoparticles and Recovery of Growth Medium

Conclusion

139

8. Biodiesel Production from Non-edible Oilseeds Introduction Non-edible Oilseeds as Biodiesel Feedstock Properties of Free Fatty Acids in Non-edible Oils Biodiesel Production Technology for Non-edible Oilseeds Fuel Properties of Biodiesel Economic Feasibility of Biodiesel from Non-edible Oils Advantages of Non-edible Oilseed Crops National Efforts to Promote Non-edible Plant Species Conclusion

149

149

151

159

163

166

167

168

169

170

9. Carbon Sequestration and Biofuel Production by Microalgae: An Integrated and Sustainable Approach

Introduction Carbon Sequestration

183

107

108

116

121

122

127

133

139

140

142

144

145

146

183

184

Contents

Carbon Sequestration by Microalgae Carbon Concentrating Mechanism Algal Species Factors Affecting Microalgal Efficiency Biosynthesis of Lipids in Microalgae Microalgal Biomass Harvesting and Processing Microalgal Lipid Extractions Production of Biofuels Chemical Conversion Thermochemical Conversion Biochemical Conversion Conclusion

xi

185

187

188

190

193

193

194

197

198

198

200

201

10. Role of Meteorological Parameters on Atmospheric Aerosols Concentration and Its

Control Through Modern Biomass Application

Introduction Aerosols Shape and Size of Aerosols Aerosol Sources Aerosol Removal Processes Implications of Aerosols Brief History of Earlier Aerosol Studies Experimental Technique Used Results and Discussion Modern Biomass Application to Control Aerosol Emission Conclusion

209

11. Application of Immobilized Algae in Water and Wastewater Treatment

Immobilization Immobilization Techniques Application of Immobilized Algae Nutrient Removal Metal Removal Removal of Organic Compounds Lipid Content of Immobilized Algae Conclusion and Future Work

237

209

209

210

210

212

212

213

214

215

232

233

237

237

246

247

248

250

252

253

xii Contents 12. Recent Biotechnological Approaches for Bioenergy Production: The Path Forward

Introduction Biotechnological Approaches in Bioenergy Production Different Biotechnological Approaches for Bioenergy Production Biotechnology and Sustainable Society Conclusion

259

13. Lipid Induction in Algal Biomass for Sustainable Bioenergy Production

Introduction Composition of Microalgal Lipid Factors Affecting Lipid Productivity in Algal Biomass Engineering Efforts of Lipid Enhancement Conclusion

273

Index



About the Editors

259

260

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Biorefinery: A Future Approach for a Sustainable Bioeconomy

CHAPTER



Arya Pandey1, Shamshad Ahmad1, Richa Kothari1,2, Vinayak V Pathak3, and V V Tyagi4 1

Bioenergy and Wastewater Treatment Laboratory, Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India 2 Department of Environmental Sciences, Central University of Jammu, Rahya Suchani (Bagla), Samba, Jammu, Jammu and Kashmir, India 3 Department of Chemistry, Manav Rachna University, Faridabad, Haryana, India 4 Department of Energy Management, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India

INTRODUCTION The global energy consumption is on the rise with increasing world population, high standards of living, and energy-use pattern. Presently, the basic feedstock to produce commodity fuel and chemicals is crude oil. The rapid depletion of crude oil is creating pressure on the transportation sector and the aviation industry as well as on the environment in terms of pollution (CO2, CH4, N2O, etc.). In this regard, Ministry of Environment, Forest and Climate Change (MoEF&CC) issued 29 warnings to Oil Refinery Limited, as these oil refineries crossed their permissible emissions rate of NOx, organic carbon, CO, and particulate matter from 2012 to 2015. Hence, it becomes important to investigate an alternative to petro-based refinery to minimize the environmental pollution load. Recently, researchers focused on the advanced biorefinery concept of using different feedstock to produce a broad spectrum of main and co-products in a single biorefinery system (Ghosh, Dasgupta, Agrawal, et al. 2015; MingXiong, Jing-li, Quin, et al. 2014; Hasunuma, Okazaki, Okai, et al. 2013). As fossil fuel-based refineries have been unsustainable, economically unviable, and limited (Han, Forman, Elgowainy, et al. 2015; Elgowainy, Han, Cai, et al. 2014), hence, efforts have been put towards developing bioenergy and bioproducts from biorefineries. Biomass-based energy and high-value product generation have huge potential towards addressing energy-related challenges and

2

Algal Biofuel: Sustainable Solution

issues of environmental security. Biorefineries have the potential to process one feedstock and produce numerous end products, including transportation fuels (biodiesel, biohydrogen, biogas, bioethanol, and biomethanol) and value added products (cosmetics, nutraceuticals, biofertilizers, bioplastics, etc.) (Kothari, Pandey, Ahmad, et al. 2017; De Jong, Higson, Walsh, et al. 2012a; Cherubini, Jungmeier, Wellisch, et al. 2009). The concept of biomass-based biorefinery (BBBR) is an integrated approach, capable of producing multiple products which are crucial for commercialization of biomass-based products and mitigating the adverse effects of local ecosystem services (De Jong, Langeveld, Van Ree, et al. 2009). By implementing the biorefinery concept, the market potential of biomass can be increased manifold and, at the same time, minimize the generated biowaste that is discarded without any treatment, and affecting the environment (Jungmeier, Hingsamer, Van Ree, et al. 2013a). The biorefinery manufacturing techniques coupled with agro-energy crops (copra, castor seed, sesame, groundnut kernel, rapeseed, palm kernel, mustard seed, sunflower, palm fruit, soybean, cottonseed, etc.) can ensure sustainable development in biomass-based bioproducts and biopower, leading to a conceptual change from petro-refinery to biorefinery (De Jong, Higson, Walsh, et al. 2012a; Sanders, Scott, Weusthuis, et al. 2007)). The relevance of biorefinery has been successfully reported by the specific IEA Bioenergy Task1 on biorefineries, production of fuel, biopower, biomaterial, high-value chemicals, etc. The upstream, midstream, and downstream processes of biomass to produce a wide range of bioproducts is an integral part of biorefineries. Various bioprocess routes have been in practice for the conversion processes – thermochemical, chemical, enzymatic, and biological (fermentation, aerobic, and anaerobic) (De Jong, van Ree, Sanders, et al. 2010; Abraham and Hofer 2012; De Jong and Gosselink 2014). In the biorefinery concept, various biomass raw materials such as agricultural crops, household biowastes, biowaste residue from industry, forestry, and agricultural-based wastes are being used in the production of multiple products such as biopower (biogas, biohydrogen, biodiesel, and bioethanol) and value added products (fertilizers, food and fodder, cosmetics, medicines, etc.). This has been illustrated in Figure 1. Biorefineries are anticipated to have benefits in an economically viable, socially valuable, and environmentally sustainable manner. They have shown promise in developing and industrialized countries (Van Putten, van der Waal, de Jong, et al. 2013). The electricity-based biorefinery processes different biomass for the production of biofuel, heat, etc., and the generated residues in the process routes can be used for internal heat system (Singh, Kothari, Gupta, et al. 2019a; Schaffenberger, Ecker, Koschuh, et al. 2012). Hence, biorefineries are sustainable approaches because they optimize the economic growth and 1

Details available at http://www.biorenery.nl.

      

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Biorefinery: A Future Approach for a Sustainable Bioeconomy

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4

Algal Biofuel: Sustainable Solution

environmental benefit. (Kokossis and Yang 2010). However, the prices and volumes of the products should be competitive in order to maximize the market potential. It is important that the biorefineries be upgraded and extended as per the current industrial infrastructure and then incorporated into the market sector. Presently, the new biorefinery concepts still need research and development (R&D) to successfully transition from pilot- or lab-scale demonstration to the commercial phase. The BBBR concept uses ecosystem services, which are nonexhaustible sources, compared to petrochemical refineries (Patel, Meesters, den Uil, et al. 2013) as shown in Figure 2. Under customary conditions, a strawfed 150 kt biorefinery in Europe is unlikely to be cost-effective. Replacement of straw by hardwood or SRC poplar increases the production of biomass. However, even in this case Harwood or SRC popular case production, an internal rate of return threshold of 25% was not surpassed. A larger biorefinery capacity (>250 kt dry biomass per year) improves the internal rate of return and is cost-effective, as is evident in a rice straw-fed 500 kt biorefinery in India.2 A BBBR is expected to expand at a theoretically calculated annual growth rate of 7.7%; as a consequence, the market value of produced renewable chemicals increased to $83.4 billion by 2018. The BBBR concept is anticipated to efficiently utilize natural resources (biomass) in a sustainable manner to fulfill the growing demands of society while mitigating the effects of climate change. In this chapter, we discuss the global and development scenarios of using biomass with regards to BBBR. The current status and progress of BBBR are mainly demonstrative and carried out within a small-scale arena. Therefore, research and technical fulfillness are extremely necessary to sustain the concept of BBBR for economic growth and development.

Classification and Multi-product Formation in Biorefinery System The biomass-based biorefinery product can be classified on the basis of direct and indirect used substrate as feedstocks and technologies employed in processing manufactured products (main and intermediate). Many existing industries such as sugar, starch, vegetable oil, pulp and paper, and feed, etc., based on the biomass, come under conventional biomass-based biorefineries (CBBBR) (Freddy, Navarro-Pineda, Robert, et al. 2016; Dinjus, Arnold, Dahmen, et al. 2009; Ekhart, Van, and Oekraïne 1999), as explained in Figure 2. The main shortcoming of CBBBR is that it focuses on the main products and neglects the broader spectrum of other value-added products that could be produced from the same biorefinery. The new BBBR optimally uses biomass at different stages and focuses on the production of main and by-products, as described in Table 1. 2

Details available at http:// www.biocore-europe.org/file/BIOCORE_D7_6_Integrated %20 assess­ ment_ 2014-03-31.pdf

 

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Biorefinery: A Future Approach for a Sustainable Bioeconomy

5

6

Table 1 Distinctive characteristics of biorefinery concept

Biorefinery type

Feedstock used

Predominant technology

Developmental scenario

Generated products

Reference

Whole crop

Grain and straw, cereals, wheat, maize, etc.

Dry or wet milling followed by consequent fermentation and distillation of grains

Pilot plant

Starch, cellulose, proteins, oil

Abraham and Hofer (2012); Zhang, Rong, Chen, et al. (2014)

Oleochemical

Oil crops (Jatropha, rapeseed, etc.)

Biological conversion, transesterification

Pilot plant

Chemicals, functional monomers, lubricants, surfactants

Kazmi (2012)

Lignocellulosic feedstock

Biomass rich in lignocelluloses

Pretreatment of biomass, hydrolysis (chemical and enzymatic), fermentation, separation

R&D/pilot plant, demo in the United States of America

Bioethanol, butanol, hydrogen, value-added chemicals, polymers

Ahmad, Pathak, Kothari, et al. (2018a); Abraham and Höfer (2012)

Thermochemical

All types of biomass

Pyrolysis, gasification, products separation, catalytic synthesis

Pilot plant

Liquid fuel, biodiesel, biochemicals, biopolymers, biohydrogen

Pathak, Ahmad, Pandey, et al. (2016); Haro, Ollero, Ángel, et al. (2013); Irshad, Anwar, But, et al. (2013)

Next-generation hydrocarbon BR

Lignocellulosic biomass

Biochemical and R&D thermochemical conversion

Green gasoline, green diesel, and green jet fuel

Subhadra and George (2011)

Cell disruption, product extraction/separation, and purification

Bio-oils, carbohydrates, starch, vitamin, protein, food and feeding material for animals, functional food for humans

Aquatic biomass-based biorefinery Aquatic and marine BBBR

Micro and macroalgae, Cyanobacteria, seaweeds

BBBR: Biomass-based biorefinery; R&D: Research and development

R&D, pilot plant

Borowitzka (2013); Tong, You, and Rong (2014); Zhu (2015); Usher, Ross, CamargoValero, et al. (2014); Zhang, Rong, Chen, et al. (2014)

Algal Biofuel: Sustainable Solution

Terrestrial biomass-based biorefinery

Biorefinery: A Future Approach for a Sustainable Bioeconomy

7

Currently, new biorefinery concepts are in developmental, pilot, or smallscale demonstration phases without large-scale commercialization and having low-market potential. Hence, there is an urgent need to overcome the hurdles in the way of implementing the biorefinery concept in market to uphold the green economy (Ghosh, Dasgupta, Agrawal, et al. 2015). A BBBR promotes several different types of biomass products which range from high-value chemicals to low-value chemicals, as given in Table 2.

Table 2 Multi-product formation through biomass-based biorefinery concept Terrestrial biomass

Technology type

Product

Woody biomass Fischer–Tropsch synthesis

Syngas

Generation of bioproducts

Power generation, lower alcohol (Fischer–Tropsch Hydrogenation Synthetic diesel), di-methyl biofuel ether, liquid Hydro-treatment of Hydro-treated hydrocarbon (ranges bio-oils renewable jet from C1 to C50 ) fuel

Biomass with moisture (manure, biowaste from food industry)

Reference Malherbe and Cloete (2002)

Thermal gasification

Syngas to methane

Anaerobic digestion

Biogas

Biopower, residue can be used as feeding substrate of animal

Mandl 2010

C6 sugar

Amino acids, enzyme, lactic acid, vitamins, antibiotics, xanthan, succinic acid, itaconic acid, adipic acid, glutamic acid, aspartic acid, 3-hydroxypropionic acid, isoprene, etc. Products through transformation of chemicals (sorbitol, furfural, glucaric acid, hydroxyl methyl furfural, and levulinic acid, animal feed (protein rich)

Ahmad, Pathak, Kothari, et al. (2018a); Janke, Leite, Batista, et al. (2016); Schaffenberge, Ecker, Koschuh, et al. (2012)

Agricultural Fermentation biomass, sugar/ starch-rich biomass (wheat, corn, cassava, sugar beet, sweet sorghum, and sugar cane)

Contd...

8

Algal Biofuel: Sustainable Solution

Table 2

Contd...

Terrestrial biomass

Technology type

Product

Generation of bioproducts

Reference

Oil-based biomass (Jatropha, coconut, palm, sunflower, rapeseed, castor, jojoba seeds, desert shrubs, etc.)

Transesterification

Seed-based oil

C12 and C18 monounsaturated and saturated fatty acids, unsaturated oil, oleic acid, bio-lubricants, biodiesel, lipid, biooils, alkyl esters, glycerol, etc.

Ahmad, Kothari, Pathania, et al. (2019); Ahmad, Pathak, Kothari, et al. (2018b); Anwar, Gulfraz, and Irshad (2014)

Bioenergy

Syngas products (methanol, ethanol, mixed alcohols), hydrocarbons (benzene, toluene, xylene, cyclohexane), phenols (catechols, resorcinols, eugenol, coniferols, guaiacols), macro-molecules, resins, composites, polymers, aromatic compounds, carbonfibres, etc.

Anwar, Gulfraz, and Irshad (2014); Kuhad, Gupta, Khasa, et al. (2011); Shahriarinour, Ramanan, Abdul Wahab, et al. (2011)

Biofuel, antimicrobial, antifungal, antiviral agents, toxins, aminoacids, proteins, sterols, MAAs for light protection, vitamins A, B1, B6, B12, C, E, biotin, riboflavin, nicotinic acid, pantothenate, folic acid, C-carotene, astaxanthin, lutein, zeaxanthin, canthaxanthin, chlorophyll, phycocyanin, phycoerythrin, fucoxanthin, human nutrition, pharmaceutics and therapeutical application, green plastics, etc.

Pathak, Ahmad, and Kothari 2019; Ahmad, Pandey, Kothari, et al. 2017; Zhu 2015; Tong, You, and Rong 2014; Usher, Ross, CamargoValero, et al. 2014

Lignin-based Fermentation biomass (wood, straw)

Aquatic biomass Blue-green algae, macroalgae, microalgae, cyanobacteria, seaweeds

Transesterification, anaerobic digestion, fermentation, etc.

Biorefinery: A Future Approach for a Sustainable Bioeconomy

9

Though a large number of bioproducts can be produced from terrestrial and aquatic BBBRs, it is also important to amplify this concept without disturbing the sustainability of the ecosystem. In comparison to the aquatic BBBR, the terrestrial BBBR has various uncertainties as it requires intensive fertile land (induces fuel verses food conflict), with an excess of nutrients (fertilizers), fresh water, electricity, manpower, etc. On the other hand, the aquatic BBBR (microalgae and macroalgae) requires barren land for cultivation, wastewater as substrate, CO2 from flue gases for growth and development of biomass, which can be further used to produce end products applicable in the food, health, cosmetics, medicine, and energy sectors (Kothari, Ahmad, Pathak, et al. 2019; Singh, Pathak, Chopra, et al. 2019b; Chisti 2012; Pittman, Dean, Osundeko, et al. 2011). Hence, aquatic biomass-based biorefinery is potentially more sustainable than terrestrial biomass. Similarly, saccharification and co-fermentation techniques have been adopted for ethanol production. In order to minimize the cost of enzyme supply, an on-site cellulose enzyme production unit can be modelled in biorefinery simulation. Kaparaju, Serrano, Thomsen, et al. (2009) have reported 9364 MJ/tonne of multiple energy (biogas, bioethanol, and biohydrogen) generation by using wheat straw through the biorefinery concept. The authors have also reported 3572 MJ/tonne of bioethanol production using wheat straw in a single production system. Moreover, Kreuger, Sipos, Zacchi, et al. (2011) have also reported the co-production of ethanol and methane by using hemp seeds (Cannabis sp.) as raw substrate. The authors have also reported a high yield of processed dry matter (11,100–11,700 MJ/tonne), which was greater than the energy recovered by ethanol, formed alone from hexoses (4400–5100 MJ/tonne).

Future Developments in Key Sectors of Biorefinery For the successful implementation of BBBR concept in global business models, integration of all components (raw material, process routes, extraction, purification, and bioproducts) with significant key sectors (technical, strategic, sustainability, exploitation, logistic, and economic) is assessed for development and integration with the environment.

Technical Sectors Technical areas such as tissue culture technology, plant genomics, breeding programmes, biotechnology, genetic engineering, and chemical engineering of desirable traits must be taken into consideration for better development of biorefinery concept. Waste produced from lignin-based biomass is the major residue derived from biofuel plants, and constitutes 30% of the weight with 40% of the energy content of biomass (Pittman, Dean, Osundeko, et al. 2011). This lignin undergoes combustion and produces energy. But lignin poses a phenolic

10 Algal Biofuel: Sustainable Solution heteropolymer chemical structure, and can be converted into several numbers of value added products (for example, aromatics, phenolic resins, carbon fibre, adhesive) (Sklavounos 2014). The technologies used for extracting the residual lignin-based high-value chemicals are under process, and further developments should be made for achieving zero-waste production. Microbial cell factories have the potential to produce desired products in large quantities (Wachtmeister and Rother 2016) but a large number of intermediate compounds within the process are usually discarded as waste due to lack of technical advancements in the processing units (i.e., pretreatment, bioprocessing, biocatalysis, and polymerization). In order to access pilot-scale facilities of biorefinery technology, bioprocessing techniques must be integrated to increase production.

Sustainable Sector The main objective of switching from petro-based biorefinery to bio-based biorefinery includes the sustainable conversion of natural resources along with environmental protection as given in Figure 3. The direct and indirect land-use change and its associated effects such as greenhouse gases emissions and food security are serious concerns. The feedstocks used in BBBR system mostly comprise biomass grown in forest and agricultural land areas which means drastic reduction in carbon sequestration (Soudham, Brandberg, Mikkola, et al. 2014). Therefore, the direct and indirect land-use strategies to produce biomassbased bioproducts may have the potential to change the sustainability of the ecosystem. To cope with this problem, proper land-use strategies must be adopted and various stakeholders should play a significant role in the proper industrialization of the biorefinery concept. If government organizations, non­ governmental organizations, and private sector support the BBBR concept by investing in the R&D sector to protect the environment as well as maintain energy security bioprocess routes (conversion process), then product distribution chain, and infrastructure, food security can also be achieved through land-use changes.

Strategic Sectors Industrialization of bioproducts involves various strategic challenges – integration of biorefinery concepts into existing value chains, government, semi-government, and private funding difficulties, and many other uncertainties associated with novel, alternative, and unconventional biorefinery concepts. The researcher community needs to tackle the strategic challenges by improving the existing technologies to integrate the single product-based biorefinery to multi-product-based biorefinery system. They need to develop efficient process routes (physical, chemical, and biological) to produce end products for high- and low-value chemicals included in the bioeconomy-based market. These products

Biorefinery: A Future Approach for a Sustainable Bioeconomy

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can then be used for distribution, storage, and innovation of new trade routes (Zhu 2015; Schaffenberger, Ecker, Koschuh, et al. 2012) for the exploitation of substrates for maximum BBBR (Gallezot 2007).

Economic Sector An efficient BBBR system will minimize expenditure in infrastructure, production of biomass-based bioproducts, cost-competitive end products, pollution cost, and distribution supply chain. A biorefinery system associated with single-product process system is not economically feasible. On the one hand, a biorefinery system that is based on multiple products’ process system is. The integrated biorefinery processes have the potential to produce biomass-based products and at the same time serve as an energy carrier, that is, biopower, which is an efficient and emerging technology for a sustainable future with specific reference to biomass-based bioeconomy, as shown in Figure 4. The economic

12 Algal Biofuel: Sustainable Solution value of BBBR system is determined by the capital cost, operational cost, and the revenue generated by various products (Sklavounos 2014; WEF 2010; Ree and Annevelink 2007). The products with high-market potential are usually allied with high-production cost and vice versa. The economic viability of BBBR also depends on the size of the market. Therefore, to make the BBBR system economically feasible, supply and distribution chain infrastructure must

               

  

    

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Biorefinery: A Future Approach for a Sustainable Bioeconomy

13

correlate with the appropriate and best-suited products. To obtain a desirable biorefinery system, a frugal approach must be adopted. This includes the use of local resources and existing infrastructures, optimum conversion efficiency rate (biomass to multiple-product generation), integration of biorefinery with single-product formation with multi-product-based biorefinery that has a flexible and modular type of biorefinery installation, a streamlined supply chain, and distribution of products in a competitive market. Therefore, the ultimate challenge is to promote an economically feasible biorefinery concept.

Environmental Implications of Biorefinery An efficient biorefinery technology should have minimum harmful emissions and optimim utilization of substrates (Watanabe, Pereira, Chagas, et al. 2016; Goedkoop, Heijungs, Huijbregts, et al. 2016). The products generated at each step of biorefinery must ensure the standards in order to prevent negative health and environmental implications. The biorefinery products must be economically competitive as well as health-based products (e.g., protein, carbohydrates) generated from the biorefinery so that they can ensure new job opportunities and social well-being of the country (Cardoso, Chagas, Rivera, et al. 2015). Biorefineries utilizing lignocellulosic biomass can produce xylite, lignin, and ethanol, whereas conventional technologies used to obtain gasoline from crude oil emit pollutants that adversely affect human health and the environment. Today the focus should be on identifying innovative business models (see Figure 5), opportunities, and perspectives integrated with corporate social responsibility (CSR) in BBBR system to enhance the biomass-based bioeconomy, as illustrated in Figure 4. For the successful integration of CSR and BBBR, four important key factors must be abided by the corporate sectors and business groups: (i) altruism – the adaptation must be flexible for equitable distribution of resources to society to improve the quality of life of the citizens; comparators must be good and responsible corporate citizens; (ii) ethics – business groups must promote the right and fair obligation; (iii) legal – the BBBR concept must follow all legal responsibilities; and (iv) economics – the products generated by BBBR in integration with CSR must be economically viable so that these eco-friendly products are easily affordable. Hence, it is crucial to infuse CSR with BBBR for better encroachment of the biorefinery concept.

ROLE OF GOVERNMENT, PUBLIC, AND PRIVATE STAKEHOLDERS According to an extensive research, the present scenario of BBBR concept is flourishing in Europe and the USA (Biopol 2009; IEA 2009). But, for most of the countries, an advanced biorefinery concept is still a nascent idea. A survey conducted in 110 industrial sites by the Biopol and Biorefinery Eurovie projects, over 16 European nations with Norway, Switzerland, and the USA

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found that only 34 are working in a well-planned manner (Asgher, Ahmad, and Iqbal 2013). These biorefineries are mainly based on various concepts such as cereal biorefinery, whole crop biorefinery, oleo-chemical biorefinery, marine biorefinery, lignocellulosic feedstock, forest-based biorefinery, multiple feed/ integrated biorefinery, etc. In Europe, various biorefineries have been identified in R&D, pilot, and demonstration projects that have to be implemented in commercial sector in time in western, northern, southern Europe, respectively. Approximately, 75% of the total biorefineries have been established in Northern France, Germany, Denmark, Belgium, the Netherlands, and the UK, and they are using a variety of suitable feedstocks for processing. A positive correlation between the existing and the planned biorefineries has also been noticed. It was found that biomass such as agricultural crop residues, algal biomass, and other organic biomass generated from different domestic and industrial

Biorefinery: A Future Approach for a Sustainable Bioeconomy

Table 3

15

Indian organizations working on biomass-based biorefineries

Organizations

Functions

1. M i n i s tr y of N ew a n d Provides funding for biofuel-based research. Renewable Energy (MNRE) Prepared a draft for national policy on biofuel. 2. Ministry of Rural Development (MoRD)

Provides financial support to enhance biodiesel plantbased agriculture

3. Department of Biotechnology Provides research (DBT) demonstration of high plants. Facilitates research organisms involved in

facility for production and quality and yielding biomass and for biomass, enzymes, and biofuel production.

4. Indian Council for Agricultural Research (ICAR)

Works for coordinating research project on biofuel­ based biorefinery. Works towards protection of seeds, germ plasm, oilyielding plants. Provides biochemical and molecular characterization of biomass and provides training to researchers and farmers.

5. Tamil Nadu Agricultural University (TNAU)

Involved in collection of high-yielding varieties of biodiesel from Jatropha and evaluated them for development of hybrids. Launched 250 kg oil/day capacity pilot plant of biodiesel, which also produced 55 kg/day glycerol as by-product.

6. Indian Institute of Technology (IIT), Delhi

Performs research on BBBR from Karanja oil. Performs biogas production from agricultural biomass and de-oiled seed cakes of Jatropha and Karanja with production of valuable fertilizers.

7. The Energy and Resources Institute (TERI)

Worked on a project on National Mission on Biofuel sponsored by MNRE, India. Working on genetic modification of Jatropha and Pongamia sponsored by various government agencies.

8. Centre for Jatropha Production and Biodiesel (CJP)

Works towards scientific commercialization of Jatropha­ based biorefinery. Provides financial support for development and establishment of non-food oil crops.

9. Tree Oil India Limited (TOIL)

Aims to establish environment-friendly and sustainable energy systems based on plant sources.

sources is being used efficiently to obtain maximum bioproducts such as food, feed, chemicals, materials, and bioenergy through the implementation of the biorefinery approach (Mussatto and Dragone 2016; Dinjus, Arnold, Dahmen, et al. 2009). The sugar industry is a perfect example of BBBR in India for sugar, fertilizers, ethanol, heat, and electricity production (Sinha, Subramanian, Singh, et al. 2019). Various biomasses such as seeds of plants like Jatropha

16 Algal Biofuel: Sustainable Solution and Pongamia as well as algal BBBRs can be successfully operated in India (Ahmad, Kothari, Pathak, et al. 2019a; Jungmeier, Stichnothe, de Bari, et al. 2013b). On estimation, 100 kg of Jatropha oil, consisting of 24 kg methanol and 2.5 kg of NaOH, produces approximately 90 kg of biodiesel and 26 kg of glycerine. Except this, seed coat biomass could be used for making packaging materials; de-oiled seed cakes are rich in protein and they could be a good feed for animals and fish. Table 3 gives a list of organizations that are working on promotion of R&D for bioenergy as well as involved in the processes of BBBRs. Many biorefineries are working well across the world. Countries such as Australia, Canada, Germany, Denmark, Turkey, the USA, New Zealand, Netherland have biorefinery plants with multiple feedstocks. The details of some biorefineries plants and their locations are given in Table 4.

Table 4

Operational biorefineries

Biorefinery plant substrate

Product

Location

Pulp, off-gas, electricity, and heat biorefineries from wood chips

Pulp, gas, electricity, and biomethanol

Alpac Forest Products Inc., Canada

A bioliq SynCrude and syngas biorefinery pilot plant from lignocellulosic residues

Customized fuels and chemicals

Bioliq, Germany

C6 sugars, C5 sugars, lignin, and Bioethanol, feed, electricity and heat biorefineries from electricity, and heat straw

Inbicon, Denmark

A sugar residue biorefinery from sugar beets

Sugar, bioethanol, and animal feed

Konya Seker San, Turkey

A sugars, lignin, and syngas biorefinery from renewable biomass and MSW

Bioethanol, power, and heat

INEOS New Planet Bioenergy (USA)

A turpentine, gum rosin, crude sulphate turpentine, and tall oil biorefinery from co-products paper industry and pine trees

Terpenes, resins, and nutraceutics

Dérivés Résiniques et Terpéniques (DRT), France

An oil production and refinery pilot plant from microalgae

Omega-3, fuels, and chemicals

Wageningen UR, the Netherlands

A biogas, bio-methane, green pressate, fibres, electricity and heat biorefinery from grass and manure

Bio-methane, lactic acid, Utzenaich, Austria biomaterials, and fertilizers

The Mackay Renewable Biocommodities Pilot Plant from sugarcane bagasse, corn stover

Bioethanol, lignin, and various chemicals

Godavari Biorefineries Limited

Producing sugar, other Mumbai, India foods, biofuels, chemicals, power, compost, waxes, and related products

MSW - Municipal solid waste

Queensland University of Technology, Australia

Biorefinery: A Future Approach for a Sustainable Bioeconomy

17

India, however, is still lagging behind in the implementation of such technologies. Though Ministry of New and Renewable Energy and Ministry of Rural Development are promoting R&D in biorefineries using agricultural residues, oil-yielding plants, and algal biomass, they are still at lab scale and not at either field or commercial scale.

CONCLUSION The advanced biorefinery concept is potentially strengthened for multidimensional product generation such as biochemicals and bioenergy with complete utilization of substrates at every step of different processing units. The technology is also helpful in mitigating biowaste generation so that the harmful effects on our environment can be reduced by adopting such an integrated approach. If the capabilities of biorefinery are exploited well, it could lead to the generation of technological models in the near future, and provide a trove of bio-based products, restoring the natural pristine environment towards the zero-waste Earth. Regulatory policies and cross-disciplinary collaborations among biotechnology, engineering, chemistry, and environmental sciences, along with life-cycle assessments could further help in the development of an advanced biorefinery concept, which is underway, and could be commercially deployed to overcome the BBBR. The advanced biorefinery concept is essentially based on lignocellulosic/waste biomass, which still requires technical maturity for a broader spectrum of product generation to sustain our bioeconomy.

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22 Algal Biofuel: Sustainable Solution Sinha, S K, K A Subramanian, H M Singh, V V Tyagi, and A Mishra. 2019. Progressive trends in bio-fuel policies in India: Targets and implementation strategy. Biofuels 10(1): 155–166 Sklavounos, E. 2014. Conditioning of SO2-ethanol-water (SEW) spent liquor from ligno-cellulosics for ABE fermentation to biofuels and chemicals, Aalto University, pp. 108–162 Sonnenberg, A, J Baars, and P Hendrickx. 2007. IEA Bioenergy Task 42 Biorefinery. Avantium, Biomass Research and Wageningen University and Research Centre, Wageningen, The Netherlands Soudham, V P, T Brandberg, T P Mikkola, and T P Larsson. 2014. Detoxification of acid pretreated spruce hydrolysates with ferrous sulfate and hydrogen peroxide improves enzymatic hydrolysis and fermentation. Bioresource Technology 166: 559–565 Subhadra, B and G George. 2011. Algal biorefinery-based industry: An approach to address fuel and food insecurity for a carbon-smart world. Journal of the Food Science and Agriculture 91: 2–13 Tong, K L, F Q You, and G Rong. 2014. Robust design and operations of hydrocarbon biofuel supply chain integrating with existing petroleum refineries considering unit cost objective. Computer and Chemical Engineering 68: 128−139 Usher, P K, A B Ross, M A Camargo-Valero, and A S Tomlin. 2014. An overview of the potential environmental impacts of large scale microalgae cultivation. Biofuels 5: 331–349 Van Putten, R J, J C van der Waal, E de Jong, C B Rasrendra, H J Heeres, and J G de Vries. 2013. Hydroxymethylfurfural, a versatile platform chemical made from renewable resources. Chemical Reviews 113: 1499–1597 Wachtmeister, J and D Rother. 2016. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale. Current Opinion in Biotechnology 42: 169–177 Watanabe, M D B, L G Pereira, M F Chagas, M P Cunha, C D F Jesus, A Souza, E C Rivera, R MacielFilho, O Cavalett, and A Bonomi. 2016. Sustainability assessment methodologies. In Virtual Biorefinery – An Optimization Strategy for Renewable Carbon Valorization, edited by A Bonomi, O Cavalett, M P Cunha, and M A P Lima. Basel: Springer International Publishing World Economic Forum. 2010. The Future of Industrial Bio-refineries. Details available at http://www3.weforum.org/docs/WEF_FutureIndustrialBiore_neries_ Report_2010.pdf Zhang, X, J Rong, H Chen, C He, and Q Wang. 2014. Current status and outlook in the application of microalgae in biodiesel production and environmental protection. Frontier in Energy Reserch 2: 1–15 Zhu, L. 2015. Microalgal culture strategies for biofuel production: A review. Biofuels Bioproduct Biorefining. Details available at http://dx.doi.org/10.1002/bbb.1576, last accessed on 4 March 2020

Algal Biomass Harvesting for Biofuel Production

CHAPTER



Ashutosh Pandey1,3, Sanjay Kumar2, and Sameer Srivastava1 1

Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad, Uttar Pradesh, India 2 School of Biochemical Engineering, Indian Institute of Technology (BHU) Varanasi, Uttar Pradesh, India 3 Department of Biotechnology, Ashoka Institute of Technology and Management, Varanasi, Uttar Pradesh,

INTRODUCTION The microalgae-based industry, including cosmetics, pharmaceuticals, transportation fuel, has evolved considerably over the last few decades (Borowitzka 2013). During this period, depletion in the natural stock of fossil fuels and its price fluctuations have shifted the focus to developing alternatives of renewable energy from a variety of feedstocks (Tapie and Bernard 1988). Microalgae are considered a renewable energy source which has explicitly shown to be a feedstock for biofuel production. However, high production costs are the biggest limitation of microalgae cultivation and harvesting. The production cost of microalgae-based biofuels are higher ($9.65–$25.22/gallon) than the production cost of non-renewable fuels ($2.32–$2.55/gallon). Several strategies are being pursued to reduce production costs, including isolation of new species, modification of microalgal species (Yu, Ansari, Schoepp, et al. 2011; Radakovits, Jinkerson, Darzins, et al. 2010), development of low-cost mediums (Ummalyma, Mathew, Pandey, et al. 2016; Chiu, Kao, Chen, et al. 2015), design of highly efficient culture devices (Soman and Shastri 2015; Bhave, Kuritz, Powell, et al. 2012), and optimization of culture conditions (Smith and Crews 2014; Roleda, Slocombe, Raymond, et al. 2013). Apart from the aforementioned strategies to reduce the production costs, lack of an economical and efficient method of separating microalgal biomass from the liquid medium poses a major challenge to engineers and scientists (Wang, Li, Wu, et al. 2008). The energy consumption in the harvesting process also represents a considerable part (20–30%) of the total production cost (Mata, Martins, Caetano, et al. 2010; Verma, Mehrotra, Shukla, et al. 2010; Grima, Belarbi, Acien-Fernandez, et al. 2003). However, estimates as high as 50% of microalgal biomass cost have been reported (Greenwell, Laurens, Shields, et al. 2010; Wijffels and Barbosa 2010). Energy consumption is considered the

24 Algal Biofuel: Sustainable Solution main factor limiting the economic feasibility of biomass valorization (Salim, Bosma, Vermuë, et al. 2011; Chisti 2007). Moreover, the previously discussed approaches could be feasible only if the biomass harvested is used for extracting high-value products such as taxanthin, polyunsaturated fatty acids, arachidonic acid, eicosapentaenoic (EPA), dihydroxyacetone (DHA), etc. (Panis and Carreon 2016; Girma, Belarbi, Acien-Fernandez, et al. 2003). Microalgae have been reported to be harvested using several methods (Chen, Yeh, Aisyah, et al. 2011), including centrifugation (Knuckey, Brown, Robert, et al. 2006), foam fractionation (Csordas and Wang 2004), filtration (Sahoo, Gupta, Rawat, et al. 2017; Zhang, Hu, Sommerfeld, et al. 2010; Rossignol, Vandanjon, Jaouen, et al. 1999), flocculation (Branyikova, Prochazkova, Potocar, et al. 2018), gravity sedimentation (Shen, Yuan, Pei, et al. 2009), and electrolytic method (Pandey, Shah, Yadav, et al. 2019; Vandamme, Pontes, Goiris, et al. 2011; Uduman, Qi, Danquah, et al. 2010). Some other harvesting techniques such as electrophoresis, electroporation, and ultrasound have also been used to a lesser extent. The choice of technology for algae harvesting has to be energy efficient and comparatively inexpensive for commercial production of biofuel. The feasibility of the harvesting technology depends on the microalgal species (Singh, Nigam, and Murph 2011; Mata, Martins, Caetano, et al. 2010) and its intrinsic properties (Figure 1) such as cell size, shape, presence/absence of flagella, oil content, and nature of the cultivation medium. Harvest of microalgae is a challenging task because of the small cell size (3–30 μm), low-cell density/mass fraction in the broth (0.02–0.05% dry solids), electrostatic repulsion between negatively charged cells, and excess amount of algogenic organic matter (AOM) (Pragya, Pandey, and Sahoo 2013; Tran, Le, Lee, et al. 2013; Zamalloa, Vulsteke, Albrecht, et al. 2011). Besides these, its higher growth rate (as compared to terrestrial crops) means that it needs frequent harvesting (Melledge and Heaven 2013). Biofuel production (biodiesel) from microalgae is a multistep process (Figure 2) that involves cultivation, biomass harvesting, drying, lipid extraction, and trans­ esterification. Harvesting is still the most energy-intensive step as compared to others. A traditional harvesting method may involve two steps: the first step would be thickening in which the microalgae (mass) to water (volume) ratio is increased to achieve 2–7% of total solid matter, and the second step is dewatering that is done to concentrate the biomass up to 15–25%, which is then followed by drying. This further concentrates the slurry and increases the solid matter up to 90–95%. To address this crucial step, we have listed and discussed the various available techniques for algal biomass harvesting and dewatering. Additionally, we have also discussed the benefits, constraints, and drawbacks of various algae harvesting techniques, along with their energy requirements.

Figure 1

 

   

       

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List of various abiotic and biotic factors af fecting microalgae cell har vesting

     

     

    

  

   

-#            "     . (    

      



         !  " !    &  '    ( !     )   *   "!     

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Algal Biomass Harvesting for Biofuel Production

25

26 Algal Biofuel: Sustainable Solution

HARVESTING PROCESS The process of separation of algal biomass from the aqueous medium is known as harvesting. It usually requires one or more solid–liquid separation steps, together with concentration and drying processes, and is considered to be a tedious task in the production process. Microalgae harvesting is a two-step process involving thickening and dewatering (Figure 1). In bulk harvesting, the target is the separation of biomass from the bulk suspension. The concentration factor for this process is usually 100–800 times to reach around 5% of the total solid matter, and depends on the technologies employed. The thickening process includes gravity sedimentation, flocculation, and flotation. The primary objective is to concentrate the slurry through technologies such as centrifugation, filtration, and ultrasonic aggregation, which is why it is generally a more energy-intensive process than bulk harvesting.

Flocculation Flocculation refers to the process of separating microalgal cells from the broth by addition of one or more inorganic/organic chemicals. Microalgal cell walls carry negative charge that prevents self-aggregation within the suspension. This negative charge is counteracted by the addition of polyvalent ions called ‘flocculants’. These non-toxic polyvalent ions flocculate the cells without affecting their composition. Flocculation is considered to be an effective and a suitable process by which suspended microalgal cells aggregate into large and loosely attached conglomerates (Vandamme, Foubert, and Muylaert 2013; Girma, Belarbi, Acien-Fernandez, et al. 2003), eventually enabling efficient gravity sedimentation, centrifugal recovery, and filtration. A variety of flocculants have been used, including inorganic multivalent metal salts (Pirwitz, Rihko-Struckmann, and Sundmacher 2015; Duan and Gregory 2003), organic polymers (Vandamme, Foubert, Meesschaert, et al. 2010), and bacterial and algal bioflocculants (Liu, Tao, Wu, et al. 2014; Kim, La, Ahn, et al. 2011; Oh, Lee, Park, et al. 2001). pH-induced flocculation has also been reported (Ummalyma, Mathew, Pandey, et al. 2016; Liu, Zhu, Tao, et al. 2013; Wu, Zhu, Huang, et al. 2012; Spilling, Seppälä, Tamminen, et al. 2011; Horiuchi, Ohba, Tada, et al. 2003) in this process. The pH of the growth medium is increased or decreased to a certain level to induce flocculation. In pH-induced methods, multivalent metal cations interfere with the negatively charged algal surfaces. As a result, neutralization reactions occur, leading to decreased electrostatic repulsion between algal cells, and, finally, multicellular flocs formation takes place. Flocculation can be considered a more suitable approach for microalgae biomass harvesting when compared to existing centrifugation and filtration methods. Flocculation can deal with enormous amount of microalgae culture (Lee, Kim, Kim, et al. 1998) and can be applied to a wide range of algal

Figure 2

Schematic presentation of an overall microalgae biodiesel production process with main inputs and outputs

Algal Biomass Harvesting for Biofuel Production

27

28 Algal Biofuel: Sustainable Solution species (Pushparaj, Pelosi, Torzillo, et al. 1993). Flocculation is performed in three ways: chemical flocculation, auto-flocculation, and bioflocculation. Chemical flocculation In chemical flocculation, specific chemicals are used, namely, inorganic flocculants (metal salts, alums, ferric chloride, aluminium sulphate, ferric sulphate, polyacrylamide, poly-aluminium chloride), organic polymers (chitosan, starch, Megnafloc LT25, Zetag, Praestol, etc.), and acids/alkalis to induce pH changes [Ca(OH)2, NaOH, CO2, etc.]. Success stories of chemical flocculants in harvesting variety of algal species are well documented, such as in Chlorella sp. (Papazi, Makridis, and Divanach 2009), Scenedesmus sp. (Chen, Wang, Wang, et al. 2013), Neochloris sp. (Beach, Eckelman, Cui, et al. 2012), Nannochloropsis sp. (Rwehumbiza, Harrison, and Thomsen 2012), and Phaeodactylum sp. (Zheng, Gao, Yin, et al. 2012). The summary of different chemical flocculants with their harvesting efficiency is tabulated in Table 1. The flocculants are added in the algal growth media, causing the algae to clump together in the solution in the form of flocs or flakes. The floc may then float to the top of the liquid or settle at the bottom of the liquid, and can be readily filtered from the liquid. Among the various flocculants, aluminium sulphate (alum) is most widely used, because of its ease of use, economical, and non-toxic nature (Ebeling, Sibrell, Ogden, et al. 2003; Schlesinger, Eisenstadt, Gil, et al. 2012). However, it cannot be applied over a wide pH range. Moreover, the floc size with alum is smaller (when compared to ferric flocs), and results in unproductive sedimentation (Ebeling, Sibrell, Ogden, et al. 2003). The main drawback associated with this method is the persistence of metals and inorganic polymers, resulting from the addition of chemical flocculants (e.g. aluminium and iron), which may further lead to an additional cost and may also induce undesired changes in the cell composition (Benemann and Oswald 1996). Residual metals in algal biomass have also been shown to affect fatty acid methyl esters (FAME) (Rwehumbiza, Harrison, and Thomsen 2012). Aluminium salts could cause cell damage that may result in loss of end product quality, whereas ferric salts affect microalgae pigment quality (Papazi, Makridis, and Divanach 2009). Organic polymer (poly-aluminium chloride and chitosan) present in the harvested biomass could significantly affect transesterification (Tran, Le, Lee, et al. 2012). Using chitosan as a flocculant for algal harvesting could benefit downstream dewatering processes, eventually saving time and cost (Xu, Purton, and Baganz 2013). Chitosan flocculation can be improved by optimizing the cell culture conditions to achieve higher cell wall polysaccharide content or selecting an algal strain with higher cell wall polysaccharide content

Algal Biomass Harvesting for Biofuel Production

Table 1

29

Different chemical flocculants used for algal harvesting

Microalgal strain Cell density (g/L)

Chemical flocculant

Chemical Flocculation flocculant efficiency dose (mg/L)

Reference

Nannochloropsis 10–20 salina

Aluminium 5.4 nitrate sulphate

>95% (30 min)

Rwehumbiza, Harrison, and Thomsen (2012)

Chlorella zofingiensis

0.5

Aluminium sulphate

100

>90% (60 min)

Chen, Wang, Wang, et al. (2013)

Chlorella sorokiniana

-

Ammonia

640–2000 >85% (180 min)

Chen, Liu, Li, et al. (2012)

Scenedesmus sp.

0.23

Ferric chloride

200

>90% (60 min)

Chen, Wang, Wang, et al. (2013)

Chlorella minutissima

2.2 × 108 Ferrous Cells/mL sulphate

1000

>99% (60 min)

Papazi, Makridis, and Divanach (2009)

Scenedesmus sp.

0.54

Polyacrylamide 50

60% (10 min)

Chen, Wang, Wang, et al. (2013)

Polyaluminium chloride

70–80% (30 Sirin, Clavero, and min) Salvadó, (2013)

Nannochloropsis oculata Dunaliella sp.

Nannochloropsis 0.132 gaditana Phaeodactylum tricornutum

0.142

Chlorella sorokiniana

2.0

20–40

-

Sirin, Trobajo, Ibanez, et al. (2011)

Chitosan

20–30

60–90% (30 Xu, Purton, and min) Baganz (2013)

Nannochloropsis 0.132 gaditana

Chitosan

20-30

60-90% (30 min.)

Sirin, Clavero, and Salvadó (2013)

Phaeodactylum tricornutum

0.105

Chitosan

20-30

60-90% (30 min.)

Sirin, Trobajo, Ibanez, et al. (2011)

Scenedesmus sp.

0.23

chitosan

20-30 min

60-90% (30 min.)

Chen, Wang, Wang, et al. (2013)

Nano chitosan

60

96% (60 min)

Farid, Shariati, Badakhshan, et al. (2013)

Nannochloropsis 6.7 × 108 sp. Cells m/L Chlorella protothecoides

0.56–0.77 Starch

20–40

79–90% (60 Leqtelier-Gordo, min) Holdt, Francisci, et al. (2014)

Chlorella vulgaris

-

Moringa oleifera seed flour

1000

88% (120 min)

Teixeira, Kirsten, Teixeira, et al. (2012)

Chlorella protothecoides

0.6

Polyglutamic acid

20

>90% (120 min)

Zheng, Gao, Yin, et al. (2012)

Nannochloropsis gaditana Phaeodactylum tricornutum

30 Algal Biofuel: Sustainable Solution (Cheng, Zheng, Labavitch, et al. 2011). The carbohydrate composition in the cell wall was found to be the most significant factor positively affecting the flocculation efficiency of Chlorella variabilis NC64A (Cheng, Zheng, Labavitch, et al. 2011). Poly-c-glutamic acid has potential as an efficient and a sustainable microbial flocculant for harvesting microalgae in biodiesel production (Zheng, Gao, Yin, et al. 2012). The use of organic polymers for algal harvesting is restricted due to their pH dependence. The use of organic polymers for harvesting is also more expensive as compared to inorganic polymers. Moreover, high ionic strength is demanded in the flocculating process; therefore, pH-induced flocculation is found to be more favourable compared to the aforementioned chemical flocculation. Bioflocculation Microalgal flocculation induced by the microorganism itself and/or due to the presence of extracellular polymeric substances, especially exopolymeric substances (EPS) and glycoproteins (flocculating agent) secreted by the microorganism, is termed as bioflocculation and has been successfully applied in wastewater treatment plants. Bioflocculation of microalgae can be done in three different ways, as given in Table 2. The success of bioflocculation depends on the production of EPS by a microorganism (bacteria/fungi) and the ability of microalgae to attach to them and form flocs/aggregates. Studies suggest that the mechanism of bioflocculation is bridging (large network of cells) and patching (closely attached cells). Bioflocculation eliminates the need to use chemical flocculants, which represent an expensive, a non-reliable, and a toxic alternative. The bacteria, fungi, and microalgae associated with microalgal harvesting result in microbiological contamination in harvested biomass and limit its use for food and feed alternatives (Vandamme, Foubert, and Muylaert 2013). It is also well documented that the use of microorganisms for algal biomass harvesting may contribute to the increase in lipid yield (Salim, Bosma, Vermuë, et al. 2011). The resulting culture broth can be efficiently recycled for microalgae growth without any productivity loss (Zhou, Cheng, Li, et al. 2012). Bacteria-assisted bioflocculation: Bacteria play an important role in flocculation by increasing the floc size, resulting in sedimentation of microalgae. A flocculating activity of 94% was achieved with xenic Chlorella sp. as compared to 2% achieved with axenic culture (Lee, Cho, Ramanan, et al. 2013). Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene of xenic C. vulgaris cultures revealed the presence of Flavobacterium sp., Terrimonas sp., Sphingobacterium sp., Rhizobium sp., and Hyphomonas sp. (Lee, Cho, Ramanan, et al. 2013). The presence of certain bacteria is the determining factor in the flocculation of certain species such as Chlorella vulgaris (Lee, Cho, Ramanan, et al. 2013). The soil bacteria (Paenibacillus sp., Solibacillus

Algal Biomass Harvesting for Biofuel Production

Table 2

31

Microalgae biomass harvesting by bioflocculation

Microalgal strain

Procedure

Flocculating agent/ dosage/time

Flocculation Reference efficiency

Nannochloropsis oceanica

Bacteriaassociated bioflocculation

Solibacillus silvestris (0.1% w/v)

88%

Wan, Zhao, Guo, et al. (2013)

Pleurochrysis carterae

Tap water bacterial inoculum (0.9% v/v), 30 min

90–93%

Lee, Lewis, and Ashman (2009)

Chlorella sp., Scenedesmus sp., Pediastrum sp., Phormidium sp. Consortium

Aerobic-activated sludge bacteria, 90 min

98%

Van Den Hende, Vervaeren, Desmet, et al. (2011)

Chlorella vulgaris

Paenibacillus sp.

93%

Oh, Lee, Park, et al. (2001)

Botryococcus braunii, Scenedesmus quasicauda, and Selanastrum capricornutum

91–95%

Anabaenaflos­ aquae and Microcystis aeruginosa

38–49%

Chlorella vulgaris Fungiassociated bioflocculation

Aspergillus oryzae 97% (1.1 × 104 (spores/mL), 3 days, light:dark cycle: 0:24

Zhou, Min, Hu, et al. (2013)

Aspergillus oryzae 93% (1.1 × 104 (spores/mL), 2 days, light:dark cycle: 24:0 Aspergillus oryzae 63% (1.1 × 104 (spores/mL), 3 days, light:dark cycle: 24:0, without carbon source (glucose)

Chlorella vulgaris Algaeassociated bioflocculation

Cunninghamella 99% echinulata (3.44 × 107 (spores/mL), 3 days, light:dark cycle: 24:0

Xie, Sun, Dai, et al. (2013)

Ettliataxensis, light:dark cycle: 16:8, 3 hours

Salim, Vermue, and Wijffels (2012)

55%

Contd...

32 Algal Biofuel: Sustainable Solution Table 2 Contd... Microalgal strain

Procedure

Flocculating agent/ dosage/time

Flocculation Reference efficiency

Scenedesmus obliquus, 34% light:dark cycle: 16:8, 3 hours Ankistrodesmus falcatus 50% light:dark cycle: 16:8, 3 hours Neochloris oleoabundans

Tetraselmis suecica

72%

Xie, Sun, Dai, et al. (2013)

silvestris, etc.) excrete a large quantity of EPS with potential flocculating ability. The Paenibacillus sp. showed the highest flocculant activity at a dose of 7–8 g/L for harvesting Batryococcus braunii and Chlorella vulgaris (Table 2). Its activity further improved with the addition of inorganic flocculant CaCl2 (Oh, Lee, Park, et al. 2001). The flocculant produced by Solibacillus silvestris showed effective flocculation of Nannochloropsis oceanica without adding any inorganic flocculants. Bioflocculants from Pestalotiopsis sp. KCTC 8637P were found to achieve satisfactory biomass recovery efficiency for Batryococcus braunii cells with a dose of 100 mg/L (Smith and Davis 2012). Moreover, such bioflocculants exhibit excellent flocculation efficiency at a dose of 3.5 g/L. Scenedesmus sp. in the presence of inorganic flocculant (Ca+2 and Fe +3) and recycling exhibited less than 8% decline in algal biomass yield (Lee, Kim, Kim, et al. 1998; Kim, La, Ahn, et al. 2011). The cocultivation of bacteria and oleaginous microalgae cells was found efficient for algal cell aggregate formation. The cocultivation of bacterium Psuedomonadales with an alga Nannochloropsis oceanica IMET1 at the cell ratio of 30:1 for 3 days resulted in good harvesting efficiency (Wang, Laughinghouse, Anderson, et al. 2012). Co-cultivation system of Tetraselmis suecica and Synechococcus WH8007 also exhibited high harvesting efficiency (Wang, Laughinghouse, Anderson, et al. 2012). Furthermore, increased biomass accumulation and lipid accumulation was also obtained in this system (Wang, Laughinghouse, Anderson, et al. 2012). The major drawback of this system is its high bacterial inoculum size and long cocultivation time. These problems can be rectified by the addition of inorganic ions (Ca+2) and organic carbon sources (acetate, glucose, or glycerine). Powell and Hill (2013) reduced the cell ratio (1:1) and the flocculation process finished in 30 minutes when employed with Bacillus sp. and Ca+2 ions to harvest Nannochloropsis oceanica. Flocculation of Pleurochrysis carterate was efficiently done in 24 hours by cocultivation with a mixture of bacteria (Pseudomonas stutzeri and Bacillus cereus) and low concentration of organic substrates (Lee, Lewis, and Ashman 2009).

Algal Biomass Harvesting for Biofuel Production

33

Fungi-assisted bioflocculation: The use of fungal palettization is well established in wastewater treatment, where they are used to trap the sludge solids. Wan, Alam, Zhao, et al. (2015) reviewed flocculation of algal harvesting and hypothesized the mechanism behind fungal-associated bioflocculation. The aggregation of microalgal and fungal cells can be accomplished by palettization and charge distribution of filamentous fungi containing polysaccharide with active sites. This polysaccharide can attract microalgal cells by changing their surface charge and co-pelletization (Muradov, Taha, Miranda, et al. 2015; Zhou, Cheng, Li, et al. 2012). The fungal-assisted microalgal flocculation is effective for both autotrophic and heterotrophic grown microalgal species. The cocultivation of fungi (Asperpergillus oryzae, Rhizopus, and Cunninghamella echinulata) with microalgae in definite rations efficiently pelletized Chlorella vulgaris (Table 2) and achieved 63–97% recovery efficiency (Zhou, Min, Hu, et al. 2013). Furthermore, when Chlorella vulgaris was cocultivated with Aspergillus sp. under heterotrophic conditions, the lipid productivity also improved (Wrede, Taha, Miranda, et al. 2014). The advantage associated with this method is that besides the reduction of harvesting cost, the biomass and lipid yield can be significantly increased. Similar results were obtained when fungus Cunninghamella echinulata and Chlorella vulgaris were cocultivated for the harvesting of microalgae cells (Xie, Sun, Dai, et al. 2013). Algae-assisted bioflocculation: Self-flocculating microalgae can be used as a source of bioflocculants to assist the harvest of target microalgae (Table 2). Three flocculating microalgae, namely Ankistodesmus falcatus, Scenedesmus obliquus and Tetraselmis suecica, were tested for the harvesting of nonflocculating microalgae Chlorella vulgaris and Neochloris oleoabundans from different habitats. The results suggest that the addition of autoflocculating microalgae induces faster sedimentation of non-flocculating microalgae and increases the recovery of biomass. It has several benefits over chemical flocculation as bioflocculation does not cause any compositional change in algal biomass and the retreating of medium/supernatant before reuse or discharge of cultivation media is not required. In addition to the improved recovery efficiencies, the presence of the self-flocculating microalgae also does not interfere with downstream processing (Salim, Bosma, Vermuë, et al. 2011). Sometimes a combination of different techniques is used for effective harvesting of biomass. Liu, Tao, Wu, et al. (2014) developed a flocculation method to harvest target microalgae (Chlorella zofingiensis and Chlorella vulgaris) with self-flocculating microalgae (Chlorococcum nivale, Chlorococcum ellipsoideum, and Scenedesmus sp.) induced by a decrease in pH. They reported higher efficiency than when the target microalgae were flocculated only by the decrease in pH. These results ascertain that the flocculation efficiencies of the target microalgae can be improved by this method. The maximum flocculation

34 Algal Biofuel: Sustainable Solution efficiencies (>90%) were achieved at pH 4.5 and were stable within the pH range of 1.5–4.5. Autoflocculation/Self-flocculation Autoflocculation is an attractive, a novel, a cost-effective, a non-toxic, and an energy-efficient harvesting method in which flocculation is induced by pH adjustment. It does not require any contaminating chemical flocculants, enabling medium reuse (Schenk, Hall, Stephens, et al. 2008; Lee, Lewis, and Ashman 2009; Salim, Bosma, Vermuë, et al. 2011). The advantage of pH-induced flocculation is that it can strongly reduce the hazard of chemical contamination and secondary pollution. Autoflocculation occurs naturally in microalgal cultures exposed to sunlight with limited CO2 supply. CO2 dissolved in the medium is removed by microalgae by photosynthesis, thus increasing its pH value. It was well documented that a variety of algal species, namely Nannochloropsis sp. (Schlesinger, Eisenstadt, Gil, et al. 2012), Scenedesmus sp. (Chen, Wang, Wang, et al. 2013), Dunaliella viridis (Mixson, Stikeleather, Simmons, et al. 2014), Phaeodactylum tricornutum (Spilling, Seppälä, Tamminen, et al. 2010), Chlorella sp. (Smith and Davis 2012; Vandamme, Foubert, Fraeye, et al. 2012), Chlamydomonas sp. (Ras, Lardon, Bruno, et al. 2011) and Chlorococcum sp. (Wu, Zhu, Huang, et al. 2012) were successfully harvested via autoflocculation (Table 3). It has greater influence in the recovery of marine microalgae than freshwater microalgae under high pH value (10–12). Ions present in the seawater react, leading to changes on the microalgal cell surfaces and accelerate cell settling (Chen, Liu, Li, et al. 2012). Schlesinger, Eisenstadt, Gil, et al. (2012) achieved 95% flocculation efficiency of Nannmochloropsis sp. (107 cell m/L) in late exponential phase by using Ca(OH)2. The harvesting cost was estimated to be $7.5/tonne microalgae biomass and even reduced to $3.5/tonne microalgal biomass when cell density reached 108 cell m/L. It was also observed that the addition of Mg+2 ions in the alkaline pH medium could facilitate the flocculating process of Chlorella vulgaris, Chlorella vulgaris UTEX395, and Chlorocuccum sp. (Vandamme, Foubert, Fraeye, et al. 2012; Smith and Davis 2013; Wu, Zhu, Huang, et al. 2012). The major drawback of pH-induced autoflocculation is that the extreme pH may damage the algal cell and could be unreliable and uneconomic at a commercial scale (Beneman and Oswald 1996). Cell self-flocculation is defined as the aggregation and adhesion of cells to each other in culture broth due to special cell surface property. Several self-flocculating microalgae have been documented by several authors, such as Chlorella vulgaris JSC-7, Scenedesmus obliquus AS-6-1, Ankistrodesmus falcatus (SA202-9) (Alam, Wan, Guo, et al. 2014; Guo, Zhao, Wan, et al. 2013; Salim, Vermuë, and Wijffels 2012). The advantages of using autoflocculation are that the process does not require any chemicals, and it is cost-effective and environment-friendly. The molecular basis of autoflocculation has been comprehensively studied in brewing yeast. However, there are only limited

Algal Biomass Harvesting for Biofuel Production

Table 3

35

Harvesting of microalgae by using auto/self-flocculation

Microalgae strains

Procedure

Flocculation efficiency

References

Nannochloropsis sp.

pH adjustment (pH 10), Ca(OH) 2

>95%

Schlesinger, Eisenstadt, Gil, et al. (2012)

Dunaliella viridis

pH adjustment (pH 10), NaOH

95%

Mixson, Stikeleather, Simmons, et al. (2014)

Scenedesmus sp.

pH adjustment (pH 11.5), NaOH

97.4%

Chen, Wang, Wang, et al. (2013)

Phaeodactylum tricornutum

pH adjustment (pH 10.5), CO2

85%

Spilling, Seppälä, Tamminen, et al. (2010)

Chlorella vulgaris

pH adjustment (pH 10.8) NaOH

>98%

Vandamme, Foubert, Fraeye, et al. (2012)

pH adjustment (pH 10.8) KOH

>98%

–

pH adjustment (pH 10.8) Ca(OH) 2

>98%

–

pH adjustment (pH 10.8) Mg(OH) 2

>98%

–

pH adjustment with Mg +2 addition

90%

Smith and Davis (2012)

Chlorella vulgaris UTEX395

reports in other microbial strains including microalgae. In the case of yeast, a family of glycoproteins encoded by flocculin (FLO) genes is responsible for cell to cell adhesion (Bauer, Govender, Bester, et al. 2010). Polysaccharides biosynthesized by Chlorella vulgaris JSC-7 and Scenedesmus obliquus AS-6-1 were reported to be responsible for autoflocculation (Alam, Wan, Guo, et al. 2014; Guo, Zhao, Wan, et al. 2013). Therefore, it could be hypothesized that overexpressing the genes responsible for certain polysaccharide biosynthesis could result in flocculation of cells.

Flotation Flotation process is a prompt and an effective method for harvesting microalgae. The process is based on the air or gas bubbles which cause the algae cells to move to the surface from where they can be collected (Chiu, Kao, Huang, et al. 2011). In the flotation process, small bubbles attach to the destabilized particles of the medium such as algae, and so the success rate of flotation depends on the instability of algae cells in the medium. The flotation process is classified according to the way the bubbles are generated in general flotation and can be performed in two ways: dissolved air flotation (DAF) and froth flotation (Hantou, Bandulasena, and Zimmerman 2012).

36 Algal Biofuel: Sustainable Solution Dissolved air flotation Dissolved air flotation uses pressurized micro-bubbles for transporting suspended particles to the surface of the reactor using features of both froth flotation and flocculation. The dissolved air flotation unit comprises a compressor and flotation cell. The compressor creates an air bubble in the water and forms supersaturated water, which is then released into a flotation cell at atmospheric pressure (Kyzas and Matis 2018; Henderson, Parsons, and Jefferson 2010). In the flotation cell, dissolved air from supersaturated water forms a small bubble in the media solution and adheres to the unstable components (such as microalgae) with minimum shear. Thus, decreased stability of algae in media increases the ease of harvest. Generally, 5–15% supersaturated water, which is pressurized in the range of 400–650 kPa, is used in the flotation cell (Torres, Araújo, de Oliveira, et al. 2017; Henderson, Parsons, and Jefferson 2010). Certain chemicals can also be used to help flocculate microalgae with fine bubbles supplied by an air compressor. Air bubbles, which adhere to the algae surface, cause the algae to float to the surface, so that it can be harvested. Major drawbacks of this technique are the utilization of high energy for creating supersaturated water and the need of additional chemicals such as aluminium, chromium, potassium, sodium, or iron that are not eco-friendly. This is a welldeveloped and widely used lab-scale method and has already been scaled up at a commercial level (Laamanen, Ross, Scott, et al. 2016). Froth flotation Froth flotation is the modification of dispersed air flotation. In this method, air is mechanically introduced to the system through an agitator or a porous medium (Garg, Li, Wang, et al. 2012; Chen, Liu, and Ju 1998). The major drawback of this process is that the bubbles formed are large (700–1500 μm), which limit their use, despite the fact that it is a less energy-consuming process in comparison to dissolved air flotation. To decrease the size of bubbles, surfactants are added to the system, which not only decrease the size of the bubbles but also the coalescence of these bubbles. The commonly used surfactants for froth flotation are CTAB (cetyltrimethylammonium bromide) and SDS (sodium dodecyl sulphate). Hydrophobicity and electrical charge of particle surfaces play important roles in the froth flotation process (Bulatovic 2007). The efficiency of froth flotation depends on various factors such as pH, surfactant type, and its concentration. Studies were also conducted by altering the surface charge of the algae and changing the pH of media to enhance the algae harvesting by froth flotation. This approach holds potential for commercial harvesting of algae due to its low cost and ease of operation (Sharma, Garg, Li, et al. 2013).

Algal Biomass Harvesting for Biofuel Production

37

Gravity Sedimentation Gravity sedimentation is most commonly used to separate microalgae from water in wastewater treatment. Due to poor compaction and low-settling velocity, this process yields a wet voluminous sludge/biomass. The settling of microalgae in the sedimentation process is influenced by algal cell size, density, type of microalgae, temperature, ageing of the cells, time, and induced sedimentation velocity (Harith, Yusoff, Mohammed, et al. 2009; Knuckey, Brown, Robert, et al. 2006; Waite, Thompson, and Harrison 1992). The success of microalgal settling is dependent on the density of the microalgae. It is, therefore, a challenge to remove microalgae from water because of the similar density of algal cell and media (Wiley, Campbell, and McKuin 2011). The rate of settling depends on the microalgae type; green microalgae showed 0.1 m/d settling rate (Mohn 1980). However, in another report, the settling rate was investigated in 24 different microalgae of different cell sizes (0.4 to 2.2 m/d) and no correlation was observed between the size of the cell and settling velocity (Cole and Buchak 1995). Choi, Lee, Kwon, et al. (2006) noted that the sinking rate of large- and small-sized algae was 0.62 m/d and less than 0.24 m/d, respectively. Knuckey, Brown, Robert, et al. (2006) studied the effect of temperature over the settling rate of microalgae and observed that major algal cells settled efficiently at 4°C after 24 hours. However, these findings were contradicted by Davis, Downs, Shi, et al. (1995) in which a slower sinking rate of algal cells at lower temperature was reported due to increased viscosity. In another report, lower settling rate was observed for exponential growth phase cells (4–10 days old) as compared to stationary phase cells (10–12 days old) (Danquah, Gladman, Moheimani, et al. 2009), suggesting the effect of cell age over sedimentation. In a similar experimental setup, Manhein and Nelson (2013) observed that there was little or no settling of Scenedesmus sp. in the exponential growth phase over 2 hours, but greater sinking was observed in the stationary growth phase. They also observed that the settling of Chlorella vulgaris was higher in an exponential growth phase as compared to the stationary growth phase. Based on these experiments, it was later concluded that the settling rate also depends on algal species. The absence of light increased the settling rate during the high- and low-growth phases. The biomass obtained in the supernatant of exponential growth phase was 0.57 g/L (with light) and 0.39 g/L (without light). A similar fashion was followed for the low-growth phase (Danquah, Gladman, Moheimani, et al. 2009). Large-scale harvesting of microalgae by using sedimentation requires long retention times, usually greater than 24 hours (24–48 h) for algal recovery (Park, Craggs, and Shilton 2011). Griffiths, Hille, and Harrison (2012) found that the fraction of biomass recovery after 24 hours of retention time for settling for S. plantensis, C. fusiformis, T. Suecica, Nannochloropsis, and Scenedesmus sp. were 95%, 96%, 80%, 59%, and 86%, respectively. Lamella separators

38 Algal Biofuel: Sustainable Solution and sedimentation tanks are commonly used to achieve enhanced microalgal harvesting by sedimentation. Lamella separators offer a better settling area than conventional thickeners. In the settling tank, denser solids settle on the bottom, leaving clear water at the surface. The addition of flocculants also enhances biomass recovery. The cost associated with sedimentation is very low, but without using a flocculating agent, the reliability of this method is less. The drawback associated with this method is its low biomass recovery (Mata, Martins, and Caetano 2010), the longer retention time for settling, and compositional change in algal biomass (Gonzalez-Fernandez and Ballesteros 2013). The harvesting of algal biomass using gravity sedimentation alone for biofuel is not the most efficient method because the recovery is too low (Ras, Lardon, Bruno, et al. 2011) and the variation in algal biomass composition may change the biodiesel property.

Filtration/Membrane Technology Membrane technology is a cheaper and quite effective technology for algal harvesting, offering advantages of almost complete retention of microalgal biomass (Bilad, Vandamme, Foubert, et al. 2012; Mouchet and Bonnelye 1998). It also offers the advantage that potential disinfection via removal of protozoa and viruses is not additionally required (Judd 2006; Vandamme, Pontes, Goiris, et al. 2011). Several membrane-based microalgae harvesting technologies have been studied so far, including submerged membrane (Baerdemaeker, Lemmensa, Dotremonta, et al. 2013; Bilad, Vandamme, Foubert, et al. 2012), crossflow filtration (Zhang, Hu, Sommerfeld, et al. 2010; Rossignol, Vandajon, Jaouen, et al. 1999), and dynamic microfiltration (Ríos, Clavero, Salvadó, et al. 2011). Among these, cross-flow mode warrants further investigation due to its potential for high biomass recovery and less energy consumption (Rossignol, Vandajon, Jaouen, et al. 1999). Micro and ultrafiltration cross-flow configurations offer a high harvesting efficiency due to the high cross-flow velocity and high shear rates exposed onto the membrane surface. However, it consumes a substantial amount of energy due to high applied pressures and liquid velocities (Le-Clech, Chen, and Fane 2006). Shear is an important consideration in algal fouling. Excessive exposure of microalgal biomass to shear, particularly pumping through a restrictive valve, may break the microalgae, resulting in smaller particles, colloids, and dissolved organic matters or the release of EPS. The small particles released after the cell breakage are best known to cause severe membrane fouling by enhancing pore blocking and producing a less porous cake layer on the membrane surface (Babel and Takizawa 2010; Ladner, Vardon, and Clark 2010). Ladner, Vardon, and Clark (2010) reported that the sheared samples cause greater reduction in flux

Algal Biomass Harvesting for Biofuel Production

39

than non-sheared samples, and rejection of AOM also diminished after shear. The cell rupture leads to the loss of targeted products from the cell interior. Therefore, application of submerged microfiltration that applies lower pressures in the absence of any cross-flow velocity is expected to be more efficient (Babel and Takizawa 2010). This system is commonly used in submerged membrane bioreactors (MBRs) for wastewater treatment. It is cheaper due to the absence of pressure-resistant membrane housings and offers lower energy consumption (Judd 2006; Le-Clech, Chen, and Fane 2006). In such an immersed system, the shear is usually provided by coarse air bubbles, which limit the enhanced shear rates to the microalgal cell. This leads to reduced EPS release and, thus, better filtration operation. The use of membrane technology to reap microalgae can be done in two ways: (i) as a separate step to concentrate the broth and (ii) coupled directly to the cultivation process in a membrane photobioreactor (MPBR). Both the ways are efficient up to a certain biomass concentration, due to the increasing fouling propensity of the concentrating broth. Membrane fouling can be severe at elevated biomass concentrations, which could be further worsened by the production of materials with high fouling properties (Bilad, Arafat, and Vankelecom 2014). However, membrane photobioreactor offers two additional advantages: biomass concentration and increased productivity. The membrane is directly immersed in the PBR; therefore, the aeration can be used not only to deliver dissolved carbon dioxide and mix the broth but also to scour the membrane for fouling control (Bilad, Arafat, and Vankelecom 2014). Another drawback of membrane filter is ‘blinding’. Blinding refers to the rate at which the deposited algal matter clogs up the pores, thereby reducing the rate of filtration across the filter membrane. Benemann (1989) reported favourable recovery rates in excess of 80% but also observed that the blinding of filters imposed serious threats in membrane filtration. Several improved designs to overcome blinding had been claimed by various manufacturers for microstrainers. A polyacrylonitrile (PAN) ultrafiltration membrane with a 40 kDaM/W cut-off was found to be suitable for the uninterrupted recovery of two marine microalgae (Haslea ostrearia and Skeletonema costatum) (Rossignol, Vandajon, Jaouen, et al. 1999). Petrusevski, Bolier, Breemen, et al. (1995) explored a tangential flow filtration system for the concentration of living freshwater phytoplankton from large volumes of reservoir water with low algal biomass. Freshwater phytoplankton were concentrated 5–40 times using a 0.45 μm pore-size membrane. Further increase in the concentration factor is difficult and costly because as biomass concentration increases, it results in an increase in membrane fouling (Chu, Zhao, Zhang, et al. 2015). Understanding membrane fouling by the biomass and developing anti-fouling strategies are significantly important for sustainable biomass concentration using membrane technology.

40 Algal Biofuel: Sustainable Solution The build-up of the algal cake layer and adsorption of AOM on the membrane cause membrane fouling. Zhang, Hu, Sommerfeld, et al. (2010) developed a competent membrane technology for algae biomass concentration and established anti-fouling strategies. They used air-assisted backwash with air scouring. In the same study, the cake layer was removed by conducting an air-assisted backwash every 15 minutes. Shorter backwash intervals led to higher permeate flow rates and higher initial flux for each filtration cycle, indicating that frequent backwashing does help in controlling fouling. Airassisted backwashing with air scouring of the lumen was found to be a better option as it resulted in the recovery of almost 99% of the initial flux in comparison to 92% of the initial flux the recovery without air scouring. An improved backwashing efficiency was observed when compressed air was injected into the feed side of the hollow fibre to scour the foulants (Zhang, Chen, Konsowa, et al. 2009; Pearce 2007). Removal of algal cakes (cleaning) also improves the membrane harvesting efficiency. Several chemicals, including 2% NaOH, 0.5% citric acid, and 200 mg/L NaClO, were used as cleaning agents. Higher NaClO concentrations resulted in better cleaning. The adsorbed AOM could be removed by soaking the membrane in 400 mg/L NaClO for 1 hour; the flux increased to 90.7 g/m2 /h, which was 98% of the average initial (pre-harvesting) flux [92.2 g/m2 /h)] of the membrane. NaClO is the most widely used chemical for membrane disinfection and biofouling control (Hung and Liu 2006). NaClO acts as a swelling agent and protein solubilizer, which makes it very effective (Kennedy, Kamanvi, Rodriguez, et al. 2008). Liang, Yang, Gong, et al. (2008) also showed the effectiveness of NaClO in cleaning membranes used for the treatment of algae-rich water. The algal suspension was concentrated to give a final cell concentration of 154.85 g/L. In the same study, the harvesting efficiency and average fluxes were 46.01 g/m2 /h and 45.50 g/m2 /h, respectively. No algae were found in the permeate, which had an average turbidity of 0.018. Bilad, Vandamme, Foubert, et al. (2012) combined submerged membrane filtration with centrifugation to minimize the harvesting cost and improve the harvesting efficiency. They investigated the applicability of submerged microfiltration as the first step of up-concentration for harvesting freshwater Chlorella vulgaris and a marine diatom Phaeodactylum tricornutum using three lab-made membranes with different porosity. The filtration performance was assessed by conducting the improved flux step method and batch up-concentration filtrations. It was found that submerged microfiltration for algal harvesting is economically feasible. To control membrane fouling, Bilad, Arafat, and Vankelecom (2014) compared an aerated and a vibrated membrane system for Chlorella vulgaris

Algal Biomass Harvesting for Biofuel Production

41

broth filtration. The results showed that the vibrated system offers considerable advantages to maintain high permeance by minimizing the fouling and thus supporting the filtration operation. A constant vibration restricts the occurrence of pore blocking, which occurs in most of the larger pores.

Harvesting by Using Magnetic Nanoparticles Magnetic nanoparticle has been extensively used due to its advantages such as fast separation, low running cost, and energy savings. Magnetic particles coated with silica or cationic polyelectrolytes were utilized for efficient harvesting of freshwater and marine algae (Xu, Guo, Wang, et al. 2011; Cerff, Morweiser, Dillschneider, et al. 2012; Lim, Chieh, Jalak, et al. 2012). Hu, Wang, Wang, et al. (2013) investigated the magnetic harvesting of marine microalgae Nannochloropsis maritima using low-cost naked Fe3O4 nanoparticles and concluded that approximately 95% of recovery efficiency was achieved for N. maritima within 4 minutes. It was also found that the recovery was dosage dependent. The functional groups onto the surface of algal cells decreased with the increase of biomass, and this resulted in fewer magnetic particles for the same harvesting efficiency (Zhang, Amendola, Hewson, et al. 2012). Harvesting efficiency was also influenced by the pH of the medium. Harvesting efficiency of N. maritima showed a slight decrease when the pH value increased from 4 to 6 and afterwards increased when the pH value was above 7. Lee, Lewis, and Ashman (2013) synthesized chitosan–Fe3O4 magnetic nanoparticle (CS/ MNP) composites and investigated the composites as biocompatible flocculants for the magnetophoretic harvesting of Chlorella sp. KR-1. They reported 99% harvesting efficiency without changing the pH of the culture medium. Farid, Shariati, Badakhshan, et al. (2013) have estimated the cost of harvesting microalgae by using nano-chitosan to be about $0.0246/kg-dry mass (minimum cost for harvesting microalgae is $0.08 /kg dry mass), which is promising based on the consideration of the economic viability of algal biodiesel.

Electric-based Process/Electrolytic Methods The electric-based approach is generally established in water and wastewater treatment. Nonetheless, such methods are also applicable to the harvesting of a wide variety of microalgal species (Table 4). There are three wellestablished electrolytic methods: (i) electroflocculation, (ii) electroflotation, and (iii) electrocoagulation. Electrical-driven harvesting methods are much more attractive today as they are energy efficient, environment friendly, and cost effective. The electrical-based approach has fewer advantages as compared to flocculation; electrochemical harvesting requires little or no chemicals such as alum, chlorides, sulphate or chitosan (generally required as traditional flocculant); performs well in a wide range of pH; and have no deteriorating

42 Algal Biofuel: Sustainable Solution effect on the quality of biomass (Guldhe, Misra, Singh, et al. 2015; Pandey, Shah, Yadav, et al. 2019). Electroflocculation [Figure 3 (A)] utilizes an inert anode (non-sacrificial electrode) and a cathode that allows the movement of algal cells towards the anode. Once the algal cells reach the anode, the negative charge is dropped from the microalgae and they form floc/aggregates without any metal ions. Poelman, Pauw, and Jeurissen (1997) used electroflocculation for harvesting microalgae for the treatment of drinking water. The removal efficiency of 95% or more was easily obtained with different microalgal strains, while energy consumption was as low as approximately 0.3 kWh/m 3. Electroflotation [Figure 3 (B)] uses a sacrificial metal cathode and a sacrificial metal anode to create hydrogen bubbles through the electrolysis of water, as well as releasing metal ions from the sacrificial metal anode. The positively charged metal ions attract the negatively charged microalgal cell to create flocs and hydrogen bubbles cling to the flocs, which are then carried to the surface from where they can be removed by a conventional skimming method. The settling of algal cell was observed with the increasing current density in the reactor vessel (Bleek, Quante, Winckelmann, et al. 2015; Barros, Gonçalves, Simões, et al. 2015; Aragon, Padilla, and Ursinos 1992). Electrocoagulation was found to be superior to chemical flocculation because of its low cost, shorter time required for separation, and lower probability of contamination with metallic hydroxides. Another advantage of electrocoagulation is that low current strength is needed to achieve effective coagulation of algal cells (Aragon, Padilla, and Ursinos 1992; Bleek, Quante, Winckelmann, et al. 2015). During the process of EC, metal ions are dispersed from the oxidizing sacrificial metal electrodes. This process involves oxidation of anode, which causes electrode depletion [Figure 3 (C)]. Thus, it requires periodic replacement of the electrode. Guldhe, Misra, Singh, et al. (2015) assessed non-sacrificial carbon electrode as the anode for harvesting small-sized microalga Ankistrodesmus falcatus achieved 91% recovery efficiency after 30 minutes with minimal energy consumption of 1.76 kWh/kg in comparison to centrifugation (65.34 kWh/kg). Alfafara, Nakano, Nomura, et al. (2002) investigated the use of electroflotation of a mixed algal population dominated by Microcystis sp. using an aluminium anode and titanium alloy cathode and found that in a batch reactor, high electrical input achieved higher and faster removal efficiencies of chlorophyll ‘a’. A small degree of external mixing followed by electroflotation was found to be useful at a lower electrical power input. Electroflotation alone could not achieve complete algae removal (maximum efficiency of 40% to 50%). In a continuous electrolysis experiment, the ratio of current density (ampdm–3) and chlorophyll ‘a’ loading (mgdm–3h–1) (charge dose required to remove a unit mass of chlorophyll ‘a’) was found to be useful for scale up and operating factor

Figure 3

– – – Cathode (–)

DC power –

OH

–

O2

H

+

H2

O2 collector H2 Collector

e + –

– + + – –– – ––– + + + + – Cathode (–) Anode (+) Alkaline electrolyte



M

+

+

Precipitation

+

(OH )

Cathode (reduction)

Flotation O H2 H2(g)

Electric supply – +

M(OH )

Anode (oxidation)



Dewatering of microalgae: (A) electrolytic flocculation, (B) electrolytic flotation, and (C) electrolytic coagulation

– –

Algal floc Migration of algal cells – – – – – –

DC power supply + –

Anode (+)



Algal Biomass Harvesting for Biofuel Production

43

44 Algal Biofuel: Sustainable Solution to balance high algal removal efficiency with a minimum release of metal ions. The 250 coulombs was found as an optimum charge dose to achieve high algal removal efficiency (around 90%). Gao, Yang, Tian, et al. (2010) reported that 100% of removal was obtained with energy consumption as low as 0.4 kWh/m3. Vandamme, Pontes, Goiris, et al. (2011) used electroflocculation for microalgae harvesting with energy consumption of approximately 0.2 kWh/kg for Chlorella vulgaris and 0.3 kWh/kg for Phaeodactylum tricornutum. Electroflocculation process carried out at 10 V for 1 minute, with an electrode separation of 5.5 cm and a height of 4 cm in the culture vessel, obtained recovery efficiency greater than 95% (Valero, Álvarez, Cancela, et al. 2015). Electroflocculation is a successful method of harvesting green algae during the blooms, obtaining the greatest amount of biomass for subsequent use as a source of biodiesel. Many factors can influence the effectiveness of electrocoagulation for microalgal biomass recovery, namely the electrodes (material, design, separation distance), the current density, operation time, temperature, initial pH of the medium, conductivity of the microalgae suspension, and the microalgae size (Zhang, Zhao, Chu, et al. 2014; Uduman, Qi, Danquah, et al. 2010; Gao, Yang, Tian, et al. 2010). Vandamme, Pontes, Goiris, et al. (2011) reported that aluminium electrodes are more efficient than the iron ones for microalgal biomass recovery. With aluminium electrodes, the oxidation and reduction reactions that occur during electrocoagulation can be written as follows (Vandamme, Pontes, Goiris, et al. 2011; Gao, Yang, Tian, et al. 2010). Aluminium oxidation at anode: Al (s) m Al3+ (aq) + 3 e –

(i)

xAl3+ (aq) + yOH(aq) m Alx(OH) z+y(s)

(ii)

As reported by Vandamme, Pontes, Goiris, et al. (2011), the composition of aluminium hydroxides formed during electrocoagulation depends on the pH of the microalgae suspension. Lower pH promotes the formation of positively charged aluminium hydroxide, while alkaline pH promotes the formation of negatively charged hydroxides. Water oxidation in the anode: 2H2O (l) m
4H + (aq) + O2 (g) + 4 e –

(iii)

Water reduction in the cathode: 2H2O (l) + 2 e– m
H2 (g) + 2OH– (aq)

(iv)

Overall reaction: xAl (s) + y H2O (l) m Alx(OH) z+ y (s) + yH2 (g) + y/2 O2

(v)

Algal Biomass Harvesting for Biofuel Production

45

Considering a harvesting time of 45 minutes, the aluminium electrode performed a total removal efficiency (100%), while only ~79% removal was achieved with iron (Fe) electrode. (Barros, Gonçalves, Simões, et al. 2015). In addition, the iron electrode is not convenient as it consumes more energy and results in brown-coloured algal slurry due to ferric oxidation. Bleek, Quante, Winckelmann, et al. (2015) performed an experiment and tested six diverse electrode materials (magnesium, aluminium, zinc, copper, iron, and brass) for electrolytic flocculation of freshwater microalgae Scenedesmus sp. and achieved more than 90% flocculation efficiency (FE) with magnesium electrode in 7.3 minutes at 40 V. The choice of the electrode also depends upon the ultimate use of microalgal biomass. In algal biomass for food and feedlike additives, metals such as magnesium are best and have other advantages besides their high flocculation efficiency, as they are relatively harmless even at a higher concentration. Current density greatly influences the harvesting efficiency, as well as the energy consumption of the operation. The current density has a reciprocal relationship with electrolysis time (Alfafara, Nakano, Nomura, et al. 2002; Gao, Yang, Tian, et al. 2010; Zhang, Zhao, Chu, et al. 2014). An increase in the current density results in shorter harvesting periods though the energy consumption increases (Gao, Yang, Tian, et al. 2010; Valero, Álvarez, Cancela, et al. 2015). In a report, microalgal harvesting using a current density of 0.5 mA/cm2 required an energy consumption of 0.2 kWh/m3; when a current density of 5.0 mA/cm2 was applied, the energy consumption increased to 2.28 kWh/m3 (Gao, Yang, Tian, et al. 2010). Thus, despite the increase in harvesting efficiency at a high current density, to make the harvesting process cost-effective, a balance must be set between energy consumption and electrolysis time. Further improvement can be done by stirring the mixture in order to achieve more particle contact and, therefore, overcome the electrical double layer that prevents their aggregation. Electroflocculation followed by flotation results in higher biomass concentration and energy with lower processing time (Lee, Cho, Ramanan, et al. 2013). The harvesting efficiency by electrocoagulation flocculation is also influenced by initial cell density. Considering the constant ratio of current density to initial cell concentration and electrolysis time (20 minutes), the collection efficiency decreased from 99% of 0.24 g/L to 30.5% of 1.17 g/L (Zhang, Zhao, Chu, et al. 2014). That may be due to the fact that higher cell concentration in the medium offers resistance to cell motion towards electrode (Show, Lee, and Chang 2014; Gao, Yang, Tian, et al. 2010; Barros, Gonçalves, Simões, et al. 2015). The microalgae recovery efficiency decreases with increasing initial cell density, as no aluminium ions are available for removal of excess cells with in a short electrolysis time. The total cell recovery was achieved in only 25 minutes for an initial cell density of 0.55 × 109/cells; and the same was achieved in 75 minutes for an initial

46 Algal Biofuel: Sustainable Solution cell density of 2.10 × 109/cells (Gao, Yang, Tian, et al. 2010). Therefore, in a high cell density, complete harvesting can be achieved only by increasing the electrolysis time (Gao, Yang, Tian, et al. 2010). The initial pH of the medium determines the type of metal ions formed during electrolysis. In the pH range of 4–7, the monomeric hydroxoaluminium cation and polymeric species such as Al3O4 (OH)24+7 are formed. Hence, the negatively charged microalgal cells are easily adsorbed onto these positively charged precipitates and facilitate harvesting. On the other hand, in alkaline conditions, the monomeric hydroxoaluminium anions dominate the solution and reduce the adsorption capacity of microalgal cells. Marine microalgae are more effortlessly separated than freshwater microalgae and need approximately half of the energy. This is due to the higher ionic strength of seawater that promotes the separation process (Gonzalez-Fernandez and Ballesteros 2013). Temperature also plays a key role in algal harvesting by electrocoagulation and flocculation; considering the electrocoagulation flocculation process time of 15 minutes, an increase in temperature from 18°C to 36°C resulted in an increase from 46% to 98% in the process recovery. It also saved electrolytic time, as it improved the dissolution rate of aluminium and lowered the energy consumption from 0.36 kWh/m3 to 0.16 kWh/m3. In a recent study, Pandey, Shah, Yadav, et al. (2019) optimized the harvesting efficiency of Scenedesmus sp. using electrocoagulation–flocculation (ECF) and achieved complete harvesting at initial pH of 5, electrolysis time of 15 minutes, electrode distance of 2 cm, sedimentation time of 60 minutes, and current density of 12 mA/cm2. The energy consumption of the ECF process was estimated much less (2.65 kW/kg) than centrifugation (16 kW/kg). The electrolytic method has several advantages, it is fast, efficient and versatile resulting in no or a little contamination of the biomass and lipid yield (Pandey, Shah, Yadav, et al. 2019). Moreover, electrolytic methods need low maintenance and minimal operation requirement, and the process can be carried out in a continuous mode with an efficiency of as high as 98% (Demirbas and Kobya 2017). Besides these advantages, the energy requirement and equipment cost remain high for large-scale applications. Operation parameters such as electrode material, electrode size, electrode distance, electrode surface area, initial cell density, initial pH of the medium, and current density are considered in the scale-up of an electrolytic method of the harvesting process. Moreover, the efficacy of ECF varies with algal species differing in oil quality and amount. Therefore, to overcome the challenges involved in the ECF process, it is of great importance that the ECF process is optimized specific to a species.

Centrifugation Centrifugation is similar to sedimentation, where the gravitational force is replaced by centrifugal acceleration to enhance the concentration of solids. It is

Algal Biomass Harvesting for Biofuel Production

47

widely used in labs and at industrial scales for algal cell removal from liquid growth medium (Benemann, Koopman, Baker, et al. 1980; Mohn 1980). It is one of the fastest methods of algal harvesting with great efficiency, but due to high energy requirement, its application is limited to high-value products such as DHA, EPA, ARA, Astaxanthin, pigments, antioxidants, nutrient supplements, etc. (Zhou, Min, Hu, et al. 2013; Grima, Belarbi, Fernandez, et al. 2013; Christenson and Sims 2011; Rawat, Kumar, Mutanda, et al. 2011). Centrifugation is suitable for almost all algal species such as Scenedesmus sp., Chlorella sp., Nannochloropsis sp., Spirulina sp., Coelastrum proboscideum, and Tetraselmis sp. (Dassey and Theegala 2013; Grima, Belarbi, Fernandez, et al. 2003). In almost all the cases, it is efficient as a one-step separation process but at larger scales, it requires more energy and time to run several batches. Hence, it is always recommended to do a pre-concentration of the algal slurry to reduce the harvesting volume (Grima, Belarbi, Fernandez, et al. 2003). Two types of centrifuges (disk-stack and decanter) are commonly used for microalgae harvesting at the industrial scale. A disk-stack centrifuge consists of a relatively shallow cylinder bowl containing several stacks of metal cones which are closely spaced together. The separation of the particulates is based on the size and density of the separating material. Sim, Goh, and Becker (1988) compared centrifugation, chemical flocculation followed by dissolved air flotation (DAF), and membrane filtration processes for harvesting algae from pilot-scale ponds treating piggery wastewater, and they found that none of these processes was completely satisfactory. Centrifugation was reported to be very effective but too costly and energy-intensive to be applied on a commercial scale. Industries mainly used conventional centrifugal sedimentation for algal biomass since it is highly efficient and independent from the type of algae. However, it is a cost-intensive process due to the presence of low biomass in a large volume of algal cultures. Among various available harvesting methods, none can compete with the performance of centrifugal sedimentation (Pirwitz, Rihko-Struckmann, and Sundmacher 2015). Heasman, Diemar, Connor, et al. (2000) assessed nine different strains of microalgae. A cell harvest efficiency of more than 95% was obtained only at 13,000 g. The harvesting efficiency declined to 60% at 6000 g and 40% at 1300 g. During centrifugation, the microalgal cells are exposed to high gravitational and shear forces, resulting in cell breakage and altered morphology (Knuckey, Brown, Robert, et. al. 2006) and the cells’ viability depends significantly on the algal species and the method of centrifugation (Heasman, Diemar, Connor, et al. 2000). Decanter centrifugation is based on the concept of using a settling tank, in which solids are allowed to settle down due to gravitational force and the liquid left after the particle has been extracted is passed through the

48 Algal Biofuel: Sustainable Solution overflow pipe (Rees, Leenheer, and Ranville 1991). The energy consumption of decanter centrifuge (8 kWh/m3) is higher than that of disk-stack/bowl centrifuge (1 kW/m3) (Mohn 1980). Grima, Belarbi, Acien-Fernandez, et al. (2003) stated that the harvesting efficiency of microalgae using decanter centrifuge is better than other harvesting techniques. The clarity of liquid obtained after decanter centrifuge was not better as compared to disk-bowl centrifuge (Smith and Charter 2009). By continuous feeding of algal cells into the centrifuge bowl, the decanter centrifuge operates continuously for large-scale applications. Dassey and Theegala (2013) used continuous decanter centrifuge with a flow rate of 18 L/min and achieved 28.5% harvesting efficiency. Mackay (1996) suggested initial microalgal biomass thickening (such as through DAF) in order to concentrate the cells to 2–3% before using decanter centrifugation. Spiral plate centrifuge manufactured by Evodos is considered a new-generation centrifuge for algal harvesting. The algal cell suspension flows outwards in thin films over vertical plates with the solid microalgae being collected on the outer bottom edge of the vanes. Besides higher capture efficiency of the centrifuge, the cost-effective microalgal harvesting may not coexist with maximum capture efficiency (Dassey and Theegala 2013). It was found that at low-feed flow rate (0.94 L/min), longer retention time, and more energy (20 kWh/m3) result in high removal efficiency (94%) whereas, only 0.80 kWh/m3 energy was required for 17% removal efficiency at a higher feed flow rate of 23 L/min (Dassey and Theegala 2013). This low energy condition results in a decrease in the overall cost per litre of produced oil (Dassey and Theegala 2013). Schlesinger, Eisenstadt, Gil, et al. (2012) suggested reducing the overall cost by lowering the energy consumption. For this, a pre-concentration of the algal slurry should be done by using flocculation/coagulation, because it reduces the volume to be processed.

FUTURE PROSPECTS AND CONCLUSIONS Accelerated consumption of natural fossil fuel reservoirs and increasing greenhouse gas emissions have made the search for an alternative form of energy imperative. Microalgae with high lipid or polyunsaturated fatty acid (PUFA) content are of great interest in the search for a suitable feedstock for the production of biodiesel (Kanaga, Pandey, Kumar, et al. 2016; Perez-Garcia, Escalante, de-Bashan, et al. 2011; Xu, Guo, Wang, et al. 2011; Vazhappilly and Chen 1998). Microalgae offer a great potential for a suitable and sustainable system for the production of biofuel. Microalgae have the ability to grow on non-arable land and also have potential for wastewater treatment (Kothari, Pathak, Kumar, et al. 2012). They convert nutrients from wastewater and atmospheric CO2 (flue gas) into biomass via photosynthesis at much higher rates than conventional crops (Rittmann 2008).

Table 4 Yields achieved by different electrolysis techniques with the use of different electrolytic conditions

Microalgal strain

Electrode material

Initial cell density

Scale

Current/ Current density/ Voltage

Experimental Harvesting Harvesting Energy Reference conditions period efficiency consumption (%)

Microcystis aeruginosa

Aluminium

1.2–1.4 × 106 cells m/L

1.0 L

-

pH 7.0, room 45 min temperature, stirring speed: 200 RPM

Chlorella sorokiniana

1 × 10 cells m/L

0.1 L

25 min

45

0.20 kWh/ m3

5.0 mAcm –1

25 min

100

2.28 kWh/ m3

45 min

78.9

-

90

-

Bleeke, Quante, Winckelmann, et al. (2015)

59.58– 79.26%

1.8–6.0 kWh/kg

Misra, Guldhe, Singh, et al. (2014)

40 V

pH 7.5, room 30 min temperature

Aluminium

9.0 min

Iron

46.9 min

Carbon

2.8 g/L

0.9 L

Gao, Yang, Tiang, et al. (2010)

0.5–1.5 pH 6.0, room 60 min A/-/5.5–8.0 V temperature

Contd...

Algal Biomass Harvesting for Biofuel Production

Scenedesmus acuminatus Magnesium

7

-

0.5 mAcm –1

1 mAcm –1

Iron

100

49

Microalgal strain

Electrode material

Chlorella sorokiniana

Carbon electrode with NaCl (2–6 g/L)

1 A/–/3.7–4.7 V

60 min

68.42– 94.52%

1.6–2.7 kWh/kg

Scenedesmus obliquus

Carbon electrode

0.5–1.5 A/–/6.10– 10.40 V

60 min

49.24– 57.16%

7.5–33.0 kWh/kg

Scenedesmus obliquus

Carbon electrode with NaCl (6 g/L)

Ankistrodesmusfalcatus

Carbon electrode

Desmodesmussubspicatus Iron

Aluminium

Initial cell density

Scale

Current/ Current density/ Voltage

Experimental Harvesting Harvesting Energy Reference conditions period efficiency consumption (%)

0.9 L

1.5 A/–/4.6 V pH 9.0, room 60 min temperature

83 ± 0.1

3.84 kWh/kg Misra, Guldhe, Singh, et al. (2015)

2.88 g/L

1.0 L

0.5–1.5 A/–/– -/ room temperature

69.71– 91.71%

0.84-3.62 kWh/kg

Guldhe, Misra, Singh, et al. (2016)

-

0.5 L

-/5.6 mA/ cm2 /­

-

Baierle, John, Souza, et al. (2015)

pH |5.5 / room temperature -

30 min

15–30 min 64.7– 89.3%

15–20 min 66.0–95.4 % Contd...

50 Algal Biofuel: Sustainable Solution

Table 4 Contd...

Table 4 Contd... Electrode material

Chlorella vulgaris

Scenedesmus sp.

Initial cell density

Scale

Current/ Current density/ Voltage

Experimental Harvesting Harvesting Energy Reference conditions period efficiency consumption (%)

Aluminium

1.0 L

-/2.9 mA/ cm2 /­

pH 4.0, room 60 min temperature, stirring speed 250 RPM

99%

1 kWh/kg

Aluminium

0.4 L

-/12 mA/cm2 /

pH 5.0, room 15 min temperature, stirring speed 200 RPM

100%

2.65 kWh/kg Pandey, Shah, Yadav, et al. (2019)

-/

80–95%

-

35 V

Algal consortium

Lead: cathode and Aluminium: anode

-

100.0 L

1 A/-/26.5 V

75 min

Fayad, Yehya, Audonnet, et al. (2017)

Poelman, De Pauw, and Jeurissen (1997)

Algal Biomass Harvesting for Biofuel Production

Microalgal strain

51

52 Algal Biofuel: Sustainable Solution Recent studies have indicated that major attempts are underway to develop new harvest technologies which are more prompt and cheaper. All the available harvesting technologies depend on microalgae characteristics, as shown in Figure 1. It is our primary goal to do a more accurate economic assessment of different harvesting methods and further feed into life cycle analyses for future algal biorefineries. Based on the present literature, it can be suggested that in order to achieve lower energy consumption and harvesting cost, it is necessary to perform optimization of pre-concentration steps before the dewatering process. However, environmental sustainability must also be considered as an important parameter during the optimization process. Past and recent reports have established the importance of bio-based harvesting methods. They not only improve the removal efficiency and operation costs, but also reduce chemical and biological contaminations after harvesting. Another alternative for algal harvesting could be genetic manipulation of the algal species to identify and overexpress Flo (Yeast)-like genes, which can increase intracellular adhesion and ultimately flocculation (Bauer, Govender, Bester, et al. 2010). This could be of use in certain species to increase the bioflocculating properties, reduce energy consumption, and improve harvesting. The harvesting processes discussed in this chapter are commonly used in microalgal biomass recovery from the culture medium. Among them, not a single method can be claimed to be a universal method which can be applied to harvest all microalgal strains with the same efficiency, as each harvesting process has its own advantages and limitations (as shown in Figure 4). Flocculation and sedimentation require the lowest energy input and these processes have been deemed suitable for large-scale operations. Beside these, centrifugation cross-flow filtration was also found to be suitable for large-scale applications because of its effective separation efficiency, low operating cost, low process time, minimal maintenance, and suitability for numerous algal species. It is also environment friendly. To improve harvesting efficiency and cost, in the present context, it would be safe to say that the combination of two or more harvesting processes (usually, a two-step separation composed of thickening and dewatering) must be adopted. Concerning biofuel application, bioflocculation/ chemical flocculation followed by gravity sedimentation seems to be a costeffective way to harvest microalgal biomass. However, microbiological/chemical contamination may limit the application of bioflocculation when the aim of the separation is for high value-added products. To avoid such contamination of inorganic and organic coagulants and microbiological agents, an electricalpower-based filtration and centrifugation processes may be suitable alternatives. It is suggested that before the selection/designing of the harvesting process, the microalgae cell characteristics/morphology, desired end products, and the factors affecting available harvesting processes (Figure 2) are considered.

Figure 4

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Different techniques applied to microalgae biomass harvesting: advantages and disadvantages

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Algal Biomass Harvesting for Biofuel Production

53

54 Algal Biofuel: Sustainable Solution With the availability of newer and more efficient harvesting systems, microalgae harvesting will become more economical, easier to manage, and more accessible to farmers, rural communities, and industries around the world. The establishment of large-scale microalgal biorefineries in certain countries is on the verge of becoming a reality. Microalgal biorefineries are expected to be first established on a large scale in countries with high irradiation, flat, non-arable, low-biodiversity land.

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Biogas as Bioenergy Option: Advances and Challenges

CHAPTER



Har Mohan Singh1, Atin Kumar Pathak1, V. V. Tyagi1 , Richa Kothari2 and Sanjeev Anand1 

1

School of Energy Management, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India 2 Department of Environmental Sciences, Central University of Jammu, Samba, Jammu and Kashmir, India

INTRODUCTION Climate change is a prominent environmental issue. Global warming has adverse impacts on flora, fauna, and humans. Anthropogenic emissions of gases are the main cause of rising global temperatures. Most of the world’s energy is generated by burning fossil fuels (Appels, Lauwers, Degrève, et al. 2011; IEA 2017). The increasing global focus on renewable energy technologies and greater sustainability aims at lowering emissions rates, minimizing the generation of waste, and increasing utilization of waste. Anaerobic digestion (AD) is a biochemical conversion process with wide acceptance, especially in developing countries, whereby biogas from agricultural wastes and animal manure is extracted. The wide range of raw materials suitable for biogas production, and their relative abundance, evince that the process has gained interest around the world (Abbasi, Tauseef, and Abbasi 2012). Due to abundance of raw materials, a disposition is seen to develop kilowatt to megawatt biogas plants. Various substrate materials can be used in AD, having their own advantages and disadvantages. The use of animal manure and slaughterhouse waste, for example, can lead to ammonia inhibition due to the presence of high nitrogen content; by contrast, plant waste is deficient in nitrogen and trace elements (Mata-Alvarez, Dosta, Romero-Güiza, et al. 2014; Sawatdeenarunat, Surendra, Takara, et al. 2015). Lignocellulosic biomass materials form the most suitable feedstock for biogas production because of their abundance. Since the direct use of crops for biogas production would require large amounts of arable land and may additionally hinder food supply, the utilization of waste and residual parts of agricultural crops is more suitable as it avoids competition

68 Algal Biofuel: Sustainable Solution between food and fuel production (Heeg, Pohl, Sontag, et al. 2014). Waste and marginal lands, forest wastelands, low lands, riparian zones, slopes, and wildlife corridors, which are unsuitable for conventional agriculture due to low-crop productivity, can be utilized for energy crop production, where tall perennial grasses can be grown naturally with minimal inputs (Awasthi, Singh, and Singh 2017). Forest waste residues, such as bark and leaves that are degraded by weathering, can also be utilized for AD to produce biogas. Bachmann, Jansen, Bochmann, et al. (2015) compiled a report on sustainable biogas production in municipal wastewater treatment plants. The production of biogas from sewage, whose benefits are been recognized worldwide, has enormous untapped potential. The biogas produced from sewage sludge can be used as a source of renewable energy; sewage sludge is generated during the wastewater treatment process. The majority of the sludge is composed of organic material. The high nutritive value makes it suitable for microbial growth. This sludge can anaerobically be digested, with microorganisms breaking down the organic materials and converting them into biogas. Primary sludge has a higher energy content than secondary sludge, (which has a smaller degradable fraction). Since biogas production is dependent on the energy content, primary sludge has greater yield. Attempts have been made to increase biogas production using various substrates compositions, microorganisms, and by improving operating parameters. Co-digestion is a hybrid method of AD that resolves the issues associated with using single substrates. It applies multiple substrates such that they are able to provide complementary nutrients, for instance, poultry waste has a high nitrogen content, while the leaves of green plants are rich in organic carbons. These two substrates can be complementary to each other. The valorization of biogas is energy efficient and environment-friendly as it minimizes the emission of hazardous pollutants. Some application technologies require highly purified biogas, which has high methane and low water vapour contents, such as combined heat and power (CHP) in heat and power applications and Bio-CNG for transport applications (Appels, Lauwers, Degrève, et al. 2011). AD produces a huge amount of slurry in the form of digestate, which has a wide variety of nutritional values. This slurry is widely accepted for use in the agricultural sector and enhances crop productivity (Tambone, Genevini, D’Imporzano, et al. 2009). After biogas production, the waste slurry is applied in agricultural fields, which is promotes the zero-waste concept where waste is not produced. AD feasibility ranges from small to large-scale industrial installations, which makes possible the utilization of biogas in the rural areas of developing countries such as India and Nepal, where agricultural residues are abundant. This chapter discusses the optimized parameters of AD, technological advancements, and applications of biogas.

Biogas as Bioenergy Option: Advances and Challenges

69

BIOCHEMICAL PROCESSES OF ANAEROBIC DIGESTION (AD) AD is an environmentally-friendly process to produce self-sustaining energy by utilizing local bioresources. In this process, organic material is converted into a gaseous mixture of methane (CH4) and carbon dioxide (CO2) by the action of microbes in an oxygen-free environment. This is a complex process – the biochemical reactions that are involved in methane formation can be broadly divided into the following four phases: hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Adekunle and Okolie 2015), with various consortia of microorganisms carrying out each phase. The process requires diverse groups of microorganisms with various degrees of metabolic capacities in the four phases, as shown in Figure 1.

Hydrolysis Hydrolysis is the first phase of AD. It involves breaking down of large and complex organic compounds into their constituent parts, through the enzymemediated transformation of the insoluble higher molecular compounds and organic materials, such as lipid, polysaccharides, proteins, fats, nucleic acid, into soluble organic material. The breakdown process requires strict anaerobic   



   

   

  





 

        

      

         

 

       

            

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Figure 1

      

  

    

 

 

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Anaerobic digestion phases (Nzila 2017)

Source Nzila (2017)

     

 

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70 Algal Biofuel: Sustainable Solution Table 1

Hydrolytic enzymes and their functions

Substrate

Enzyme

Breakdown product

Reference

Proteins

Proteinase

Amino acids

Cellulose

Cellulase

Cellobiose and glucose

Hemicellulose

Hemicellulose

Sugars (glucose, xylose, mannose, and arabinose)

Adekunle and Okolie (2015); Odnell, Recktenwald, Stensén, et al. (2016)

Starch

Amylase

Glucose

Fat

Lipase

Fatty acids and glycerol

Pectin

Pectinase

Sugars (galactose, arabinose, and polygalacturonic acid)

conditions and involves anaerobic microorganisms, which secrete various types of extracellular enzymes, which break down macro-molecules into small pieces. These small pieces are further used as sources of energy and nutrition by the microorganisms. Each microbial community is specific to its nature and various microbes secrete several types of enzymes, which break down different kinds of organic materials such as saccharolytic for sugar and proteolytic for proteins. Microbial diversity depends on the type of substrate applied for the production of biogas. For example, cow dung contains a higher presence of proteolytic microorganisms, while poultry waste substrates contain amylolytic microorganisms (Adekunle and Okolie 2015). Similarly, about 17 fermentative microbial communities are identified and their number depends on the chemical composition of the substrate materials used during AD.

Acidogenesis The hydrolytic phase forms a wide variety of monomers that are further acted on in the acidogenesis phase by various facultative and obligatory anaerobic bacteria. In this phase, short chain and volatile fatty acids such as butyric acid, propionic acid, acetic acid that have C1-C5 molecules, alcohols, hydrogen, and CO2 are formed. Hydrogen ion concentration alters the products of fermentation and their higher concentration reduces the formation of acetate-like products (Deublin and Steinhauser 2008; Arif, Liaquat, and Adil 2018). This is the fastest phase in the process of AD of complex organic matter that gives a higher energy yield for the microorganism. The final products of this phase consist of approximately 51% acetate, 19% H2 or CO2, and 30% reduced products such as higher volatile fatty acids, lactate, or alcohols (Kumar, Sridevi, Rani, et al. 2013). These sub-complex compounds are further utilized in the next phase of AD.

Biogas as Bioenergy Option: Advances and Challenges

71

Acetogenesis The production of acetic acid (especially through the action of acetogens) is called acetogenesis. In this process, the interim products are converted in to acetic acid, which is followed by methanogenesis. Intermediates of carbohydrates, fatty acids, and amino acids formed during the acidogenesis phase are not directly used in the methanogenesis phase. These intermediates are further oxidized to acetate and hydrogen in the acetogenesis phase by proton-reducing microorganisms. Acetogenic bacteria consume the necessary energy for their survival and grow at a very low hydrogen ion concentration. Methanogenic bacteria require higher hydrogen concentration. At higher hydrogen concentrations, butyric acid, propionic acid, caproic acid, valeric acid, and ethanol are formed and at lower hydrogen concentrations, acetate is formed by acetogenic bacteria (Li, Chen, and Wu 2019; Arif, Liaquat, and Adil 2018). Therefore, the hydrogen concentration maintains the equilibrium of action by acetogens and methanogens, and these microorganisms live symbiotically, as shown in Table 2. 2CO2 + 4H2

Table 2

CH3COOH + 2H2O……(Equation 1)

Substrate degradation reactions in the acetogenesis phase

Substrate

Reaction

Reference

Propionic acid

CH3 (CH2)COOH + 2H2O CH3COOH + CO2 + 3H2

Butyric acid

CH3 (CH2) 2COO – + 2H2O 2CH3COO – + H + + 2H2

Valeric acid

CH3 (CH2) 3COOH + 2H2O CH3COO – + CH3CH2COOH+H + + 2H2

Deublin and Steinhauser (2008); Zhang, Zhao, Zhang, et al. (2018); Salama, Saha, Kurade, et al. (2019)

Isovaleric acid

(CH3) 2CHCH2COO – + HCO3 – + H2O 3CH3COO – + H2 + H +

Caproic acid

CH3 (CH2) 4COOH + 4H2O 3CH3COO – + H + + 5H2

Carbon dioxide/ hydrogen

2CO2 + 4H2

Glycerine

C 3 H 8 O3 + H 2O CH3COOH + 3H2 + CO2

Lactic acid

CH3CHOHCOO – + 2H2O CH3COO – + HCO –3 + H + + 2H2

Ethanol

CH3 (CH2)OH + H2O CH3COOH + 2H2

CH3COO – + H + + 2H2O

72 Algal Biofuel: Sustainable Solution

Methanogenesis The final phase of AD is the formation of methane by bacteria, wherein the substrate produced in the acetogenesis phase is used. In this phase, various species of methanogenic microorganisms degrade the substrate. Acetoclastic methanogens use acetate to produce methane and CO2 ; hydrogenotrophic methanogens reduce CO2 using hydrogen to produce methane; and methylotrophic methanogens produce methane using methanol (Kumar, Paritosh, Pareek, et al. 2018). The breakdown of acetic acid is given in Equation 2 and the final product of AD is methane. CH3COOH

CH4 + CO2….……(Equation 2)

FEEDSTOCK MATERIALS For biogas production, liquid and liquefied excrement and animal manure are the most suitable substrates because of their biochemical properties. With its high buffering capability and the abundant availability of micronutrients and of digestive forms of carbohydrate, proteins, fats, cellulose, and hemicellulose, animal manure provides ideal conditions for the survival of methanogens. Use of plant biomass as a substrate for AD creates problems as it faces competition with food crops and finally creates a food versus fuel situation. Therefore, it is better to utilize waste materials for AD as described in Table 3. The powder from the leaves of leguminous plants such as Gulmohar, Leucaena leucocephala, Acacia auriculiformis, Dalbergia sissoo, and Eucalyptus tereticornis has augmented the biogas production from 18% to 40%. The augmentation was due to the adsorption of the substrate, which created favourable conditions for the growth of microbes. Similarly, the co-digestion of alkali-treated plants residues of lantana, wheat straw, apple, and peach leaves with cattle dung almost doubled the biogas production, and enhanced the methane production as well. The partially digested organic materials enhanced 10%–18% of the biogas production by enrichment with crop residues of maize stalks, cotton stalk, rice straw, wheat straw, and water hyacinth. The partially digested cattle dung with ageratum and Euphorbia tirucalli also produced 14% more biogas (Kumar, Sridevi, Rani, et al. 2013). With their higher growth rates, greater methane content, and potential to replace food crops such as maize, algae can provide a new horizon of AD such as Chlamydomonas reinhardtii and Scenedesmus obliquus, which are good substrates for biogas production (Mussgnug, Klassen, Schlüter, et al. 2010). Biomass of wild algal strains (algal bloom), which are major contributors of eutrophication in fresh waterbodies such as ponds, lakes, and rivers, are also potential sources of anaerobic co-digestion (Kothari, Ahmad, Pathak, et al., 2018). Algal bloom can easily provide nutrient-rich and higher amount of substrate materials in the form of algal biomass.

Biogas as Bioenergy Option: Advances and Challenges

73

The following are the special features of substrate materials for AD as delineated in literature so far: (i) The substrate materials should be biodegradable. (ii) The substrate materials should be free from pathogens. (iii) The substrate materials should have high nutritional values. (iv) The substrate materials should have the potential to maintain the ideal composition of biogas after AD. (v) The substrate materials should only have trace amounts of toxic elements so that they do not affect the methanogens. (vi) The substrate materials should have the ability to produce compost that can be further utilized.

Table 3

Feedstock materials of anaerobic digestions

Supply sector

Type

Example

Reference

Energy crops

Dry lignocellulosic wood energy crops

Poplar, eucalyptus, acacia sp., mango, azadirachta, banyan, and so on

Gissén, Prade, Kreuger, et al. (2014);

Dry lignocellulosic herbaceous energy crops

Miscanthus, switch grass, Indian shrub

Whittaker, Hunt, Misselbrook, et al. (2016);

Starch energy crops

Wheat, potatoes, maize, barley, amaranth, and so on Flax (linum), hemp (cannabis), and so on Algae, water hyacinth, and so on

Kothari, Ahmad, Pathak, et al. (2018)

Primary sludge – high organic content

Demirbas, Taylan, and Kaya, et al. (2016)

Aquatic and marine biomass

Wastewater

Sludge

Secondary sludge – smaller degradable fraction than primary sludge Industrial residues

Wood industry residues

Waste of foodprocessing industries

Fibrous vegetable waste from virgin pulp production and from production of paper from pulp, (including black liquors) Milk, food, beer

Salehian, Karimi, Zilouei, et al. (2016);

Wu, Dong, Yao, et al. (2011)

Contd...

74 Algal Biofuel: Sustainable Solution Table 3

Contd...

Supply sector

Type

Example

Reference

Municipal residue

Organic waste materials

Paper, wood, food scraps, cotton, wool, leather, and so on

Ofoefule, Nwankwo, and Ibeto (2010); Priebe, Kipper, Gusmão, et al. 2016; Patel (2017)

Agricultural waste

Dry lignocellulosic agricultural residues

Cattle manure Landfilling

Municipal solid waste deposited leachate

Straw rice)

(maize,

cereal,

Gissén, Prade, Kreuger, et al. (2014)

Sugar beet leaves Residue flows from bulbs Dung of cow, buffalo, sheep, goat, and so on Degrading sediments of sewage sludge and municipal solid waste

Xu, Qin, and Ko (2019)

MICROBIAL COMMUNITY There are many different microbes participating in the various phases of AD. Bacteria are responsible for the hydrolysis of high-molecular organic substances, leading to the formation of oligo- and monomers, and these intermediates are further converted into volatile fatty acids (VFAs) in acidogenesis phase. In the acetogenesis phase, acetic acid, CO2, and hydrogen are formed. In the methanogenesis phase, acetic acid, CO2, and hydrogen are converted into methane, biogas, and allied matter by acetoclastic and hydrogenotrophic archaea (Ahring 2003). Both hydrolytic/acidogenic bacteria and archaea differ in nature and their requirements are varied such as pH, nutrient, and growth metabolism. The physical separation of acid-forming and methane-producing microbes is best suitable in two separate containers which explore ideal conditions for individual microbial conversion phases (Heeg, Pohl, Sontag, et al. 2014). Individual microbial strains have the capacity to enhance the production of biogas by stimulating the activity of particular enzymes such as cellulolytic strains of bacteria (actinomycetes). Table 4 illustrates the effects of various parameters on the microbial community.

IMPORTANT PARAMETERS There are several important parameters that affect the AD process. From the optimization point of view, temperature, C/N ratio, pH-value, organic loading rate, and hydraulic retention time are the key parameters.

Biogas as Bioenergy Option: Advances and Challenges

Table 4

75

Effects of various parameters on microbial community

Operational parameter

Microbial community

Reference

Temperature

Mesophilic

Firmicutes, Bacteroidetes, Proteobacteria phyla

Thermophilic

Firmicutes, Thermotogae phyla

Pap and Maróti (2016); Wang, Wang, Qiu, et al. (2018); Jha and Schmidt (2017)

Firmicutes phylum, Clostridium genus Methanosarcina, Methanothermobacter genera Methanosarcina genus Methanoculleus genus Volatile fatty acid

Low acetate

Methanosaeta genus

High acetate

Methanosarcina genus

Low ammonia

Methanosaeta genus

High ammonia

Methanosarcina genus, Methanoculleus genus

Substrate

Low C/N ratio

Bacilli class, Thermotogae phylum

Other parameters

High salt

Methanocelleus genus

Aeration

Methanoculleus genus

Ammonia

Temperature Temperature is a significant parameter in biogas production because of the involvement of microorganisms that show sensitivity with the change in their surroundings. There are three different kinds of microbial communities classified on the basis of the range of temperature, which are as follows: s 0SYCHROPHILIC TEMPERATURE FROM  # TO  # s -ESOPHILIC TEMPERATURE FROM  # TO  # s 4HERMOPHILIC TEMPERATURE FROM  # TO  # 4HE OPTIMUM TEMPERATURE RANGE FOR THE ACIDIFYING BACTERIA IS n # Most of the methanogens fall in the mesophilic category. Mesophilic bacterial digestion produces more biogas (30% more for cattle manure and 22% for sewage sludge) than psychrophilic digestion. Thermophilic conditions are mostly applied in large-scale centralized co-digesters biogas plants. Thermophilic

76 Algal Biofuel: Sustainable Solution digesters have lower retention times because of the higher catalytic activity of thermophiles, as well as reduced risks of contaminants (Kothari, Pandey, Kumar, et al. 2014). Thermophilic digestion has a 50% higher degradation rate, and due to this, fat-containing substrates easily break on higher temperature range, provide optimum raw materials for microorganisms food, and obtain a higher yield of biogas production. The solubility of oxygen at thermophilic range is very slow, which is best suited for the AD. Although mesophilic range of temperature is most favourable for the hydrolysis phase and similarly thermophilic range of temperature for the methanogenesis, substrate materials can change the operating optimized temperature ranges of various biogas production phases.

C/N ratio The C/N ratio represents the relationship between the amount of carbon and nitrogen in the feedstock. AD requires an optimized C/N ratio for the degradation of organic materials. The presence of nitrogen in the feedstock materials has two advantages: it is the main constituent of amino acids, proteins, and nucleic acids; and it is easily converted to ammonia, which regulates the optimum pH level. The lower C/N ratio substances increase ammonia formation that elevates pH level at 8.5. This is the main cause of toxicity in the digester and subsequently reduces the rate of biogas production. It can be maintained by an analysis of the chemical composition of substrate materials and proper agitation in the digester. It has been observed that the C/N ratio in the range of 20–30 is suitable for AD, with 25 representing an optimized ratio. It also varies with feedstock such as organic solid waste, animal manure, poultry waste, and so on, as shown in Table 5 (Kothari, Pandey, Kumar, et al. 2014). Wang, Lu, Li, et al. (2014) studied the relationship between temperature and C/N ratio performance in the co-digestion of dairy manure, poultry manure, and rice straw. Increasing the temperature from the mesophilic to the thermophilic range reduces ammonia production, and it can be controlled by increasing the C/N ratio of mixed feedstock at an appropriate level. In the anaerobic co-digestion of DAIRY MANURE POULTRY MANURE AND RICE STRAWS THE #. RATIO WAS  AT  # AND  AT  # 4HEREFORE IT WAS OBSERVED THAT TEMPERATURE CAN ALSO CAUSE fluctuations in the C/N ratio, and plays an important role in its regulation.

pH values pH is the activity of hydrogen ion concentration and a measure of the acidity and alkalinity of a solution. The optimized pH value for methanogenesis is 7. Lee, Behera, Kim, et al. (2009) reported that the optimum range of pH for methanogenesis is 6.5–8.2. The pH value continuously changes in various phases of AD, that is, hydrolysis, acidification, acedogenesis, and methanogenesis, and

Biogas as Bioenergy Option: Advances and Challenges

Table 5

77

C/N ratio of various feedstock materials.

Feedstock material

C/N ratio

Reference

Dairy manure, poultry manure, and rice straws

26–30

Cattle manure

15–26

Matheri, Ndiweni, Belaid, et al. (2017); Siddique and Wahid (2018)

Chicken manure

3–10

Straw

50–150

Sugar cane waste

139–151

Oat straw

47–51

Sheep manure

20–34

Slaughterhouse waste guts

21–26

Distillery waste

8

Food waste

3–17

Swine manure

7–15

Fruit and vegetable waste

8–36

Wheat straw

51–151

Corn waste

21

Algal sludge with waste paper

51–57

depends on the retention time of the digestive substrate. The acidogenesis phase lowers the pH below 5 because of the accumulation of a large number of organic acids (butyric, propionic, capronic, and valeric acids). Hydrogen transportation takes place through nicotinamide adenine dinucleotide (NAD) and various products are formed in the fermentation process, as shown in Equation 3, and it takes place during the phase of hydrolysis and acidification of protein and hydrocarbons. Protein degradation does not produce pH-buffering ions but helps to acidify hydrocarbons. Thus, the degradation of carbohydrates reduces the pH value (Deublin and Steinhauser 2008). The thermophilic acidogenic phase occurs at pH 6–7. The pH reduction can be controlled by the addition of lime and other alkaline substances, and the ammonia produced during the methanogenesis phase also helps in elevating the pH level to 8 in the digester (Kothari, Pandey, Kumar, et al. 2014). The pH values of the various phases of AD also depend on the feedstock, which can alter the whole AD process, and consequently, biogas production. NADH + H+

H2 + NAD + ……(Equation 3)

78 Algal Biofuel: Sustainable Solution

Organic loading rate Microorganisms decompose substrate materials continuously for biogas production in an anaerobic digester. When the substrate material gets completely decomposed/consumed by microorganisms, the digestion process is complete with end-products. Therefore, it is necessary to add an optimized dose of organic materials into the anaerobic digester for continuing the digestion process. Loading is a term that indicates the addition of new organic materials to the AD process per unit time is commonly known as organic loading rate (OLR). The OLR needs to maintain the microbial growth rate in a substrate. For this, an optimized OLR is required in both types of digesters (continuous flow and batch): 1–6 kg COD/m3 reactor volume/day and 0.5 kg/m3/reactor volume/day for the continuous flow and batch, respectively (Angelidaki, Chen, Cui, et al. 2006; Nielsen 2009). The optimized OLR values were reported by Zhang, Loh, and Zhang (2018) as 7.5 gVS/L/d, 3 gVS/L/d, 11.2 gVS/L/d, and 23.6 gCOD/L/d for co-digestion of manure and food waste. A substrate rich in digestible materials, such as residues from the food processing industry, with high sugar content allows easier microorganism digestion as compared to fibrerich materials, such as plant residue, while woody intermediates are hard to digest and take more time. Like other parameters of AD, OLR also depends on substrate materials, microorganism, pH, and other factors, and may take longer or shorter periods. Because of the acclimatization factor, microorganisms are directly affected by the growth parameters.

Hydraulic retention time Hydraulic retention time (HRT) is defined as the time taken to replace organic materials in the digester, that is, the average time that the organic substrate stays in the digester (Siddique and Wahid 2018). During the AD process, microorganisms continuously consume organic materials to produce biogas, thus, a feed-up of organic substrate materials in the digester at regular intervals is required. HRT of AD is generally 10–30 days, but it can also change. Kaosol and Sohgrathok (2012) analysed various properties of biogas production in continuous stirrer reactors at different HRTs, as shown in Table 6. Fibre-rich substrates such as plant residue, which involve a long hydrolysis phase, can have a long HRT, while fruits and starch-rich substrates, which are more easily broken down, have a short HRT. Therefore, an optimized HRT depends on the substrate materials’ composition, temperature, and microbes.

PROPERTIES OF BIOGAS The final products of AD of organic materials are methane, CO2, digested slurry, small amount of hydrogen sulphide, and water vapour, as presented in Table 7. Hydrogen sulphide’s odour (like that of rotten eggs) is a characteristic

Biogas as Bioenergy Option: Advances and Challenges

79

Table 6 Hydraulic retention time and biogas production (Kaosol and Sohgrathok 2012) Property

HRT (days)

Unit

10

20

30

Biogas production

2.99

2.88

1.83

L/day

Maximum biogas production

5.50

3.53

2.35

L/day

Methane production

1.57

1.86

1.18

L/day

Maximum methane production

3.27

2.29

1.46

L/day

Average methane

51.7

64.6

63.6

%

Maximum methane

60.2

66.7

67.4

%

feature of digester gas. It causes a variety of problems which are health-related and it is a highly corrosive gas that over acidifies the engine oils during combustion in the gas engine. This may lead to a deterioration of materials and structures. The composition of biogas varies depending on the origin of the AD process, for example, landfill gas has 50% methane concentration and advanced waste treatment technologies can produce 55–75% methane.

UPGRADATION OF BIOGAS Although biogas is a clean and an eco-friendly fuel, in addition to the 55–65% of CH4 content, it also contains a number of other contaminants (such as water vapour, traces of H2, H2S, siloxanes, and so on). These contaminants render biogas unsuitable for use in more sophisticated technologies such as CHP and vehicle engines unless they are purified (Appels, Lauwers, Degrève, et al. 2011). The main purpose of biogas upgradation is purification for targeted

Table 7 Specific features and composition of biogas (Rao, Baral, Dey, et al. 2010; MNRE) Composition of biogas

60%–65% methane, 35–40% CO2, 0.5%–1.0% hydrogen sulphide, and rest includes water vapour, traces of other gases

Energy content

6.0–6.5 kW/m3

Fuel equivalent

0.6–0.65 L oil/m3 biogas

Explosion limits

6–12% biogas in air

Ignition temperature

650–750oC

Critical temperature

–82.1oC

Critical pressure

75–89 bar

Normal density

1.214 kg/m3 (assuming about 60% methane and 40% CO2)

Liquefied pressure

47.4 kg/cm2

Calorific value

20 MJ/m3 (4700 kcal)

80 Algal Biofuel: Sustainable Solution technology. CO2 reduction in biogas enhances the heating value and reduces the density of biogas. Therefore, the upgradation of the quality of biogas is needed. Many biogas purification technologies have been developed, which are primarily based on the removal of CO2 and H2S, in order to attain more purified methane. These are adsorption-based, membrane-based, and cryogenicbased techniques. Water absorption and chemical absorption are the most common technologies for biogas upgradation, while pressure swing adsorption and membrane technologies are gaining attention. Cryogenic biogas purification technology is in a developing stage and most of the purification work has been done at the laboratory scale (Yousef, El-Maghlany, Eldrainy, et al. 2018). The selection of suitable upgrading technology depends on the specific application, site specification, and cost. The biogas upgradation techniques discussed by Awe, Zhao, Nzihou, et al. (2017) are given in Table 8. Water vapour also poses problems in the downstream application of biogas. Water vapour content depends on the temperature of the digester. Lower temperature results in greater accumulation of water vapour in biogas, which must be dried-up before the introduction of biogas into energy conversion technologies. Water vapour is mostly removed by condensation and chemical adsorption methods.

Table 8 Biogas upgradation technologies: advantages and disadvantages (Awe, Zhao, Nzihou, et al. 2017) Technology

Advantage

Disadvantage

Absorption

High efficiency (97–99%) Simultaneous removal of H2S, NH3, HCN, and H2O Low-cost and energy requirements

Requires higher initial cost Clogging problems Toxic waste production

Membrane technology

High efficiency (96%) H2S and H2O are removed

Requires high power and gas Multiple steps required to reach higher degree of purity of CH4

CO2

Simple construction and operation High reliability Low cost Cryogenic separation

High degree of purification, 90–98% of CH4

Higher capital cost because of higher equipment cost

H 2S Absorption

High purification rate Low-operating cost Compact technology

Various problems in controlling temperature and pressure during operation Toxic waste production

Membrane technology

Higher removal rate up to 98% and CO2 Expensive operation and is also removed maintenance Highly complex technology

Biogas as Bioenergy Option: Advances and Challenges

81

TYPES OF DIGESTERS A wide variety of anaerobic digesters have been developed and utilized across the world based on their requirements and availability of substrate material. Among these, the selection of a suitable digester is a complex task. Anaerobic digesters are of two types: batch and continuous flow. The different types of biogas plants commonly used are given in Table 9 and Figure 2.

APPLICATIONS OF BIOGAS Biogas has a wide range of direct and indirect applications. Biogas can be used for direct combustion (for heat production), power production by fuel cells and micro-turbines, as vehicle fuel, and in combined heat and power production, as shown in Figure 3. The low calorific value is an important factor as it hinders the conversion of biogas into chemical and thermal energy, thus, upgradation is a necessity. The applications of biogas are:

Table 9

Advantages and disadvantages of biogas plants

Biogas plants type

Advantage

Disadvantage

Fixed-dome plant (Deenbandhu, Janata, and CAMARTEC models) (Figure 2A)

Low cost and long life-span Less metalwork, thus free from rusting

Leakage problems because of requirement of special kinds of sealant and dome made of brickwork, which is at higher risk of cracks Volume of produce cannot be easily analysed

Floating drum plant (Khadi and Village Industries Commissions [KVIC], BORDA, and Ganesh models, Pragati model, ASTRA model) (Figure 2B)

Compact design – saves space No moving parts

Fluctuation in gas pressure that makes gas utilization complex

Thermal comfort index is high because of underground construction Construction of plants provides local employment opportunities

Underground construction makes reaching optimum temperature difficult because the inner temperature of the dome does not easily change

Volume of gas produced is easily observed

High material cost

Gas pressure remains constant

Metalwork increases the chances of corrosion and rusting, therefore, reducing the life-span Needs regular maintenance and high technical expertise

Construction is comparatively easy, reducing construction mistakes

Contd...

82 Algal Biofuel: Sustainable Solution Table 9

Contd...

Biogas plants type

Advantage

Disadvantage

Balloon plant (Figure 2C)

Produced gas is easy to transport

Low-gas pressure across the anaerobic digestion Plastic balloons have short life-span

Free from deep underground burying and can be easily installed on ground High digester temperature results in higher rate of methanogenesis

Earth pit plant

Horizontal plant (Figure 2D)

Ferrocement plant

More susceptible to damage

Easy to clean

Requires high technical expertise during control of leakage and repair

Low cost of installation

Short life-span

Self-sustainability of cleaning

Construction is costly because of high price of plastic gasholder

Requires small space because of shallow construction Easy to remove slurry

Gas leakage problems

Low cost of construction

Difficult to remove scum from digester Gas leakage problems Requires high cost of raw materials, for example, ferrocement, wire mess

Lighting Biogas is most commonly used for lighting purposes in rural and remote regions, where electricity has not reached yet. The light output from biogas (maximum 400–500 lm achieved) is comparable to a normal 25–75 W light bulb, which is sufficient for basic lighting needs (Kossmann and Pönitz 2011). Nevertheless, biogas lighting lamps are not yet as developed as the presently available electricity-based light appliances, therefore, more research and development are required in this domain.

Cooking Direct burning is one of the most prominent applications of biogas. The biogas produced from digesters can easily be used for cooking purposes with domestic burners, and there is no need to advance the technology. Biogas use for domestic and community cooking applications in rural parts of South Asian and African countries allows the fuel to be supplied by the utilization of waste manure as feedstock. Biogas burners supply gas at a pressure of 10 mbars and 4:1 air–fuel

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83

 

  







  

 



    



 

  



      

  

 



Figure 2 Types of digesters: (A) fixed-dome plant; (B) floating dome plant; (C) balloon plant; (D) horizontal plug-flow digester

84 Algal Biofuel: Sustainable Solution

Figure 3

Applications of biogas

ratio (Kadam and Panwar 2017). After lighting, cooking is the main application of biogas in various parts of the world. In India, biogas applications are widely accepted for cooking purposes because the direct burning of biogas is a lowcost and pollution-free source of energy.

Others Presently, developed countries utilize biogas for mechanical energy generation in controlled combustion systems by heat engines and electricity production. Germany increased the number of biogas fuel-based power plants by approximately 20%, from 1050 in 2001 to 6000 in 2010, and the Mexican landfill gas model predicted that the electricity production would increase by 2.4 MW in 2019. Biogas-based power production is in a nascent stage and needs technical advancements for development of other applications based on biogas as a fuel material. Technical deficiencies are the bottlenecks in this biogas development utilization. Sweden uses biogas in trains and buses and their refilling stations are established on the streets (Kadam and Panwar 2017; Scarlat, Dallemand, and Fahl 2018). Multi-dimensional applications of biogas depend on a biogas-dedicated policy and the development of low-cost technology. Both mbar and MPa used to indicate Pressure, please use one convention and standardise. Biogas can be compressed up to 20–25 MPa pressure in

Biogas as Bioenergy Option: Advances and Challenges

Table 10

85

Typical biogas and Bio-CNG composition

Compound

Composition (%) Raw biogas

Bio-CNG

Methane (CH4)

40–75

93

Carbon dioxide (CO2)

25–55

4

Ammonia (NH3)

0–1

0.

Nitrogen (N2)

0–5

2.94

Hydrogen (H2)

0–1

0.06

Hydrogen sulphide (H2S)

50–5000*

20*

Oxygen (O2)

0–2

-­

Water (H2O)

0–1

-­

Reference

Kadam and Panwar (2017); Khan, Othmanb, Hashim, et al. (2017)

*ppm – part per million

steel cylinders. The clean and upgraded biogas can be compressed for value addition and this bio-compressed natural gas (Bio-CNG) can be utilized as a fuel for transport vehicles. It can be a suitable replacement for petrol (gasoline), diesel, and LPG. The Bio-CNG can be stored in cylinders. Three-stage biogas purification and bottling plants are also being established, which are solving the complexities of transportation and reducing the cost (Vijay, Chandra, Subbarao, et al. 2006). Table 10 shows the properties of Bio-CNG which are similar to that of raw properties. Vapour absorption machine and radiant heater (up to n # HAVE BEEN DEVELOPED TO UTILIZE BIOGAS AS FUEL +OSSMANN AND Pönitz 2011). These are path-breaking applications that are raising new hopes for the utilization of biogas. After the digestion of feedstock material, a large amount of digestate is left, which has potential use in the agricultural sector due to its nutritional values. Apart from energy production of AD, it acts as an organic fertilizer because digestate material is easy to decompose in the agricultural field and liberates various nutrients, which are necessary for plant growth. This is an interesting way for digestate to be reutilized as an organic fertilizer. Thus, digestate provides various applications – restores soil quality, provides conditioning, enhances the fertility of degraded agricultural lands, and increases crop productivity. Hence, the AD process improves environmental sustainability.

CHALLENGES/BOTTLENECKS There are various technical and non-technical challenges that have been identified in the production of biogas. These challenges not only hinder the growth of biogas, but also create wastes that can cause various diseases and pollute the environment. Mittal, Ahlgren, Shukla, et al. (2018) described a broad spectrum of bottlenecks associated with biogas in the Indian context,

86 Algal Biofuel: Sustainable Solution Table 11 Challenges associated with biogas production (Mittal, Ahlgren, Shukla, et al. 2018) Challenges for biogas production

Remark

Economical and financial challenges

High capital cost of biogas technologies is a challenge for rural application where opportunities of biogas application are much higher Average cost of family-size biogas plant of capacity 1 m3 is about $348 Government provides 20–40% of the cost of the plant and their capacity as subsidy Monthly expenditure of rural India is only around $150

Market challenges

Abundance and low-cost availability of firewood, cattle dung, solid biomass Biogas faces competition from liquid petroleum gas distribution systems

Socio-cultural challenges

Rural population is reluctant to use night soil (human excreta) in biogas plants because of social and cultural habits Women who take care of cooking, are not the decision-makers in rural areas and they have no say in choosing either wood or biogas for the cooking purposes, which is the main barrier

Regulatory and institutional challenges

Family size biogas plants require manure from two to three cattles and every family cannot afford it due to low income Lack of coordination among various implementing agencies involved in the development of biogas programmes

Technical challenges

Adoption of unscientific behaviour such as inaccurate mixing ratio of water and substrate, improper installation, and so on, thereby promoting malfunctioning of biogas plants due to lack of awareness and unskilled users

which can be a representative for both developed and developing economies. The challenges, as presented in Table 11, are discrete and subject to regional, social, and climatic considerations; a factor applied to a particular region may not be globally acceptable. Mitigation of these challenges can help disseminate biogas production in rural and urban areas. Additionally, the climatic variations pose prominent challenges in India – these conditions range from extremely hot deserts to cold mountainous topographies. India possesses six climates zones – among these, the Himalayan states such as Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, and Arunachal Pradesh have cold climate; central India and the southern states have hot and dry climatic conditions; and seashores are hot and humid. Low

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temperature in the cold climatic states increases the HRT and retards the rate of AD because in below optimum temperatures, the rate of microbial activity reduces. Hot and dry climatic states such as Rajasthan and Gujarat, where temperature remains high, they experience a reduction in HRT and an increase in AD, but higher temperature that is more than the optimum level also reduces the biogas production. The main cause of the reduction rate of AD is the OPERATIONAL TEMPERATURE OF BIOGAS PLANTS n n # n WHICH IS HIGHER THAN THE OPTIMUM TEMPERATURE n n # ,OHAN +UMAR $IXIT et al. 2015). Therefore, AD requires greater attention for the development of region- or space-specific digesters, which can withstand various climatic conditions.

CONCLUSION Anaerobic Digestion is a robust process and most of the feedstock material is both locally and abundantly available. It can be directly used for lighting, cooking, space heating, thermal energy, and electricity production applications. Bio-CNG can act as a substitute for fossil fuels (petrol, diesel, and LPG) with higher calorific values. Continuous improvement of biogas production and ‘technologies’ upgradation are the light of hope for providing technoeconomically viable energy security to the world. However, more research is needed to improve operational parameters of biogas plants to enhance production. s !VAILABILITY AND QUALITY OF FEEDSTOCK MATERIAL INFLUENCE THE PERFORMANCE of AD technology, its cost, and the use of biogas; s 4HERE IS A NEED FOR DEEPER RESEARCH ON OPTIMIZED PARAMETERS OF !$ AS these vary from place to place, and negatively affect the production of biogas; s 5PGRADATION OF BIOGAS IS STILL A CHALLENGING AND COST INTENSIVE PROCESS which retards sophisticated applications such as CHP and transport as these applications require a higher degree of purified biogas or BioCNG; s 4HERE IS A REQUIREMENT OF A UNIVERSALLY ACCEPTABLE BIOGAS DIGESTER WHICH is compatible to the contemporary challenges of the AD process; s #OOKING AND LIGHTING ARE THE BASIC APPLICATIONS OF BIOGAS !PART FROM these, research and development of biogas-adoptive technologies can create more applications of biogas in the near future.

88 Algal Biofuel: Sustainable Solution

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Application of Algal Biomass as a Feedstock for Fermentative Biohydrogen Production

CHAPTER



Veeramuthu Ashokkumar1,2 , Wei-Hsin Chen2, and Chawalit Ngamcharussrivichai1,3 1

Center of Excellence in Catalysis for Bioenergy and Renewable Chemicals (CBRC), Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, Thailand 2 Department of Aeronautics and Astronautics, National Cheng Kung University, Tainan, Taiwan 3 Center of Excellence on Petrochemical and Materials Technology (PETROMAT), Chulalongkorn University, Pathumwan, Bangkok, Thailand

INTRODUCTION Today, extensive efforts are being made worldwide to find sustainable and renewable energy sources that could replace fossil fuels and mitigate global warming and other environment-related issues. But owing to the rapid increase in the human population globally and combustion of fossil fuels, greenhouse gas (GHG) emissions have increased extensively. Also, greenhouse effect is responsible for arctic ice melting, rising sea levels, extreme heat waves, and frequent occurrences of droughts. Currently, the CO2 emission concentration is above 350 parts per million (ppm), which will possibly increase the greenhouse effect with the rising global temperature. The majority of GHGs are emitted from motor vehicles, and this alone accounts for more than 20% of CO2 emissions and 70% of carbon monoxide (CO) (Goldemberg 2008). Therefore, alternative renewable energy sources have become one of the key solutions of this century in attaining sustainable and green energy for the future. Different types of renewable energy sources have been identified by researchers: solar, hydro, wind, and bio-energy (Lam and Mohamed 2010). In addition to this, the energy produced from the combustion of biomass and wastes has made a significant contribution to society as it has helped in lowering the GHG emission, thus indicating an immense opportunity for biomass in the energy sector. Among the different biofuels, hydrogen (H2) fuel is considered as one of the cleanest renewable energy sources because in its combustion, zero CO2

94 Algal Biofuel: Sustainable Solution is emitted and water vapour is produced as a by-product (Brentner, Peccia, and Zimmerman 2010). Usually, H2 gas contains a higher energy value of 142 MJ/kg, whereas methane gas holds 56 MJ/kg and gasoline 47 MJ/kg (Lam and Lee 2011). Researchers believe that biohydrogen produced from microalgae is an energy-dense alternative energy source with clean-burning properties, which can reduce the GHG emissions to a great extent. Although H2 is the most common element present in the earth, it does not occur in elemental form. Currently, 96% of H2 energy is derived from chemicalbased and non-renewable resources, particularly from steam methane reforming (SMR) process, petroleum refining, and coal gasification (Srirangan, Pyne, and Perry Chou 2011). Hydrogen produced from methane through the SMR process is an endothermic reaction, and the energy utilized for H2 production comes from fossil fuel resources (Mun Sing Fan, Abdullah, and Bhatia 2009). Besides, H2 production from methane is unsustainable and emits GHGs into the atmosphere. Hydrogen production exceeds 1 billion m 3/day, of which 48% of H2 is produced from natural gas, 30% from oil refineries, 18% from coal, and 4% from water electrolysis (Jong 2009). Moreover, H2 produced from fossil fuels leads to an increase in the emission of GHGs, namely, CO2, CH4, etc., to the atmosphere (Jong 2009). Therefore, to address the aforementioned issues, H2 produced from biological sources will be renewable, sustainable, and environment-friendly (Oncel and Sabankay 2012; Oncel 2013). Generally, biohydrogen is produced through different groups of microorganisms, namely bacteria (Srirangan, Pyne, and Perry Chou 2011), microalgae, and cyanobacteria (Ghirardi and Prasanna 2010; McKinlay and Harwood 2010; Oncel 2013). However, H 2 produced from photosynthetic microorganisms holds several benefits towards environmental impact and low-production cost (Eroglu and Melis 2011). Gaffron and Rubin (1942) first reported H2 metabolism through green microalgae. Subsequently, Gest and Kamen (1949) studied H2 synthesis using photosynthetic bacteria. Later, researchers developed easier techniques to produce H2 which involved using microorganisms grown under aerobic and anaerobic conditions (Melis 2007). Also, the major advantage of biological H2 production is that it does not compete with agricultural land for food production like other biofuels (biodiesel and bioethanol). Currently, several methods have been adopted to produce biohydrogen from microalgae. They include indirect biophotolysis, direct biophotolysis, dark fermentation, and photofermentation (Hallenbeck and Benemann 2002). Hydrogen production through anaerobic fermentation is a relatively simple and low-cost technique, which can be used in a broad spectrum of substrates, particularly microalgae species such as Chlamydomonas sp., Scenedesmus sp., Nannochloropsis sp., Chlorella sp., Dunaliella sp., etc. Recently, researchers have focused on establishing a lowcost techniques for biological H2 production at a commercial scale (Mathews and Wang 2009). Therefore, this chapter discusses the detailed survey about the

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application of algal biomass as a potential feedstock material for fermentative biohydrogen production.

MICROALGAE Microalgae are commonly found in freshwater and marine water. They are the most primitive and simple organized groups in the plant kingdom and refer to all microscopic oxygenic phototrophs. Although microalgae lack leaves, stems, and roots, like plants, they have chlorophyll pigment for photosynthesis. They use water, CO2, and sunlight to synthesize their own food, and produce biomass and oxygen as by-products. Microalgae are grouped as prokaryotic (Cyanobacteria), eukaryotic green algae (Chlorophyta), red algae (Rhodophyta), and diatoms (Bacillariophyta), and have a robust growth due to their simple structure. Researchers have demonstrated that the microalgae can be easily cultivated in seawater, freshwater, and wastewater resources. These organisms are cultivated at a large scale through two different cultivation methods: open condition (open raceway pond) and closed condition (photobioreactor) (Ashokkumar, Chen, Al-Muhtaseb, et al. 2019). Microalgae are also considered as one of the most attractive feedstocks for food, medicine, and energy production. These organisms are commonly of heterotroph, mixotroph, and autotroph in nature. The autotrophic microalgal groups require inorganic carbon sources such as light energy, CO2, salts for their growth, whereas the heterotrophic microalgal species are non-photosynthetic organisms and cannot synthesize their own food. At the same time, these heterotrophic microalgal species use organic compounds present in the growth medium as carbon and energy sources. In the mixotrophic mode of cultivation, the microalgae can grow with both light and organics as energy sources, and CO2 and organic carbon as carbon sources. Additionally, these algae groups have the ability to do photosynthesis and acquire exogenous organic nutrients. The mixotrophy nature makes the microalgae better adapted to the environment. Microalgal biomass is generally composed of three major cellular components: lipids (5–65%), carbohydrates (10–50%), and proteins (15–40%). Meanwhile, the percentage of cellular components is mainly dependent on the microalgal species, environmental conditions, and cultivation methods. In addition, a nutritional limitation plays a vital role in the synthesis of cell components.

Microalgal Biomass and its Potential Applications Microalgae and seaweeds play a crucial role as sources of nutrients, and countries such as China, Korea, and Japan widely use algal biomass in several applications (John, Anisha, Nampoothiri, et al. 2011). Microalgae are a group of photosynthetic organisms like plants; they evolve O2 during photosynthesis and take CO2 for growth. Also, microalgae are the primary

96 Algal Biofuel: Sustainable Solution source material for understanding the fundamentals of photosynthesis in higher plants. Scientists have found that microalgae are potential biomass sources of proteins, carbohydrates, and lipids, which can be easily converted to various biofuels such as biodiesel, bioethanol, and biogas (Chang 2016) to solve the energy appetite of the world. On the earth, microalgae are considered as the most primitive photosynthetic organism, because they are primary producers of the ecosystem. Algae are the highest photosynthetically efficient (10–50 times higher) organisms with a short life cycle and they are high biomass producers in comparison to terrestrial crops. Moreover, they do not compete with land and water resources like other food crops and can be grown at a large scale using open raceway and closed photobioreactor methods. Higher plants need lignin and hemicellulose, the structurally complex compounds, for their growth. Microalgae do not require lignin for their growth as they are unicellular and buoyant type of organisms. Thus, the aforementioned characteristic features make algae a potential candidate for an alternative source for bioenergy production. Compared to macroalgae (seaweeds), microalgae hold several advantages, as the algal cells are composed of high-value compounds such as pigments, vitamins, antioxidants, polyunsaturated fatty acids, etc. They can also play a vital role in the pharmaceutical and nutraceutical industries (Yun, Jung, Kim, et al. 2013). Scientists believe that microalgal cultivation that adopts a biorefinery approach could be the possible way to reduce the production cost and this will make bioenergy production from microalgae commercially viable. Generally, microalgal biomass is rich in lipids, carbohydrates, and proteins, which can be easily converted to various biofuels. Further, biomass residues obtained after primary extraction can be utilized for several processes such as thermochemical conversion and anaerobic digestion. Recently, researchers have started focusing on biorefinery approaches as cost-effective technologies using microalgal species. Also, microalgae are considered a potential source for atmospheric CO2 sequestration. Green microalgal species (e.g., Chlamydomonas reinhardtii) are considered as the best feedstock for biohydrogen production. Thus, microalgal species with high amount of carbohydrates could be used for fermentative H2 production through anaerobic bacteria. They are discussed in detail in the next section.

Microalgae for Fermentative Biohydrogen Production Fermentative H2 production is a conversion process of organic substrates to H2 through a fermentative organism (e.g., bacteria). These organisms can utilize the carbon sources and release H2 as a by-product via the anaerobic pathway. This method has been exploited for decades by researchers for biological H 2 production. During biohydrogen synthesis, the fermentative organisms utilize

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proton as terminal electron acceptors and help to produce biohydrogen (Lee, Show, and Su 2011). Mostly, biohydrogen production is carried out through biophotolysis process; nevertheless, this method has some hurdles. Biohydrogen production through some microalgal species is limited by efficient growth with higher photosynthetic efficiencies. Generally, microalgae can absorb and use photon energy to generate H2 from sunlight and water. Besides, the other major problem of biohydrogen conversion is the efficient conversion of photons, and it was measured in terms of H2 released and the photons used in the process. However, it seems to be lower in microalgal H2 production methods. Nonetheless, compared to biophotolysis process, H2 production from fermentative method is simple and free of all the aggravations of an algal cultivation system (Eroglu and Melis 2011). Generally, microalgae have tremendous capacity for producing lipids and carbohydrates which can be easily converted into biofuels. Microalgae grow faster compared to other land plants and produce high-biomass yield, because of the absence of lignin. The absence of lignin in microalgae holds many advantages because processing lignin is currently a significant impediment for bioethanol production. In microalgae cultivation, specific nutritional stress such as sulphur and nitrogen depletion enhances carbohydrate productivity by up to 50%. Researchers have studied nitrogen starvation during the cultivation of microalga Chlorella vulgaris and found that 53% of the carbohydrate content increased (Ho, Lee, Su, et al. 2012). Thus, microalgal species with highcarbohydrate content attracts many researchers to the conversion of various biofuels, particularly for biohydrogen production. Therefore, fermentative H2 production through microalgal species could be a promising method for biohydrogen production at a commercial level. The most promising way to produce biohydrogen was well established through a fermentative process. In this process, the conversion of microalgal carbohydrates into biohydrogen involves a large number of H2-producing mesophilic bacteria (Citrobacter, Clostridium, and Enterobacter), thermophilic anaerobic fermentative bacteria (Caloramator, Clostridium, Thermoanaerobacter, etc.), and facultative bacteria (e.g., Escherichia coli). Biohydrogen production from microalgal species has possible advantages in the dark fermentative pathway. In this process, the energy metabolism does not require any specific energy for glucose transportation into the cell, however, without expending adenosine triphosphate (ATP), and they produce glucose-6-phosphate. Hence, biohydrogen production from carbohydrates obtained from microalgae through dark fermentation requires lowenergy input. Further, biohydrogen production from microalgal biomass through dark fermentation takes place at a higher rate compared to photofermentation, microbial electrolysis, and biophotolysis. Researchers have reported that biohydrogen synthesis has been successfully done through dark fermentation

98 Algal Biofuel: Sustainable Solution using facultative anaerobes such as Enterobacter aerogenes, E. cloacae, and obligate anaerobic bacteria (Clostridium beijerinckii and Ruminococcus albus) (Mutanda, Ramesh, Karthikeyan, et al. 2011).

Biohydrogen and Biomass Pretreatment Technologies To improve the efficiency of biohydrogen production from microalgal biomass via fermentative process, the basic pretreatment stages are considered as the best option to release the cell components easily. Currently, researchers have established various pretreatment methods for microalgal biomass and improving the maximum bioenergy extraction. The most important pretreatment methods are: (i) physical methods which include mechanical, heat, and ultrasonication treatments; (ii) chemical methods which include acid, base, and ozone treatments; (iii) biological methods including enzymatic and microbiological treatments, and (iv) a combination of different pretreatment methods. Researchers found that the most common method used for the pretreatment of microalgal biomass which releases carbohydrates were milling, ultrasonication, steam explosion, microwave radiation, chemical method, and enzymatic hydrolysis. Notwithstanding, the aim of all these pretreatment methods is mainly designed only for the purpose of breaking the cell wall of the microalgal biomass and to release the cell wall components which can be easily converted into different forms of bioenergy such as biohydrogen. By these methods, the microalgal cell wall gets lysed and releases carbohydrates, which can be efficiently utilized by the H2 producing bacteria. Some studies have demonstrated pretreatment technology and biohydrogen production from microalgal species. The microalgae Chlamydomonas reinhardtii contains high amount of carbohydrates, and this can be easily converted to biohydrogen through the fermenting organism Thermotoga neopolitana. A study was conducted with the previously mentioned fermenting organism using microalgal biomass as a substrate and the results show that no biohydrogen production was perceived when the microalgae were used as a substrate without the pretreatment process. Nevertheless, the ultrasonication pretreatement of microalgal biomass showed significant bioethanol yield, and this was due to cell wall breakage and release of sugar (Nguyen, Kim, Nguyen, et al. 2010). Some studies showed that biomass without undergoing any basic pretreatment process yields low biohydrogen (474 mL/L), while the biomass after pretreatment produced high amount of biohydrogen yield (1424 mL/L) (Liu, Chang, Cheng, et al. 2012). Generally, prior to the fermentative H2 production from microalgal biomass, the algal cell wall needs to be disrupted first and stored carbohydrates released which is present in the cells. After that, the next step involves the saccharification of converting the accumulated sugars into monomeric units. Through this technique, fermentable sugars, such as monosaccharides and

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disaccharides, usually pentoses and hexoses, were converted to bioethanol or biohydrogen with the help of fermenting organisms. The sugar obtained from the microalgal biomass can be easily used by anaerobic biohydrogen-producing microorganism through glycolysis process, and the metabolite pyruvate can be supported for biohydrogen production (Liu, Chang, Cheng, et al. 2012). Today, several pretreatment methods such as mechanical, chemical, enzymatic, and thermal are widely applied to lysis the cell wall of microalgae. Each method has its own advantages and limitations, and discussed in detail in this chapter. Usually, the mechanical pretreatment technique follows the bead beating and milling process; in this method, the cell wall of the microalgal species breaks down through shear force and liberates cellular components, particularly sugars for hydrolysis. A biorefinery concept was explored in the marine microalgal species Nannochloropsis. In this study, after biodiesel extraction, the residual biomass was homogenized by milling followed by supercritical fluid extraction for biohydrogen production. Most of the researchers widely follow the ultrasonication pretreatment technique to extract various biofuels and bioproducts. The ultrasound wave techniques release the shear forces, which are generated by acoustic cavitation at the frequency of 20 kHz (Yun, Jung, Kim, et al. 2013), and this energy-intensive technique was used for the maximum extraction of cell wall components. In thermal pretreatment techniques, heat energy is utilized and applied in several thermal processes which include pyrolysis, liquefaction, and gasification, respectively. The microalgae Nannochloropsis oceanica biomass was investigated for fermentative H2 production using combined thermal and chemical treatments and gave high yield (Xia, Cheng, Ding, et al. 2013a). In addition, microwave heating was also preferred as a pretreatment process in fermentative H2 production. It has been reported that thermal pretreatment alone did not show any significant role in the extraction of sugar from the microalgal biomass. Nevertheless, the combined chemical and heat treatment methods improved the biohydrogen production from the microalgal biomass (Xia, Cheng, Lin, et al. 2013b). Mostly, the pretreatment process was conducted through chemical methods, because it is an effective and a low-cost technique widely used by researchers. In this technique, different solvents, acids, and alkalis were used to lysis the cells of microalgal biomass and release the components. Notwithstanding, the alkali pretreatment method was extensively used by researchers to treat the lignocellulosic biomass materials, and, at the same time, the alkali pretreatment techniques were ineffective when they were applied to the microalgal biomass (Liu, Chang, Cheng, et al. 2012). In this case, acid hydrolysis is considered as the most preferred and efficient method. The pretreatment of hemicellulose biomass through a strong acidic condition can lead to the development of fermentative inhibitors that are toxic in nature. In addition, higher concentration inhibits the fermentation process and gives low

100 Algal Biofuel: Sustainable Solution yield. Studies show that during hydrolysis of microalgal biomass using acid, the fermentative inhibitors are found to be low or non-toxic. Moreover, after the acid pretreatment method, the mixture is generally neutralized by base catalyst and used for fermentation process. Some researchers observed that during acetone–butanol–ethanol (ABE) fermentation by Clostridia in the presence of acid during heat treatment at 121°C, biohydrogen productivity reduced by three folds (Efremenko, Nikolskaya, Lyagin, et al. 2012). The most common technique used in thermal pretreatment is a simple autoclaving method and when combined with dilute acid is considered a potential method used to lysis the cell wall of the microalgal biomass. In this method, the operation is easier and simpler, besides, it also yields high amount of reducing sugar which can be easily converted into various biofuels such as bioethanol and biohydrogen (Kumar, Gupta, Kumar, et al. 2013). The other important method used in biomass pretreatment technology is enzymatic hydrolysis. Generally, enzymatic hydrolysis is a process in which enzymes facilitate the cleavage of bonds in the molecules with the addition of the elements of water. This technique has been widely used in the food industry. Currently, most researchers are using enzymatic hydrolysis process in bioenergy sectors for fermentation biohydrogen and cellulosic bioethanol production. However, the microalgal cell wall structure and carbohydrate storage vary from species to species based on the enzyme cocktail design for a particular strain. Some research studies reported that biohydrogen synthesis was evaluated using microalgae Chlamydomonas reinhardtii biomass and the results revealed that amylolytic enzyme Termamyl in simultaneous saccharification and fermentation (SSF) process supported maximum H2 production. In this technique, the lysed microalgal cells were taken and the enzyme was added to the culture medium and incubated. Further, the glucose and cellobiose obtained after the hydrolysis process were completely metabolized by using the bacteria (Thermotoga neopolitana), and the volumetric biohydrogen production was estimated up to 32 mL H 2 /h/L (Nguyen, Kim, Nguyen, et al. 2010). According to the literature survey, the application of hydrolytic enzyme technology for fermentative biohydrogen production is considered to be a high-cost method and it is not recommended by the research for commercial production. Nevertheless, to reduce the cost, some reports show that the enzymes which are partially purified, or crude can be involved during the fermentative process. Today, the production of biohydrogen through anaerobic fermentation process is an area of comprehensive research and attract many researchers globally. The microalgal species and fermentative biohydrogen production are listed in Table 1. The table details how biohydrogen production can be carried out by utilizing different microalgal species at different pretreatment processes and productivity.

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ADVANTAGES AND LIMITATIONS OF BIOHYDROGEN FROM MICROALGAE Today, microalgal biomass is used globally for many purposes, because of its highly valuable components. In bioenergy synthesis, the lipids and carbohydrates obtained from the microalgal biomass have lot of advantages in the bioenergy sector, and these lipids and carbohydrates can be used for the production of bioethanol/biohydrogen and biodiesel. Mostly, microalgal biomass contains high amounts of sugar, which can exist in the form of glucose and some polysaccharides such as carrageenan, starch, and agar, and it can be easily converted to various fermentative products. Microalgal lipids are mainly composed of triglycerides which can be transesterified to produce biodiesel. Besides, the microalgal biomass produces long-chain fatty acids, pigments, and protein molecules which hold numerous benefits in pharmaceutical and nutraceutical industries. In this connection, the residues’ biomass after lipid or biodiesel extraction can be employed for biohydrogen production through a fermentative process with high yield (Figure 1). In this emerging scenario, microalgae are also used to treat different levels of wastewater, such as domestic, industrial and municipal, and convert them into valuable products. The main advantages of microalgae are that they can grow fast when compared to higher plants and play an important role in CO2 sequestration. Microalgal biomass contains approximately 60% carbon on a dry weight basis and they are mostly derived from CO2. For example, to produce 1 kg     $ %&

          

# 

           

    

                

    !   ! "      #  

Figure 1

Schematic diagram of fermentative biohydrogen production from microalgae

102 Algal Biofuel: Sustainable Solution of biomass, it requires 1.83 kg of CO2. One of the significant shortcomings of microalgae for biohydrogen production is the cost involved in large-scale cultivation and harvesting process. In addition, due to the more extensive water content in microalgal cells, biomass drying is considered to be an expensive process. Besides these, microalgal cultivation at the commercial level requires an additional cost-effective technology. For example, the growth nutrient cost is the major issue that has led to algal biofuels fail at the commercial level till now, and it should be replaced by some other alternative resources such as wastewater. Nevertheless, to address the aforementioned problems, technologies must be improved in various aspects. Thus, researchers have focused on applying an integrated cost-effective biorefinery technology to make microalgal bioenergy (e.g., biohydrogen) successful at the commercial level (Figure 1).

Table 1 Microalgal species and fermentative biohydrogen production Microalgal species

Fermentative organism

Pretreatment method

Biohydrogen production

Reference

Chlorella sp.

Heat treated anaerobic sludge

–

7.13 mL/g VS

Sun, Yuan, Shi, et al. (2011)

Microwave heating with diluted H2SO4

183.9 mL/g VS

Xia, Cheng, Ding, et al. (2013a)

Nannochloropsis Heat treated oceanica anaerobic sludge Chlorella pyrenoidosa

Mixed microbial Steam heating 83.3 mL/g VS consortia with diluted acid

Xia, Cheng, Lin, et al. (2013b)

Chlamydomonas Thermotoga reinhardtii neopolitana

Enzymatic hydrolysis

Chlorella sorokiniana

Steam heating 9 ± 2 mol H2 /kg Kumar, Gupta, with diluted HCl COD Kumar, et al. reduced (2013)

Enetrobacter cloacae IIT-BT 08

311.1 mL H2 /g Nguyen, Kim, monosaccharide Nguyen, et al. (2010)

Chlorella vulgaris Clostridium ESP6 butyricum CGS5

Steam heating 1476 mL/L with diluted HCl cumulative H2

Liu, Chang, Cheng, et al. (2012)

Nannochloropsis Immobilized sp. Clostridium acetobutylicum B-1787

0.1 mM H2SO4 at 108°C for 30 minutes

8.5 ± 0.2 mmol/L/day

Efremenko, Nikolskaya, Lyagin, et al. (2012)

Scenedesmus obliquus

Clostridium butyricum

Dry biomass, autoclaved

7.3 g H2 /kg biomass

Ferreira, Ortigueira, Alves, et al. (2013)

Arthrospira platensis

Anaerobic sludge

Heating at 135°C with 2.5% H2SO4

85.0 mL/g VS

Xia, Jacob, Tabassum, et al. (2016)

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CONCLUSION Microalgal species are capable of producing high amount of lipids and carbohydrates (cellulose or starch) as stored materials, and this makes algae a potential feedstock for biohydrogen production. Recently, significant breakthroughs have been made to improve the biohydrogen productivity from microalgae, which includes genetic engineering and cultivation methods. In order to make the microalgal biohydrogen production a success, this technology can be integrated with wastewater treatment, industrial flue gas (CO2) mitigation, and high value-added bioproducts production. Recently, significant attention focused on microalgae as the renewable feedstock for biohydrogen production. However, in comparison to terrestrial biofuel-producing feedstocks, microalgae hold a number of advantages and can easily convert solar energy into various biofuels with higher photosynthetic efficiency. According to the research published on microalgal biohydrogen production, the results revealed that the fermentative H2 production from microalgae shows excellent potential in sustainable energy generation. Biohydrogen production from microalgal cells can be improved through proper cell disruption techniques, especially pretreatment methods. For example, the microalgae Chlamydomonas reinhardtii seems to be the most promising feedstock for biohydrogen production. However, the biohydrogen production rate is still far lower when compared to chemical-based processes such as steam methane reforming, which hampers commercialization. Therefore, to address these issues, the following factors need to be considered carefully before scaling up microalgae-based biohydrogen production, and they are growth nutrient, cultivation methods, harvesting techniques, and suitable pretreatment processes. In addition, the integrated bio-refinery approaches could be the possible solution for improving the overall energy balance and economic feasibility of biohydrogen production.

References Ashokkumar, V, W-H. Chen, Ala’a H Al-Muhtaseb, G Kumar, P Sathishkumar, S Pandian, F N Ani, and C Ngamcharussrivichai. 2019. Bioenergy production and metallic iron (Fe) conversion from Botryococcus sp. cultivated in domestic wastewater: Algal biorefinery concept. Energy Conversion and Management 196: 1326–1334 Brentner, L B, J Peccia, and J B Zimmerman. 2010. Challenges in developing biohydrogen as a sustainable energy source: implications for a research agenda. Environmental Science and Technology 44(7): 2243–2254 Chang, J S, P L Show, T C Ling, C Y Chen, S H Ho, C H Tan, D Nagarajan, and W N Phong. 2016. Photobioreactors. Current Developments in Biotechnology and Bioengineering. Elsevier (Chapter 11) in press

104 Algal Biofuel: Sustainable Solution Efremenko, E N, A B Nikolskaya, I V Lyagin, O V Senko, T A Makhlis, N A Stepanov, O V Maslova, F Mamedova, and S D Varfolomeev. 2012. Production of biofuels from pretreated microalgae biomass by anaerobic fermentation with immobilized Clostridium acetobutylicum cells. Bioresource Technology 114: 342–348 Eroglu, E and A Melis. 2011. Photobiological hydrogen production: recent advances and state of the art. Bioresource Technology 102(18): 8403–8413 Ferreira, A F, J Ortigueira, L Alves, L Gouveia, P Moura, and C Silva. 2013. Biohydrogen production from microalgal biomass: energy requirement, CO2 emissions and scale-up scenarios. Bioresource Technology 144: 156–164 Gest, H, and M D Kamen. 1949. Studies on the metabolism of photosynthetic bacteria IV: photochemical production of molecular hydrogen by growing cultures of photosynthetic bacteria. Journal of Bacteriology 58(2): 239–245 Ghirardi, M L and M Prasanna. 2010. Oxygenic hydrogen photoproduction – current status of the technology. Current Science 98(4): 499–507 Goldemberg, J. 2008. Environmental and ecological dimensions of biofuels. In Proceedings of the Conference on the Ecological Dimensions of Biofuels. Washington, DC. Hallenbeck, P C and J R Benemann. 2002. Biological hydrogen production; fundamentals and limiting processes. International Journal of Hydrogen Energy 27(11): 1185–1193 Ho, K-L, D J Lee, A Su, J S Chang, 2012. Biohydrogen from lignocellulosic feedstock via one-step process. International Journal of Hydrogen Energy 37(20): 15569–15574 John, R P, G S Anisha, K M Nampoothiri, and A Pandey. 2011. Micro and macroalgal biomass: A renewable source for bioethanol. Bioresource Technology 102(1): 186–193 Jong, W. 2009. Sustainable hydrogen production by thermochemical biomass processing. In Gupta, R B. (Ed.), Hydrogen Fuel: Production, Transport, and Storage. Boca Raton: CRP Press. Details available at https://doi.org/10.1201/9781420045772, last accessed on 27 April 2020 Kumar, S, R Gupta, G Kumar, D Sahoo, and R C Kuhad. 2013. Bioethanol production from Gracilaria verrucosa, a red alga, in a biorefinery approach. Bioresource Technology 135: 150–156 Lam, M K and K T Lee. 2011. Renewable and sustainable bioenergies production from palm oil mill effluent (POME): Win–win strategies toward better environmental protection. Biotechnology Advances 29(1): 124–141 Lam, M K, K T Lee, and A R Mohamed. 2010. Homogeneous, heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: A review. Biotechnology Advances 28(4): 500–518 Lee, D-J, K-Y Show, and A Su. 2011. Dark fermentation on biohydrogen production: Pure culture. Bioresource Technology 102(18): 8393–8402 Liu, C-H, C-Y Chang, C-L Cheng, D-J Lee, and J-S Chang. 2012. Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich

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microalgal biomass as feedstock. International Journal of Hydrogen Energy 37(20): 15458–15464 Mathews, J and G Wang. 2009. Metabolic pathway engineering for enhanced biohydrogen production. International Journal of Hydrogen Energy 34(17): 7404–7416 McKinlay, J B and C S Harwood. 2010. Photobiological production of hydrogen gas as a biofuel. Current Opinion in Biotechnology 21(3): 244–251 Melis, A 2007. Photosynthetic H2 metabolism in Chlamydomonas reinhardtii (unicellular green algae). Planta 226: 1075–1086 Mun-Sing Fan, A Z Abdullah, and S Bhatia. 2009. Catalytic technology for carbon dioxide reforming of methane to synthesis gas. ChemCatChem 1(2): 192–208 Mutanda, T, D Ramesh, S Karthikeyan, S Kumari, A Anandraj, and F Bux. 2011. Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production. Bioresource Technology 102(1): 57–70 Nguyen, T-A D, K-R Kim, M-T Nguyen, M S Kim, D Kim, and S J Sim. 2010. Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods. International Journal of Hydrogen Energy 35(23): 13035–13040 Oncel, S and M Sabankay. 2012. Microalgal biohydrogen production considering light energy and mixing time as the two key features for scale-up. Bioresource Technology 121: 228–234 Oncel, S S. 2013. Microalgae for a macroenergy world. Renewable and Sustainable Energy Reviews 26: 241–264 Srirangan, K, M E Pyne, and C Perry Chou. 2011. Biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria. Bioresource Technology 102(18): 8589–8604 Sun, J, X Yuan, X Shi, C Chu, R Guo, and H Kong. 2011. Fermentation of Chlorella sp for anaerobic biohydrogen production: influences of inoculum-substrate ratio, volatile fatty acids and NADH. Bioresour. Technol 102: 10480–10485 Xia, A, J Cheng, L Ding, R Lin, R Huang, J Zhou, and K Cen. 2013a. Improvement of the energy conversion efficiency of Chlorella pyrenoidosa biomass by a three-stage process comprising dark fermentation, photofermentation, and methanogenesis. Bioresource Technology 146: 436–443 Xia, A, J Cheng, R Lin, H Lu, J Zhou, and K Cen. 2013b. Comparison in dark hydrogen fermentation followed by photo hydrogen fermentation and methanogenesis between protein and carbohydrate compositions in Nannochloropsis oceanica biomass. Bioresource Technology 138: 204–213 Xia, A, A Jacob, M R Tabassum, C Herrmann, and J D Murphy. 2016. Production of hydrogen, ethanol and volatile fatty acids through co-fermentation of macro- and microalgae. Bioresour. Technol 205: 118–125 Yun, Y-M, K-W Jung, D-H Kim, Y-K Oh, S-K Cho, and H-S Shin. 2013. Optimization of dark fermentative H production from microalgal biomass by combined (acid + ultrasonic) pretreatment. Bioresource Technology 141: 220–226

Bioethanol Production from Lignocellulosic/Algal Biomass: Potential Sustainable Approach

CHAPTER



Surajbhan Sevda1, Vijay Kumar Garlapati2, Poulami Datta3, Anuj Kumar Chandel4, Lalit Pandey3, Dheeraj Rathore5, Anoop Singh6, and T R Sreekrishnan7 1

Department of Biotechnology, National Institute of Technology Warangal, Telangana, India 2 Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India 3 Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam, India 4 Department of Biotechnology, Engineering School of Lorena, University of São Paulo, Lorena, Brazil 5 School of Environment and Sustainable Development, Central University of Gujarat, Gandhinagar, Gujarat, India 6 Department of Scientific and Industrial Research, Ministry of Science and Technology, Government of India, Technology Bhawan, New Delhi, India 7 Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, India

INTRODUCTION The demand for sustainable energy supply is increasing due to declining fossil energy resources, increasing environmental pollution and climate change, and increasing dependency on oil-exporting countries. Bioethanol is one of the most important biofuels preferred over fossil fuels because of its high octane number and higher oxygen content, which produces less greenhouse gas (particularly, CO2) after burning (Ganguly and Garlapati 2017). It is miscible with petrol so it can be utilized directly in the automobile industry without any further modification in engine. There are three generations of bioethanol based on the production source. The first-generation bioethanol is usually produced from food crops by fermentation of glucose and sugar-derived substrates (starch) (Nail, Goud, Rout, et al. 2010). The second-generation bioethanol is derived from lignocellulosic biomass, wood residue, sawdust, low-value timber, straw,

108 Algal Biofuel: Sustainable Solution forest, and agricultural residues (Chandel, Garlapati, Singh, et al. 2018; Aditiya and Mahlia 2016). The most recent, third-generation bioethanol production is an emerging technology using microalgae feedstock and cyanobacteria. The microalgae can be heterotrophic and autotrophic, and are capable of generating a large amount of biomass-containing carbohydrates, proteins, and lipids (Alaswad, Dassisti, Prescott, et al. 2015). Microalgae are unicellular photosynthetic microorganisms having cellulose-based cell walls and can be grown using CO2 as their primary carbon source and solar light as their energy resources (Lee, Cho, Chang, et al. 2017; Menetrez 2012).

BIOETHANOL FROM LIGNOCELLULOSIC MATERIAL The food chain conflict of the first-generation bioethanol (from food crops) paved the way for second-generation bioethanol. The second-generation bioethanol mainly revolves around every available lignocellulosic biomass. The lignocellulosic biomass includes different types of grasses, weeds, and woody material, which have very limited or no usage. The main structural components of lignocellulosic material include cellulose, hemicellulose, and lignin. The main components for bioethanol production are cellulose and hemicellulose fractions. To produce bioethanol, lignocellulosic material has to undergo different processing steps, which are pretreatment/delignification, hydrolysis/saccharification, fermentation, and downstream processing. The pretreatment technique helps in disrupting the complex lignocellulosic structure, which results in the release of polysaccharides from lignin. In the hydrolysis/ saccharification step, the released polysaccharides undergo hydrolysis to C6 sugars (glucose) and C5 sugars (xylose) by cellulolytic enzymes. The released sugars are further fermented to produce bioethanol through C6 and C5 pathways. The produced bioethanol is further purified through downstream processing steps (Chandel and Silveira 2017). The typical steps in the production of bioethanol from lignocellulosic material are depicted in Figure 1.

          

  



  

   

  

Figure 1

Schematic representation of bioethanol production from lignocellulosic material

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Pretreatment Pretreatment/delignification is primarily aimed at the separation of lignin from cellulose and hemicellulose. The step facilitates the most of the cellulose crystallinity by removing the lignin and preserving the hemicelluloses fractions. Various physico-chemical and biological techniques are used for the pretreatment step. The physico-chemical techniques are rapid but suffer from harsh environment and inhibitory compounds produced. The produced inhibitory compounds will affect the hydrolysis step, usually by inhibiting the enzymatic activities. The biological laccase-mediated pretreatment method is a selective and an eco-friendly process. During the enzymatic conversion, no new inhibitory compounds were produced. The main drawback of the biological pretreatment is that it takes more processing time than the acid-based pretreatment (Sharma, Garlapati, and Goel 2016; Roy, Garlapati, and Banerjee 2015). Hence, the usual practice in lignocellulosic industries is a combination of physico-chemical techniques. The advantages and disadvantages of different pretreatment techniques are tabulated in Table 1.

Hydrolysis The higher hydrolysis ratio is obtained based on the adopted pretreatment techniques. These include crystallinity and degree of polymerization of cellulose and inhibitory compounds (5-hydroxymethyl-furfural [HMF], levulinic acid). The hydrolysis step is usually carried out through the acid or enzymatic/cellulase. Mostly, enzymatic is preferred over acid hydrolysis due to selectiveness and associated mild-reaction conditions lesser by-products being formed. The inhibitory compounds produced during the pretreatment step may have adverse effects on the cellulase activity. The latter takes higher hydrolysis duration than acid hydrolysis and has reduced hydrolysis efficiency. Moreover, a successful hydrolysis also depends on the adsorption of cellulase on pretreated lignocelluloses material. The cellulase adsorption on pretreatment is mainly affected by the presence of inhibitory compounds and by-products produced in the process. The overall products from the hydrolysis step are C6 sugars (glucose) and C5 sugars (xylose) (Kuila, Sharma, Garlapati, et al. 2017).

Fermentation In this, the released C6 and C5 sugars in the hydrolysis step are fermented to produce bioethanol through C6 and C5 fermentation pathways, respectively. Generally, C6 sugars are fermented by Saccharomyces cerevisiae and C5 sugars by bacterial/yeast species to produce bioethanol. Having more unit operations in the production of bioethanol from lignocellulosic materials and fermentation of C6 and C5 sugars while utilizing a single microbe is an economically viable

110 Algal Biofuel: Sustainable Solution Table 1 Various pretreatment strategies for lignocellulosic biomasses: advantages and disadvantages Pretreatment

Advantage

Disadvantage

Mechanical

No inhibitors production

High power consumption, energy intensive

Alkaline

Changes cellulose crystallinity and ameliorates enzymatic accessibility

Carbohydrate degradation, requires high capital

Oxidative

High selectivity towards lignin

Loss of hemicellulose and cellulose, if selective oxidant is not selective

Thermal

Effective solubilization hemicellulose

of Low sugar yield, high amount of enzyme needed

C5 sugars separation, cellulose modification High amount production

of

Inhibition or production, corrosion issues sugars Phenolic inhibitors generation

High biomass digestibility Pyrolysis

Rapid decomposition cellulose into gases

Sugars loss, high capital requirement of

Volatile products generation

Steam explosion High hemicellulose solubilization, better enzymatic hydrolysis

High energy demand, inhibitors production

Hydrodynamic cavitation

High sugars recovery, mild process conditions

High energy consumption and difficulty in scale up in continuous process systems

Liquid hot water

Efficient C5 sugars recovery

Sugars-derived inhibitors generation, partial solubilization of lignin

CO2 explosion

No inhibitors generation, low environmental impact

High energy requirement

Ammonia fibre explosion

Low inhibitors, high solubilization of lignin

Environmental concerns, energy intensive

Ozonolysis

Low inhibitors, High c onsumption of lignin, and hemicellulose environmental concerns breakdown

Ionic liquids

Efficient lignocellulose breakdown

High cost and ionic liquid recovery concerns

High cellulose solubilization

Retainment of lignin in solution, maintenance of microwaves

High solubilization of hemicellulose and lignin

High cost of solvents and solvent recovery

Lignin degradation by fungi and solvent-mediated hydrolysis of hemicellulose

High amount of ethanol and recovery concerns

Produc tion of enz ymes, environment friendly

Very slow process, difficulty in scale up

Organosolv

Biological

Source Chandel and Silveira (2017)

ozone,

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option (Chandel, Junqueira, Morais, et al. 2014). The fermentation ability of different microbes and the fermenting abilities of glucose and xylose are summarized in Table 2.

Downstream Processing: Separation and Distillation The obtained bioethanol from the fermentation step needs further purification and ethanol has to be separated from water through fractional distillation or continuous distillation. In this process, the alcohol–water mixture is boiled and based on the volatilities, water and ethanol will separate from each other (Chandel and Silveira 2017).

Table 2 Advantages and disadvantages of different microbial species in the fermentation of hexose and pentose Microbial species Advantage Saccharomyces cerevisiae

Naturally adapted fermentation

Disadvantage to

ethanol Not able sugars

High ethanol tolerance

to

ferment

pentose

Unable to withstand the higher hydrolysis temperature

Amenable to genetic modifications Zymomonas mobilis

Amenable to genetic modifications Not able to ferment xylose sugars Does not require additional oxygen

Low tolerance to inhibitors

Candida shehatae

Ferments xylose

Low tolerance to ethanol

Pichia stipitis Recombinant

Ferments xylose

Does not work at low pH

Able to ferment glucose, galactose, and cellobiose

Sensitive to inhibitors

Low yields Does not work at low pH

Requires microaerophilic conditions for better performance Recombinant Escherichia coli

Ferments both pentose and hexose sugars

Limited ethanol tolerance

Amenable to genetic modifications Narrow pH and temperature range Low tolerance to inhibitors and ethanol Kluyveromyces marxianus

Ferments broad spectrum sugars

Excess sugars affect the alcohol yield

Reduces contamination

Low ethanol tolerance

Amenable to genetic modifications Probable chance towards formation of xylitol Source Limayem and Ricke (2012)

112 Algal Biofuel: Sustainable Solution Algae: the third-generation biomass and bioethanol production Microalgae can grow in any natural environment including freshwater, seawater, wastewater in open ponds, and closed photobioreactors (Sambusiti, Bellucci, Zabaniotou, et al. 2015; Vassilev and Vassileva 2016). Due to their intense growth profile and high protein, pigment, vitamins, lipid, and carbohydrate-accumulating capacity (Bastos 2018) the low lignin content and their saccharification is much easier, and hence can be used as a significant source for bioethanol formation. Microalgal biomass can be converted to a different form of energy (bioethanol, biobutanol, biogas, biohydrogen, and biodiesel) by various thermal, chemical, and biological methods. The starch may be employed for bioethanol production through a standard fermentation process considering its economic feasibility by Chlorella vulgaris (GarcíaCubero, Moreno-Fernández, Acién-Fernández, et al. 2018). Microalgae could produce 23,400 kg of bioethanol per hectare per year (Noraini, Ong, Badrul, et al. 2014). The main pathways for bioethanol production using microalgae are yeast fermentation using microalgal inoculum, enzymatic hydrolysis, metabolic pathway in dark environment, and photo fermentation (conversion of organic substrate to biohydrogen by photosynthetic bacteria). Bioethanol production can be carried out through the following steps: preliminary biomass treatment, hydrolysis technique, fermentation processes, and product recovery mechanisms. (a) Biomass pretreatment: Alkaline pretreatment of microalgal inoculum (Chlorococcum infusionum) was investigated for bioethanol production using NaOH. This process helped to release the polysaccharides entrapped in microalgal cell wall and it was converted to fermentable sugar. For the determination of effectiveness of the pre-treatment process, glucose concentration, bioethanol concentration, and inoculum size were considered as the crucial parameters. The highest bioethanol yield was 0.26 g/g microalgae using 0.75% NaOH (w/v) at 120°C for 30 minutes (Harun, Jason, Cherrington, et al. 2011). The pretreatment intensification of cell disruption and sugar release from Scenedesmus obliquus (23.7% starch content) microalgae for bioethanol production has been described by Miranda, Passarinho, and Gouveia (2012). The pretreatment process included physical process preparation (bead beating, homogenization, sonication, and autoclaving) as well as chemical disruption by some acids and bases (NaOH, HCl, and H2SO4). The optimized conditions were achieved through acid hydrolysis by 2N H2SO4, at 120°C for 30 minutes. Sugar extraction was 95.6% in comparison with quantitative acid hydrolysis. The effect of other variables such as biomass loading, temperature, concentration of acid, and the number of extraction cycles was also analysed. Chlamydomonas reinhardtii mutant cw15 was used

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as the microalgal biomass to produce bioethanol by Scholz, Riley, and Cuello (2013). Liquid hot water (LHW) pretreatment was utilized along with the enzymatic hydrolysis for bioethanol production from microalgae. The optimum conditions for the bioethanol production from Scenedesmus sp. WZKMT were determined by response surface methodology and the parameters were found to be – solid and liquid ratio 1:13 (w/v), 147°C, and 41 minutes. The 14.22 g/L of glucose with the conversion rate of 89% was achieved, which was five-fold greater than the control (Yuan, Li, Xiao, et al. 2016). (b) Hydrolysis: Chlamydomonas reinhardtii UTEX 90 microalgal biomass, which contains 44% starch accumulation, was transformed into fermentable substrate by using two commercially available enzymes (B-amylase and amyloglucosidase). Separate hydrolysis fermentation (SHF) method resulted in 235 mg bioethanol generated from 1 g microalgal biomass. The most favourable conditions for B-amylase (0.005%, pH 4.5)-based SHF method were found to be 0.5 h incubation at 90°C. In case of glucoamylase (0.2%, pH 4.5)-based SHF, the conditions seemed to be equal to 30-minute incubation time at 55°C using orthogonal analysis technique (RenJie 2008). This work proved that the enzymatic hydrolysis of microalgal biomass could be used as a possible substrate for subsequent fermentation process by S. cerevisiae S288C (Choi, Nguyen, and Sim 2010). A feasible approach for bioethanol production utilizing lipid-extracted microalgal biomass of Scenedesmus dimorphus was studied by Chng, Chan, and Lee (2016). The microalgal biomass was first saccharified and then fermented without any costly and energyintensive pretreatment step, which reduced the contamination risk in the biorefinery process. The best conditions for maximum amyloglucosidase activity were analysed, which were found to be 60 units/mL enzyme concentration, at pH 5 and 36°C and yeast loading concentration of 3 g/L. Theoretically, 90% conversion was achieved and the highest bioethanol production was 0.26 g/g of lipid-extracted microalgal biomass (96%). Carbohydrate and lipid-rich oleaginous microalga Tribonema sp. was used for bioethanol and biodiesel production. The 50 g/L microalgal biomass was acid hydrolysed with 3% H2SO4, at 121°C for 45 minutes for prominent hydrolysis efficiency of 81.48%. A maximum bioethanol production of 56.1% was obtained from 14.5 g/L glucose hydrolysate by yeast fermentation (Wang, Ji, Bi, et al. 2014). (c) Fermentation: The selection of microorganisms for the conversion of sugars to bioethanol is very crucial. The maximum activity of a microorganism is dependent on several process variables such as pH, temperature, growth rate, specificity, salinity, and alcohol tolerance (Balat, Balat, and Öz 2008). Chlorella vulgaris FSP-E (51% carbohydrate

114 Algal Biofuel: Sustainable Solution content) was isolated from the freshwater area of southern Taiwan and it was employed as the carbohydrate-rich feedstock for bioethanol generation via different hydrolysis strategies and fermentation using Zymomonas mobilis. The enzyme mixture of amylase and cellulose could hydrolyse the microalgal biomass for alcohol production via simultaneous saccharification fermentation (SSF) and SHF processes. The glucose yield was 90.4% and the enzymatic hydrolysis of the microalga resulted in 79.9% and 92.3% bioethanol with SHF and SSF, respectively. Acid hydrolysis with 1% H2SO4 resulted in a better glucose yield of 93.6% with the initial biomass concentration of 50 g/L. The 11.7 g/L bioethanol with 87.6% yield could be achieved within 12 hours by acid hydrolysis of C. vulgaris FSP-E and SHF (Ho, Huang, Chen, et al. 2013). The 0.16 g bioethanol production per gram of residual biomass was carried out by Chlorella sp. by Lee, Oh, and Lee (2015). Chng, Lee, and Chan (2017) conducted separate experiments of SHF and SSF for bioethanol production using Scenedesmus dimorphus via different fermentation configurations. It was observed that a combination of both the treatments was very efficient in producing fermentable sugar, whereas the organosolv treatment with SSF resulted in a theoretical bioethanol yield of 90%. However, hydrothermal acid-hydrolyzed fermentation and enzymatic hydrolysis SHF resulted in 80% and 84% theoretical bioethanol yields, respectively. The microalgae inoculum loading was 18 g/L and the other process parameters were pH 5 and 34°C. The results proposed an affirmative relationship between the preliminary treatment methods and the fermentation processes for maximum bioethanol production. The productivity of the bioethanol in the flat-plate photobioreactors was 2.76 g/L/day using horizontal system of ID 3 cm with Phaeodactylum. The productivity of bioethanol in tubular photobioreactor was 4.3 g/L/ day with inclined system of ID 1.3 cm with Spirulina platensis (Chen, Min, and Chen 2009; Ugwu, Aoyagi, and Uchiyama 2008). (d) Product recovery: Chlorococcum biomass was utilized as the elementary source for bioethanol generation using Saccharomyces bayanus and maximum 3.83 g/L bioethanol concentration was procured from 10 g/L microalgal biomass (Harun, Danquah, and Forde 2010). Chlorella sp. KR1 could accumulate 36% carbohydrate and 38% lipids. The lipid was transformed to biodiesel whereas bioethanol production was carried out by employing the lipid-extracted biomass. The residual carbohydrate (49.7%) was saccharified using Pectinex at pH 5.5 and 45°C, and 0.3 N HCl at 121°C for 15 minutes in enzymatic and chemical methods, respectively, giving a yield of 76.9% and 98.2%. The bioethanol was extracted from fermentable sugar using SHF with 79.3% yield. The 0.4 g ethanol/g fermentable sugar and 0.16 g ethanol/g residual biomass

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were produced (Lee, Oh, and Lee 2015). The bioethanol generation using local microalgal isolates from Pearl River Delta coastal water was performed by Guo, Daroch, Liu, et al. (2013). Among all the 18 identified isolates, Mychonastesafer PKUAC 9 and Scenedesmus abundans PKUAC 12 were screened for bioethanol production analysis. The biomass production was carried out in three different conditions (static, agitated, and aerated) and the maximum dry biomass production was observed in the aerated mode (0.09 g and 0.11 g dry weight/day/L for Mychonastesafer PKUAC 9 and Scenedesmus abundans PKUAC 12, respectively). Saccharification of microalgal biomass produced 10.75 g/L total sugar and 5.73 g/L glucose. After fermentation, the bioethanol yield was 0.103 g/g of dry microalgal biomass. Bioethanol production and its kinetics and sensitivity study were performed by enzymatic hydrolysis of microalgal cellulose content. The model considered two reactions: algal cellulose hydrolysis to its simplest components of cellobiose and glucose, and conversion of cellobiose to glucose. After 72 hours of fermentation at pH 5 and 50°C, 57% glucose yield could be attained from 50 g/L microalgal biomass. Saccharomyces cerevisiae produced 12.87 g/L bioethanol with 0.46 g ethanol/g glucose yield (Shokrkar, Ebrahimi, and Zamani 2018). Bioethanol was produced from microalgal starch by SSF in combination with enzymatic hydrolysis. Cycloheximide treatment enhanced the amount of starch in Chlorella sp. (from 19.3% to 38.2%). Amylase activity was employed for microalgal starch hydrolysis, and 0.116 g bioethanol/g of microalgal biomass was recovered. A numerical model was obtained to elucidate the behavioural expression of SSF with time and interaction of substrate–enzyme–microorganism. The model could predict starch depletion, biomass growth, glucose concentration, and bioethanol production (Singh, Chakravarty, Pandey, et al. 2018). The main focus area in microalgal bioethanol technology, which needs to be optimized to be used in industry, is screening of proper microalgal biomass, pretreatment of the efficient fermentation process, and selection of proper fermenting microorganism (Khan, Shin, and Kim 2018). (e) Bioethanol production under stressed conditions: Cultivation under nutrient-stress condition was performed to increase the amount of carbohydrate of Chlorella vulgaris, where it was observed that nitrogenstressed condition enhanced the carbohydrate percentage from 16 to 22.4. Saccharification yield was also tried to be improved by varying the pretreatment methods and enzymes, loading proportion, duration of hydrolysis, and loading volume of microalgae. Bead-beating pretreatment method was found to be 25% more efficient than the processes with no such pretreatment method. In enzymatic hydrolysis assay, the pectinase

116 Algal Biofuel: Sustainable Solution could release more fermentable sugar from carbohydrates. Pectinase derived from Aspergillus aculeatus showed 79% of saccharification efficiency after 72 hours at 50°C with 10% loaded algal biomass. Immobilized yeast fermentation could convert 89% of microalgal hydrolysate into bioethanol within 24 hours in continuous fermentation (Kim, Choi, Kim, et al. 2014). Doan, Moheimani, Mastrangelo, et al. (2012) have discussed bioethanol fermentation from microalgal biomass with its implications in hypersaline conditions.

CONCLUSION The second- and third-generation bioethanol production processes have tremendous scope for fulfilling the existing requirements of bioethanol such as for mixing with gasoline, direct use as fuel, for portable purpose, etc. Currently, second-generation industrial plants are in operation in various parts of the world. They have become economical with the increased efficiency. More work needs to be done to operate this process in continuity and with a reduction in the overall process, which ultimately reduces the production cost (Banerjee, Kumar, Mehendale, et al. 2019). Further studies have to be conducted on lignocellulosic bioethanol production with focus on process development using efficient enzymes and bioreactors. In developing countries, with higher population growth rates, the demand for food is high, thus foodgrains cannot be utilized for bioethanol production; therefore, there is a need to utilize other kinds of biomass (Chandel, Bhatia, Garlapati, et al. 2017). Once a new process is developed at a research centre, changes are required to be carried out at the bioreactor and factory setup for higher bioethanol generation. This new development should be carried out at both fermentation process and at downstream processing part. Improvement is also required in downstream processing where algae are used as feedstock. The algae biomass also has different bacteria, mold, yeast, biological toxins, carcinogen produced by microbes, antibiotics, chemicals (wastewater high in biological oxygen demand and nutrients), acid, and bases, so, the downstream processing needs to be operated in a highly efficient manner. The bioethanol produced by algae can be directly used as a replacement for motor fuel or blend with respective fossil fuels to meet the standards and compete on the market price of existing fossil fuels. Hence, more research must be carried out by focusing on high-volume production with a competitive price compared to the second generation of bioethanol and gasoline products. Now is the time to initiate the commercialization of bioethanol production from algae that allows for new methodology and knowledge tools, which will enable higher efficiency in bioethanol generation, which can be used as renewable fuel for transportation. It will also reduce the dependency on gasoline and related products.

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118 Algal Biofuel: Sustainable Solution Ganguly, R and V K Garlapati. 2017. Comparative account on carbon footprints of burning gasoline and ethanol. In Sugarcane Bio-refinery: Technologies, Commercialization, Policy Issues and Paradigm Shift, edited by A K Chandel and M H L Silveira, 241–252, USA: Elsevier Science Publishing Co, Inc. García-Cubero, R, J Moreno-Fernández, F Acién-Fernández, and M García-González. 2018. How to combine CO2 abatement and starch production in Chlorella vulgaris. Algal Research 32: 270–279 Guo, H, M Daroch, L Liu, G Qiu, S Geng, and G Wang. 2013. Biochemical features and bioethanol production of microalgae from coastal waters of Pearl River Delta. Bioresource Technology 127: 422–428 Harun, R, M K Danquah, and G M Forde. 2010. Microalgal biomass as a fermentation feedstock for bioethanol production. Journal of Chemical Technology and Biotechnology 85(2): 199–203 Harun, R, W Jason, T Cherrington, and M K Danquah. 2011. Exploring alkaline pre-treatment of microalgal biomass for bioethanol production. Applied Energy 88(10): 3464–3467 Ho, S-H, S-W Huang, C-Y Chen, T Hasunuma, A Kondo, and J-S Chang. 2013. Bioethanol production using carbohydrate-rich microalgae biomass as feedstock. Bioresource Technology 135: 191–198 Khan, M I, J H Shin, and J D Kim. 2018. The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microbial Cell Factories 17(1): 36 Kim, K H, I S Choi, H M Kim, S G Wi, and H-J Bae. 2014. Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation. Bioresource Technology 153: 47–54 Kuila A, V Sharma, V K Garlapati, A Singh, L S Roy, and R Banerjee. 2017. Present status on enzymatic hydrolysis of lignocellulosic biomass for bioethanol production. In Advances in Biofeedstocks and Biofuels. Volume 1: Biofeed stocks and their processing, edited by L K Singh and G Chaudhary, 85–96. Austin: Wiley–Scrivener Publishing LLC Lee, O K, Y-K Oh, and E Y Lee. 2015. Bioethanol production from carbohydrateenriched residual biomass obtained after lipid extraction of Chlorella sp. KR-1. Bioresource Technology 196: 22–27 Lee, S Y, J M Cho, Y K Chang, and Y-K Oh. 2017. Cell disruption and lipid extraction for microalgal biorefineries: a review. Bioresource Technology 244: 1317–1328 Limayem, A and S C Ricke. 2012. Lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects. Progress in Energy and Combustion Science 38: 449–467 Menetrez, M Y. 2012. An overview of algae biofuel production and potential environmental impact. Environmental Science and Technology 46: 7073–7085 Miranda, J, P C Passarinho, and L Gouveia. 2012. Pre-treatment optimization of Scenedesmus obliquus microalga for bioethanol production. Bioresource Technology 104: 342–348

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CHAPTER



Crop Residues as a Potential Substrate for Bioenergy Production: An Overview Vinayak V Pathak1 and Richa Kothari2 1

Department of Chemistry, Manav Rachna University, Faridabad, Haryana, India 2 Department of Environmental Science, Central University of Jammu, Samba, Jammu and Kashmir, India

INTRODUCTION According to the Tracking SDG 7: The Energy Progress Report 2019 (IEA, IRENA, UNSD, WB, WHO 2019), the world is making progress towards achieving the Sustainable Development Goal (SDG) 7. But the current rate of achievement is insufficient to meet the SDG target 7.1 which ensures universal access of clean and affordable energy services (World Energy Outlook 2019). Undeveloped and developing countries with high-population density, and limited access to natural resources do not have stable access to electricity and liquid transportation fuel. Approximately, 2 billion people are dependent on solid fuels such as burning of biomass and coal. Currently, coal as a solid fuel is considered the largest source of energy in the world and is extensively used for electricity generation (Huaman and Jun 2014). Combustion of coal leads to the emission of toxic gases such as carbon monoxide, sulphur dioxide, sulphur trioxide, nitrogen dioxide, and nitric oxide (Munawer 2018). These emissions are directly or indirectly related to many health problems such as skin, cardiovascular, brain, blood and lung diseases, cancer, and autism spectrum disorders (Raz, Roberts, Lyall, et al. 2015; Munawer 2018). On the other hand, biomass is estimated to be the third-largest natural source of energy in the world with an annual consumption of 211.5 petajoule (PJ) that fulfils 4.72% of the total primary energy demand (CSO 2014). Although biomass is undoubtedly a renewable source of energy, its direct combustion is not ecologically viable (Wielgosinski, Łechtanska, Namiecinska, et al. 2017). Direct combustion of biomass emits a significant amount of organic pollutants such as polycyclic aromatic hydrocarbons, polychlorinated dibenzo-p-dioxins, and dibenzofurans (Wielgosinski, Łechtanska, Namiecinska, et al. 2017). Emission of PM2.5 from coal and biomass combustion is associated with 2.7 million premature births in developing countries (Malley, Kuylenstierna, Vallack, et al. 2017).

122 Algal Biofuel: Sustainable Solution In view of the potential of biomass for energy supply, many researchers have assessed biomass from different origin and found that biomass from the agriculture sector (crop residue and animal manure) is the most promising feedstock (Cutz, Haro, Santana, et al. 2016). India being an agrarian country, with diverse climatic conditions, has huge potential for biomass production. Among various crop systems, paddy and wheat are the extensively practised farming systems in the north-western states of India, with huge amounts of crop residue generated in the form of straw and stubbles. The share of wheat and rice stubble in the total stubble load was found to be 11% and 36%, respectively (Kumar, Kumar, and Joshi 2015). Management of agricultural waste is carried out in various ways, for instance, the waste can be used as animal feed, fish feed, or as a constituent in feed preparation. However, consumption of raw agricultural waste as feed is found to be harmful and requires mechanical and chemical treatments to make it edible. The chemical treatment method is found superior over the mechanical method. Burning of agricultural waste is commonly practised by the farmers of Punjab and Haryana, which results in the loss of 3.85 tonnes of organic carbon, 59,000 tonnes of nitrogen, 20,000 tonnes of phosphorus, and 34,000 tonnes of potassium. Emission from burning of farm waste is one of the major contributors of air pollution. The extent of pollution, generated by burning of farm waste, causes health hazards such as asthma, chronic bronchitis, and pulmonary diseases. Among various crops, waste from wheat and rice is the major contributor to the total stubble load. Utilization of agricultural waste biomass for bioenergy generation is the most sustainable way of biomass management. Biogenic material obtained from crop residue and livestock manure can be used to produce heat, mechanical energy, and electricity, utilizing different energy conversion pathways. Bioenergy as a mainstream method of energy supply is now being promoted by many countries to reduce dependency on fossil fuel as well as to decrease greenhouse gas emissions (Haberl, Beringer, Bhattacharya, et al. 2010).

AGRICULTURAL RESIDUES FOR BIOENERGY PRODUCTION Researchers have demonstrated bioenergy production (biogas, bioethanol, hydrogen, etc.) by using agricultural waste through different conversion pathways. Agriculture waste and waste emitted from agro-based industries are non-woody biomass, highly degradable, and frequently available, which present suitable feedstock for bioenergy production. The important crop residues for bioenergy production are as follows:

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Sugar Cane It is the most widely cultivated crop. In 2016, the estimated production of sugar cane was about 1.8 × 109 tonne in more than 100 countries. Sugar cane plant is a rich source of sugar and contributes to 80% of the worldwide sugar production. Sugar cane is a rich source of biomass and is composed of stalk, roots, and leaves, and provides 0.17 tonne of residue from 1 tonne of cane. Apart from field-based residue generation, manufacturing of sugar from cane also generates significant amount of energy-rich residue, which is termed as bagasse. About 540 million tonnes (MT) per year of bagasse are produced by the top three sugar cane-producing countries: Brazil, Mexico, and Colombia (Bezerra and Ragauskas 2016). Sugar cane bagasse is a potential substrate for bioethanol production, and when integrated with sugar cane ethanol mill, it lowers the production cost. Bioethanol is produced from fermentation of sugar obtained by enzymatic hydrolysis of sugar. It is estimated that from 1 tonne of bagasse, 149.3 L ethanol is produced (Bezerra and Ragauskas 2016). The yield of bioethanol can be increased by following the pretreatment process prior to hydrolysis. A number of pretreatment processes are investigated to enhance the yield of bioethanol production such as steam explosion, hot water, acidic and alkaline treatment, microwave-assisted acidic treatment. Pretreatment steps enhance the availability of glucose for fermentation, thereby increasing the bioethanol yield. Recently, Raj and Krishnan (2019) reported high concentrated ethanol production (72 g/L) from sugar cane bagasse soaked in ammonia solution. It was reported that bagasse, pretreated with ammonia solution, is promising for cellulosic bioethanol production. Apart from sugar cane bagasse, vinasse is another important residue released during ethanol production. Vinasse is considered as the largest source of contamination in the ethanol production industry. Initially, vinasse was used as a nutrient supplement to improve soil quality. Later, however, it was found that vinasse is highly toxic to aquatic and terrestrial animals (Christofoletti, Escher, Correia, et al. 2013). Vinasse contains low levels of macro and micronutrients, and is a significant resource for biogas production. It has been estimated that about 22.4 g/L of vinasse is produced throughout the world with a potential to yield about 407.68 g/L biogas (Parsaee, KianiDeh, and Karimi 2019). Production of vinasse is expected to increase globally with the development of ethanol production sites (Hoarau, Caro, Grondin, et al. 2018).

Maize It is the third-largest crop in terms of cultivation, after wheat and rice. Maize is an annual plant with high productivity and adaptability under various geographic conditions. On the basis of colour and test, maize can be classified as yellow and white. Yellow maize contributes to the bulk of the world’s total

124 Algal Biofuel: Sustainable Solution maize production while white maize is cultivated in fewer countries since it requires favourable climatic conditions. It has been estimated that about 1.06 × 109 tonnes of maize was produced in 2016. Since the last two decades, the global maize production has increased by nearly 50% or with 1.8% annual compound growth rate. Maize has been found as a suitable substrate for bioethanol production. Globally, 65% of maize is used for livestock feed, 15% is consumed as food by humans, and the remaining is destined for multiple industrial applications. In contrast to sugar cane, maize produces field-based residues. After collection of grains, the maize plant consists of stalk (42%), leaves (20%), cobs (14%), husk (8%), and others (16%). Kadam and McMillan (2003) estimated that about 80–100 MT per year of dried maize stover can be sustainably collected and majority of that can be used as feedstock for ethanol production. Ethanol production from maize depends on the variety and starch content in maize grain, and it was observed that higher starch content caused lower efficiency of starch saccharification (Gumienna, Szwengiel, Lasik, et al. 2016).

Wheat Wheat grains are widely consumed by humans and they make for animal feed as well. The current production of wheat is approximately 700 MT, making it the third-largest crop in the world. Straw is generated as a field-based residue during the cultivation of wheat, and for 1 tonne of grain production about 1.5 tonne of wheat straw is generated. The European Union, China, India, and the USA are the top wheat cultivators. According to Otero, Panagiotou, and Olsson (2007), surplus wheat straw has a potential to produce about 120 billion litres of bioethanol annually, which can substitute 93 billion litres of gasoline. Wheat straw is composed of cellulose, hemicellulose, and lignin in a range of 33% to 40%, 20% to 25%, and 15% to 20% (w/w), respectively. It has been found that ash content of wheat straw is almost three to four times lower than rice straw, making it more suitable for bioenergy generation (Prasad, Singh, and Joshi 2007). Wheat straw can be used as a substrate for generation of various bioenergy products (Table 1). Application of wheat straw for biogas production is demonstrated by various researchers. Mancini, Papirio, Lens Piet, et al. (2018) reported a cumulative biomethane potential of 274 mL methane/g VS (volatile solids) from untreated wheat straw. Singh, Behera, Yadav, et al. (2014) investigated the potential of wheat straw for biogas production and reported a maximum yield of 0.178 m3/kg VS at 50°C operational temperature.

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Table 1 Worldwide wheat straw potential for biogas Country

Total straw production 100% (million tonne/ annum)

Straw potential for energy generation (million tonne/annum)

Methane potential (million cubic metres/ annum)

Electrical energy potential (megawatt)

Reference

Germany

30

13.0

2980

1530

Weiser, Zeller, Reinicke, et al. (2014)

European Union

110

44.03–50.63

10,355–11,907

5162–5936

FAO (2015)

China

53.3

76.7

18,000

9000

Antonczyk, Arthur, and Scherer (2011)

Worldwide

680

340

80,000

40,000

Antonczyk, Arthur, and Scherer (2011)

Rice Rice straw is an abundant lignocellulosic material globally. It is the third most important crop cultivated in the world, after wheat and corn, with an annual production of 650 MT (Binod, Sindhu, Singhania, et al. 2010). For each kilogram of grain production, about 1–1.5 kg straw is produced. Thus, globally about 650–975 MT straw is produced annually (Garrote, Dominguez, and Parajo 2002). A major portion of the rice straw is consumed by animals and the remainder is waste. Rice straw is a potential feedstock for bioethanol production. The high cellulose and hemicellulose content in rice straw can be readily hydrolysed into fermentable sugar. Rice straw composition involves cellulose (32% –47%), hemicellulose (19% –27%), and lignin (5% –24%) (Garrote, Dominguez, and Parajo 2002). The chemical composition of the residue strongly influences the bioenergy-generation process. High-ash content (10%–17%) in rice straw indicates the low quality of feedstock, however, rice straw has low levels of alkali contents (