Extremophiles: Wastewater and Algal Biorefinery 1032370807, 9781032370804

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Extremophiles Wastewater and Algal Biorefinery

Editors

Pratibha Dheeran

Department of Botany Maharaj Singh College Saharanpur, Uttar Pradesh, India

Sachin Kumar

Biochemical Conversion Division Sardar Swaran Singh National Institute of Bio-Energy Kapurthala, Punjab, India

p,

A SCIENCE PUBLISHERS BOOK A SCIENCE PUBLISHERS BOOK

Cover credit: Dr. Anuchaya Devi, Banaras Hindu University, Varanasi, India

First edition published 2023 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 and by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN © 2023 Pratibha Dheeran and Sachin Kumar CRC Press is an imprint of Taylor & Francis Group, LLC Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission 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. Library of Congress Cataloging-in-Publication Data (applied for)

ISBN: 978-1-032-37080-4 (hbk) ISBN: 978-1-032-37081-1 (pbk) ISBN: 978-1-003-33522-1 (ebk) DOI: 10.1201/9781003335221 Typeset in Times New Roman by Radiant Productions

Preface The microorganisms dwelling into the extreme conditions not only on earth but into the extremes of space are known as extremophiles. They have unique ability to survive under the extremes of temperature, pH, pressure, radiations, vacuum and gravity. They can sustain in toxic wastes where no other living organism can thrive. These extremophiles/extremotolerant organisms (bacteria and microalgae) have tremendous potential in biomass biorefineries and waste water treatment. The book ‘Extremophiles: Wastewater Treatment and Algal Biorefinery’ covers the applications of extremophiles for the production of value-added products, including biofuels, extremozymes, wastewater treatment, electricity generation, biofilms, microbial corrosion, etc., and has twelve chapters as described below: Chapter 1 articulates the outlook of present trends in the extraction of efficient bioenergy from wastewater resources for sustainability concerns with special emphasis on the application of extremophiles. A number of studies have been reported indicating the energy potential of different kind of waste streams, since the concept of integrated wastewater biorefineries was first deciphered. Researchers have streamlined numerous methods to bridge the shortfalls arising in realization of integrated wastewater treatment and biorefineries. Chapter 2 describes the potential of extremophiles in bioremediation and the treatment of different types of wastewaters. The chapter also discusses the specific extremophiles which are being employed for the treatment of waterbodies contaminated with specific pollutants and the specific nature of wastewaters. Chapter 3 highlights the application of different types of extremophiles as biocatalysts in microbial electrochemical system (MES) and its future prospectus. This chapter covers the principle of different MESs, microbes associated with anode and catabolic pathways (exocellular electron transfer) responsible for microbial oxidation of the substrate. Besides, it discusses the physicochemical factors affecting energy harvesting and challenges in MFC operation during electrogenesis. Chapter 4 understands the promising and sustainable technical approaches which are being employed to treat the wastewater for harnessing energy through biological route and environment protection, and also futuristic scope for their industrial growth. Globally, scientific fraternity have put their efforts tirelessly for the recognition and development of wastewater treatment technologies not only for the generation of carbon-neutral, and eco-friendly energy but also for resolving the issue of wastewater disposal that are given paramount importance to fulfil the burgeoning demands of energy, and environment protection. Chapter 5 discusses the various metabolic adaptations that enable the survival of extremophilic microalgae under harsh environmental conditions and further elucidates their phylogenetic evolution based on the16S rDNA sequences. Subsequently, the salient features of large-scale cultivation of extremophilic microalgae are discussed. The chapter also examines the different products that can be derived from extremophilic microalgae and the possibility of engineering them for enhanced product recovery.

iv Extremophiles: Wastewater and Algal Biorefinery Chapter 6 provides insight on seaweeds and their applications, different types of seaweed degrading enzymes obtained from marine extremophiles and their applications, current research, and developments on marine extremophiles and future prospective. The enzymes from marine extremophiles have gained global interest due to their competence to tolerate extreme harsh conditions of salinity, pH, temperature, pressure, etc. Chapter 7 elaborates the various studies based on wastewater bioremediation and biofuel production under large-scale studies for various microalgal species under adverse and challenging environmental conditions. These microalgal strains are depicted to be halophiles (salt-loving), alkaliphiles (survive under elevated pH levels), thermophiles (grow under high temperature), psychrophiles (grow at low temperature), barophiles (survive under high pressure), etc., with their growth environments and potential tolerance capacity. Chapter 8 discusses about the morphological diversity of extremophilic microalgae, followed by the commercially employed growth engineering systems to generate massive biomass. The chapter highlights the significance of extremophilic algae in the biological, pharmaceutical and industrial sector and also covers the recent developments on EPA and DHA (Omega 3-PUFAs) production, that can reduce excess demand from fish stock. Chapter 9 deals with a base footstep towards understanding an algal-based bioeconomy and, consequently, helps the global energy demands. Chapter 10 discusses and recapitulates the various applied strategies against biofilm formation, and reduction in the economic losses caused by microbial corrosion. In recent years, various new technologies such as bacteriophages, quorum sensing inhibitors, boosters of biofilms, plant extracts, etc., have been proposed for the treatment of problematic biofilms in the industries. Chapter 11 deliberate the investigations on biotechnologically relevant attributes of extremophiles by tapping into biofilm’s advantages. Recent biotechnological advancements have explored biofilm formation as a natural route for immobilization. Chapter 12 highlights the importance and potential avenues of extremophiles in industrial applications, especially in textile processing. Textile desizing, bioscouring, degumming of textile fibers, bleaching and wastewater treatment are some of the major areas where extremophiles and their products can be well incorporated and such studies should be aggressively pursued.

Acknowledgements We thank all the authors, who made painstaking contributions in the making of this book. Their contributions brought the desired lustre to the quality of this book. Their patience and diligence in revising the initial draft of the chapters after incorporating the comments/suggestions of the reviewers are highly appreciated. We would also like to acknowledge the contributions of all the reviewers for their constructive and valuable comments and suggestions to improve the quality of the contributions of various authors.

Contents Preface Acknowledgements 1. Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources: An Insight to Application of Extremophiles Bhumica Agarwal and Lalit Kumar Singh

iii v 1

2. Extremophiles for Wastewater Treatment Keshav Rajarshi, Karri Sudharshana and Shantonu Roy

23

3. Role of Extremophiles in the Microbial Electrochemical Cell: Recent Advances Shreya Gavas, Prajakta Pawar, Soumya Pandit, Namita Khanna, Abhilasha Singh Mathuriya and Ram Prasad

43

4. Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy Nidhi Sahni, Meenu Hans and Sachin Kumar

61

5. Potential of Extremophilic Algae for the Synthesis of Value-added Products Aishwarya Atakkatan, Sandra Innesent, Shreya Padmesh Prajapat, Soumya Pandit and Namita Khanna

80

6. Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes Dhanshree Mone and Nitin Trivedi

115

7. Extremophilic Microalgae: The Potential Source for Wastewater Treatments and Biofuel Productions Sourav Kumar Bagchi, Reeza Patnaik, Navneet Sharma and Ramasare Prasad

151

8. Extremophilic Microalgae as a Potential Source of High-Value Bioproducts Meenakshi Singh, Nitin Trivedi, Navonil Mal and Sanjeet Mehariya

167

9. Value-Added Products and Biofuels from Extremophilic Microalgae Biomass Usharani K., Lakshmanaperumalsamy P. and Jayesh M.S.

187

10. Microbial Corrosion and It’s Current Mitigation Strategies Reena Sachan, Ajay Kumar Singh, Madan Sonkar and Shiv Bharadwaj

219

11. Extremophile: Biofilm Behavior, Characterization and Economical Applications Shashi Bhushan, Jayakrishnan U., Shaon Raychaudhuri and Halis Simesk

243

12. Extremophiles for Textile Industry Shalini Singh, Sujata Das and Charu Khanna

277

Index

299

1 Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources An Insight to Application of Extremophiles Bhumica Agarwal 1,* and Lalit Kumar Singh 2

1. Introduction Economy of any developing nation is largely estimated from its net energy balance (consumption vs. generation). According to BP Energy outlook 2019, India’s share of global primary energy demand was estimated to jump from 6% in 2019 to 11% by 2040, most of which will be met through coal, resulting in doubling of CO2 gas emission. Envisioning unexceptionally high demand for primary energy coupled with fluctuating crude oil prices, commercial scale production of third generation biofuels is the priority of Government of India as well as across the globe. Indian Biofuel Policy 2018 dictates a bioethanol-gasoline blending target of 10 and 20% by year 2022 and 2030, respectively (India Biofuels Annual 2019). As for biodiesel blending, no nearterm target has been defined, scrapping the previously set aim of 20% blend procured from Jatropha (Jatropha curcas) as the most suitable inedible oilseed owing to numerous agronomic and economic constraints. Despite immense advances in biorefinery technologies, factors such as costs and reliable supply of feedstocks, energy intensive process and generation and disposal of secondary byproducts renders commercial-scale biorefinery operations economically unfeasible. Chemical and thermochemical processes have always been the choice methods for synthesis of biofuel (Barnard et al. 2010). Microbial routes are preferred over chemical catalysis owing to no or little requirement of extreme process conditions, thus decreased expenditure. Although, use of these biological agents often augment some additional processing steps in lieu of their sensitivity, thus adding to overall process complexity. However, increasing use of a class of microorganisms namely extremophiles, has evolved as a green solution. Extremophiles, as the name suggests, refer to the organisms capable of surviving in one or more extreme environmental conditions of

Water Testing Laboratory, Indian Institute of Technology Jammu, Jagti-181221, Jammu, Jammu and Kashmir, India. Department of Biochemical Engineering, School of Chemical Technology, Harcourt Butler Technical University, Kanpur-208002, Uttar Pradesh, INDIA. * Corresponding author: [email protected]

1 2

2

Extremophiles: Wastewater and Algal Biorefinery

high temperature (thermophiles), low temperature (psychrophiles), low pH (acidophiles), high pH (alkaliphiles), salinity (halophiles), high hydrostatic pressure (barophiles or piezophiles) or low water content (xerophiles). Some extremophiles are adapted simultaneously to multiple stresses (polyextremophile); common examples include thermoacidophiles (for e.g., Thermoplasma, Thermococci sp.) and haloalkaliphiles (for e.g., Chromatium and Thiospirillum sp.). Extremophilic organisms are primarily prokaryotic (archaea and bacteria), with few examples of eukaryotic classification. They have aroused interest of biochemical engineers due to secretion of extremozymes which are capable of functioning under extreme process conditions generally employed in biofuel production. Given the ubiquitous application array of extremophiles in wastewater treatment as well as bioenergy generation, uncountable excellent reviews exist (Barnard et al. 2010, Schneider et al. 2012, Bibra et al. 2015, Varshney et al. 2015, Coker 2016, Zhu et al. 2020). However, this is a first attempt to summarize the literature available on the application of wastewater-biorefinery focusing on the use of extremophiles, to the best of our knowledge.

2. Background The unprecedented increase in the concentration of atmospheric carbon dioxide (CO2) followed by notoriously high global warming has forced the focus of world policy makers towards developing and utilizing carbon-negative energies. In 2015, United Nations Framework Convention on Climate Change (UNFCCC) Conference of Parties (COP21) in Paris drafted an agreement to keep global warming below 2℃. Particularly considering the Indian stance on rising dangers of emissions from conventional fuels, we committed to reduce emission intensity of gross domestic product 33–35% by 2030 as compared to 2005 level (Singh 2019). The only way forward to achieve the set goals lies in elevated use of bioenergy over conventional energy. Therefore, India proposed to ensure at least 30% of blending of Jatropha oil and bioethanol to diesel and ethanol, respectively by the end of 2020. The choice of raw material for biofuel production ranges from molasses for bioethanol and nonedible oil for biodiesel depending on geographic and economic status of nations. The Indian approach to biofuels is based solely on non-food feedstock to be raised on degraded/waste lands which are not suitable for agriculture, thus avoiding a possible conflict of fuel versus food security (MNRE 2009). Apparently, biofuels are classified into generations based on the technology and raw material employed. First-generation biofuels are largely produced from plant-based starch, sugars and oils. The oil extracted from oil-seed crops, for, e.g., canola, soy, Pongamia and Jatropha as well as from animalbased tallow, can be converted into biodiesel through transesterification. The sugar (produced from sugarcane and sweet sorghum) and starch (produced from crops such as maize, corn and sorghum) can be readily fermented into bio-ethanol. Clearly, synthesis of bioethanol from food crops was not sustainable especially in countries like India where policies are to be made keeping in mind large food demands of its vast population. Further concern was based on low theoretical as well as practical yields of bioethanol given various inhibitory factors including substrate and product inhibition of the microorganism used. Although numerous advances in the field of genetic engineering have led to improvement in product yields, commercial production of bioethanol is still lagging behind. As for biodiesel, arguments were presented that cultivation of nonedible oil-yielding crops on wastelands disturbed the ecological balance in the long run. For instance, the German Plant Nutrition Society raised the issue of nitrogen imbalance in soil and groundwater recently (Agarwal and Kumar 2018). Thus, the need for development of second-generation biofuels arose from global concerns regarding food security. In contrast to first generation biofuels, the second-generation was based on exploitation of lower-value biomass residues, such as biological material from living or recently living organisms. The candidate material for production of second-generation biofuels fell into the categories of forestry, agriculture and dedicated energy crops (Ren et al. 2009). The major pathway

Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources 3

postulated to achieve fuel sufficiency corresponded to biochemical processes which produce fermentable sugars from lignocellulosic biomass. Lignocellulosics are abundant, inexpensive and a renewable resource of organic material composed of cellulose, hemicellulose and lignin and has served as a substrate in microbial processes for the production of alcoholic beverages for thousands of years. Some of the lignocelluloses containing species include wheat straw, corn husks, prairie grass, discarded rice hulls, grasses, paper, etc. Apart from cellulose component of lignocellulosic material, its hemicellulosic fraction (particularly xylan, a polymer of D-xylose) could also be hydrolyzed for development of novel products (Agarwal et al. 2010). Complete substrate utilization is one of the prerequisites to render lignocellulosic xylitol process economically viable. But the closely knitted structure of cellulose, hemicellulose and lignin in lignocelluloses offers resistance to microorganisms from acting on fermentable sugars. Therefore, the hydrolysis of lignocellulosic biomass is crucial. Various options are available to fractionate, solubilize, hydrolyze and separate cellulose, hemi-cellulose and lignin components which include treatment with concentrated acid, hydrogen peroxide, steam explosion, auto-hydrolysis, ammonia fiber explosion (AFEX), wet oxidation, lime, hot water, CO2 explosion and use of organic solvents. The major drawback of lignocellulosic biomass hydrolysis includes low yields and generation of inhibitory compounds which appears to be toxic to the microorganisms employed in further fermentation steps (Agarwal et al. 2010). This phenomenon results in inefficient utilization of sugars, increased fermentation time with decrease in product yield. The complete removal or at least decrease in concentration of such inhibitory compounds is thus, necessary. A number of detoxification methods: chemical, physical or biological could be used to overcome the inhibitory effects of such compounds. The suitability of any detoxification method depends on the composition of hemicellulose hydrolysate which in turn depends upon the raw material and the hydrolysis conditions employed. It has also been demonstrated that a combination of various physical, chemical and biological methods is more effective than detoxification by single treatment process. Overall, the process of hydrolysis of lignocellulosic biomass followed by fermentation of sugars is expensive and energy-intensive process. The key to commercialization of lignocellulosic biofuels lies in optimizing the process of hydrolysis in terms of expenditure and yields. The third-generation of biofuels evolve from mass cultivation of algal biomass for extraction of bio-oil from lipid rich algae. Additionally, oil-extracted algae could be further used for bioethanol generation employing fermentation (Duan et al. 2020, Shen et al. 2020). Algae is also a rich source of various pigments and nutraceuticals (John et al. 2011). Applicability of algae as a sustainable biofuel source will be discussed in detail later. The fourth-generation biofuels essentially correspond to fuels produced through genetically engineered microorganisms and aim towards increasing yields of already existing processes. Although advantages and disadvantages of latest techniques will unfold in a few decades, the major concern remains the sustainability of any process for biofuel generation with the view of unburdening of nature earth rather than the reverse. Currently, the blending of ethanol in petrol and biodiesel in diesel is only about 2 and 0.1%, respectively. Clearly, the reason for failed targets lies in the complex process and high-cost input associated with biofuel development on commercial scale (Biofuels Annual 2019). On June 4, 2018, India revamped its National Policy on Biofuels, with a few modifications in the earlier bioenergy policies and almost the same target of 20% blending of ethanol in petrol and 5% biodiesel oil in diesel by 2030. In order to meet the revised targets, India needs to level up its game and work towards the sustainable production of biofuels. One process to achieve the goal seems to look for cheap sources that are easy to process. Undoubtedly, waste streams from domestic and industrial processes as well as generated solid waste is a rich source of valuable precursors for biofuel production. Generally, waste streams are loaded with organic carbon which could be converted to energy-laden products. The rich concentrations of nitrogen, phosphorus and other macro- or micro-nutrients in waste streams

4

Extremophiles: Wastewater and Algal Biorefinery

serve as growth promoters to various commercially important microorganisms. Thus, the concept of integrated wastewater-biorefineries (WWBR), i.e., simultaneous remediation of wastewater along with generation of valuable products with special emphasis on biofuel production is gaining popularity across the globe.

3. Current Development A task force on waste to energy created by the National Institute for Transforming India (NITI Aayog) has estimated an annual generation of 62 MMT of Municipal Solid Waste (MSW) in India. Similarly, industrial and domestic wastewater are highly energy and nutrient laden. Speaking from an economic point of view, this waste has enormous potential to support the agricultural sector by producing compost and the industrial sector in form of drop-in fuels and energy, including refusederived fuel and biogas/electricity. To this effect, many technologies are available for converting waste into biofuels and other value-added biochemicals. Although they are in the nascent stage and continuous efforts are being employed to carry them on a commercial scale. Conversion of such waste into fuels is envisaged as a crucial instrument in meeting the energy demand across the globe along with addressing the surmounting environmental issues (Biofuels Annual 2019). Exploiting the potential of microorganisms presents a favorable scenario in development of bioenergy from waste with special focus on extremophiles. Extremophiles and their enzymes, in particular, are predicted to play important roles in many kinds of bioprocessing, including conversion of nonfood biomass into biofuels owing to their superior survival capacities. Extremophiles grow under extreme conditions of temperature, acidity, alkalinity or salinity and have been found to produce not only biofuels but also value-added products with improved yields. The processes employing extremophilic microorganisms to produce biofuels presents to be sustainable as they have (i) low risk of unwanted microbial contamination, (ii) increased reaction kinetics, (iii) higher yields of products, and (iv) minimal environmental hazards. Current technological developments in production of biofuels/bioenergy from waste resources through the use of extremophiles is summarized next.

3.1 Biogas Biogas can be produced from a large number of substrates including animal manure, slaughter house waste, organic household and garden waste or municipal solid waste. Production of biogas as the main source of rural power from one of the largest byproduct of wastewater treatment plants, Waste Activated Sludge (WAS), has recently evolved (Coelho et al. 2011, Go et al. 2019). Sewage sludge, commonly known as biosolids, is either stabilized or dewatered before disposal to meet regulatory requirements (EPA 1999). Stabilization is essentially the process of reduction in pathogen levels, odor and volatile solid contents. One of the common methods of biosolids stabilization is anaerobic digestion wherein microorganisms break down the organic matter contained in the sludge and convert it into biogas, a mixture of mainly methane and carbon dioxide, which can be used for electricity, heat and biofuel production. With advances in studies pertaining to microbial consortia, a number of extremophiles have been evaluated for their methane production capacity, viz., thermophilic Methanobacterium sp., Methanosarcina thermophila and Methanothermococcu okinawenis along with some psychrophilic Methanosarcina lacustri, Methanolobus psychrophilus, etc. (Barnard et al. 2010). Studies involving mesophilic bacteria have been reported, however, mesophilic digestion is less preferred over the thermophilic one owing to extended Hydraulic Retention Time (HRT) and ineffective pathogen removal mechanism (Bibra et al. 2015). Several researchers have reported effects of WAS pretreatment on reduced digester HRT, increased biomass production and enhanced dewatering properties. Pretreatment could be achieved with biological, thermal, mechanical or chemical methods either alone or in combination. A number of good reviews are available summarizing the increased biomethane production after pretreatment

Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources 5

(Carrere et al. 2010, Tyagi and Lo. 2011). Although biological pretreatment methods are always preferred; they are often overlooked due to additional cost burdens. For instance, Lu et al. 2008 achieved 48% increase in methane yields utilizing thermostable enzymes. Later, Carrere et al. 2010 achieved 50% increase in biogas production employing a hyper-thermophilic aerobic bacterium namely Geobacillus stearothermophillus in a two stage (aerobic-anaerobic) digestor. Efforts need to be diverted towards studying the diversity of these mixed cultures found in WAS for the optimization of biomethane production. Results have demonstrated that biogas synthesis from waste biosolids should not only be considered as a source of renewable energy but a concerted effect of resolving large-scale environmental hazards with respect to toxic waste handling. Thermophilic processing plants are considered to be less stable than mesophilic ones, thus there are limited number of thermophilic biogas plants. Consequently, studies on the associated microbial trophic networks are very few. Hence, thermophilic microbial consortia appear to be less well understood than mesophilic ones (Maus et al. 2016). Schaefer and Sung 2008 carried out anaerobic digestion of corn ethanol thin stillage at thermophilic temperature (55℃) with two stirred tank reactors. Steady-state was achieved at 30-, 20- and 15-d HRT with significant reduction of volatile solids occurring at the 20-d HRT with a maximum reduction of 89.8%. Methane yield were obtained in the range of 0.6 to 0.7 L methane/g volatile solids. The authors also evaluated ultrasonic pretreatment for one digester to study the effect of pretreatment on digestion rate, however no significant improvement was observed. Technoeconomic evaluation suggested that natural gas consumption of ethanol plant could be reduced from 43 to 59% with the methane produced, while saving an estimated US$7 to US$17 million on the basis of production of 360 million L ethanol/yr. Although the authors did not carry studies on characterization of biocatalysts responsible for anaerobic digestion of stillage, the study revealed the importance of thermophilic digestion in wastewater treatment and biogas production. The process of thermophilic anaerobic digestion is marred by the additional heat requirement increasing the cost and thus decreasing the viability of the process. However, ethanol stillage temperature lies above thermophilic levels when it exits the process stream, thus alleviating the shortfall and making the process economically attractive.

3.2 Microalgae as Feedstock for Bioethanol and Biodiesel Microalgae are unicellular plants that are either autotrophic or heterotrophic, capable of growth in a diverse environment. Autotrophic algae harness sunlight and fix atmospheric CO2 into carbohydrates such as starch and cellulose via photosynthesis, while heterotrophic algae species convert small organic carbon compounds into lipids, protein and oils (John et al. 2011). Laboratory scale production of microalgae is carried out in closed systems such as vertical bubble column or horizontal tubular photobioreactors, flat panels, bio‐coils and bags whereas large scale production relies on open systems such as turf scrubber system, raceways and open pits. Although closed systems allow for controlled growth conditions, open systems are predicted to be more efficient when using wastewater as well as include low energy costs as compared to open systems. Also, the oil level of the biomass produced in closed systems is greater than in open systems thus favoring the possibility of using all the biomass. In its simplest and least attractive form, dried algal biomass may be used to generate energy by direct combustion (Kadam 2002). Plants rich in lipids or carbohydrates are used to produce derivatives of fatty acids and alcohols, respectively. As microalgae contain a large amount of lipids, an application of microalgae is production of biodiesel through transesterification. In addition, after hydrolysis, the residual biomass can potentially be used for bioethanol production through a biochemical process such as fermentation or into methane through anaerobic digestion. Although acid hydrolysis is an energy efficient process, alternatives such as enzymatic digestion and gamma radiation offer more sustainable pretreatment protocols. In addition, hydrogen can be produced from algae by biophotolysis (Pittman et al. 2011). Producing biofuel from microalgae offer numerous advantages,

6

Extremophiles: Wastewater and Algal Biorefinery

such as: higher growth rate and lower demand for water than commercial crops; high efficiency in CO2 mitigation; requirement of smaller areas for cultivation than conventional crops, etc. On the contrary, disadvantages of the process include, availability of light incidence and penetration into the culture to ensure constant biomass yield, and expensive procedures for conversion of lipids into biodiesel. Table 1.1 presents a list of value-added products that are derived from extremophiles through biotechnological routes. Table 1.1. Value added products obtained from extremophiles. Microorganism

Product

Type of Extremophile

Source of Extremophile

Reference

Coccomyxa melkonianii SCCA 048

Luetin

Unicellular green algae

Heavy metal polluted river

Pasqualetti et al. 2015

Haloferax mediterranei

Polyhydroxyalkanoate (PHA)

Halophile

–

Bhattacharyya et al. 2014

Mesotaenium berggrenii

Pigments

Psychrophilic green algae

Alps

Remias et al. 2009

Raphidonema sp.

Tocopherol (pigment)

Psychrophilic algae

Arctic snow and permafrost

Leya et al. 2009

Galdieria sulphuraria 074G

Phycocyanin pigment

Thermophilic red algae

–

Eriksen 2008

Chlorella sorokiniana UTEX 2805

Ammonia removal

Thermophilic green algae

Wastewater stabilization ponds

De-Bashan et al. 2008

Chlamydomonas nivalis

Astaxanthin (vitamin E) Psychrophilic algae

Alpine snow

Remias et al. 2005

Dunaliella salina

β-carotene

Halophilic microalga

–

García-Gonzalez et al. 2005

Arthrospira Platensis

Food and feed additive

Filamentous alkaliphilic Hypersaline lakes cyanobacterium

Berry et al. 2003

To qualify as a biodiesel producer, composition of Fatty Acid Methyl Esters (FAME), the major lipid fraction with main composition (C16–18), of the algal biomass should be at least 92% of the total algal biomass. Additionally, sustainability of the process depends upon characteristics of the selection species such as appropriate production time and yield of biomass and lipids, ease of separation from the culture medium, adaptation to low-cost cultivation conditions and resistance to contaminating organisms. Advent of extremophile microalgae have emerged as a prominent solution to all these problems owing to their capacity of growth under extreme conditions. The fatty acid composition of biodiesel produced from microalgae (14 to 22 carbon atoms) is found to be similar to vegetable oils used for biodiesel production. The composition and proportion of fatty acids in the microalgae oil could be optimized through species used, the nutritional composition of the medium and other cultivation conditions (Guschina and Harwood 2006). Various other parameters, viz., oil unsaturation levels, the productivity of the microalgae in the respective effluents, the growth rate and the total biomass composition are paramount in deciding the techno-economic feasibility of waste treatment and biofuel synthesis. Other than biodiesel, microalgae serve as a source of bioethanol through fermentation, organic liquids (such as acetic acid, acetone and methanol) through pyrolysis, bioelectricity through fuel cells, photosynthetic pigment, food additive (e.g., lutein is used to ameliorate cardiovascular diseases, some types of cancer and age-related diseases), nutraceuticals (such as Eicosapentaenoic acid, an omega-3 poly unsaturated fatty acid used) (Fig. 1.1). The yield of oil and biomass could be optimized through variations in growth conditions thus altering the biochemical composition of the algal biomass, for instance, stress may increase

Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources 7

Fig. 1.1. Utilization potential of microalgae cultivated in wastewater (adapted from Schneider et al. 2013).

the lipid content thus favoring biodiesel yield (Qin 2005). Soru et al. 2019 reported that heavymetal-resistant Coccomyxa melkonianii SCCA 048 increases the total lipid content under nitrogen manipulation while the green alga Pseudochlorella sp. YKT1 isolated from an acidic mine drainage accumulated a fairly large amount of storage lipids (30% of dry weight) under nitrogen-depleted condition and low pH (Hirooka and Miyagishima 2016). As it is known, two kinds of lipids are found in nature: polar and neutral. Polar lipids include phospholipids and glycolipids, while the latter include acylglycerides (tri, di- and monoglycerides) and free fatty acids. It is neutral lipids that are used as an energy source by microalgae, while polar lipids form cell membranes. Additionally, microalgae constitute fatty-acid free components that are not converted into biodiesel, such as steroids and pigments albeit are commercially important themselves (Halim et al. 2012). Furthermore, microalgae producing large amounts of polyunsaturated fatty acids are useless for biodiesel production as they reduce biofuel oxidative stability. Consequently, the higher yield of pigments corresponds to lower production of biodiesel. In practice, some microalgae may produce high lipid content, yet low biodiesel production (Bogen et al. 2013). Nevertheless, appropriateness of any strain for biodiesel synthesis relates to its lipid content. The percentage of lipid content per dry weight of microalgae may range from 1.5 to 75% (Table 1.2). Scenedesmus obliquus is one of the most widely studied microalgae species for wastewater treatment as well as biomass accumulation. Ruiz-Marin et al. 2010 carried out extensive studies to compare the growth patterns of free and immobilized cells of S. obliquus and C. vulgaris in alkaline environments and removal of nitrogen and phosphorus in artificial as well as urban wastewater. Contrary to earlier studies, the authors observed a shorter lag period with an immobilized cell with S. obliquus offering better adaptability than C. vulgaris. Previous studies have reported the disadvantage of immobilization as restricting nutrient diffusion; however, the present study found no P and N restrictions and thus complete removal of both pollutants was observed. It is worth mentioning that the lipid content of S. obliquus was lower than some other species such as C. vulgaris, the former was better suited for organic carbon pollutants reduction than the latter, establishing its predominance during real wastewater treatment. Brennan and Owende 2010 reported production of algal biomass from CO2 produced in industrial flue gases. Nitrogen and phosphorus concentration in the effluent of a wastewater treatment plant vary abruptly, which could affect the biomass growth and chemical composition of microalgae when such wastewater is used as a cultivation medium. This effect was studied by Arbib et al. 2013 who reported lipid and crude protein content of S. obliquus reaching the maximum lipid content (34%) at the lowest N:P (1:1) and the maximum crude protein content (34.2%) at the highest N:P (35:1) at highly alkaline process conditions. Selvaratnem et al. 2014 carried out remediation of effluent from a primary settling tank of municipal wastewater treatment facility, New Mexico

8

Extremophiles: Wastewater and Algal Biorefinery Table 1.2. Lipid content of some extremophile microalgae. Microalgae Scenedesmus obliquus Scenedesmus species Novo Desmosdesmus sp. Scenedesmus obliquus

Lipid Content (% Dry mass) 18–25

Reference Duan et al. 2020

33

Durvasula et al. 2015

13.3

Komolafe et al. 2014

34

Arbib et al. 2013

Chlamydomonas sp. TAI-2

18.4

Wu et al. 2012

Ankistrodesmus falcatus

1.58

Abubakar et al. 2012

Desmodesmus sp. F2

58

Pan et al. 2011

Botryococcus braunii

25–75

Mata et al. 2010

Scenedesmus dimorphus

16–40

Demirbas 2010

Chlamydomonas rheinhardii

21

Scenedesmus obliquus

12–14

Chlorella vulgaris

14–22

Prymnesium parvum

22–38

Dunaliella bioculata

8

Euglena gracilis

14–20

Porphyridium cruentum

9–14

Gymnodinium sp.

29.6

Chlorella sorokiniana

20

Mansour et al. 2003 Illman et al. 2000

using an acidophilic and moderately thermophilic Galdieria sulphuraria CCMEE 5587.1 and demonstrated encouraging results both at the laboratory and large-scale. After 7-days treatment protocol, removal efficiencies were 88.3% for ammoniacal-nitrogen and 95.5% for phosphates. The authors also achieved higher biomass yield (27.42 g biomass/g nitrogen removed) as compared to average reported in literature (25.75 g/g) at the laboratory scale and 1.32 times higher cell densities under natural light establishing the practical utility of the strain in wastewater remediation and bioenergy generation. The corresponding theoretical yield estimated from the empirical molecular formula of algal biomass was recorded as 15.8 g/g. Higher biomass yields are preferred to maximize energy production through anaerobic digestion or hydrothermal processing route (Chakraborty et al. 2012). To further clarify the mechanism and intensify wastewater treatment and microalgal biofuel production, Shen et al. 2015 evaluated the effect of organic and inorganic carbon on growth of S. obliquus and nutrient removal by varying total organic carbon concentrations of 20–120 mg/L in wastewater while feeding CO2 concentrations in the range of 0.03–15%, respectively. Maximal biomass and average lipid productivity obtained with 5% CO2 aeration were 577.6 and 16.7 mg/L. d, respectively. The total nitrogen, total phosphorus and Total Organic Carbon (TOC) removal efficiencies were reported as 97.8, 95.6 and 59.1% respectively within 6 days when cultured with secondary municipal wastewater. The importance of this work lies in the observation that S. obliquus could be utilized for simultaneous remediation of organic pollutants, N and P removal along with lipid accumulation. Due to increased attention on potential applications of microalgae, several researchers have carried out the life cycle assessment of microalgal productions to reveal its economic, social, energy and environmental feasibility (Wang et al. 2016). Through common consensus, sustainable cultivation of microalgae has been envisaged through usage of some wastes such as municipal wastewater, food industry wastes and forestry wastewater as cultivation medium. As a result, microalgae cultivation in wastewater is considered to be the most-effective way of biofuel production today (Cheah et al. 2016).

Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources 9

Biodiesel: Energy recovery from algal biomass is a solid to liquid/gas transformation process and could be carried out through hydrothermal liquefaction (HTL), anaerobic digestion, fast pyrolysis and gasification (Elliott 2008, McCarty et al. 2011, Chakraborty et al. 2012, Wang et al. 2016). Detailed coverage of these methods is out of the scope of this chapter and readers are advised to follow Demirbas 2009 for detailed literature. HTL has been demonstrated and thus preferred as an energetically favorable thermochemical conversion method over other biomass conversion technologies (Vardon et al. 2012). Biocrude oil yields from HTL of WWT algae have achieved 30–50 wt% on a dry, ash-free basis, with higher heating values (HHV) of 35–39 MJ/kg (Zhou et al. 2013). Wu et al. 2012 investigated assimilation of nitrogen and phosphorus and accumulation of lipid in Chlamydomonas sp. TAI-2 when grown in industrial wastewater. The optimal growth and lipid accumulation of Chlamydomonas sp. TAI-2 were tested under different nitrogen sources, nitrogen and CO2 concentrations and illumination periods in modified Bold’s Basal Medium (BBM). At optimal CO2 aeration of 5%, Chlamydomonas sp. TAI-2 achieved maximal lipid accumulation under continuous illumination. When industrial wastewater was used as a growth medium, Chlamydomonas sp. TAI-2 could remove 100% of total nitrogen and 33% of total phosphorus and accumulate lipid up to 18.4%. Over 90% of total fatty acids were 14:0, 16:0, 16:1, 18:1, and 18:3 fatty acids, which qualify the quality test for biodiesel production. Piggery effluent is one of the most highly produced wastewaters throughout the world, containing high levels of inorganic nutrient species. Abou-shanab et al. 2013 employed fivefold diluted piggery effluent fortified with synthetic media to reduce the toxicity of (in)organic components and demonstrated improved microalgal growth, nutrient removal and lipid productivity using S. obliquus YSW-14. It was observed that a 40% concentration of wastewater effluent was most effective for microalga lipid productivity (0.13 mg/L) along with 46 and 78% removal of total nitrogen and total phosphate respectively. Komolafe et al. 2014 carried out sustainable synthesis of biodiesel using Desmodesmus sp., which is a thermotolerant microalgae using ozone-flotation harvesting and reported FAME of greater oxidation stability along with 80 and 99.8% nitrogen and total coliform removal. Ji et al. 2015 evaluated the effect of Food Wastewater (FW) on the biomass, lipid and carbohydrate production by S. obliquus cultivated in Bold’s Basal Medium at different dilution ratios (0.5–10%). Dilution factor of 1% was found to promote the highest growth (0.41 g/L), lipid productivity (13.3 mg/L. d), carbohydrate productivity (14.7 mg/L. d) and nutrient removal (38.9 mg total nitrogen/L and 12.1 mg total phosphorus/L) after 6-days of cultivation. It was observed that FW promoted algal auto-flocculation due to the formation of inorganic precipitates at an alkali pH, thus, improving the growth, lipid/carbohydrate productivity and biomass recovery efficiency of S. obliquus. The increasing popularity of Galdieria sulphuraria was examined by Cheng et al. 2019 who tested HTL efficiency of G. sulphuraria 5587.1 and SOOS grown on municipal wastewater under various conditions of temperature (310–350℃) and time (5–60 minutes). Energy recovery in total bio-crude oil and char at 350°C was 17–28% and 14–19%, respectively for the 5587.1 strain, and 23–27% and 14–25%, respectively for the SOOS strain. From the studies, the authors inferred that despite higher ash content and lower bio-crude oil yield in the wastewater-grown G. sulphuraria when compared to media-grown G. sulphuraria, the total energy and nutrient recovery potential of the bio-crude oil, char and aqueous phases produced by HTL may be high enough for the net positive energy system development. They further predicted the necessity of pilot-scale Continuous Flow Reactor (CFR) as the next step in commercialization of the process with the aim of ameliorating the productivity and thus economic feasibility of entire WWBR-HTL process. In a next-step research, Russel et al. 2020 explored a novel algae-bacteria partnership system to strengthen the resistance of algae and improve the treatment effect of domestic wastewater. Their

10

Extremophiles: Wastewater and Algal Biorefinery

efforts were based on reports that bacterial presence can enhance microalgal metabolism by releasing growth-promoting factors, like vitamins and siderophores which are prominent chelating agents of algal growth or even by reducing oxygen concentration in the growth medium. Further, CO2 is released by bacteria which could be used by algae for photosynthesis reaction. However, studies on the asymbiotic relation between bacteria and algae also exist indicating competitive interactions (Gonçalves et al. 2017). The system proposes decreased operational costs and minimized risks for pollutant volatilization under mechanical aeration thus improvising wastewater treatment protocols using microalgae. Shen et al. 2020 recently investigated the effects of nitrogen and phosphorus supply on biodiesel production from S. obliquus in heterotrophic cultivation conditions with glucose as the carbon source. They obtained 72, 95 and 54% removal efficiencies of COD, total nitrogen and total phosphorus after 8-days cultivation, respectively. Inspiringly, after 8-days of cultivation, the fatty acid productivity reached as high as 99.3 mg/L. d, which was surprisingly 1.15 times higher than the highest efficiency using a glucose culture. It was also observed that even under nitrogen starvation, sufficient phosphorus could further improve biodiesel production. Table 1.3 summarizes research on some extremophilic microalgae used in wastewater treatment and bio-oil synthesis. Table 1.3. Extremophilic microalgae employed in wastewater treatment and bio-oil synthesis. Species Name

System

Parameter Studied Nitrogen Removal (%)

Phosphorus Removal (%)

Reference

Scenedesmus obliquus

Na2CO3 fortified synthetic wastewater

52

67

Duan et al. 2020

Scenedesmus obliquus

Soyabean processing wastewater

95

54

Shen et al. 2020

Co-culture of Scenedesmus obliquus and Acinetobacter Pitti

Synthetic domestic wastewater

86

91

Russel et al. 2020

Desmodesmus sp.

Facultative lagoon wastewater treatment plant

80

–

Komolafe et al. 2014

Scenedesmus obliquus YSW-14

Piggery effluent

46

78

Abou-shanab et al. 2013

S. obliquus (SAG 276-10)

Pretreated urban wastewater

100

100

Arbib et al. 2013

Chlamydomonas sp. TAI-2

Untreated wastewater from Taichung science industrial park

100

33

Wu et al. 2012

Alginate immobilized S. obliquus

Urban wastewater

96.6

–

Ruiz-Marin et al. 2010

Green Algae

Municipal Centrate wastewater

78.3

85.6

Wang et al. 2009

Scenedesmus obliquus

Urban wastewater

100

98

Martinez et al. 2000

In addition to eutrophic waters, algae-based removal of heavy metals from industrial wastes is a well-established practice. Remediation of heavy metals through algae works on two major processes, either through metabolism-dependent uptake or biosorption. A number of algal species, viz., Scenedesmus acutus, Chlorella vulgaris, Lemna minor, Nostoc muscorum, Phormidium ambiguum, Pseudochlorococcum typicum, Scenedesmus quadricauda and Spirogyra hyaline have been found to have excellent abilities to extract toxic metals from water and wastewater (Ankit et al. 2020). However, detailed discussion on heavy metal accumulation be extremophilic microalgae is out of the scope of this chapter and readers are advised to refer to Ankit et al. 2020.

Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources 11

Bioethanol: Large scale cultivation of microalgae as a bioethanol source after exhaustive exploration of first- and second-generation feedstocks provides important support for development of renewable energy worldwide. The productivity per unit area of microalgae is higher compared to conventional biomass cultivation for production of biofuels. Although the major barrier remains the high cost of nutrient requirements for ample cultivation on synthetic medium. Various researchers have advocated the utilization of different waste feeds as growth medium for such potential algae. Despite advantages such as microalgae need less cultivation area than terrestrial crops, can be cultivated in range of waters including saline water, brackish water or even wastewater and possess high CO2 fixation capabilities, the popularity of cultivating microalgae on industrial wastewater for bioethanol synthesis is yet to be spear-headed. In a recent study, Reyimu and Ozcimen 2017 performed cultivation of Nannochloropsis oculata and Tetraselmis suecica microalgae on combined growth medium containing seawater and wastewater from Istanbul Water and Sewerage Administration (ISKI). From extensive growth kinetics, the authors achieved the specific growth rate of the cultures can up to 0.5430 per d (on 75% of wastewater) for N. oculata and 0.4778 per d (on 25% of wastewater) for T. suecica. Further, bioethanol production studies were carried out on alkaline pretreated samples with maximum carbohydrate content which was 75% wastewater medium for N. oculate and 100% wastewater for T. suecica. Owing to a higher carbohydrate content accumulation, T. suecica offered better bioethanol yields (~ 4, 100% wastewater) than N. oculate (3.68, 75% wastewater) after 48 hours. The authors concluded that in stoichiometric aspects, the higher carbohydrate content will increase the bioethanol yield. Undoubtedly microalgae cultivation opens up golden gates towards sustainable biorefinery concept. Albeit the fact remains that despite more than half a century of commercial potential recognition, only a handful number of micro-algal species are currently being produced sustainably on a large scale (Richmond 2004). The major constraint remains difficulties in maintaining constant growth conditions like high light, temperature as well as problems arising from seasonal and diurnal fluctuations in light and temperature and contamination by other organisms which adversely affect growth and productivity in outdoor algal ponds (Torzillo et al. 2003). Furthermore, exploitation of extremophilic microalgae for biofuel synthesis is even marginal despite several reports on their bioremediation capacities of wastewater along with exceptional growth rates (Varshney et al. 2015).

3.3 Bioelectricity Biomethane synthesis in itself is not a sustainable solution for waste treatment or biofuel production owing to cost considerations. Although concepts of such CHP (Combined Heat and Power) plant have evolved, complete energy autonomy is achieved only by few highly advanced and sophisticated plants (IEA Bioenergy 2015). Energy autonomy refers to the balance between energy generated to energy used in the WWTP. To offset the disbalance, advent of fuel cells (typically Solid Oxide Fuel Cells (SOFC)) to generate direct electricity from the chemical energy contained in the biomethane, with an electric efficiency of 50 to 55% is being envisaged. Microbial Fuel Cell (MFC) is a novel green technology that generates energy and simultaneously removes the pollutant during the treatment of waste/wastewater. Compared to CHP, fuel cells emit lower pollutants such as CO, NO and hydrocarbons. However, fuel cells are highly sensitive to poor gas quality and requirement of stringent security measures owing to the involvement of hydrogen. Simultaneous electricity generation and wastewater treatment using a thermophilic or halophilic MFC or Microbial Electrolysis Cell (MEC) have evolved as a sustainable solution for the energy crisis. Additionally, proper deployment of MEC serves to improve overall energy efficiency of ethanol biorefineries by liberating hydrogen, which can be utilized in the biorefinery for improving fuel yield or as a separate product itself (Akinosho et al. 2015).

12

Extremophiles: Wastewater and Algal Biorefinery

Simply stated, an MFC is a bio-electrochemical device that harnesses the power of respiring microbes to convert organic matter (chemical energy) in wastewater directly into electrical energy using oxidation-reduction reactions. The success of an MFC is appraised in terms of power output, Coulombic efficiency, stability and longevity and is dependent on the design. MFCs can be optimized in two types on the basis of number of compartments of chambers. A standard two chambered MFC (H-shaped) is connected by a separator which is normally a salt bridge or a Cation Exchange Membrane (CEM), to allow passage of protons to the cathode while blocking the diffusion of oxygen into the anode. The two-chamber design of MFCs is generally operated in batch- and fed-batch mode can function in various practical shapes such as cylindrical, rectangular and miniature. A single chamber MFC, as the name suggests, consists of one anodic compartment with porous cathode located on the wall of the chamber utilizing oxygen from the atmosphere and allowing protons to transfer through them. In typical MFC reactors, electrons are harvested from organic matters (including various biodegradable wastes and biomasses) by using a group of microorganisms, collectively referred to as exoelectrogens, capable of extracellular electrons transfer. Exoelectrogens possess the capacity of anaerobically oxidizing organic content of wastewater and extracellularly releasing electrons, which are collected at a solid anode for the current generation along with the release of protons and carbon dioxide. The generated protons (H+) and electrons (e−) are transferred and combined with electron acceptors such as oxygen, potassium ferricyanide, etc., present in the cathode compartment completing the energy circuit (Shrestha et al. 2018). A major component of MFC is toxic and hazardous chemical catholytes which are harmful to the environment as well as economically unviable for large scale operation. Another important limitation of conventional MFC is the need for aeration at the cathode chamber, which act as an electron acceptor. CO2 is a major gaseous end product during degradation of wastewater at the anode (Mohan et al. 2014). To offset all these drawbacks presented by MFCs, carbon sequestration is an important tool to mitigate the adverse effects associated with CO2 emission (Shahbazi and Rezaei Nasab 2016). An economic sequestration technique is the cultivation of microalgae using CO2 as a nutrient source in the cathode chamber. Microalgae are productive autotrophic microorganisms and have the ability to alleviate emission issues through the transport of bicarbonate into cells (Mohamed et al. 2020). Most of the research on MFCs are focused on mesophilic microorganisms despite advantages offered by the use of thermophilic fuel cells. It is well-stated in literature that thermophilic anaerobic digestion provides higher substrate degradation rate, efficient heat utilization of high-temperature wastewater and lower risk of contamination from ubiquitous mesophilic microorganisms (Dai et al. 2017). Thermophilic strains such as Bacillus licheniformis, Bacillus thermoglucosidasius, Thermincola ferriacetica, Thermincola potens and Calditerrivibrio nitroreducens have been successfully employed for electricity generation in MFCs using various substrates. Further, mixed culture has been evaluated and appeared to be more advantageous in the treatment of wastewater than pure culture. Lower operation costs, wider substrate ranges, no or minimal sterilization requirements and high adaptability to environmental conditions are few of the benefits reported by various authors (Ha et al. 2012, Zhang et al. 2014). Although bioremediation of organic wastewater through MFCs is promising, the degradation of aromatic substances, especially aromatic derivatives, involving dehalogenation and nitrogroup reduction pose specific resistance owing to the adverse effects of high-temperature on more specific consortia. It is difficult for recalcitrant substances to supply sufficient energy for the survival and maintenance of microbial growth and community evolution under high temperature. Zhang et al. 2015 investigated the feasibility of cooperative selection of electrical stimulation and thermophilic conditions to induce evolution of communities suitable for remediation of p-fluoronitrobenzene (p-FNB) laden wastewater such as pesticide, dye and pharmaceutical industries. Complete removal of 0.4 mmol/L of p-FNB in simulated wastewater was achieved in

Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources 13

96 hours using mixed thermophilic culture. Microbial culture screening revealed Anaerolineae was the predominant population (46.9%) in the thermophilic bioelectrochemical system (TBES), followed by Betaproteobacteria (16.0%), Clostridia (10.1%), Gammaproteobacteria (6.8%) and Ignavibacteria (4.7%). In a recent study, Mohamed et al. 2020 carried out bioelectricity generation accompanied bioremediation of kitchen wastewater from a student’s mess through Synechococcus sp. and Chlorococcum sp. mediated biocatalysis at the cathode. Food waste contains a high content of biodegradable organic compounds, oils, protein, moisture, etc., and therefore presents itself as a potential source of bioelectricity generation. In comparison, Synechococcus sp. showed the higher power density (42.5 ± 0.5 mW/m2) than the latter (32.1 ± 0.5 mW/m2) at 1600 lux with CO2 supply. Operation at two different COD concentration (2500 and 5000 mg COD/L) indicated that both COD removal efficiency and peak current density decreases with increasing COD indicating prominent effect of substrate inhibition. Lu et al. 2019 studied the possibility of applying bioelectrochemical systems (BESs) at low temperatures (4–15°C) utilizing directly and indirectly acclimatized and enriched psychrophilic biofilms. Although several researchers have reported physiological characteristics, microbial growth rate and microbial activity to be negatively affected by a decreased temperature of wastewater, psychrophilic treatment conditions are advantageous in lower operating costs, wide application and advancement in enriching psychrophilic microbes compared with mesophilic conditions. Prior to this attempt, most studies reported the operation of psychrophilic BESs at 10–15°C, and only few of the studies were operated at 4°C. Psychrophilic bioreactors can operate in direct start-up and stable mode at 4°C with a sequential inoculation method with a mixed culture or with the indirect method where BES is started at a mesophilic condition (mostly 25°C and 30°C), followed by decreasing the temperatures to 20°C, 10°C and 5°C. Other methods could also be employed such as use of psychrophilic microbes as the inocula of BESs to treat wastewater at 15°C. In this study, the effects of inocula pretreatment at different temperatures on the start-up and operation of MFCs at 4°C were estimated and microbial community structures of the anode biofilms from second sedimentary sludge collected from the Wenchang Wastewater Treatment Plant (Harbin, Heilongjiang, China) were analyzed. Both treatment methods resulted in high COD removal (73–91%) with maximum removal by MFCs with 10°C, 7-days pretreated inocula. The Coulombic Efficiencies (CEs) of the MFCs fluctuated varying from 41 to 51%, the highest being achieved with 4°C, 14-d pretreated inocula. The studies indicated a trade-off between COD removal and CE as MFCs with a 7-day pretreated inocula demonstrated higher COD removal, but lower CEs than a 14-day pretreated system. Hierarchical clustering and heatmap analysis of nine anode biofilms community structure indicated Thauera, Thermomonas, Sphingobium, Legionella and Rhodanobacter were the differential populations in the control MFCs. Desulfobulbus, Methylibium, Nitrospira, Candidatus Accumulibacter and Caldilinea were the most differential populations in MFCs with 4°C, 7-day pretreated inocula, while differential populations in MFCs with 4°C, 14-day belonged to Devosia and Fusibacter. The majority of psychrophilic populations demonstrated the potential of the enrichment of Geobacter in MFCs with pretreated inocula. Successful application of extremophiles in bioelectricity generation from wastewater is summarized in Table 1.4. Currently, real-world applications of MFCs are limited due to their low power density level of meager several thousand mW/m2. However, efforts are being made to improve the performance and reduce the construction and operating costs of MFCs. It is postulated that unique membrane structures of extremophilic microalgae will play a crucial role in decoding of higher power densities from MFCs using wastewater.

3.4 Biohydrogen Hydrogen (H2) is considered to be a clean energy carrier owing to its ability to be converted into electrical energy through fuel cells. It offers a high energy density of 143 MJ/kg and could be developed

14

Extremophiles: Wastewater and Algal Biorefinery Table 1.4. Application of extremophiles in bioelectricity generation from wastewater.

Strain

Substrate

Process Conditions

Current (mA/ m2)

Coulombic Efficiency

Mixed culture of psychrophiles

Reference

Second sedimentary sludge

Pretreated inocula at 4°C for 14 days

–

51

Lu et al. 2019

Isolates of Firmicutes sp.

Saline starch water

58.44 g/L NaCl

2050

–

Vijay et al. 2018

Caldanaerobacter subterraneus

Oil field formation water

> 80℃

350

–

Fu et al. 2015

Stenotrophomonas spp.

Seafood processing wastewater

Salinity: 100 g/m3

–

28

Jayashree et al. 2016

Desulfuromonas Geoalkalibacter

Olive brine wastewater

100 g/L NaCl

7500

30

Marone et al. 2016

Halotolerant extremophile bacterial isolates

Hyper saline wastewater (Great Salt Lake)

> 35 g/L NaCl

820

–

Grattieri et al. 2016

Facultative anaerobic sludge from desalination plant

Produced water

Salinity > 200 g/L

2.8

0.2

Naraghi et al. 2015

Caloramator australicus

Anaerobic sludge

55℃

1.3

–

Fu et al. 2013

Bacteroidetes from thermophilic anaerobic digester

Alcohol distillery wastewater

55℃

2300

89

Ha et al. 2012

as a promising alternative fuel to fossil fuels (Kumar et al. 2012). Commercially, hydrogen production is carried out through various physical, chemical, physiochemical and biological processes such as water electrolysis, steam reformation, catalytic steam gasification of biomass, biomass pyrolysis, supercritical water gasification, photolysis of water and fermentation (Rittman and Herwig 2012). Despite numerous production technologies available, 96% of the current H2 is obtained from fossil fuels (49%-natural gas, 29%-crude oil and 18%-coal) through steam reforming. The remaining 4% H2 is generated by electrolysis. Considering the non-renewable nature of fossil fuels along with emission of green-house gases, the biohydrogen production provides an attractive alternative as the process is eco-friendly and renewable. Biohydrogen produced through biotechnological routes can also be diverted to MFCs for direct electricity generation and the process is gaining recognition (Verhaart et al. 2010, Pant et al. 2012, Dessi 2018). Biotechnological routes for production of hydrogen pertains to bio-photolysis of water through algae and cyanobacteria or through the use of photosynthetic bacteria for the photo-fermentation of organic substances and via dark fermentation of organic substances by anaerobic organisms. Photosynthetic bacteria are prone to high concentrations of ammonium and oxygen toxicity, making the process unsuitable for commercial hydrogen production, especially from industrial wastewater. In contrast, the dark fermentation can degrade a wide range of organic waste from complex lignocellulose, food waste and industrial wastewater to non-toxic, simpler monomers. Dark fermentation is however, associated with a disadvantage of relatively lower COD removal efficiency (~ 33%) and may require additional treatment before safe discharge into the system (Sivagurunathan et al. 2017). Nevertheless, the technique of dark fermentation is preferred over others owing to its simple reactor design and operation, non-reliability on light source, availability of wide range of feedstocks and comparatively higher H2 production rates compared to other methods (Saripan and Reungsang 2013). Similar to most of the other bioprocesses, thermophilic biohydrogen fermentations have higher H2 yields as compared to mesophilic ones owing to suppression of hydrogen-consuming bacteria,

Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources 15

such as methanogens and sulfur-reducing bacteria at extreme temperatures (Ren et al. 2009). Mohan et al. 2008 carried out biohydrogen production from distillery wastewater in anaerobic sequencing batch biofilm reactor at two operating pH values and observed better productivity at adopted acidic micro-environment. As start-up inoculum, selectively enriched anaerobic mixed consortia which was sequentially pretreated with repeated heat-shock (100°C; 2 h) and acid (pH of 6 and 7; 24 hours) methods. At ambient temperature, the set-up showed the feasibility of hydrogen production along with pollutant degradation. At acidophilic microenvironment (pH 6.0) H2 production rate of 26 mmol H2 d–1 with specific H2 production rate of 6.98 mol H2/kg COD. d which was considerably higher than those obtained at neutral microenvironment (H2 production rate of 7 mmol H2/d and specific H2 production of 1.63 mol H2/kg COD. d). In contrast to H2 productivity, COD removal efficiency (69.68%) was found to be effective in operated neutral microenvironment (pH 7) as compared to 56.25% at pH 6.0. Sompong et al. 2011 carried out production of biohydrogen from Palm Oil Mill Effluent (POME) utilizing anaerobic sludge from POME treatment plant as the inocula. During optimization studies on temperature and initial pH for biohydrogen production from POME it was observed that at a temperature of 60ºC and initial pH of 5.5, maximum hydrogen production of 4.820 l H2/l POME corresponding to hydrogen yield of 243 ml H2/g sugar can be obtained along with 46% removal of COD. Almost comparable results were obtained at continuous scale at HRT of 2 days. The effect of decreasing HRT on H2 production was also evaluated and found to have a negative effect. Phylogenetic analysis of the mixed culture revealed that Thermoanaerobacterium thermosaccharolyticum was majorly responsible for conversion, both in batch and continuous mode. Luo et al. 2011 studied the effect of two-stage anaerobic hydrogen and methane production process with the aim of enhancing bioenergy production stillage of an ethanol plant employing Thermoanaerobacterium thermosaccharolyticum and Clostridium thermocellum-like species for biohydrogen and methane generation, respectively. They were able to obtain 11% higher energy returns using two-stage reactor as compared to single stage process. Dessi 2018 carried out extensive studies on biohydrogen production using synthetic and real wastewater in a thermophilic Fluidized Bed Reactor (FBR) with biofilm-coated activated carbon as inocula. On varying operating temperature between 30–80℃, maximum H2 yield of 3.6 mmol H2/g CODadded (from thermomechanical pulping wastewater) was obtained at 70°C. Although the yield was two times lower than that achieved when pure xylose was used, the results are exciting as the yields were comparable to those obtained by direct fermentation of industrial sugar-containing wastewater under similar conditions. On further increasing the temperature, negligible H2 production was observed, indicating inability of dominating Thermoanaerobacterium sp. at these temperatures. Regardless of the temperature, the total COD removal efficiency was surprisingly higher than the 30–40% as expected for dark fermentation, most likely owing to the adsorption of volatile fatty acids (mainly butyrate) on the activated carbon. Numerous studies have been conducted to justify the application of extremophiles in biohydrogen production through dark fermentation. However, for the process to be economically sustainable extensive studies on complete conversion of substrates, higher yielding microbes, efficient bioreactors, process parameter optimization, etc., which make this technology very challenging need to be carried out. Further, molecular identification and genetic manipulation of efficient microbes resulting in least pre-treatment requirements will also be an good approach to make the process economically viable. Needless to say, with the proper implementation of crucial factors and strategizing enhanced biohydrogen production, ‘wastewater to biohydrogen production’ technology is the potential solution to reduced pollution as well as energy security.

3.5 Macroalgae as Fuel Source Recent studies have focused on conversion of wastewater to macroalgae, an important instrument for carbon dioxide assimilation, nitrogen and phosphorus removal along with potential biofuel

16

Extremophiles: Wastewater and Algal Biorefinery

feedstock (Chen et al. 2015, Yun et al. 2015). Macroalgae (red, brown and green) are large multicellular marine algae and are mainly harvested to produce hydrocolloids that constitute 10–40% of their biomass. In contrast to microalgae, macroalgae has a low concentration of lipids and primarily contains 35–74% carbohydrates and 5–35% proteins (Ito and Hori 1989). A concerted mechanism of cultivating macroalgae from wastewater and using thermophilic bacteria for conversion of inherent mannitol to bioethanol has been described (Ge and Champagne 2017, Chades et al. 2018). Ge and Champagne 2017 efficiently cultivated Chaetomorpha linum on primary, secondary and diluted centrate wastewaters using a step feeding approach with 26.5% increased biomass productivity as compared to single feed along with attractive nitrogen and phosphorus removal efficiencies of 86.8 and 92.6%, respectively. Economic feasibility studies carried out by the author on daily wastewater flow of approximately 6.09 × 104 m3 revealed 6.92 × 106 kg of protein for soil enrichment or animal feed, 1.86 × 106 kg of carbohydrates for bioenergy as well as 1.14 × 106 kg of lipids (oil) on per annum basis. Chades et al. 2018 carried out extensive studies on two macroalgae biomass namely, Ascophyllum nodosum and Laminaria digitata for effective mannitol extraction and consequent conversion of sugar alcohol to bioethanol through 11 strains within the genus Thermoanaerobacter. The highly ethanologenic strain of Thermoanaerobacter pseudoethanolicus (DSM 2355) offered ethanol yield of 82% with A. nodosum extract as compared to 70% on pure mannitol although the longer fermentation time of 195 hours could be problematic for commercialization of bioethanol production via algal mannitol. These two recent studies hold the potential of large scale WWBR coupled with utilization of lower value fractions such as mannitol. Although the road ahead is long and application of increasing cell density through immobilization or continuous culture, optimization of growth conditions and genetic engineering needs to be explored.

4. Future Prospects Evidently, the combination of tertiary wastewater treatment and microalgal lipid production is considered to be a promising approach to alleviate water pollution as well as the energy crisis. The carbon sequestration potential of algae can be further enhanced by introducing genetic engineering and manipulating key metabolic enzymes (Bajhaiy et al. 2017). Improved yields are expected if the mechanism of organic carbon removal in the coupling system could be decoded. Many researches have been focused on the symbiotic interaction between microalgae and bacteria as co-existing bacteria are found to be beneficial to algal growth as well as pollutants removal. Therefore, more studies need to be carried out to explore the co-culture systems containing microalgae and bacteria, specifically focusing on characterization of spices and their individual contributions in the system. More value-added products can be obtained by exploiting the advantage of lipid accumulation by optimizing growth medium conditions to make integrated algae-based process for wastewater treatment more economically viable. MFCs are predicted to be the future of bioelectricity generation if few hiccups such as low power densities could be overcome. In other words, future research should be focused on optimizing the performance of the device. According to one view, new electrode materials must be developed to facilitate the electron transfer between microorganisms and electrodes, thus decreasing the activation energy. Further, degradation of a wide range of pollutants will be of critical interest for the large-scale application of the device. An emerging field of interest have been presented as Constructed Wetland Microbial Fuel Cells (CW-MFCs) or phyto-power systems. These are the integrated bioelectrochemical systems that can sustainably harvest electricity during the anaerobic respiration of rhizospheric bacteria. This integration of techniques shows a promise in phytoremediation of wastewater along with bioenergy generation (Saba et al. 2019). CW-MFCs are useful in processing non-food plants resulting in high yield of biomass that can be applied for mass bioenergy production and bioaccumulation of

Strengthening Bioenergy-Based Economy Through Conversion of Wastewater Resources 17

pollutants. CW-MFCs are also expected to be considerably efficient in wastewater treatment, CO2 assimilation, power generation and air purification. However, it is a long road and various studies including microbial interaction with rhizosphere, isolated species from the phyto-systems, with soil particles and pollutants as well as critical operational parameters and their effect on power generation output efficiency need to be extensively reviewed. Evidently, cultivating microalgae on industrial/municipal wastewater is an economical technique to produce biofuel; however, sustainable efficiencies are marred by microbial contamination and needs to be controlled strictly. As described previously, microalgae lipid accumulation and recovery can be triggered by environmental pressures and thus optimization of relevant triggers holds a key for biofuel synthesis via microalgae. Recently Zhang et al. 2018 studied the possibility of microbial contamination posing as a pressure point for microalgae cultivation and lipid accumulation. The authors focused on the effects of the presence or external addition of Quorum-Sensing Molecules (QSMs) in activated sludge on biomass accumulation as well as lipid synthesis in microalgae. A pressure trigger was forced via cell-to-cell QSMs resulting in increased lipid content of Chlorophyta sp. by ~ 84%, while a negligible decrease in biomass production was observed. These results revealed the synergistic effects of microbial contamination on microalgae biofuel production. However, more research needs to be focused on application of QSMs as effective tools for enhancing lipid content of microalgae when grown on wastewaters, thus yielding commercially significant productivities of corresponding biofuels.

5. Conclusion Utilization of waste streams to generate energy molecules is the need of the hour. This has the dual benefit of minimizing pollutant load on the environment as well as reducing dependency on the non-renewable fossil fuels. In most of the developing countries like India, heavy growth in the industrial sector as well as increased population are the major factors responsible for environmental pollution and the large demand of energy. The concept of developing WWBR is one of the most promising solutions to address both these problems along with synthesis of bioethanol, biodiesel, bioelectricity and biohydrogen. However, sustainable and economically viable technology is still awaited. Also, research and development of robust and efficient consortia of bio-converters especially extremophiles are essentially required for the conversion of different organic molecules to the energy molecules and recovery of valuable metal ions from different industrial and municipal waste streams. Amongst varied extremophiles, microalgae have emerged as most proficient producers of bioenergy from waste streams. Their high lipid content (up to 80% of their weight), high efficiency, eminent growth and the possibility of being cultured on inappropriate/waste farmlands has led to elevated research interest on their use for biofuel synthesis and need to be further exploited.

Acknowledgement The authors would like to acknowledge the contribution of Dr. Neetu Singh, Assistant Professor, Department of Chemical Engineering, Ujjain Engineering College, Ujjain, Madhya Pradesh, in reviewing this book chapter and providing her valuable feedback for improvement of its quality.

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2 Extremophiles for Wastewater Treatment Keshav Rajarshi,# Karri Sudharshana# and Shantonu Roy*

1. Introduction The products derived from microorganisms, which can be used for specific applications, are the essence of a biotechnological process. The predominance of microbes that are exploited in industrial biotechnology mainly thrives in environmental conditions somewhat similar to those that support living of human cells, i.e., the mesophiles. The presence of oxygen, moderate temperatures, atmospheric pressure, absence of high salt concentrations and xenobiotic compounds and nearly neutral pH are the ideal conditions for the growth of these organisms. However, in many cases, the physicochemical environment generally does not seem to be ideal due to the severity of the factors like low or poor solubility of products or/and substrates, high viscosity, high volatility, high instability or due to the special or unusual conditions encountered in environmental conditions. This prevents the normal biodegradation/biotransformation along with reverse hydrolysis under low water activity. The starch-degradation process requires high temperatures in order to make the substrate accessible for enzymatic degradation as well as to decrease the viscosity of the medium or the decontamination of hydrocarbon polluted Arctic waters are classic examples. Almost all the ecological niches on earth have been colonized by living organisms. Extremophiles are those organisms that seem to ‘love the extreme.’ They are known to grow and survive under conditions that are considered not favorable and unusual from the viewpoint of ambient living conditions. These metabolically active microorganisms have now been extensively studied, and various microbiological techniques have been developed for their isolation and characterization. The identification, isolation and characterization of extremozymes, i.e., the enzymes derived from these organisms, has gained a lot of attention in recent years. Thermophilic prokaryotic organisms can survive and thrive at temperatures above 60°C, whereas the archaea dominate the hyperthermophilic organisms, which show optimal growth in temperatures above 80°C. Methanopyrus kandleri is known to live at the highest temperature of 122°C under 200 bar pressure (Stetter 2011). Arguments can, of course, be placed against the economic efficiency of large scale production of these enzymes due to the requirement of special conditions for the development of these microorganisms and their limited direct usage in some applications. Nevertheless, most of the potential of these extremophiles lie in their metabolic versatility.

Indian Institute of Engineering Science and Technology, Shibpur. * Corresponding author: [email protected] # Both the authors have contributed equally

24 Extremophiles: Wastewater and Algal Biorefinery Extremophiles have piqued the interest of researchers in the field of biotechnology due to their potential to subside environmental pollution under extreme conditions and extraordinary conversion capability (Hough and Danson 1999, Antranikian et al. 2005, Aislabie et al. 2006, Podar and Reysenbach 2006, Ferrer et al. 2007). Due to the vivid enzymatic activities possessed by these microorganisms, they are resistant to several toxic agents, capable of carrying out biodegradation of various xenobiotic compounds, detoxification of harmful metal compounds and production of specific metabolites (Kalin et al. 2005). For environmental biotechnologists, extremophilic microorganisms are of special interest as they can be exploited to degrade toxic compounds and materials which are present in the environment under extreme conditions (Maloney et al. 1997, Kalin et al. 2005, Aislabie et al. 2006, Brakstad and Bonaunet 2006, Takeuchi and Sugio 2006). For instance, the nature of wastewater released by a large number of industries such as cement, paper, beverage and pulp industries are alkaline. Hence, the extremophilic bacteria that are showing optimal growth in such environments can be exploited for the treatment of wastewaters.

2. Characterization and Classification of Extremophiles Extremophilic organisms can easily sustain the extremities and are able to grow under conditions where normal organisms are unable to survive. The extremophilic organisms are often attracted towards temperature conditions which are extraordinarily low or high, extremely high salinity, high radiation exposures, extremely acidic or basic pH, growth in the presence of hazardous or toxic waste materials, heavy metals and organic solvents, extremely high and low atmospheric pressures and several other environments which are considered harsh for normal survival. In accordance with these growth conditions, extremophiles are classified as extremophilic and extremotolerant organisms. The extremophilic organisms are which are able to grow and live under one or more extreme environmental conditions. On the other hand, extremotolerant organisms are those who usually grow under normal conditions and can also sustain if exposed to extremities (Rampelotto 2013). The extremotolerant organisms are also known as extremotrophic organisms or simply extremotrophs (Mueller et al. 2005). Apart from these, there are certain organisms that can withstand more than one extreme condition, such as extreme radiations, pH, metals and temperatures, etc., and are called polyextremophiles.

2.1 Prokaryotic and Eukaryotic Extremophiles Extremophiles are comprised of archaea, prokaryotic bacteria and several other eukaryotic organisms. Most archaebacteria are extremophilic, among the prokaryotes, due to their adaptive behavior and high versatility towards the extreme environments (Fig. 2.1). These archaea are usually strict anaerobes, high acid and temperature tolerant and saltloving. Pyrolobus fumarii is a type of archaea, which is considered a hyperthermophile as it can withstand temperatures up to 121°C. Geothermobacterium ferrireducens is another bacteria that can survive a temperature up to 95°C (Kashefi et al. 2002, Stetter 2006). The Picrophilus torridus and Methanopyrus kandleri are the identified archaebacteria, known to grow at a pH as low as 0.06 and a temperature as high as 122°C, respectively. Likewise, cyanobacteria are known to survive, thrive and withstand extremities such as high alkalinity, metal and salt concentrations, less water in dry regions, but cannot survive in acidic or low pH conditions (Rampelotto 2013). The Gloeocapsa sp., which is extremotolerant, is known to survive in extremities of space, including vacuum exposures, temperature shifts and exposures to radiation. Correspondingly, Helicobacter pylori, which has a spiral shape, can withstand the extremely acidic environments of the stomach. Earlier, only the prokaryotic and unicellular organisms were considered to be extremophiles, but the following studies reported that all extremophiles are not necessarily unicellular, and therefore the term extremophile began to include both prokaryotic and eukaryotic microorganisms (Bull 2010).

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Fig. 2.1: Ecological niche of extremophiles (Adapted from Merino et al. 2019).

2.2 Classification of Extremophiles Microorganisms which dwell in extremities of the environment which in the human frame of reference considered as the hostile territories of existence (Gupta et al. 2014). The extremes here refer to physical, biological as well as geochemical. Physical extremes refer to temperature, salinity, pH, redox potential, radiations and pressure; biological extremes are like nutritional, intraspecific or interspecific competition, parasites and prey. In general, the diversity of extremophiles is high and really advanced to check. Some orders or genera contain extremophiles only, whereas different orders or genera contain few extremophiles and non-extremophiles. They can be classified in line with the conditions in which they grow, which are described next. 2.2.1 Thermophiles Thermophilic microorganisms have an optimum growth temperature of 50℃ or higher. They have attracted good attention among extremophiles as a result of their sources of thermostable enzymes. These are isolated from the various ecological zones of the world (e.g., hot springs, deep-sea). Most members of those are Archaea. They are usually classified into three categories, moderate thermophiles (growth optimum: 50–60℃ ), extreme thermophiles (growth optimum: 60–80℃) and hyperthermophiles (growth optimum; 80–110℃). To date, an outsized range of enzymes degrading chemical compounds (e.g., amylases, cellulases, chitinases, pectinases, pullulanases, xylanases, proteases, isomerases, esterases, lipases, phytases, dehydrogenase) enzymes are characterized from numerous thermophilic and hyperthermophilic microorganisms which have significant application in biotechnology (Lasa and Berenguer 1993, Turner et al. 2007). 2.2.2 Psychrophiles Psychrophilic or psychrotolerant organisms have the optimum growth temperatures of −20°C to +10°C or lower. These will able to grow at the brink of low temperatures. These microorganisms are found inhabiting extremely cold environments of the world: like polar regions, glaciers, ocean deeps, shallow subterranean regions, higher atmosphere, cold appliances and on and in plants and animals inhabiting cold regions (Feller and Gerday 2003). These organisms are distributed in many

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families of microorganism bacteria (e.g., Pseudoalteromonas, Vibrio, Arthrobacter and Bacillus), archaea (e.g., Methanogenium and Halorubrum), fungi (such as Penicillium and Cladosporium) and yeasts (Cryptococcus). The enzymes from these cold-adapted microorganisms show activity at extremely low temperatures that gives them sizable potential application in detergent, textile, food, pharmaceutical, leather, brewery and paper and pulp industries (Cavicchioli et al. 2011, Feller 2013). The use of psychrophiles and their enzymes is found in the bioremediation of soils and wastewaters. 2.2.3 Alkaliphiles and Acidophiles The alkaliphiles/acidophiles have great potential for biotechnological application (Horikoshi and reviews 1999). The alkaliphiles square measure are the category of extremophiles that thrive in alkalic environments at a pH of 8–11 and above, such as in soda lakes and carbonate-rich soils. Acidophiles, on the other hand, tend towards acidic conditions with a pH optimum for growth at or below, pH. They can be found in drains of coal mines and many such low pH environs. One of the foremost hanging properties of such organisms is their proton pump mechanisms, which help to maintain a neutral pH internally (Matin 1999). Alkaliphiles find great industrial applications as they provide a smart supply of pH stable enzymes like proteases, amylases, cellulases, lipases, xylanases, pullulanases, pectinases and chitinases (Fujinami and Fujisawa 2010). Alkaliphilic cyanobacterial species were discovered in salt lakes (Gerasimenko et al. 1996). The Nostoc calcicola is an alkaliphilic diazotrophic cyanobacterium (Singh 1995). Alkalophilic bacteria are generally from the genera like Bacillus (Bacillus pseudofirmus, B. Haloduran), Micrococcus, Pseudomonas and Streptomyces. Fungal alkalophiles (Acremonium, Fusarium, Paecilomyces, Stilbella, Fusarium, Metarhizium and Scopulariopsis spp.) (Kanai et al. 1995) and one of the archeal examples of this category is Natronococcus amylolyticus sp. which is a haloalkaliphilic archaeon (Sharma et al. 2012). As such, acidophiles are also widespread in all the archaeal, bacterial, fungal and algal populations and are found to have many potential applications (Horikoshi 1998, Kladwang et al. 2003). 2.2.4 Piezophiles/Barophiles Microorganisms that prefer high-pressure conditions are known as piezophiles (or barophiles). These are found in oceans or sea (Yano et al. 1998). They are found to have inbuilt adaptations that help them to maintain proper fluidity of membrane lipids under high pressure (Misra et al. 2012). It is believed that enzymes isolated from piezophiles square measure stability at high pressures. Piezophilic microorganisms and their enzymes have sizable potential to be used in biotechnology, specifically for food industries, wherever high pressure employing processing conditions prevails. 2.2.5 Radiophiles Microorganisms that grow in or which are highly resistant to ionized and ultraviolet illumination are referred to as radiophiles. These radiation-resistant microorganisms show high potential within the treatment of radioactive environmental wastes. Radiophiles are attaining importance due to their ability to survive conditions of starvation, aerobic stress and high amounts of DNA damage. Deinococcus radiodurans is a well known radio-tolerant bacterium. Extensive research is going on in using this species in the bioremediation of radio-active wastes (Gabani et al. 2013). These radiophilic microbes are found to exhibit significant applications in biotechnology and the therapeutic industry (Dighton et al. 2008). There were also many findings of radiotolerance in fungal and cyanobacterial populations (Kraus 1969, Rivasseau et al. 2016). A new radiotolerant extremophilic algae has also been discovered in a nuclear reactor. The found microalgae is of the genus Coccomyxa belonging to the class Trebouxiophyceae, and was named Coccomyxa actinabiotis sp. Nov. (Grant 2004).

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2.2.6 Xerophiles The organisms that have the ability to grow in extremely dry conditions or in the environs of terribly low water activity are known as xerophiles. These organisms are mainly responsible for the spoiling of dry foods. Few specialized genera among yeasts, fungi, lichens and algae are able to survive below such conditions. Some filamentous fungi and yeasts (Zygogaccharomyces rouxii) are capable of growth at a drastically low water activity of about 0.61. High salt environments are characterized by very low water activity in which haloarcheal species can sustain. There are abounding findings of life at low water activities from all domains (Jerez Guevara 2017). 2.2.7 Metallophiles Microorganisms that grow at high metal concentrations are referred to as metallophiles. Since pollution by serious metals (Cu, Cr, Zn, Cd, Co, Pb, Ag and Hg) pose a threat to public health and natural environments. There has been increasing interest in employing metallophiles for removal of the noxious metals from soils, sediments and wastewaters. Metallophiles also show high potential in the bio-mining of important metals from effluents of commercial processes (Khan et al. 2019). Metallotolerant fungi like those belonging to Aspergillus and Penicillium genus are used in bioremediation of heavy metal polluted soils (mycoremediation) (Khan et al. 2019). 2.2.8 Halophiles Halophiles are extremophilic microorganisms which thrive optimally in saline conditions. Depending on the requirement of NaCl for optimal growth, these organisms are classified into three different categories: slightly halophilic (1–3%), moderately halophilic (3–15%) and extremely halophilic (15–30%). Such microorganisms are found altogether in three domains of life: Archaea, Bacteria and Eukarya. There is increasing interest in the application of halophiles as a result of their best activities in the presence of high concentration of salts (Corral et al. 2020). Halophilic organisms accumulate salts (NaCl or KCl) up to concentrations which are isotonic with the surroundings (osmotic balance) (Siglioccolo et al. 2011).

3. Wastewater: Characteristics and Treatment Approaches The treatment approaches for industrial effluents should be planned and developed in such a way that it meets the goals of saving various kinds of life, restoring and conserving the recreational and outstanding value of surface waters, safeguarding the surface water’s assimilative capacity and protection of human beings from detrimental effects of worsening water quality conditions. Based on the knowledge of several factors, such as biological, chemical and physical characteristics of the wastewater, the wastewater treatment facilities should be selected. The quality of the wastewater after its treatment should match the environmental standards so that it can be reused for several purposes or the quality of water reservoirs (streams, ponds and rivers) must be maintained in which the wastewater is to be released. The primary physical characteristics of industrial wastewaters are their color, temperature, odor the content of solid materials and particles, etc., whereas the chemical characteristics of the wastewaters are classified into two main categories, i.e., inorganic characteristics and organic characteristics. The inorganic chemicals allow one to opt for the appropriate methodology for the treatment of wastewater, differ in accordance with the origin of wastewater. Inorganic factors are comprised of nitrites, nitrates, free ammonia, organic nitrogen, heavy metals, hydrogen sulfide, organic and inorganic phosphorus, chloride, sulfate, carbon dioxide and methane. For assessment of the organic content of the wastewaters, VOC (Volatile Organic Compounds), TOC (Total Organic Compound), BOD (Biological Oxygen Demand) and COD (Chemical Oxygen Demand) must be meticulously determined because they influence the selection of appropriate biological treatment technology and strategy (Amoozegar et al. 2015).

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Headsprings of wastewater are primarily micro-industries (like laundries, lodgings, hospitals and so on), macro-industries (industrial wastewaters) and normal household procedures like washing, latrine flushing, clothing, dishwashing, and so on from residential or domestic sources (domestic wastewater). Commercial wastewater originates from non-household sources, for example, beauty salons, taxidermy, furniture resurfacing, instrument cleaning, business kitchens, energy generating plants and for the most part, from heavy industries. This wastewater may contain risky materials and requires a particular treatment or removal. Wastewater is likewise created from agriculture sectors. Water is used for cleaning in animal homesteads, washing harvest crops and cleaning farming accouterments. The nitrogen and phosphorus in manure, composts and fertilizers are important to cultivate crops. In any case, when these nutrients are not completely used by plants, they can be lost from the fields and unfavorably sway into the air and also affect the downstream water quality. Nutrient contamination influences air and water around. The unfavorable effects of abundant nutrients are found in wide range of polluted water bodies. The contaminants frequently enter upstream waters like brooks and streams and afterward stream into bigger water bodies like lakes, rivers and oceans. Excess nitrogen and phosphorus can likewise travel a huge number of miles to coastal areas. It is an essential driver of eutrophication of surface waters, wherein the nutrients, generally nitrogen or phosphorus, favor algal development. Storm water is another important wastewater. At the point when it rains in cities and towns, it runs across surfaces - like housetops, walkways and streets - and carries contaminations, including nitrogen and phosphorus into local waterways. The drain channels get water from the street gutters on most motorways, expressways and other occupied streets, just as towns with heavy precipitation that prompts flooding and areas with heavy storms. Indeed, even canals from houses and buildings connect with storm drains. Storm drainage channels untreated water drains into waterways or streams, which is profoundly unsuitable as pouring dangerous substances into the water bodies will seriously influence life in there. Every phase of production of fossil fuel’s and their usage generates a large group of harmful substances which have a deleterious impact on the environment. Fossil-fuel production, transmission and use can defile water bodies with hydrocarbons, heavy metals, nutrients and salts and a large group of chemicals, including benzene, toluene and hexavalent chromium. The water that rises to the top alongside extricated crude oil is known as produced water. Produced water can contain hydrocarbon deposits, metals, hydrogen sulfide and boron, etc., which poses harm to the environment. The composition of wastewater is very complex. Depending on its origin, it can contain a wide variety of contaminants such as agro-residues, farm wastes, pesticides, insecticides, organic salts, aliphatic and aromatic hydrocarbons, oils and greases, metals and occasionally radioactive materials. Here is a classification on the basis of the source of wastewaters.

3.1 Types of Wastewaters-based on Source 3.1.1 Agro-food Industry Wastewater During or in the wake of handling of crops, huge amounts of lignocellulose-rich wastes are generated. Animal farm handling and the organic farm also generate large amounts of wastewater. The dairy industry and cheese industries are one of the significant producers of slightly alkaline and whitish wastewater, which mainly contain suspended solids, soluble organics, chlorides, sulfates, huge amounts of sodium oil and grease, which lead to high levels of BOD and COD (Shete et al. 2013). Sewage fungus growth is found to be encouraged by the lactose in the effluent. Fruit juice production involves various steps washing, cleaning, juice extraction and treatment, peel removal and emulsion, etc., in which large quantities of wastewater are generated (Thevendiraraj et al. 2003). As such, every food processing industry produces plenty of wastewaters.

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3.1.2 Pulp and Paper Industry Wastewater Effluents Paper and pulp industries are a portion of the fundamental water-and energy-intensive industries which are answerable for a large scope water contamination. The issues related with pulp and paper factory effluents are pH, chemical dyes, elevated levels of BOD, COD and so on. The paper industry discharges chlorinated lignosulfonic acids, chlorinated acids, halo-phenols and hydrocarbons into the squander water (Nunes and Malmlöf 2018). These are the significant contaminants emanating from pulp and paper factories. A few compounds in the effluents are impervious to biodegradation and can aggregate in the water bodies. 3.1.3 Oil Industry Effluents The oil industry is a significant source of contaminants that corrupt nature, with the possibility to influence it at all levels: air, water, soil, and therefore, all living creatures on the planet. Naphthenic Acids (NAs) are one class of mixes in wastewaters from oil enterprises that are known to cause poisonous impacts, and their expulsion from oilfield wastewater is a difficult task when it comes to remediation of huge volumes of petrochemical effluents (Wang et al. 2015). Polycyclic hydrocarbons and different natural and inorganic compounds, normally present or included during preparative phases, are destructive toxins that can be treated with advanced membrane technologies, oxidation techniques and with biological treatments (Wei et al. 2019). Produced Water (PW) is salty in nature that is trapped underground. This water is brought out during oil and gas extraction. Produced water usually contains plenty of pollutants, including hydrocarbons and metals, requiring appropriate treatment before its removal. 3.1.4 Wastewater from Power Generation Plants The enormous use of water in cooling condensers of thermal power plants leads to changes in temperature and physicochemical properties. This water is now discharged from the plant into nearby water bodies, which attribute to changes in the chemical nature of them and negatively impact the environment. The effluents from the coal-fired thermal power plant were found to be slightly alkaline. The ash effluents contained from these plants contained more suspended and dissolved solids, and there was a considerable amount of chloride, sulfate and phosphorus found to be present (Ajmal and Khan 1986). There is a reported increase in the number of nuclear reactors for power generation, due to which the amounts of radioactive effluents are increased substantially. Tritium is generated from the neutron activation process. This is used as a coolant in pressurized heavy water reactors. The liquid tritium effluent discharged from the plants is the major radionuclide causing radiological pollution of water bodies (Son et al. 2013). 3.1.5 Textile Industry Effluents The wastewater from several textile industries uses tremendous quantities of synthetic and organic dyes. The discharge of these colored compounds into the environment causes considerable pollution and serious health-risks. Various steps in textile processing, like carbonising, felting, dyeing, bleaching, printing and rinsing, involve the use of different chemicals, which later end up in the effluents in considerable quantities (Bisschops and Spanjers 2003). In textile manufacturing, wastewaters from preparation processes such as mercerising and caustic scouring are reported to have high alkalinity (Tomasino 1992, Bisschops and Spanjers 2003). 3.1.6 Steel Industry Effluents A complex range of organic compounds like Polycyclic Aromatic Hydrocarbons (PAH), ammonia, cyanide, thiocyanate, Benzene Toluene Xylene (BTX), phenols and cresols containing large amounts of toxic, hazardous effluents are produced at different stages of steel manufacturing

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operations (Das et al. 2018). Steel plant effluent discharges from the Bhilai Steel Plant were reported to have polluted the river in the region, causing an increase in dissolved and suspended solids, toxic chemical elements. There was also a drastic reduction in pH values (from 8.9 to 3.9) and dissolved oxygen levels (1 to 3 ppm). As a result of offloading toxic acidic effluents into river bodies, the aquatic life are becoming completely demolished (Satish et al. 2012). 3.1.7 Effluents from Chemical Industries The chemical industry is a greatly expanded area, and one can typically categorize this sector into groups based on the type of chemical being produced. Like, petrochemicals, inorganic chemicals, fine chemicals, pharmaceuticals, fertilizers, etc. Essential chemicals (commodity chemicals) are an expansive chemical category that consists of pharmaceutical products, polymers, bulk petrochemicals and intermediates, other derivatives and basic industrials, inorganic/organic chemicals and fertilizers. The chemical industries are monitored with the utmost significance in terms of its impact on the environment. Chemical industrial wastewater effluents usually contain organic and inorganic compounds in varying concentrations, depending on the chemical product being developed. Numerous compounds discharged from the chemical industry are poisonous, mutagenic, cancer-causing or almost non-biodegradable. Therefore, a legitimate earlier treatment should have been done to these effluents before releasing into the environment in order to forestall the consequent risks (Awaleh and Soubaneh 2014).

4. Exploitation of Extremophiles In medicine and the biotechnology industry, extremophilic microorganisms have several applications. Extremozymes are considered as one of the most crucial products extracted from extremophiles because they are essentially required in industries. Apart from their applications in industries, they are advantageous due to the fact that they can be used as a model system for studying the mechanisms of stabilization and activation of protein structure-functional properties by enzymes (Demirjian et al. 2001). The most promising enzymes known for their applications in various industries are obtained from alkaliphilic, psychrophilic, thermophilic and hyperthermophilic groups of extremophilic microorganisms (Van Den Burg 2003). Hydrolases with enhanced thermostability and wide industrial applications like amylases, chitinases, cellulases, lipases, pullulanases, xylanases, glucose isomerases, esterases, alcohol dehydrogenases and proteases can be derived from thermophilic and hyperthermophilic microorganisms. Extremophilic microorganisms are also known to produce several other thermostable enzymes such as restriction enzymes, DNA polymerases, DNA ligases and phosphatases, which play an essential role in molecular biology and medicine (Gomes et al. 2004, Egorova and Antranikian 2005). One of the most intriguing applications of the extremophilic microorganisms is their potential bioremediation, which is one of the most efficient and lucrative techniques of cleaning and removal of pollutants from contaminated environments (Kumar et al. 2011). Extremophilic microorganisms provide vigorous enzymatic and whole-cell biocatalytic systems that are appealing under conditions that restrain the efficacy of typical bioconversions.

4.1 Extremophiles in Wastewater Treatment 4.1.1 Extremophiles Employed in Treating Alkaline Wastewaters The extreme environments are colonized by several archaea, including sulfur-metabolizing thermophiles, methanogens, hyperthermophiles and extreme halophiles. Since extremophilic microorganisms have uncommon and remarkable characteristics, they are regarded as a potentially valuable resource in developing novel and innovative biotechnological processes. Even with the extensive research going on, there are very few confirmed existing industrial applications of either

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alkalophilic archaeal enzymes or biomass. Evaluation of halophilic archaea has also been done for degradation of organic pollutants, treatment of concentrated wastewaters from textile industries and for carrying out bioremediation in harsh environments (Margesin and Schinner 2001). Affirmative results for hydrocarbon pollution control at a self-sufficient system representing marine sites were obtained (Banat et al. 2000). The degradation of hydrocarbons by archaeal microorganisms under anoxic conditions have also been examined (Lovley 2001). The sulfate-reducing microorganisms which come under the archaea domain have also been isolated due to their known potential to carry out anaerobic degradation of alkanes. The oxidation of methane anaerobically with sulfate as an electron acceptor has also been reported and is presumed to take into account the reversal of archaea catalyzed methanogenesis with sulfate-reducing bacteria scavenging an electron-carrying metabolite (Spormann and Widdel 2000). A gram-positive, non-spore-forming, rod-shaped and non-motile strain was identified, which was capable of lowering the pH of high alkaline wastewaters ranging from 12.0 to 7.5 within approximately 2 hours. This strain of the facultatively alkaliphilic bacterium was isolated from industrial wastewater drain sludge of the beverage industry. On the biochemical characterization and 16s rRNA genome sequencing, it was found that the isolate belonged to the Exiguobacterium genus. It was found from the results of FT-IR (Fourier transform infrared) spectroscopy that the bacterium produced carboxylic acid as a metabolic product for neutralizing alkaline wastewater. Treatment of alkaline wastewaters by the process of neutralization and without the addition of an external carbon source points towards the potential of this isolate and regards it as an alternative for the conventional techniques of acid neutralization of wastewater (Kulshreshtha et al. 2010). The C. matritensis and A. fumigatus showed unchanged capacity to grow in the concentration ranges of 0–10% NaCl in a study to determine the ability of fungi isolated from olive brine wastewater for producing extracellular phenoloxidases (EPO). Among them, Citeromyces matritensis (syn. Candida globosa) and Aspergillus fumigatus displayed laccase and Mn-peroxidase (MnP) activities. They are found to maintain EPO-producing ability and removed phenols from the effluent. These results suggest the possibility of using these strains in the treatment of other saline phenol-rich effluents, such as pickling and tannery wastewater (Crognale et al. 2012). A study by Vadlamani et al. demonstrated that extreme alkalophilic microalgae Chlorella sorokiniana (SLA-04), on account of its origin in the Soap Lake, was well-adapted to thrive in an unusually high-pH environment. The cultures were capable of growing at extreme alkaline pH (pH > 10) without CO2 supply in both indoor and outdoor conditions. It is also reported with high lipid productivities. Overall, results demonstrate that employing such extremophilic alkalophiles will be a novel strategy of microalgae cultivation in extremely high-pH effluents with reduced costs of algal cultivation due to no additional CO2 sparging (Vadlamani et al. 2017). 4.1.2 Extremophiles Employed in the Treatment of Heavy Metal Polluted Wastewaters with Low pH Abandoned coal mines are the primary sources of acidic polluting wastewaters. The acidic nature of the water is mainly due to the oxidation of sulfide minerals being catalyzed by the specialized bacteria in surface waste rock heaps and tailings or flooded underground workings (Younger et al. 2002). Acidic environments that are naturally occurring are home for a diverse range of extremophiles, acidophiles to be precise, including algae, which can tolerate and grow well in low pH (Souza‐Egipsy et al. 2011). Low pH leads to enhanced solubility of metals, and ultimately, high metal levels cause toxicity (Novis and Harding 2007). Usually, at the contaminated site where several metals are present, extremophiles either have a physiological or genetic adaptation that enables them to tolerate several metals at once and thus enhancing their ability for remediation (Gaur and Rai 2001). The cellular response towards a metal ion can either be barring of metal from the cell, the absorption/uptake/incorporation and transformation of the metals to a relatively less

32 Extremophiles: Wastewater and Algal Biorefinery toxic form via several modifications inside the cell, followed by internal sequestration or by metal’s expulsion or release out of the cells (Hall 2002). The Reactive Oxygen Species (ROS) levels are increased on the metal intoxication, as a significant role is played by them in several ROS-producing mechanisms, including Fenton’s reaction, reduction of the glutathione pool, Haber-Weiss cycle and disruption of the photosynthetic electron chain leading to O2˙– (Pinto et al. 2003). The role of signaling molecules is also played by the ROS, which brings about the production of a network of several stress-related molecules, including antioxidants and antioxidant enzymes (Panchuk et al. 2002). The metal stress-induced antioxidative response is composed of metal-chelating molecules such as glutathione, metallothioneins, urate, phytochelatins and low molecular weight antioxidants. Acidophilic extremophiles are considered as one of the potent tools for biological-based remediation as they can tolerate metals under already extreme conditions (Whitton 1971); however the mechanism by which these extremophilic organisms tolerate the oxidative metal stress is yet not fully understood. The antioxidative response and the ROS signaling pathways may hold the key for understanding how extremophilic organisms tolerate these stresses. For instance, it was shown in an extremophilic bacterium, Pseudomonas fluorescens, that a cascade of biochemical reactions was triggered by ROS, which led to an enhancement in the NADPH level, and therefore increased the aluminium-induced tolerance and reductive capacity to the oxidative stress (Singh et al. 2005). In a comparative analysis of acidophilic alga Chlamydomonas acidophilia and a neutrophilic alga Chlamydomonas reinhardtii, the acidophilic C. acidophilia exhibited an enhanced basal level of Heat Shock Proteins (HSPs), which was considered to be the mechanism of adaptation into the extremities (Gerloff-Elias et al. 2006). In another study involving Chlamydomonas acidophilia and Chlamydomonas reinhardtii, it was found that during metal stresses, higher levels of ascorbate peroxidase (APX), an antioxidant enzyme was produced by the acidophile (Panchuk et al. 2002, Garbayo et al. 2008). In a study conducted to isolate and assess the tolerance to high concentrations of copper, lead and zinc by resistant fungal strains of five filamentous fungi; Aspergillus clavatus, Penicillium chrysogenum, Aspergillus terreus, Fusarium oxysporum and Trichoderma viride from a polluted beach revealed that they showed tolerance and uptake of high concentrations of copper (1.6 mg/g), lead (3.3 mg/g) and zinc (4.8 mg/g). Some of the strains were reported to exhibit multi-tolerant behavior. Finally, through the results of this investigation, it is evident that using these kinds of fungi is an alternative way to resolve some issues of environmental pollution by heavy metal-containing effluents (Bourzama et al. 2018). It was reported that some of the fungi removed a notable amount of heavy metals, particularly lead, cadmium, chromium and nickel. This shows the propensity of these organisms to expel metal elements from wastewater. The highest uptakes of Pb, Cd, Cr, Ni was observed in A. Terreus, Trichoderma viride, Trichoderma longibrachiatum, A. niger Ni27, respectively. This indicated that there are more binding sites on the cell wall of the fungi and their potential as biosorbent to remove the respective heavy metal from industrial wastewaters (Joshi et al. 2011). An acidophilic micro-algae, Chlorella protothecoides var. acidicola from acidic mine waters, has indicated great heterotrophic development on glycolic acid. It was likewise found to invigorate the development of acidophilic heterotrophic microorganisms, Acidiphilium and Acidobacterium which could help in the reduction of ferric iron, by giving them organic substrates intermittently. Such technologies can be scaled up for remediation of mine-related metal pollution (Johnson 2012). 4.1.3 Extremophiles Employed in the Treatment of Hypersaline and Petroleum Wastewater Extremophiles are also used for the degradation of PAHs (Polycyclic Aromatic Hydrocarbons) present in petroleum wastewater produced from petroleum refineries. The effluents from the

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petroleum refineries are composed of various hydrocarbons as the water comes in contact with several hydrocarbons at different stages of processing. The desalter effluent’s spent caustic, sour water and tank bottom are some of the main sources of hydrocarbons in the water. In order to withstand considerable water stress and the ionic strength, two main osmoregulatory mechanisms are executed by the halophiles. The mechanism through which the thermodynamic adjustment of the cell can be achieved is using the salt-in-cytoplasm mechanism. The salt concentration inside the cytoplasm is kept similar to the salt concentrations of the surrounding environment. The second mechanism is the osmolytic mechanism, in which, due to the accumulation of highly water-soluble, uncharged, organic solutes leads to the reduction of the chemical potential of cell water and the cytoplasm is kept free of NaCl. Several halophiles such as Paenibacillus sp., Bacillus napthovorans, Cycloclasticus sp., Novosphingobium pentaromativorans, Ochrobactrum sp. and Pseudoalteromonas sp., etc., grow and thrive under hypersaline conditions and can efficiently degrade PAHs in crude oil and effluents from petroleum refineries (Geiselbrecht et al. 1998, Daane et al. 2001, Zhuang et al. 2002, Sohn et al. 2004, Hedlund and Staley 2006, Arulazhagan and Vasudevan 2011). Several microalgal species are able to grow under hypersaline conditions and therefore possess a lot of potential for application in the biotechnological industries. Dunaliella salina, which is a green alga, grows well in hypersaline environments, i.e., up to 3 M and higher. Three basidiomycetes recovered from marine sponges (Tinctoporellus sp. CBMAI 1061, Marasmiellus sp. CBMAI 1062 and Peniophora sp. CBMAI 1063) were isolated, and the production of some ligninolytic enzymes under saline and saline free conditions and the decolorization of Remazol Brilliant Blue R (RBBR) dye were observed. This suggests that these fungi derived from marine environs can possibly be applied in the bioremediation of saline and alkaline wastewaters, like the colored pollutants from the textile industries, distilleries and paper and pulp industries (Johnson 2012). The deep sea is an extreme habitat due to low temperatures, high pressures and salinity. Three halotolerant fungi isolated from the deep-sea sponge Stelletta normani (Cadophora sp.TS2, Emericellopsis sp. TS11 and Pseudogymnoascus sp. TS 12) (Batista-García et al. 2017), displayed high CMCase and xylanase activities. Marine-derived fungi, because of their notable thermal stability and halotolerance, probably discover other applications relating to extreme salinity, high pH, algid temperatures and high hydrostatic pressures. C. matritensis and A. fumigatus showed unchanged capacity to grow in the concentration ranges of 0–10% NaCl in a study to determine the ability of fungi isolated from olive brine wastewater for producing extracellular phenoloxidases (EPO). Among them, Citeromyces matritensis (syn. Candida globosa) and Aspergillus fumigatus displayed laccase and Mn-peroxidase (MnP) activities. They were found to maintain EPO-producing ability and removed phenols from the effluent. These results suggest the possibility of employing these strains in the treatment of other saline phenol-rich effluents, such as pickling and tannery wastewater (Crognale et al. 2012). In a study performed to assess the impact of salinity on biomass production and nutrient removal inferred that the ideal algal growth was seen at 30 ppt salinity level and that D. salina has incredible capacity with regards to nutrient take-up while giving high-esteem by-products. It was also indicated that D. salina could essentially remove nitrate, alkali and phosphorus from urban wastewater. It can accordingly be suggested that it could be utilized in hypersaline wastewater treatments combined with high-esteem by-products production, for example, beta carotene (Liu and Yildiz 2018). Chlorococcum sp., Desmodesmus communis, Chlorella vulgaris, Chlorella sorokiniana, Desmodesmus spinosus, Monoraphidium Pusillum, Scenedesmus Obtusus, Scenedesmus obliquus and Monoraphidium Komarkove are freshwater algae explored to study their halotolerance and nutrient reducing ability. It was reported that they were able to proliferate in conditions with high salt concentrations and exhibited good phosphorous, chloride and nutrient removal abilities. It very well may be inferred that collections of the surveyed microalgae species might adjust to salt-rich wastewaters and can be used in bioremediation of saline wastewaters (Figler et al. 2019).

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4.1.4 Extremophiles Employed for Treatment of Wastewaters Polluted by Lignocellulosic Biomass (LCB) The production of lignocellulose‐degrading enzymes like cellulases, xylanases, lignases, lignin peroxidases and manganese peroxidases have been identified in a vast variety of extremophiles. Hyper thermophilic cellulose degrading enzymes have been identified in archaebacterial strains like Pyrococcus furiosus, Pyrococcus horikoshii, Haloarcula sp. (Ando et al. 2002, Li et al. 2013, Shin et al. 2013, Kataoka et al. 2014). These cellulases are of great use in biofuel production from agricultural and industrial lignocellulosic wastes, and these enzymes also have a great role to play in treating paper and pulp industry effluents (Singh et al. 2016). Halophilic archaeon, Haloarcula sp. strain S-1, was reported in producing alpha-amylase, which is found to be tolerant to solvents like benzene, toluene and chloroform and found to have exhibited optimum activity at 50ºC temperature (Fukushima et al. 2005). These amylases are useful in the degradation of lignocellulosic waste in the environment (Kumar and Chandra 2020). The commercial xylanases are prone to high temperatures and pH. Therefore extremophilic xylanases are best to be used as an alternative to these native xylanases as they display high thermostability and alkali stability. In this regard, alkaliphilic and thermophilic bacteria are found to be very important sources of xylanases with great thermostability and alkali stability. Thermotoga sp., Bacillus stearothermophilus, Caldicellulosiruptor sp., Rhodothermus marinus and Clostridium thermocellum and many such strains of thermophilic and hyperthermophilic bacteria are found be producing xylanases with thermotolerance (Lüthi et al. 1990, Khasin et al. 1993, Winterhalter et al. 1995, Abou-Hachem et al. 2003). These xylanases come into play in the lignocellulose waste-based biorefineries in the production of valuable biofuels where LCB (Lignocellulosic Biomass) is needed to be broken down into simple molecules (Hu et al. 2011). Exiguobacterium antarcticum, Micrococcus antarcticus B isolated from Antarctica, produced psychrophilic β-glucosidase. They are promising enzymes for applications where low temperatures are required (Crespim et al. 2016, Miao et al. 2016). Thermophilic fungi are best known to deliver significant lignocellulolytic compounds, which assume a large job in procedures of treatment of lignocellulosic biomass in municipal and other organic wastewaters. The thermophilic fungi break down the soluble-protein fractions at a greater rate compared to bacterial degradation (Maheshwari et al. 2000). The most commonly utilized microorganisms for the creation of hydrolytic enzymes have a place within the genera Aspergillus, Trichoderma and Penicillium (Yadav 2017). A study was conducted by Paramjeet Saroj et al. (Saroj et al. 2018) on 15 thermophilic fungi isolated from the soil. A. fumigatus JCM 10253 was found to display high cellulase activity and xylose hydrolysis. The optimum activity of the enzymes was at 60 and 50°C for crude cellulase and xylanase, respectively. This investigation discusses a way to deal with issues related to the bioconversion of lignocellulosic biomass from the wastewater into biofuels by choosing profoundly proficient producers of extracellular hydrolytic and ligninase enzymes. P. rhizinflata cellulase with exoglucanase and endoglucanase activity was expressed in A. niger, soft rot fungi. The cellulase activity greatly increased in A. niger and it also showed halostability, which efficiently hydrolyzed cellulose substrates such as wheat straw under free or high concentration of NaCl condition. It suggests that it could be used for lignocelluloses degradation (Xue et al. 2017). 4.1.5 Exploitation of Extremophile for Bio-electricity Generation The fact that microbial metabolism has the potential to provide energy in the form of electrical current has drawn a lot of attention in the biotechnology field and has piqued the interest of many researchers (Potter 1910, Potter 1911). These microbial systems are very compliant and hold great assurance to provide energy sustainably. Microbial Fuel Cells (MFCs) are an alternative that exploits the ability of microbes to combine anaerobic respiration to the reduction of external acceptors of electrons (Franks and Nevin 2010, Hamelers et al. 2010). The organic matter is oxidized on the anode, which results in the release of

Extremophiles for Wastewater Treatment 35

electrons and protons. Through the anode and an external resistor, the electrons travel to the cathode, whereby pairing the protons, the circuit is completed, generating a current in the MFCs. MFCs can be considered as a fuel cell or a battery that has both its electrodes through an electrical wire. The only difference between the batteries and MFCs is that the latter utilizes the organic substrates on the side of the anode as fuels in order for the production of electricity. The organic matter is oxidized on the anode, which results in the release of electrons and protons. The electrochemical reactions occurring between the organic matter and the final electron acceptor, for, e.g., oxygen is responsible for the ideal performance of the MFC. Due to the three irreversible losses, i.e., the concentration polarization, the ohmic losses and the activation polarization, the actual or the real potential of the MFC is always lower than its ideal or theoretical value. Conventionally, opposite to the flow of the electrons, a current which is positive flows from the positive to the negative terminal. Shuttles or electron mediators can be used to transfer electrons to the anode. Electrons can also be transferred via direct membrane-associated electron transfer, by nanowires and by several other methods that are yet to be discovered (Bond et al. 2003, Rabaey et al. 2004, Gorby et al. 2005, Rabaey et al. 2005, Reguera et al. 2005, Gorby et al. 2006). The electrons reaching the cathode combine with protons that diffuse from the anode in most MFCs via a separator and oxygen supplied from the air, resulting in the production of water (Kim et al. 1999, Bond et al. 2002, Kim et al. 2002, Min et al. 2004). 4.1.5.1 Potential Applications of MFCs The utilization of an anode as the ultimate acceptor of an electron by the bacteria has led to the possibility of a broad range of implementations. Amongst the active research areas involving the MFCs, the most intriguing one is the production of energy from wastewaters together with the oxidation of compounds which are inorganic or organic in nature. Several studies have demonstrated that any compound which can be degraded by bacteria can be transformed into electrical energy (Pant et al. 2010). Several types of substrates from wastewaters can be glucose, cellulose, acetate, starch, wheat straw, phenol, p-nitrophenol, pyridine and several other complex solutions like landfill leachate, brewery waste, domestic waste, mixed fatty acids, petroleum contaminates and chocolate industry waste (Franks and Nevin 2010). Less biomass is usually produced within these systems than their equivalent aerobic processes, and without the requirement for an energy-intensive aeration process, less energy is needed (Tender et al. 2008). Problems such as slow rates of substrate degradation and scale-up from laboratory experiments are still faced by the MFCs for large scale management of wastewaters. The potential of the microbial communities of the MFCs for degradation of a broad range of contaminants and environmental pollutants may be more precious than the generation of electricity in certain conditions, particularly when the technology of MFC can be used for cleaning up of the environment in situ. In the anaerobic degradation of landfill leachate as well as petroleum contaminants in groundwater, the species Geobacter has proven to be essential (Lovley et al. 1989, Anderson et al. 1998, Rooney-Varga et al. 1999, Röling et al. 2001, Lin et al. 2005). The contaminant’s oxidation is directly linked to the Fe (III) reduction. The processes of reduction and oxidation can be enhanced via the addition of electron shuttles and Fe (III) chelators (Lovley et al. 1994, Lovley et al. 1996, Lovley et al. 1996) in order for the promotion of enhanced transfer of electrons between the insoluble Fe (III) and the cells. Utilizing an electrode as the ultimate electron acceptor, toluene and benzoate can be efficiently oxidized using the pure cultures of Geobacter metallireducens (Bond et al. 2002, Zhang et al. 2010). Several experiments have shown that MFCs are potent tools for removal of fermentation inhibitors that accumulate in the process water after the pre-treatment of cellulosic biomass (Borole et al. 2009). 4.1.5.2 Microorganisms in MFCs Several microorganisms are found associated with electrodes in MFC systems, primarily when the MFC is seeded by an environmental inoculum (Franks and Nevin 2010). Biofilm is the general

36 Extremophiles: Wastewater and Algal Biorefinery term used for bacteria that is associated with the surface. It is believed that all the organisms in association with the anode biofilm do not interact directly with the anode, but may interact via other members of the community of the electrode, i.e., indirectly. For instance, it was found that Brevibacillus sp. PTH1 is an abundant member of a microbial fuel cell community. Brevibacillus sp. PTH1’s power production is low until Pseudomonas sp. is cultured along with it, or supernatant from a Pseudomonas sp. run MFC is added (Pham et al. 2008). The Geobacter sulfurreducens transfer electrons to a surface directly through redox-active proteins such as cytochrome-c, present on the outer surface of the membrane or through conductive pilli, also known as nanowires (Snider et al. 2012, Kumar et al. 2016). Multi-layered structured biofilms having nanowires connected with the different cells is developed by extremophiles like G. sulfurreducens which enables the transfer of the electron to anode (Bonanni et al. 2012). The microbial cells are penetrated by mediators, which are in their oxidized form. These mediators become reduced during cellular metabolism. They, then, diffuse out of the cell and again become oxidized, and therefore can be reused (Kumar et al. 2016). Mediators like pyocyanin are produced endogenously by some species like Pseudomonas sp. (Rabaey et al. 2005). Other organisms present in the mixed culture system can also use the mediators to transfer their electrons to the anode as soon as the mediators are produced (Pham et al. 2008). Electrochemically active microbes like Geobacter sp. and Shewanella sp. and their pure cultures have demonstrated power production from simple substrates such as sugars and volatile fatty acids at conditions which are mesophilic (approx. 37°C) and pH neutral (6.8–7.3) (Nevin et al. 2008, Kim and Lee 2010). For wastewater treatment, mixed cultures are considered more practical due to the fact that they contain a consortium of electroactive, fermentative and hydrolytic microbes that can efficiently produce electricity from complex substrates (Nevin et al. 2008). The MFCs offer a wide range of economic, operational, energy and environmental benefits. Clean electricity directly from the organic matter is produced by the MFCs. They do not require any purification, separation or conversion of the energy products. Techniques involved in the MFCs are environmentally friendly and can efficiently function at ambient temperatures and mild operating conditions. When compared to a conventional activated sludge treatment process, only around 10% of the external energy is used by the MFCs for their operation (He et al. 2017). This suggests the potential of MFCs for saving energy and possible recovery of energy from wastewater treatment. 4.1.5.3 Thermophilic Microbial Fuel Cells (TMFCs) The thermophilic microbial communities usually possess much less diversity as compared to the consortia of mesophiles, and therefore these thermophiles are thought of as more vulnerable and less resilient to potential process disturbances (Carballa et al. 2015). The development and further application of the thermophilic microbial fuel cells are obstructed due to a lack of information on thermophilic electroactive microbial communities. As of now, Thermincola, Caloramantor and Thermoanaerobacter from Firmicutes phylum together with the Deferribacters (Pyrococcus) and the Euryarchaeota (Calditerrivibrio) are among the few bacterial genera that have been reported to produce electricity without the addition of any mediators, and at temperatures higher than 50°C. Amongst them, Thermincola potens, Pyrococcus furiosus and Thermincola ferriacetica are the only microorganisms with which the transfer of electrons to the anode has been demonstrated. In spite of the fact that MFCs have been successfully operated at higher temperatures like 98°C, most of the studies have been conducted at temperatures ranging from 55–60°C. The probable reason for such an observation could be due to the fact that at higher operation temperatures, the loss of electrolyte due to evaporation from anodic or cathodic chamber would decrease the performance of the MFC. The dominant bacterial phyla enriched from mixed cultures at 55–60°C in MFC anodes utilizing distillery wastewater or acetate as substrates were Firmicutes, Deferribacteres and Coprothermobacterota. For the studies of thermophilic MFCs, the H-type MFC designs have been used most often with cultures of mixed species. A sediment MFC which functions at 60°C together with MFC

Extremophiles for Wastewater Treatment 37

designed to manage and control evaporation (which is a prime operating challenge for TMFCs) are among the few exceptions. For mesophilic MFCs, efficient, systematic and conveniently scalable configurations like flow-through and up-flow MFCs were proposed, but they have not yet been implemented under extreme thermophilic conditions. Due to the high rates of the biochemical reactions resulting in high rates of electron production from thermophilic microorganisms, thermophilic electricity production could be advantageous (Du et al. 2007). New insights and better understandings for thermophilic microbial consortia would encourage innovations in the application of thermophilic MFCs.

5. Conclusions Microorganisms are extensively distributed in extreme environments pertaining to acidic or alkaline pH, low or high temperatures, high concentration of metals, etc. Several studies have reported the abundance of microbes residing in these extremities. These extremophilic organisms are regarded as shrouded reservoirs, and their metabolic potential is yet to be explored. The extremophiles can be considered as a potent tool for the treatment of water bodies contaminated with different sorts of components. These organisms need to be studied for their specific and accurate usage in the biotechnological fields.

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Zhuang, W.-Q., J.-H. Tay, A. Maszenan, S.J.A.m. Tay and biotechnology. 2002. Bacillus naphthovorans sp. nov. from oil-contaminated tropical marine sediments and its role in naphthalene biodegradation. Applied Microbiology and Biotechnology 58(4): 547–554.

3 Role of Extremophiles in the Microbial Electrochemical Cell Recent Advances Shreya Gavas,1,# Prajakta Pawar,1,# Soumya Pandit,2,* Namita Khanna,3,4 Abhilasha Singh Mathuriya2 and Ram Prasad 5

1. Introduction: Extremophiles RD McElroy, in 1974, first coined the term “extremophiles” (from Latin extremus meaning "extreme" and Greek philiā meaning "love") which refers to any organism that thrives under extreme conditions. The category of extremophile comprises of organisms from Archaea, Bacteria and Eukarya domains. These microbes are classified on the basis of the extreme environmental conditions they can thrive in, for example thermophiles – organisms growing optimally at higher temperature range than normal, halophiles – organisms that thrive at high salt concentrations and so on (Ha et al. 2012). The discovery of thermophiles by Thomas Brock in 1966 from boiling springs of Yellowstone National Park changed our perspective towards diversity of life and it’s sustenance in the harshest conditions. Since then these organisms and their metabolic pathways have been extensively studied. Archaea have more versatile survival mechanisms than that of Bacteria and Eukarya. Among Bacteria, Cyanobacteria are best adapted to extreme conditions from ice to hot springs. Among eukaryotes Fungi (mostly in symbiosis with cyanobacteria) show good tolerance to acidic and metal concentrated conditions (Albarracin et al. 2011). Extremophiles can be divided into two broad categories—extremophilic and extremotolerant organisms. On one hand, extremophiles are organisms that require an extreme condition(s) to thrive, for example, Methanopyruskandleri strain 116 grows at 122°C. On the other hand, extremotolerants can withstand, but not necessarily require extreme conditions to grow, for example: strains of Staphylococcus spp., Escherichia spp. are metal tolerant which means these can withstand high concentrations of copper, nickel and mercury which are otherwise lethal for cells.

Amity Institute of Biotechnology, Amity University, Mumbai, Maharashtra 410206, India. Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida-201306. 3 Department of Biotechnology, Birla Institute of Technology and Science, Pilani, Dubai Campus, Dubai, UAE. 4 Nuclera, 137 Cambridge Science Park, Cambridge CB4 0GD, UK. 5 Department of Botany, Mahatma Gandhi Central University, Motihari-845401, Bihar, India. * Corresponding author: [email protected] # Both the authors have equal contributions 1 2

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Extremophiles: Wastewater and Algal Biorefinery

Extremophiles survive in extreme conditions by modifying the cell membrane structure which enhances its integrity and prevents stress-mediated cell rupture. The cell membrane of thermophiles, for example, has ether and ester-linked lipid structures along with diether and tetraether polar lipids. Membranes of mesophiles and psychrophiles have unsaturated fatty acids in them (Liu et al. 2012). Halophiles compensate the osmotic stress by pumping in potassium and pumping out sodium out of the cell through the membrane (Xu et al. 2014). These extremophiles belonging to Archaea, Bacteria and Eukarya synthesize extremolytes, extremozymes and various primary and secondary metabolites to cope with the harsh environments. Extremolytes are metabolites that help the organism achieve homeostasis under extreme situations. Extremozymes are enzymes with characteristic features of remaining active and stable at extreme conditions like high (or low) pH, high (or low) temperature, etc. (Kong et al. 2014). Extremophiles are not just important for the ecosystem but can be used for various industrial applications due to their distinct genetic make-up. These genes are activated, transcribed and translated to give proteins of interest which help them survive through the harsh conditions, even radiations. These proteins and enzymes can be used in biotechnology, for example, Taq polymerase from thermophile Thermus aquaticus, an enzyme that is used in PCR and can withstand temperatures above 90°C. Another such potential application of these extremophiles is used in Microbial Electrochemical Systems to give eco-friendly biological outputs. Microbial Electrochemical Systems (MESs) are various types of apparatus comprising interactions between electrodes and microbial consortia to give valuable products. MES can be of various types depending upon their function and output. A few areMicrobial Fuel Cells (MFC) – This technology uses microorganisms like bacteria to convert chemical energy from organic molecules, present in the wastewater, to electrical energy in an electrochemical cell. Microbial Electrosynthesis Cell (MEC) – Is the exact opposite of MFC where the microbial consortia are provided with electrons that reduce CO2 to organic products like methane, acetate and ethanol in a electrochemical cell. Microbial Desalination Cell (MDC) – These are bioelectrochemical systems that utilize organic material present in wastewater to use them as energy source for desalinating saltwater (Tapia et al. 2009). Extremophiles have the potential to be used at highly acidic pH to oxidize sulphuric compounds as bioremediation of wastewaters. They can be used to generate electricity from highly alkaline marine waters at low temperatures and can very efficiently desalinate wastewater for its reuse and repurposing (Li et al. 2013).

2. Microbial Electrochemical Systems (MES) Microbial electrochemical systems comprise of associations between microorganisms and electrodes conducting electricity. The efficiency of these systems depends on the availability of energy for microbial activity, the electrode potential, the strain of microbes and their electron transport mechanism along with the coping mechanism to deal with environmental conditions. The microbial electrochemical system is a broad term which encompasses various bio-electrochemical applications as described next.

2.1 Microbial Fuel Cells (MFCs) These are bioreactors consisting of microbial biofilms associated with electrodes that convert organic (or inorganic) matter to electricity (Chaturvedi and Verma 2016). The MFCs not only aim at generating electricity, but they also target the biodegradation of organic waste. An MFC apparatus

Role of Extremophiles in the Microbial Electrochemical Cell: Recent Advances 45

consists of an electric circuit of an anode chamber and a cathode chamber both partitioned by an ion-exchange membrane (Fig. 3.1) (Li et al. 2013). The anodic chamber is subjected to anaerobic conditions whereas the cathodic chamber is aerated. The anaerobic chamber hosts a consortium of microorganisms that is capable of electron transfer without the use of mediators (Table 3.1). The basic mechanism is that the bioanode absorbs electrons and passes them through an external circuit to the cathode. The protons at the same time cross the Ion Exchange Membrane/separator (IEM) to the cathodic chamber where they react with available oxygen molecules to give water (Dopson et al. 2016).

Fig. 3.1. Microbial fuel cell apparatus.

The reactions for both the chambers are as followsAt anode: CH3COO– + 2H2O → 2CO2 + 7H– + 8e– At cathode: O2 + 4e– + 4H+ → 2H2O The by-products of these reactions are free electrons with flow via the circuit to give energy in the form of electrical energy. There are various components in MFCs and factors that influence the efficiency of the system. The electrode material should be biocompatible as it should not be toxic for microflora. For example, heavy reactive metal like copper will show an oligodynamic effect on the microbial biofilm. Apart from that the material with the least corrosive properties is preferred. The distance between the electrode is also a crucial factor, the closer the electrodes, the lesser is the chance of leakage, hence the efficiency is better. The most preferred material for electrodes is carbon as it is inexpensive, stable and biocompatible and shows good conductance of electricity. Carbon is usually available in the form of graphite rods or plates and as glassy carbon. Ferricyanide is also considered as a good cathode due to its observed good efficiency in microbial fuel cells (Merino-Jimenez et al. 2016). The most common and widely used proton exchange membranes for partitioning the chambers are Nafion and Ultrex CMI7000. Proper optimization of these factors and components can lead to highly efficient microbial fuel cells and the system can be hence used on a commercial scale to generate electricity out of domestic and industrial waste.

46

Extremophiles: Wastewater and Algal Biorefinery Table 3.1. Extremophiles in MFCs. Extremophile

Inoculum

Condition

Acidophiles

Acidithiobacillus ferrooxidans

pH < 2

Acidiphilium cryptum

pH < 4

Bacillus pseudofirmus

pH > 10

Corynebacterium sp.

pH < 9.5

Thermincola potens

60°C

Thermincola ferriacetica

60°C

Haloferax volcanii

158 g L−1 NaCl

Geoalkalibactersubterraneus

35 L−1 NaCl

Alkaliphiles Thermophiles Halophiles

2.2 Microbial Electrosynthesis Cells (MECs) An attractive alternative is to reduce carbon dioxide to give multi-carbon compounds which act as precursors of commercially important products like biofuels and organic chemicals. This kind of reduction reaction can take place in Microbial Electrosynthesis Cells (MECs) in which carbon dioxide is reduced to various multi-carbon compounds at the cathode by microorganisms in the presence of electricity. The electricity provided for the working of MECs can be generated from renewable and sustainable energy resources. The availability and intensity of renewable energy are irregular which makes it difficult to keep up with the market demand for energy. MECs can essentially mediate the storage of this electrical energy as chemical energy between bonds of valueadded products. Apart from this, the MECs can be coupled with environmentally friendly anodic processes like biological oxidation reactions and wastewater treatment. In biological oxidation reactions, the electrons are transferred from the bioanode, where microbial catalysts metabolize available substrates, to the cathode collecting electricity. Studies have proved that electrons from biological oxidation reactions can fuel MECs. Such reactions have wide applications in wastewater treatment, desalination, bioremediation, microbial fuel cells and many more. Figure 3.2 portrays the reactions taking place in a microbial electrosynthesis cell.

Fig. 3.2. Microbial electrosynthesis cell (MESC).

Role of Extremophiles in the Microbial Electrochemical Cell: Recent Advances 47

2.3 Microbial Desalination Cell The need of fresh and consumable water is rising for both livelihood as well as industrial production of goods with the increasing population. This has led to the invention of an eco-friendly cost-effective technology called Microbial Desalination Cell (MDC) that uses organic matter from polluted water as an energy source in order to desalinate the salt water (Pandit et al. 2018). The cell usually comprises of a couple of membranes that divides the system into three compartmentsan anode, a cathode and a desalination chamber (Fig. 3.3). The organic matter is degraded in the anode chamber where the released electrons produce electricity. The chamber in the middle, the desalination chamber, hosts the reaction of desalination whereas the cathode chamber completes the electrical circuit. The exoelectrogenic extremophilic bacteria present in the system tends to establish an electric potential across the MDCs that facilitate the ion transport through ion exchange membranes. An external electron acceptor like O2 (in the cathode chamber) uses the free electrons to produce H2O causing the said potential gradient across the chambers. This causes the movement of anions and cations of the salt particles from the middle chamber to the anode and cathode respectively. Such a process removes around 99% of salt from the wastewater while producing energy (Saeed et al. 2015).

Fig. 3.3. Microbial desalination cell (MDC).

3. Various Extreme Conditions Over the last decade, researchers have been fascinated and intrigued by extremophiles that inhabit extreme conditions. Extremophiles have been isolated from depths of 6.7 km inside the Earth’s crust, around 10 km deep inside the sea, at pressures of up to 110 Mpa. Such organisms have been found from extremely acidic conditions, i.e., at pH 0 to extremely basic conditions, i.e., at pH 13; from hydrothermal vents at 122°C to frozen seawater, at −20°C (Albarracin et al. 2011). A few examples have been enlisted of the Table 3.2.

48

Extremophiles: Wastewater and Algal Biorefinery Table 3.2. Extremophiles present at harsh environmental conditions.

Sr. No.

Environmental Parameters

Type

Characteristics

Microbes

1.

Temperature

Hyperthermophiles Thermophiles psychrophiles

> 80°C 40°C–70°C –16°C–25°C

Pyrococcusfuriosus Sulpholobusacidocaldarius Pseudomonas aeroginosa

2.

Salinity

Halophiles

3.4–5.1 M NaCl

Halobacterium sp.

3.

Requirement of oxygen

Aerobes Anaerobes

Tolerate oxygen Cannot tolerate oxygen

Clostridium difficile Actinomycetes

4.

Pressure

Piezophiles

52 Mpa at 98°C

Pyrococcusyayanosii

5.

pH

Acidophiles Alkaliphiles

pH 5.5 pH 7.5–11

Acidithiobacillusferroxidans Arthrospora sp.

6.

Dessication

Xerophiles

Anhydrbiotic

7.

Nutrient

Oligotrophs

Low nutrient requirement

Spingomonas sp. Strain RB 2256

8.

Water activity

Osmophiles

Low water activity

Aspergillus, Saccharomyces, Micrococcus

9.

Presence of damaging agents (organic solvents)

Toxitolarent

Tolerate organic solvents

Pseudomonas putids 1H 2000

3.1 Temperature Temperature is an essential parameter that affects the growth of microorganisms. Microbes can dwell in areas of variant temperature range, i.e., from sub-zero to boiling point and above. Extreme cold conditions, both natural like glaciers, fresh and marine waters and man-made environments like refrigerators and industrial freezers are hosts to a huge range of microorganisms. On the other hand, even habitats of elevated temperatures, be it natural (geothermal sites) or man-made (furnaces) house microbes. Scientists have discovered various species of extremophiles in such areas. In addition to temperature, other parameters like nutrient availability, ionic strength, etc., are also responsible for variation in extremophilic organisms. Temperature dominates the metabolic activities of life. It governs factors like keeping water in its fluidic state and dealing with the diminishing dissolvability of oxygen, etc. Another crucial way in which the temperature governs metabolism is by regulating enzyme activity. The rise in temperature results in increased enzymatic activity, i.e., slight elevation of temperature leads to an increase in the rate of conversion of the substrate to the product in an enzymatic reaction. But if increased beyond optimum, the proteins lose their conformation and the enzyme denatures losing its functions. Based on the temperature conditions they can thrive in, extremophiles are categorized into psychrophiles (ideal temperature for growth –20°C–25°C), mesophiles or facultative thermophiles (ideal temperature for growth 20°C–45°C), extreme or obligate thermophiles (40°C–70°C) and hyperthermophiles (70°C–106°C). Dry valleys of Antarctica and Indo-Burma regions are popular locations from where researchers have isolated low temperature loving microorganisms. Different examinations stated that the dirt of dry valleys contains minimal natural organic matter, high salt focus and low fluid availability. Low temperatures are characteristics of mountains where snow persists all year round. The maximum known temperature at which microbes can grow is 122°C. Many well-known areas like Yellow Stone National Park, USA, and Manikaran, India are hotspots for studies of hightemperature tolerant life forms. Bacteria like Bacillus alveayuensis, Thermocrinisruber, etc., have been isolated from such otherwise lethal conditions. Studies state that extremophiles have a large number of biological applications that are useful for human welfare.

Role of Extremophiles in the Microbial Electrochemical Cell: Recent Advances 49

3.2 Pressure Pressure plays an important role in the growth kinetics of microorganisms. With an increase in pressure, the solubility of gases increases causing changes in the metabolic pathways. It is also observed that with an increase in pressure at a specific temperature the doubling time of bacterium like a barophilic strain of E. coli increases. It is hypothesized that this occurs due to membrane phospholipid structural changes. Pressure tolerant organisms can undergo morphological changes as a coping mechanism. Peizotolerance is a resultant of physiological changes in microbes which leads to loss of normal catabolite repression. Extremophiles are classified into barophiles and piezophiles based on the pressure required for their growth. Barophiles are found under high atmospheric (barometric) pressure and piezophiles live deeper in oceans (Ha et al. 2012). The optimal pressure required is between 70 to 80 Mpa (700–800 atm). The deep-sea has a high-pressure environment (hydrostatic pressure environment). Almost all barophiles isolated from the sea are from the depth of the sea at 2000 m. High-pressure conditions are created in soil because of high temperature, high salinity and nutrient limitations. These factors exert stress on the microorganisms. In addition to barophiles and piezophiles, there is another type of extremophile that is more interesting and requires high pressure for growth “hyperpiezophile” (Satyanarayana et al. 2005). Hyperpiezophiles require 100–200 Mpa.

3.3 pH The medium pH is a parameter that conveniently measures hydrogen ion concentration hence the acidity or alkalinity of an aqueous solution on a scale of 0–14. If the pH of a solution ranges from 0 to 6.8 it is said to be acidic, if between 7.2–14, it is called basic and 7 is termed as a neutral. pH is a crucial factor that influences the rate and efficiency of any chemical and biochemical process. The enzymatic activity is largely impacted by moderate changes in pH of the environment of cells as it results in the modification of ionization of amino acid functional groups and disruption of H-bonds. This, in turn, leads to changes in protein folding, ultimately resulting in denaturation. Acidophiles are extremophiles that thrive in acidic conditions, pH < 4, which are otherwise hostile or even lethal conditions for cells. Extremely acidic conditions are results of natural geochemical processes (e.g., sulphurous gas production from hot springs) and metabolic activities of certain microorganisms. These acidic conditions arise due to oxidation of H2S and SO2 giving strong sulphuric acid (Albarracín et al. 2012). Crater Lake, Java, and Bowland Lake, Canada are hosts to acidophiles. These areas have pH value as low as 1 and host numerous spontaneous combustions providing a suitable environment for thermophiles as well. Such illuminated regions also support algal proliferation. A few examples of acidophiles are Alicyclobacillus acidocaldarius, Sulpholobus acidocaldarius, Dunaliella acidophila (Albarracín et al. 2014). Alkaliphiles are extremophiles that flourish in highly alkaline conditions, i.e., pH > 9. High alkalinity is often coupled with high salinity. Naturally occurring Soda Lakes are characterized by a high concentration of carbonate and chloride salts. Despite their hostility, these lakes have higher gross primary production rates than average lakes and streams (Albarracín et al. 2014). Significant red blooms of phototrophic cyanobacteria together with Ectothiorhodospira mobilising microbial community has been isolated from Soda Lakes. Soda lakes often host a population of phototrophs such as Cyanospira (Anabaenopsis) sp., Chlorococcum sp. and Pleurocapsa sp. Other than soda lakes, many alkaliphilic strains of microorganisms like Exiguobacterium aurantiacum were isolated from the man-made alkaline environment such as potato waste processing units (Di Capua et al. 2011).

50

Extremophiles: Wastewater and Algal Biorefinery

3.4 Water Activity Water activity (w/a) is the availability of water for hydration of materials where the maximum value of (w/a) is 1 indicating maximum water availability of substance hydration and the minimum is 0 which means no water/moisture is present. Since water is crucial for most metabolic pathways its availability for cell proliferation is essential. Below 0.85 water activity, microbes grow only in highly concentrated syrups which are tolerant of high osmotic pressure. These microorganisms are often termed as osmophiles or osmotolerants. Some osmophiles like Saccharomyces bailii are common spoiling agents or highly concentrated sugar syrups which have w/a lower than 0.8. Other microbes like Aspergillus spp., Enterobacter spp., Micrococcus spp., etc., are potential opportunistic pathogenic osmophiles. Dehydrating and desiccating situations lead to extreme osmotic stress and low w/a. Many microorganisms like Deinococcus spp. belonging to the Deinococcaeae family are resistant or tolerant to such drying conditions. This resistance is speculated to be the result of a thick cell wall ensuring the integrity of the cell membrane under extreme conditions.

3.5 Radiations Radiation is emissions in the form of waves or particles that carry the energy and are of different types depending upon their wavelengths. The radiations are mainly found to show an impact on microbial growth are ultraviolet radiations (200–500 nm) and ionizing radiations (Krisko and Radman 2013). Alpha and beta emission of radioactive decay or accelerated protons and heavier atomic nuclei that constitute cosmic rays are low wavelength but contain high energy. This variety of radiations affects the survival of organisms (Angel et al. 2012). DNA damage chemistry by UV and ionizing radiation is different from one and other. Ionizing radiation exposure leads to DNA base damage and breaks. UV radiations are of three types—UV-A (320–400 nm) UV-B (280–320 nm) and UV-C (100–280 nm). Out of these three, UV-B produces very much lower levels of damage as compared to UV-A and UV-C because of the lower energy of UV-B, it is less harmful to microorganisms. UV-A is lower energy as compared to UV-B. It acts indirectly on the DNA by producing photo oxidizing compounds. It also produces reactive O2 species that damage DNA, proteins and lipids (Zenoff et al. 2006). Direct damage to macromolecules such as DNA, protein and lipids is possible through radiolysis and free radical chemistry (Rasuk et al. 2016). Scientists have discovered and isolated radiation-resistant strains of bacteria like Dienococcus spp. and E. coli.

3.6 Low Nutrient Availability Oligotrophs are organisms adapted to use low-nutrient concentration efficiently. They can survive in extremely nutrient-depleted environment, i.e., they are capable of growing in a medium containing 0.2–16.8 mg dissolved organic carbon per litre. Oligotrophs and eutrophs exist in the same place but their proportion is dependent on the ability of an individual to dominate in a particular environment (Zeikus and Wolee 1972). Spingomonas sp. Strain RB2256 isolated from the Resurrection Bay, Alaska maintains its ultra-micro size irrespective of the growth phase and carbon concentration. Cycloclasticusoligotrophicus has similar characteristics as well. Pramanik et al. 2003 have recently reported oligophiles to be abundant in nature. Klebdsiellavariicola is facultative oligotrophic strains from the River Mahananda, Siliguri, India which produces exopolysaccharide in oligotrophic media. These have a substrate uptake system that can acquire nutrients from its surroundings; it is an important consideration for oligotrophic microbes for survival. Oligotrophic microbes have a large surface area to volume ratio, high-affinity uptake system with broad substrate specificities and an inherent resistance to environmental stress for absorbing the nutrient from the

Role of Extremophiles in the Microbial Electrochemical Cell: Recent Advances 51

surroundings. They also produce appendages to enhance their surfaces. Some examples include Caulobacter spp., Hyphomicrobium spp., Prosthecomicrobium spp., Analmicrobium spp. The theory also suggests that oligotrophs produce exopolymorphic substances that help in concentrating the nutrient.

4. Extremophiles and their Potential Applications in MEC 4.1 Acidophiles Acidophiles are microorganisms belonging to Archaea, Bacteria and Eukarya which survive and thrive under highly acidic conditions. They range from low temperature-adapted acidophiles to thermophilic acidophiles. There are two types of acidophiles, namely extreme acidophile and moderate acidophiles. The former grows in extremely low pH (< 3 pH), whereas the latter thrive at moderate pH conditions, i.e., 3–5 pH (Johnson and Schippers 2017). The isolated acidophiles belong to Actinobacteria, Proteobacteria, Firmicutes and Nitropsora phyla. Acidophiles tend to show certain physiological traits including chemolithortrophy, diazotrophy, secretion of cellular polymeric substances and a global trait amongst this group of extremophiles is that they are metal tolerant, i.e., they can tolerate and survive in conditions with high metal concentrations (e.g., copper, nickel). These organisms have various strategies to cope with the harsh acidic environment which mainly includes active, passive and mitigation mechanisms. Acidophiles keep the internal pH almost neutral hence maintaining proton gradient across the membrane. This is achieved by increasing the influx of K⁺ ions creating positive conditions inside the cell and hence reducing proton influx across the cell membrane. The first acidophile used for MESs was Acidophilium cyrptum with ferric ion coupling, however, this combination restricted current output which leads to further modifications. More Acidophilium spp. were discovered and isolated from Rio, Spain, which eliminated the use of external mediators to transfer electrons increasing the efficiency and decreasing additional involvement (Zenoff et al. 2006). This strain showed versatile traits like growth on graphite cloth and secreted extracellular polymeric substances along with interactive biofilmforming traits. They have the capability of treating waste streams from anthropogenic activities like nitrification, metal mining, etc. Acidophiles show a biofilm-forming lifestyle which enhances their survival rate and increases efficiency in MESs (Zenoff et al. 2006). Though the organism is an aerobe thick biofilm is shown to follow an anaerobic lifestyle since oxygen penetration through the consortia becomes difficult. Hence the bio-electrode tends to have an anaerobic condition in the chamber in MFCs. Overall performance of acidophiles in electrochemical systems shows that the oxidation rate of the substrate is high resulting in high rates of the current generation. Table 3.3 shows different acidophiles and alkaliphiles in MESs Table 3.3. Table 3.3. Acidophiles and Alkaliphiles in MFC and MEC. MES

Extremophile

Organism

Condition

Efficiency Parameter

MFC

Acidophile

Acidithiobacillusferrooxidans

pH 2

5.0 A m−2

Acidiphiliumcryptum

pH 4

12.7 mW m−2

Pseudomonas alcaliphila

pH 9.5

70 mA m−2

Corynebacterium sp.

pH 9.8

33 mA m−2

Acidophile

Clostridium aceticum

–

–

Alkaliphile

Geoalkalibacterferrihydriticus

pH 9.3

84–95%

Current Density

Alkaliphile

Coulombic Efficiency MEC

52

Extremophiles: Wastewater and Algal Biorefinery

4.2 Alkaliphiles Alkaliphiles are organisms that thrive under highly alkaline pH ranging from 8.5–13. They are widespread across the globe as alkaline conditions arise at various places due to biological and geothermal activities along with anthropogenic environments. Alkaliphiles can hence be found and isolated from soda lakes, cement industry vents, etc. They are categorized into obligate, facultative and haloalkaliphiles. They belong to Archaea, Bacteria and Eukarya. Alkaliphiles belong to sulphuroxidizing bacteria, nitrifiers, lithotrophs, etc. Alkaliphiles and alkali-tolerant are two subcategories. The latter can tolerate and thrive at an elevated pH but are restricted to an optimum value. For example, Virgibacillus spp. grows at pH 9 but cannot do so at pH 10. The former grows in a range of high pH without much physiological change or any change in the rate of metabolism (e.g., Bacillus pseudofirmus). Alkaliphiles maintain a slightly alkaline or near-neutral pH in their cytoplasm. H⁺/Na⁺ antporter proteins in the membrane take care of the sodium exchange which maintains the required conditions in the cytoplasm. H⁺ is imported into the cytoplasm at the expense of Na⁺ export. But Na⁺ dependent pH mediation requires Na⁺ influx which is ensured by Na⁺ driven flagellar rotation. Also, peptidoglycan of the cell wall of alkaliphiles at elevated pH is tightly cross-linked which leads to a protective shield effect under stressed conditions. Combined actions of these mechanisms assure balanced pH according to the external environment of the extremophile for its survival. Alkaliphiles are beneficial for MES due to many versatile traits. During MFC operation the anodic chamber becomes acidic and the cathodic chamber turns alkaline which results in lower power output. Bacillus spp. was the first alkaliphile used in MFCs, at pH 9.5, as redox mediator. Geoalkalibacterferrihydriticus was later used in further studies as it was found that alkaliphiles have the potential to form reversible bioelectrodes that alternate between the addition of substrate and aeration mediators were added to exploit proton accumulation to enhance the biocathode efficacy. Many MFCs have been designed using alkaliphiles to not just produce energy but to also treat food waste that consists of 40–50% of the total urban waste. This led to the generation of electricity at 63% columbic efficiency at pH 9 (Flores et al. 2009). Table 3.3 shows the various alkaliphiles in MESs.

4.3 Thermophiles Thermophiles are microorganisms which thrive at elevated temperature conditions ranging from 40°C to 122°C. Hyperthermophiles belong to two phylogenetically different domains for survival in harsh conditions. These extremophiles use mechanisms often based on the production of extremozymes which are thermostable enzymes and other primary and secondary metabolites like extremolytes which help attain homeostasis. The extremolytes allow bacterial cell protein to adapt their conformation and help in stressful conditions (Rodrigues and Silva 2016). According to Kashefi and Lovely 2003 thermophiles that grow in maximum temperatures are aerobic and many are chemolithotrophs. Usually, metabolites, proteins and nucleic acids of other living cells are susceptible to the temperature range at which the thermophiles or thermo-tolerant grow (Angel et al. 2012). The structural and functional membrane fluidity of the extremophiles is governed by a large amount of saturated straight-chain fatty acids. For the protection of DNA in maximum temperature histone-like proteins binds to the DNA which can conserve it. Apart from histones, the supercoiling of DNA by type1 DNA topoisomerase enzyme can also stabilize DNA. Heat shock proteins, chaperones take part in stabilizing and refolding of proteins. A higher proportion of thermophilic amino acids established in certain proteins of thermophilic microorganism stabilizes the thermophiles in an extreme environment. For example, proline residues have a lesser degree of freedom. Researchers have also mentioned that thermostable protein character can be a result of more arginine and lower lysine content. 2,3-diphosphoglycerate, polyamines and accumulation of

Role of Extremophiles in the Microbial Electrochemical Cell: Recent Advances 53

intracellular potassium aid protein stability (Zeikus and Wolee 1972). Scientists have also stated that other than the presence of thermostable amino acids, a higher degree of structure in hydrophobic cores, an increased number of hydrogen bonds and salt bridges are also found in thermophiles (Angel et al. 2012). Sometimes chemolithoautotrophic mode of nutrition is followed by the thermophiles. In this way of nutrition the organisms use carbon dioxide as the carbon source and inorganic redox reactions as organic cell matter and energy source respectively. Therefore, thermophilic organisms are designated as chemolithotrophs (Sarkar and Ghosh, n.d.). Hydrogen serves as an important e-donor. Like mesophilic organisms use oxygen for respiration, hyperthermophiles use oxygen as e-acceptor (Angel et al. 2012). Few hyperthermophiles which follow the chemolithoautotrophic mode of nutrition are capable of utilizing inorganic sources of nutrition as and when they are provided by their surroundings. The other way of energy generation for chemo-autolithotropes is fermentation or anaerobic respiration using organic material as the electron donor (Sarkar and Ghosh, n.d.). Hyperthermophiles belong to different domains of life: Bacteria and Archaea. The strategies of molecular mechanism including heat adaptation may be dissimilar depending upon the phylogenetic position of corresponding organisms. Membrane lipids of the bacterial hyperthermophile T. maritima contain glycol ether lipid 15,16-dimethyl-30-glyceryloxy triacontanedioic acid. It helps in maintaining structural integrity. In mesophiles, the ester lipids play a major role in maintaining the stability of the membrane. It significantly increases the stability of the membrane against hydrolysis of the membrane in high temperature, i.e., it also helps in maintaining the structural integrity of the cell. Other than ester lipids archaea also contain ether lipids obtained from diphytanyl-glycerol or its dimer di(biphytany1)-diglycerol which exhibit significant resistance against hydrolysis of the membrane at high temperature and low pH (Sarkar and Ghosh, n.d.). The earlier study stated that the secondary structure of RNA occurs in the stabilized condition against thermal destruction by an increased content of GC within steam areas. At 108°C, about 80% of the soluble protein of a crude extract of Pyrodictiumoccultum contained heat-inducible molecular chaperone designated thermosome (Sarkar and Ghosh, n.d.). The last few years have experienced an increased interest in using MFCs for accelerating waste to energy conversions. Extremophiles synthesize enzymes that facilitate the electron transfer leading to production of electricity generation by degrading organic molecules. Various studies of MFCs using extremophiles have shown that species of thermophiles like Thermincolaferriacetica, T. firmicutes, etc., have the potential of producing stable currents (Marshal and May 2009). Thermophiles isolated from the coal refuse piles, domestic and industrial hot water systems, when used for power generation, had operational temperatures of MFCs at 65°C. Thermophilic conditions speed up the activity of MFCs by enhancing substrates solubility mass transfer characteristic and microbial activity. Thermincolaferriacetica and Thermoanaerobacterpseudethanolicus have been used in MFCs. The thermophiles have applications in H2 production. A set-up of MES for hydrogen production operated at 55°C had a rate of 377 mmol days–1m–1 (check unit) and had a cathodic recovery of approximately 70%. Studies showed that Firmicutes mainly contributed towards the production of hydrogen (Bergquist et al. 2014). Another application of thermophiles in MFCs is in the treatment of distillery wastewater. Distillery water contains a high level of organic carbon and sulphate, which are essential nutrients for the growth of thermophiles in the temperature range from 70°C to 80°C. Availability of nutrient and suitable environment for the growth of thermophiles gave better results in MFCs to achieve higher power output and columbic efficiency as compared to previous wastewater studies conducted using MFCs at ambient temperatures. The steady current rate was maintained by Thermincolaferriacetica by donating electrons from acetate to anode. Biofilms grow to a thickness of approximately

54 Extremophiles: Wastewater and Algal Biorefinery 380 µm and contain extracellular appendages similar to Geobacter spp. According to literature, Thermincolaferriacetica besides T. potent is another organism that is used in MFCs. It transfers e- to the anode by using several multi-heme e- type cytochromes and total of 32 cytochromes are suggested to be located on its cell surface. B. lichenifermus also gave efficient performance results when used in MFC power generation studies (Bergquist et al. 2014).

4.4 Halophiles Halophiles are extremophiles that survive under high salt conditions. The salinity of water where halophiles are situated is tenfold of seawater. Halophiles are found in all the three domains of life: archaea, bacteria and eukarya (Battista 1997). High salinity diminishes the water movement resulting in upsetting the circulations of charges of DNA and proteins, compelling them to denature and drop out of the arrangement (Angel et al. 2012). Halophiles are classified on the basis of salinity requirement for growth: slight (grow optimally at 200–500 mM of NaCl 1 to 5%), moderate (grow optimally at 500–2500 mM NaCl 5 to 20%), extreme halophiles (grow at optimally at 2500–5200 mM NaCl 20–30%) (Angel et al. 2012). Halophiles also tolerate wide ranges of pH. DHABs, deep-sea hypersaline anoxic lakes are suitable for the growth of halophiles; the Eastern Mediterranean Sea and the Gulf of Mexico are some of the hotspots for growth of halophiles (Segura and Ramos 2014). These brackish waters are liable for saltiness. It additionally supports high pressure (16 approximately...35 Mpa) atmosphere, a near absence of oxygen and profoundly diminishing conditions of light. Halophiles exhibit high metabolic assortment and may be anoxic phototrophic, fermentative, aerobic heterotrophic, sulphate reducers, denitrifiers, ormethanogenic in nature. Halophiles adapt themselves to survive in salty environments. Some extremophiles adjust a sort of ‘salt-in’ methodology to adjust the osmotic weight, whereby they gather inorganic particles (Na+, K+, Cl–) in the cytoplasm (Segura and Ramos 2014). Moderate halophiles have certain modifications that permit them to biosynthesize explicit osmolytes in their cytoplasm. The aggregated osmolyte behaves as an osmoprotectant and helps to adjust the osmotic pressure and low salt concentrations in the cytoplasm to prevent the disruption of other metabolic activities of cells due to the presence of excess salt ions (Angel et al. 2012). Halophiles produce enzymes that are stable and active in a highly salty environment. This enzyme shows different adaptions which will help them to survive in a salty environment and help to carry out the other metabolic activities. The surface modification of proteins or protein arrangement is the main difference between the halophilic and non-halophilic organisms. The presence of certain amino acids like serine, threonine, a certain higher proportion of amino acids such as aspartate, glutamic acid and certain lower proportion of amino acids such as lysine plays important role in the survival mechanism (Segura and Ramos 2014). The steadiness of these enzymes relies on the negative charges of the acidic amino acids on the protein surface. The negative charge of the surface is significant for the solvation of the halophilic protein. These surface charges prevent the denaturation, aggregation and precipitation of the protein. For instance, Bacillus sp. Dsh 19-1 and Zunongwangiaprofunda produce salt-tolerant hydrolytic enzymes. Amylase is one of the hydrolytic enzymes that can endure both cold and saline conditions. The alphaamylase shows a great potential application in the industry especially in its use for the treatment of wastewater. The halophilic enzymes include polysaccharide-hydrolysing enzymes for the lysis of xylan and starch. Halophilic enzymes are dynamic and stable in media with low water action, as they are capable of maintaining charges at a dynamic site. The high salt concentration is suitable for the performance of the MESs. High salt concentration diminishes the interior opposition of the framework as the obstruction of the electrolyte is proportional to the electrolyte conductivity. Protons are expelled from the biofilm and the proton dissemination obstruction diminishes. In any case, expansion of salt or expansion of a buffering specialist is an exorbitant strategy to expand the exhibition of MESs. S. marisflavi is categorized as a slightly halophilic species. In the domestic wastewater treatment, power production increased by 30% after

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the addition of 20 mg/L of salt in an acetate-fed MFC. H. volcanii has the most noteworthy force and current generation capacity. Geoalkalibactersubserraneus shows great outcome as it structures 76 µm thick biofilm on the anode and it legitimately moves electrons. This pure culture also produces a high current output. Halophiles utilized in MES for the desalination of saline water can be used as business models as MES can desalinate up to 95% of salt from the ocean water. A three-chamber microbial saline wastewater electrolysis cell was planned that used natural carbon in the anode compartments in the nearness of 0.34 M NaCl while at the same time desalinating wastewater.

4.5 Psychrophiles Psychrophiles are those which grow and develop in a cold environment. The first species of psychrophile was discovered from the Arctic area of the world. Besides the Arctic, Antarctica and deep sea are also a source of psychrophiles. Their temperature of growth ranges between –20°C to + 20°C with optimal growth temperature < 15°C. Their low temperature activity demonstrates that psychrophiles have overcome key hindrances to maintain their metabolic activity under such temperature extremes. For instance, they prevent protein denaturation, maintain cell division, and so forth (Angel et al. 2012). Though they are unaffected by low temperatures, they are very extremely sensitive to higher temperature changes. To survive under low temperature, they start adapting. Cell survival is carried out by synthesizing glycoprotein and peptides with antifreeze properties. These compounds are found in many bacterial sp. isolated from Antarctica as well as in fish and some insects. Micrococcus cryophiles, Rhodococcus erythropolis, Marinomonasprotea are some most studied bacteria. AFPs (Antifreezing Proteins) are ice restricting protein, they repress the development of ice in two unique circumstances before freezing, and they have warm hysteresis movement. In a solidified state, they show ice crystallization hindrance, whereby the proteins repress the development of huge precious stones to the detriment of little gems at high below zero temperature. Another adjustment to deal with the cold is the ascent layer smoothness. A layer is expanded by enlarging the extent of unsaturated fatty acids in their lipids. This helps the membrane to remain in a semifluid state. For example, psychrophiles contain fatty acids with up to five double bonds. Deep-sea psychrophiles also maintain their membrane fluidity by increasing the number of fatty acids in the lipid molecules (Angel et al. 2012). Carotenoids additionally appear to control membrane fluidity. Other than the membrane fluidity and AFPs, there are some other adjustments like the creation of cold-stun proteins and RNA chaperones upgraded tRNA flexibility, generation of cold dynamic auxiliary metabolites, enzymes, pigments, amongst others. Psychrophile protein has a higher extent of alpha-helix than beta-helix sheets which are basic for keeping up the adaptability of a layer even at low temperatures. For a high response rate, the cold-dynamic chemicals are additionally careful because cold-active enzymes keep high reactant rate at low temperature through the enlargement of the dissolvable associations and basic adaptability (Segura and Ramos 2014). Alcaligenes, Alteromonas, Aquaspirilum, Arthrobacter, Bacillus, Bacteroides, Brevibactrium, Gelidibacter, Methanococcoides, Methanogenium, Microbacterium, etc., are some reported psychrophiles. Psychrotolerant are more widely distributed in nature than psychrophiles. Optimum temperature range for the growth of psychrotolerants is between 20°C–40°C. They can be isolated from soil and water in temperate regions as well as from meat and dairy products. Numerous microscopic organisms can be isolated from troposphere and stratosphere where temperature lies between –20°C and –40°C. Besides AFPs, early studies revealed that there are some kinds of genes present which are active at low temperature. Some sea microbes produce cryoprotectants which are polymeric substances and that protect both the bacteria and enzymes. In light of temperature vacillation, psychrophiles produce the cold stun and cold acclimation proteins, which work as transcriptional enhancers and RNA binding protein. The test is to comprehend and disentangle the physiological and molecular basis of development of psychrophiles (Angel et al. 2012).

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Psychrophiles and psychrotolerant microorganisms are both helpful in MES. The temperature has an extraordinary effect on the working of microorganisms just as MES. The ideal temperature for growth of psychrophiles is below 20°C and the performance of MES increases with the rise in temperature, bringing down the temperature, power creation is diminished while expanding the generation the response rate is diminished. An advantage of low-temperature MES leads to the production of higher coulombic efficiencies. Geopsychrobacter electrodiphilus develop by using the anode as a sole electron acceptor and have high columbic effectiveness of 90% while donating 90% of the accessible from its substrate oxidation to the anode. Methane rich fluids (a form of cold seeps) and sulphide are other electron donors for marine sediment MFC (Ha et al. 2012). Another example of low-temperature MES is H2 production in MES in the presence of glucose, acetate, activated sludge and acetate or molasses rich wastewater as electron contributors, for example, these substrates are available in the anode chamber and psychrophiles oxidize the substrate at lowtemperature to generate hydrogen. Contending methanogenic and homeostatic response pathways are commonly inert at low temperature. Acetate also acts as an electron donor and decreases the methanogenic reactions, while the growth of G. psychrophiles is promoted, which act as the electron acceptor and produces high columbic efficiency. According to literature, the trehalose acts as a catalyst. This increases the reaction rate in the MESs. The expansion of trehalose to squander initiated slime and acetic acid derivation encourages hydrogen creation; MES working at 0°C builds the response rate and coulombic efficiencies. The biodegradation of the anti-toxin chloramphenicol in a biocathode MES has been determined at 10°C.

5. Exocellular Electron Transfer in MES The main function of MFC is the production of electricity using wastewater as a substrate via the redox reactions where microorganisms act as catalysts. These microorganisms or electrogens have the ability to transfer the electrons in and out of the cell. Such organisms lead to the formation of the biofilm on the anode. In the anode chamber, the microorganisms oxidize the substrate which follows the release of CO2, protons and electrons. The transfer of these released electrons outside to the cell is known as Exocellular Electron Transfer (EET) (Fig. 3.4). Some electron transfers

Fig. 3.4. Extracellular electron transfer from cathode to microorganisms.

Role of Extremophiles in the Microbial Electrochemical Cell: Recent Advances 57

can catalyse bioelectrochemical reactions such as reduction reactions; one such transfer is from cathodes to associated microbes. Before being transferred out, the electrons are transported to outer membrane proteins by diffusible intracellular electron carriers (NAD+/NADH). This type of EET can be found in the various species of microbes such as Gram-positive bacteria, Archaea and eukaryotes (Satyanarayana et al. 2005). The electron transport out of the cell involves three fundamental steps-substrate oxidation, electron transfer to Outer Membrane Proteins (OMPs) and exocellular electron transport (Choi and Sang 2016). Three modes have been discovered by which the microorganisms transfer electrons from electrodes and vice-versa, these are 1. Indirect transfer of electrons 2. Through biological projections 3. Direct electron transfer Indirect transfer of electrons involves the transfer of electrons mediated by soluble electron carriers (shuttles) which transfers the electrons to the electrode. This carrier-mediated transport of electrons can improve the rate of electron transfer such as in Shewanella oneidensis MR-1 (Zuo et al. 2008). The second mode is the transfer of electrons through nanowire or pili (hair-like extracellular appendages). This is a unique strategy for electron transport. These nanowires help to produce a high amount of current. This mechanism involves the production of an electron from microbial species, followed by the transport of electrons via a pili or nanowire to the electron carrier protein or the insoluble electron acceptor. The production of nanowire or pili is dependent on the microbial species. Sometimes this pili leads to the accumulation of cells in the biofilm but sometimes it may be harmful to MES. Initial studies on pili filaments isolated from the anode biofilm showed metallike behaviour, increased temperature and decreased conductivity. However, recent studies revealed that the nanowire produced by the Shewanella sp. contains an extension of cytochrome rich outer membrane and periplasm rather than pili (Lee et al. 2019). In the redox conduction or transfer of electrons through redox proteins involves a chain of c-type cytochrome aligned through a nanowire; this contributes to the high current density. In this mode of EET, the multiheme c-type chromosome (MHCs) play an important role (Choi and Sang 2016). The covalent bonds between MHC hold them together to form the polypeptide chain. The distance between the two MHC is approximately 1–2 nm. This special arrangement of MHC facilitates the EET. Bioelectrochemical synthesis, based on the direction of electron flow, has two categories (as discussed earlier)—Microbial Fuel cell, where the flow of electrons is generated, and Microbial electrosynthesis cell, in which electricity is consumed. The electrode acts as an electron acceptor in the former and donor in the latter situation. This has gained great attention of researchers as it can be coupled with not just energy production but also with bioremediation and heavy metal immobilization. As mentioned earlier transfer of electrons is mediated via MHCs, Geobacter and Shewanella have shown good results when poorly soluble metal oxides and hydroxides are present in electrode chamber. Very few exoelectrogenic organisms have been isolated from the BES directly but some research experiments like U-tube MFC design have shown good results to identify organisms like O. anthropic which require metal oxides for respiration (Zuo et al. 2008).

6. Recent Trends and Applications of Extremophiles in Microbial Electrochemical Cells Recent studies involving M. thermoautotrophica, a thermophile, showed significant acetate production and columbic efficiency at a wide range of temperatures in MEC. This is credited by their exceptional carbon fixation abilities making them a valuable asset for producing industrially

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important biomolecules (Rana et al. 2020). Hyperthermophiles like Thermincolacarboxydophila has a simple respiratory mechanism that is being used in for harnessing electrical energy even at boiling temperatures. A novel strain of Knallgas bacteria, a thermoacidophilic electroautotroph, was characterized using metagenomic and 13C-labelling methods. This bacteria formed cathodic biofilm that led to enhanced electron uptake which is credited to its hydrogenases. This organism also naturally produces polyhydroxybutyrate (PHB) and hence is being coupled with carbon fixation on electrode surfaces (Reiner et al. 2020). Better understanding of the mechanisms and characteristics of extremophiles like Geoalkalibacter spp. has led to their application in pivotal microbial electrochemically driven technologies. These bacteria tend to dominate in the biofilms (up to ~ 80%) causing production of current densities of 548 + 23 µA/cm2 with acetate substrates (Yadav and Patil 2020). The MES technologies dependent on extremophilic bacteria are still at their initial stages and have immense potential to lay a platform for large scale applications. Exploration of mechanisms associated with specific electron transfer in each type of electrochemical cell can pave way for development and implementation of the cells using extremophiles.

7. Conclusion Extremophiles are versatile organisms and are widespread across the globe. They can be potentially applied in MESs to replace the conventional expensive and polluting non-renewable processes to harness power and materials. More studies on bio-compatibility and optimization are required, but it can be concluded that there is immense potential in extremophiles of all kinds that can be of use for purposes like bioremediation, bioenergy production, bio-fuels, biosynthesis, to name a few.

Declaration of Competing Interest The authors declare no conflict of interest.

Acknowledgement The authors wish to thank the financial support from Sharda University, Greater Noida, Delhi NCR, India.

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4 Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy Nidhi Sahni,1 Meenu Hans 1,2 and Sachin Kumar 1,*

1. Introduction The rising world population demands have led for the supply of food, fresh water and fuel along with a goal to restrict the atmospheric carbon dioxide (CO2) levels. Thus, the world energy forum has predicted that fossil-based oil and coal reserves are going to be exhausted in less than the next few decades (Sen et al. 2016, Lwo et al. 2016). Fossil fuels account for over 79% of the primary energy consumed in the world, out of which 57.7% is used in transport sector (Barpatragohian 2015). Achieving energy security in this strategic sense is of fundamental importance not only for economic growth but also for human development. Renewable energy is one of the best alternatives of non-renewable energy, which is derived from the resources that are regenerative, and not depleted over time. Renewable energy offers a chance to reduce carbon emissions, cleans the air and put our living system towards a more sustainable footing. Some of the renewable energy sources are bioenergy, wind energy, solar energy, tidal energy, geothermal energy, etc. (Sen et al. 2016, Lwo et al. 2016). Bioenergy or biomass energy is the energy derived from organic matter (or biomass), and has been used for thousands of years, since people started burning wood to cook food in ancient times. Wood is our largest biomass energy resource even today. Other biomass including plants, residues from agriculture or forestry and the organic components can also be used as a source of energy. The net emission of CO2 will be zero as long as plants continue to be replenished for forming building blocks for their growth. Burning of plants or animal excreta causes air and water pollution due to incomplete combustion and formation of toxic gases such as CO2, sulphur dioxide (SO2), nitrous oxide (NOx) and unburnt carbon. Therefore, it is more useful to convert biomass into liquid or gaseous biofuels such as biogas, biohydrogen, bioethanol, biobutanol, etc. (Kumar et al. 2009, Singh and Kumar 2019, Chandel et al. 2020).

Biochemical Conversion Division, Sardar Swaran Singh National Institute of Bio-Energy, Kapurthala, Punjab-144601, India. 2 Department of Microbiology, Guru Nanak Dev University, Amritsar, Punjab, India-143005. * Corresponding author: [email protected], [email protected] 1

62 Extremophiles: Wastewater and Algal Biorefinery Wastewater is one of the sources of organic matter, which is the residual water from domestic, industrial, commercial or agricultural activities, surface runoff or storm water and sewer wastewater. The characteristics of wastewater vary and depend on the source. Wastewater contains physical, chemical and biological pollutants. Households produce wastewater from flush toilets, sinks, dishwashers, washing machines, bath tubs, showers and are conveyed into a sanitary sewer. Alternatively, it is transported in a combined sewer which includes storm water runoff and industrial wastewater. Wastewater that is discharged to the environment without suitable treatment causes water pollution (Tilley et al. 2016). To avoid this pollution, wastewater has to be treated to eliminate the hazardous pollutants to be safely released into the environment. After treatment, wastewater is discharged into a receiving water body. Conventional methods used for industrial, municipal or agricultural wastewater treatment are described below. Keeping in view, the potential of wastewater as a sustainable source of energy, and sustainability of some biochemical processes of renewable energy production, advanced processes for the wastewater treatment are also proposed and discussed.

2. Wastewater Treatment Wastewater treatment is a process used to reduce the Biological Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) that can be discharged into the environment with minimum impact or can be recycled. Recycling and reuse of treated wastewater would reduce the requirement of water and would be helpful in prohibiting the water crisis in the near future. The treatment process takes place in a wastewater treatment plant (WWTP), often referred as Sewage Treatment Plants (STP). Pollutants in municipality and industrial wastewater are removed or broken down in STPs. Generally, two types of processes are used for treatment of wastewater, i.e., aerobic and anaerobic process after the physical treatment (primary step) of wastewater to remove the physical contaminants, e.g., stones, grits, sand, etc. (WWAP 2017).

2.1 Aerobic Treatment Aerobic wastewater treatment methods gained importance over the past decades due to less time consumption, easy process, use of less equipment, etc. Aerobic processes are easy to operate, and can tolerate more fluctuations during the process. The main pollutants in the wastewater include ammoniacal nitrogen, pathogens, organic matter, inorganic matter, physical factors (sand, stones), etc. To promote the aerobic biochemical reaction, an adequate supply of oxygen is required to avoid oxygen limitation (Andersson et al. 2016). Some of the commonly available technologies for aerobic treatment are described below: (a) Trickling or biological filter process involves the downward flow of wastewater over an inert material such as rock, slag, sand, etc., causing the generation of biofilm over the bed of media (made of coke, plastic matrix, polyurethane foam, gravel, sand, etc.). The system is supplied with aerobic conditions by splashing, diffusion or by forced-air through the porous medium. The developed biofilm is thick and formed of a gelatinous matrix consisting of a variety of aerobic and anaerobic microbial species (e.g., protozoa, cilliates, helminthes, aschelminthes, arthropoda larvae, bacteria, etc.) performing oxidative as well as reductive processes. Usually, industrial effluent is treated by two types of trickling filters; large concrete tanks or vertical towers filled with plastic packing or other media (Buchanan 2014). The treated water is further processed for sludge removal in a clarifier. Trickling filters proved to be promising devices, and have the capacity of high rate of removal of hexavalent chromium. Hexavalent chromium is poisonous and a carcinogenic agent is present in industrial wastewater. It is necessary to remove Cr (VI) before disposing wastewater to the environment (Tilley et al. 2016).

Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy 63

(b) Fluidized bed reactor (FBR) works on the fluidization of a solid granular material packed in the fluidized bed reactor supported by a distributor (a porous plate), by the wastewater to be treated when it passes through it at a minimum fluidization velocity, and faces multiple chemical reactions carried out by the fluidized catalytic granular material (Gopalakrishnan et al. 2019). This process is used widely throughout global industries due to the advantageous criteria of FBR for instance, uniform particle mixing, uniform temperature gradients and continuous state operation however, the requirement of the large reactor size, higher pumping power and pressure drop, particle entrainment, erosion of internal components, loss of pressure during the process increases the capital costs, energy supply and overall maintenance of the system. (c) Rotating biological contactor (RBC) involves the assimilation of organic matter with the help of a biofilm of microorganisms (along with air provided by a rotating action) arranged in closely spaced, parallel discs mounted on a rotating shaft (Wang et al. 2009). RBC is sturdy and requires low maintenance, low power and creates little noise by rotor of slow rotation (2–5 RPM). (d) Sequencing batch reactor system (SBR) consists of a series of reactors connected to reduce the organic matter in wastewater in batches with the help of oxygen bubbled through the mixture (Ergas and Aponte-Morales 2014). Basically, the process comprises five steps; filling, reaction, settling, decanting and idle, which is the only step involving aeration of the mixture. Depending on the design, SBR has some positive attributes, i.e., short Hydraulic Retention Time (HRT) that allows high Organic Loading Rate (OLR) and low production of sludge (Tilley et al. 2016). (e) Microbubble aerator exhibits useful characteristics such as a large gas liquid interfacial area, long residence time in the liquid phase and fast dissolution rate which allow dissolving the oxygen into water. (f) Aerobic granulation technology The poor settling properties of sludge in the conventional activated sludge systems causing them to require large surface areas and separation units (for biomass), which could be resolved by the advanced system of aerobic granules; a type of sludge, which is capable of immobilizing flocs and microorganisms into strong compact structures (Sarma and Tay 2018). The settleability, high biomass retention, concurrent nutrient removal and toxicity tolerance of aerobic granulated sludge provide this system a potential to treat highly polluted wastewaters.

2.2 Anaerobic Treatment Aerobic treatments as discussed above are no doubt efficient and practically used methods for wastewater treatment on a large scale, however their installation and maintenance cost affect the process economy, and require some convenient techniques, with comparable efficiency, which attract the anaerobic treatments as they are not only economic but environment friendly, and generate a large range of valuable renewable products particularly highly demanding biofuels. Anaerobic treatment of wastewater means breakdown of complex polymers to simpler molecules in the absence of oxygen. Unlike an aerobic process, an anaerobic process is less stable under fluctuations, expensive and requires long HRT, which is due to the lack of a proper design of a reactor for Anaerobic Digestion (AD). However, the technology has become advanced, and various types of reactors have been designed to overcome these drawbacks. Today, the anaerobic process has emerged as a practical and economical alternative to the aerobic process due to the significant advantages over the aerobic process such as no oxygen requirement, low production of biological sludge, oxygen independent organic loading, production of more valuable products such as methane (CH4) and no effect of non-feed conditions for a few months to the system, which

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make it a suitable option for seasonal industrial wastewater treatment (Tilley et al. 2016, Andersson et al. 2016). Anaerobic conversion of organic waste to CH4 is a complex process involving a number of steps. These can be differentiated into four steps, i.e., hydrolysis, acidogenesis, acetogenesis and methanogenesis, and overall, it is known as anaerobic digestion (AD). The main products of anaerobic digestion are CH4, CO2, H2S, hydrocarbons, etc. The first three steps are similar to Dark Fermentation (DF), which are mainly used for the production of Volatile Fatty Acids (VFAs) and Hydrogen (H2) along with CO2 (Hans and Kumar 2019). The last is methanogenesis, where methane is produced from VFAs, CO2 and H2. There is another process which is also used for production of H2, known as photo fermentation. Photo fermentation is the next step to dark fermentation, where VFAs produced in the dark fermentation are converted into H2 and CO2 in the presence of light (Luo et al. 2011, Banos et al. 2011, Budiyono et al. 2013, Forgas et al. 2012). Individual processes have will be discussed later. 2.2.1 Anaerobic Digestion The digestion process begins with the hydrolysis of complex biopolymers (carbohydrates, proteins, lipids) to their monomeric forms (sugars, amino acids, fatty acids) by the action of hydrolytic microorganisms (Hans and Kumar 2019). Acidogenic bacteria then convert the broken down polymers, i.e., sugars and amino acids into CO2, H2, ammonia (NH3) and complex organic acids. Later acetogens convert the resulting complex organic acids into acetic acid, along with additional NH3, H2 and CO2. Finally, methanogens convert these products to CH4 and CO2 by using acetic acid and CO2 (acetoclastic methanogens) or using H2 and CO2 (hydrogenotrophs) (Mathew et al. 2015, Patil and Deshmukh 2015, Hans and Kumar 2019) (Fig. 4.1). During AD, H2 is also produced as a by-product along with CH4 via an acetoclastic route.

Fig. 4.1. Complete process of anaerobic digestion, dark fermentation and photo fermentation.

Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy 65

AD is used as a part of the process to treat biodegradable waste and sewage sludge. The anaerobic treatment of liquid waste or wastewater provides the opportunity to rapidly reduce the organic content of the waste, while minimizing the energy consumption and production of microbial biomass or sludge because conversion of organic compounds into sludge produces by-products which require further disposal or treatment. Reduced sludge and energy consumption are the two attributes which have considered the direct anaerobic pre-treatment of wastewater economically attractive for municipal and industrial waste streams. Irrespective of aerobic treatment, the energy savings can be achieved by avoiding the cost of aeration which is quite significant. However, effluents from anaerobic treatments are often not suitable for direct discharge into receiving waters without further treatment which may require aerobic polishing. Yet, the reduced aeration demand and sludge production after anaerobic treatment may justify this process. 2.2.2 Dark Fermentation DF is a fermentative conversion of organic substrate into biohydrogen under anoxic or anaerobic conditions. It is a complex process involving a series of biochemical reactions and diverse group of bacteria having three similar steps to AD. The key pathway is to breakdown the carbohydrate rich substrates by bacteria to H2 and other intermediate products such as VFAs and alcohols. These substrates provide building blocks and metabolic energy for bacterial growth. Unlike photo fermentation, it occurs in the absence of light and hence, it is called dark fermentation. Hydrolytic microorganisms hydrolyze the complex organic polymers to monomers, which are further converted to a mixture of low molecular weight organic acids and alcohols by acidogenic bacteria. Then acetogenic bacteria act on these VFAs and alcohols to form acetate, H2 and CO2 (Rai et al. 2012). This fermentation process produces a mixed biogas, which may contain H2, CO2, CH4, carbon monoxide (CO) and H2S. The amount of H2 depends on the pathway resulting into different VFAs. Acetate and butyrate are the most common VFAs produced in dark fermentation, followed by propionate (Liu et al. 2013). Equations (1)–(3) describe the production of acetate, butyrate and propionate generated from glucose. From these equations, it is shown that the maximum theoretical value of H2 that can be produced using DF is 4 moles H2 per mole glucose when acetate is the end product, while it is 2 moles of H2 per mole glucose when butyrate is the end product. However, generally a mixture of acetate, butyrate and propionate are formed which make it a challenge to reach the desired H2 yield (Redwood and Macaskie 2006). C6H12O6 + 2H2O → 4H2 + 2CH3COOH + 2CO2

(1)

Acetate C6H12O6 + 2H2O

→ 2H2 + 2C3H7COOH + 2CO2

(2)

Butyrate C6H12O6 + 2H2

→ 2H2O + 2C2H5COOH

(3)

Propionate In practice, high H2 yield is associated with the production of acetate and butyrate while low H2 yield is associated with the production of propionate and other reduced end products such as alcohols and lactic acid (Levina et al. 2004). DF is considered as the simplest process of H2 production. Fermentation occurs in the dark at normal pressure, and it usually shows high H2 production rates as compared to the photosynthetic methods (Das and Veziroglu 2001, Liu et al. 2013). Besides the use of glucose and simple carbohydrates or polymers such as starch and cellulose, DF may be processed using a wide range of organic compounds as a substrate, which has led to great interest due to the possibility of using organic wastes for H2 production (Sinha and Pandey 2011). Utilization of wastewater as a potential

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Extremophiles: Wastewater and Algal Biorefinery

substrate for H2 production has been drawing considerable interest in recent years especially through the DF. Industrial wastewater as a fermentative substrate for H2 production meets most of the criteria required for the substrate selection in terms of its availability, cost and biodegradability (Angenent et al. 2004, Kapdan and Kargi 2006). Apart from industrial wastewater, cattle wastewater (Tang et al. 2008), dairy process wastewater (Venkata Mohan et al. 2007a, Rai et al. 2012), starch hydrolysate wastewater (Chen et al. 2008) and designed synthetic wastewater (Venkata Mohan et al. 2007b, 2008a) have been reported to produce H2 through DF using mixed cultures under acidic conditions. Different waste effluents such as paper mill wastewater (Idania et al. 2005), starch effluent (Zhang et al. 2003), food processing wastewater (Shin et al. 2004, van Ginkel et al. 2005), domestic wastewater (Shin et al. 2004), rice winery wastewater (Yu et al. 2002), distillery and molasses based wastewater (Ren et al. 2007, Venkata Mohan et al. 2008a), wheat straw wastes (Fan et al. 2006) and palm oil mill wastewater (Vijayaraghavan and Ahmed 2006) have also been studied for wastewater treatment and H2 production. The efficiency of the dark fermentative H2 production process depends on the consortia used, pH, and organic loading rate along with other wastewater characteristics (Venkata Mohan et al. 2007c, 2008b, c, Vijaya Bhaskar et al. 2008, Hay 2013). In spite of its advantages, the main challenge with fermentative H2 production is the relatively low conversion efficiency from the organic source. Typical H2 yield ranges from 1 to 2 mole of H2 per mole glucose. About 80–90% of the initial COD remains in the wastewater in the form of various VFAs and solvents such as acetic acid, propionic acid, butyric acid and ethanol. About 60–70% of the initial organic matters are reported to remain in the solution using highly efficient process. Bio-augmentation with the selectively acidogenic consortium for enhanced H2 production is also reported by Venkata Mohan et al. (2007b). A sharp drop in pH was observed due to the generation and accumulation of soluble acid metabolites, thus inhibits the process. Use of unutilized carbon sources present in the acidogenic process for biogas production sustains the practical applicability of the process. One way to utilize the remaining organic matter in a usable form is to produce additional H2 by terminal integration of photo fermentative processes (Venkata Mohan et al. 2008d, Rai et al. 2012, Hay 2013, Kumar et al. 2015, Roy et al. 2014) and CH4 by integrating acidogenic processes to terminal methanogenic processes. 2.2.3 Photo Fermentation Photo fermentation is a fermentative conversion of organic substrate to H2 carried out by anoxic photosynthetic bacteria that use sunlight and biomass to produce H2 and CO2 by a series of biochemical reactions involving three steps similar to the anaerobic conversion as described in Equation (4). Purple Non-Sulphur (PNS) and Green Sulphur (GS) bacteria such as Rhodobacter spheroids and Chlorobium vibrioforme, respectively are capable of producing H2 gas by using solar energy and reduced compounds. Their photosynthetic systems differ from oxygenic photosynthesis due to their requirement for reduced substrates and their inability to oxidize water. Photosynthetic bacteria have long been studied for their capacity to produce H2 through the action of their nitrogenase system. Photo fermentation differs from dark fermentation as it operates in the presence of light (Patel et al. 2012). In a study, soluble metabolites resulting from DF consisting of VFAs and alcohols were used for H2 and CO2 production in the photo fermentation (Redwood and Macaskie 2006). 2CH3COOH + 4H2O + light → 8H2 + 4CO2

(4)

The theoretical reaction for the conversion of those VFA into H2 is further explained through Equations (5)–(7). 2CH3COOH + 4H2O → 8H2 + 4CO2 Acetate

(5)

Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy 67

2C3H7COOH + 4H2O → 10H2 + 4CO2

(6)

Butyrate 2C2H5COOH + 2H2

→ 7H2O + 3CO2

(7)

Propionate The major advantages using photo fermentative process are high theoretical conversion yield of H2, potential to use a wide spectrum of light and the ability to consume organic substrates derivable from waste (Srikanth 2009, Roy et al. 2014). However, there are some disadvantages of this process, e.g., low light conversion efficiency, high energy demand by nitrogenase and the high cost of construction and maintenance of H2 photo bioreactors (Vazquez et al. 2009, Kumar et al. 2015).

3. Renewable Energy by Fermentative Treatment of Wastewater There are three major products formed after fermentation of wastewater via AD, DF and photo fermentation are CH4, H2 and CO2. Biohythane, which is a mixture of H2 and CH4 is also formed during fermentation of wastewater via a two-stage fermentation. All of these three major products are explained later.

3.1 Biomethane During treatment of the wastewater in WWTP, the organic matter breaks down into simple molecules and a semisolid slurry is formed, termed as sludge (Patil and Deshmukh 2015, Bajpai 2017). The sludge then undergoes anaerobic fermentation to produce biogas, which is mainly a mixture of CH4 and CO2. The CH4 can be used directly or after purification as a source of heat and power generation (Fig. 4.2) (Singh et al. 2021).

Fig. 4.2. Schematic illustration of treatment of wastewater using anaerobic digestion (Bachman 2015).

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Extremophiles: Wastewater and Algal Biorefinery

The sludge contains particles removed from wastewater, which is rich in nutrients and organic matter. The anaerobic treatment of liquid wastes or wastewaters provides the opportunity to rapidly reduce the organic content of the waste while minimizing the treatment process, energy consumption and less production of microbial biomass or sludge. Reduction in sludge and energy consumption are the two attributes which have led to direct anaerobic treatment of wastewater economically attractive for municipal and industrial waste streams (Geng et al. 2010). The organic matter can be degraded by sequential action of hydrolytic, acidogenic, acetogenic and methanogenic microorganisms in the absence of oxygen to produce biogas as discussed earlier. Biogas is a mixture of CH4 (50–60%), CO2 (30–40%), H2 (1–5%), N2 (0.5%), CO, H2S and traces of water vapours (Mathew et al. 2015, Chong et al. 2009, Geng et al. 2010). Properly functioning biogas system can yield a whole range of benefits for their users including application in heat, light and electricity generation, transformation of organic waste into high quality fertilizers, improvement of hygienic conditions through the reduction of pathogens, worm eggs and flies, clean fuel for cooking, in firewood collection and cooking, environmental advantages through protection of soil, water, air and woody vegetation, micro- and macro-economic benefits through energy and fertilizer substitution and decentralized energy generations, import substitution and environmental protection (Ismail et al. 2010). 3.1.1 Factors Affecting Methane Production The important factors which affect CH4 production are: (a) Hydraulic retention time (HRT) is a parameter that describes the period for which sludge stays in the reactor. Too short HRT means less digestion and thus low biogas yields. HRT depends upon digester volume (m3) and daily feedstock input (m3/day). Typical range of HRT is 16–25 days for sludge as feedstock (Kind and Levy 2012). (b) Temperature Most of the AD for WWTPs occurs at mesophilic conditions at 37–42°C (Zhang 2010). (c) Degradation of organic dry matter (ODM) There is a direct relationship between net biogas production and degradation of ODM. The proportion of ODM and its degradation depends upon the type of sludge, HRT the process used for treatment and age of sludge. Around 450–500 L of biogas is produced per Kg of organic matter when > 50% of organic matter is degraded (Bachman 2015, Montgomery and Bachmann 2014). To improve the organic matter degradation, different pre-treatment methods such as mechanical, thermal, biochemical or combined are practiced to the feedstock. Mechanical pre-treatment involves disruption of the microbial cells by using high force. Most commonly ultrasound and extruders are used for this. Thermal pre-treatment uses high temperature (up to 200°C) to destroy the microbial cell wall and release proteins which become accessible for the degradation (Montgomery and Bachmann 2014). Generally, pre-treatment methods increase the biogas yield by 30% (Kind and Levy 2012).

3.2 Biohydrogen Biohydrogen is the H2 that is produced biologically. Interest is high in this technology because it is a clean fuel and can readily be produced from certain kinds of biomass. H2 is the most promising in the succession of fuel evolution with several technical, socio-economic and environmental benefits to the credits. It has the highest energy density of 142 MJ/kg whereas CH4 has 50–55.5 MJ/kg (Hans and Kumar 2019, Roy and Das 2016). H2 is a more environment friendly gas as its combustion releases only water as an end product. The conventional methods for H2 production are chemical methods, most of which are based on the reforming reactions of hydrocarbons such as steam reforming, CO2 reforming and partial oxidation resulting into the significantly high H2 yield (Fig. 4.3). However, reforming of hydrocarbons is energetically

Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy 69

Fig. 4.3. Different routes of hydrogen production.

expensive as their reactions are endothermic, occurring at high temperatures (about 800ºC). In addition, reforming processes release a significant amount of greenhouse gases such as CO and CO2. Also, hydrocarbons reforming use non-renewable materials and fossil feedstocks (Navarro et al. 2007, Liu et al. 2012). In the present scenario, electro-chemical and biological methods are in the research and developmental stages (Momirlan and Veziroglu 2002, Li and Yu 2011, Kumar et al. 2015). Biological H2 production methods are becoming important mainly due to the (i) utilization of renewable energy sources and (ii) their operation at ambient temperature and pressure (Sinha and Pandey 2011). Biological H2 production is advantageous over other techniques in terms of the high energy efficiency, e.g., energy of the fuel cell conversion into H2 would be twice the burning biofuel in an internal combustion engine with nearly zero levels of air pollution as compared to the other biofuels (Hallenbeck et al. 2009). 3.2.1 Biological Methods of Hydrogen Production The biological H2 production process can be divided into two major categories, i.e., fermentation and photolysis. Fermentation is further divided into two groups, i.e., dark and photo fermentation. In this section, dark and photo fermentation have been discussed as shown in Fig. 4.4. H2 production using agro-residues as feedstocks becomes advantageous in terms of the economic and environmental perspectives as theses residues are abundant, cheap, renewable and biodegradable (Wukovits et al. 2013, Guo et al. 2010). Various metabolic pathways can either be promoted or inhibited, depending upon the operating conditions, which govern the production of specific VFAs and alcohols including acetate, propionate, butyrate, lactate and ethanol. Carbohydrates fermentation as described earlier, is typically associated with the production of 2 or 4 moles of H2 per mole of glucose. However, propionate, ethanol and lactic acid adversely affect the H2 production, e.g., propionate is a metabolite of the H2 consuming pathway, while ethanol and lactic acid are associated with zero H2 pathways (Guo et al. 2010). In order to improve the H2 production, culture conditions such as carbon sources, Carbon-to-Nitrogen ratio (C/N) and C/P ratios, pH and temperature have been widely studied. Metal ions can significantly influence enzyme activities related to H2 production (Zhao et al. 2012).

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Extremophiles: Wastewater and Algal Biorefinery

Fig. 4.4. Biological hydrogen production routes.

Biological H2 production is associated with various challenges such as storage and transportation of this non-condensable gas. Moreover, H2 producing microorganisms are poisoned by the presence of oxygen. Most importantly, as discussed earlier, H2 production is accompanied by CH4 due to the existence of hydrogenogens and methanogens in nature (Hans and Kumar 2019), thus complete biological conversion of substrate into H2 can be accomplished by cutting-off the methanogenic activity and preparing the environment for conversion of VFAs into H2. This can be achieved by using a multi–step treatment system, for example, by having DF as the first step and continued by the photo fermentation to convert the VFAs produced into H2 as the second step (Wang et al. 2009). The overall reactions of this hybrid system could be represented as: Stage I: Dark fermentation (facultative anaerobe) C6H12O6 + 2 H2O → 2 CH3COOH + 2 CO2 + 4 H2 Stage II: Photo-fermentation (photosynthetic bacteria) 2CH3COOH + 4 H2O → 4 CO2 + 8 H2 So, theoretically it is evident that using glucose as the only substrate in DF, where acetic acid is the predominant metabolite product, a total of 12 moles of H2 could be expected in a combined process from one mole of glucose (Guo et al. 2010, Liu et al. 2013). 3.2.2 Limitations of Dark and Photo-fermentation There are several factors which limits the H2 production during dark and photo fermentation: (i) Source of substrate: The source of substrate for H2 production using DF includes (i) municipal food waste/sewage sludge (ii) food waste and sewage sludge (iii) food waste/wastewater (iv) wheat straw hydrolysate (v) activated sludge (vi) dairy wastewater (vii) wastewater affect H2 yield using different microbial culture/consortia (Table 4.1). (ii) Inoculum: The selection of the culture or inoculum is very important for improved H2 production. Chong et al. (2009) categorized the inoculum suitable for H2 production via DF as anaerobic, facultative and thermophilic bacteria and co- and mixed cultures. Table 4.2 shows microorganisms used in the dark and photo fermentation for H2 production. (iii) Reactor operational parameters: Operational parameters such as pH, temperature, HRT, substrate concentration and source of light are important factors for optimized H2 production. The operating pH plays a critical role in governing the metabolic pathways of microbial H2

Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy 71 Table 4.1. Different sources used for H2 fermentation. Substrates

H2 Yield

Inoculum/Conditions

References

Municipal food waste, sewage sludge

35.8 l/kg TS

Primary digested sludge

Chong et al. 2009

Food waste and sewage sludge

130 l/kg VS

Acclimatized sludge

Siddiqui et al. 2011

Food waste/wastewater

33 l/kg COD

Granular sludge

Di Stefano and Palomar 2010

Wheat straw hydrolysate

89 l/kg VS

Granular sludge

Kongjan et al. 2011

Activated sludge

240.8 l/kg TS

Clostridium bifermentans

Wang et al. 2003

Waste water sludge

16 l/kg TS

Clostridium sp.

Ting and Lee 2007

Dairy wastewater

47.6 mmol/day

Anaerobic sequencing batch culture

Rai et al. 2012

Table 4.2. Micro-organisms used for H2 production using dark and photo fermentation. Type of Bacteria

Strain

H2 Yield

References

Anaerobic

Clostridium saccharoperbutylacetonicum; Clostridium butyricum EB6

3.1 mole H2/mol glucose; 31.95 ml H2/g COD

Chong et al. 2009, Mandal et al. 2010

Facultative

Enterobcater cloacae

3.9 mol H2/mol glucose

Prasertsan et al. 2009

Thermophilic

Thermobacterium spp.

0.27 l H2/g COD

Geng et al. 2010

Co- and mixed culture

Clostridium thermocellum and C. thermopalmarium

1387 ml H2/l culture

Wang et al. 2010

Photosynthetic

Rhodobacter sphaeroides

250 ml H2/g VFA

Shi and Yu 2006

Rhodopseudomonas palustris

2.61 mmol H2/l/h

Ismail et al. 2010

Rhodopseudomonas capsulata

37.8 ml H2/g dry wt./h

Noike et al. 2005

production and it affects the effluent composition of the acidogenic reactor (Won and Lau 2011). By-products such as organic acids formed during acidogenesis cause a drop in pH. As pH decreases, the H2 production will also decrease. This happens due to the dissociated form of non-polar acid filling the cell membrane, leaving more protons within the cell causing increased energy requirements for the co-enzyme A and phosphate to maintain its neutrality (Ginkel and Logan 2005). Changes in temperature may cause bacteria to expand their energy for adaptation to low or high temperatures for their survival, which could affect the H2 yield. Since, temperature affects the growth rate and metabolic pathways of microorganisms, studies on fermentative H2 production have been done under mesophilic (37°C) and thermophilic conditions (55°C) conditions (Ozgura et al. 2010, Singh et al. 2010, Ozmihci et al. 2011). HRT is one of the key factors that affect the performance of fermentation in a continuous operation. Shorter HRT changes the fermentation pattern by inhibiting the methanogenic bacteria which require longer time to grow as compared to the acetogenic bacteria (Won and Lau 2011). In addition, shorter HRT also reduces the operating costs and substrate utilization by bacteria affecting the overall process efficiency. A study conducted by Badiei et al. (2011) showed that longer HRT allows the development of a non-H2 producing bacteria while shorter HRT reduces the overall H2 yield. Thus, an optimized HRT is necessary for an efficient H2 production system.

3.3 Biohythane Hythane has attracted attention in an era of enormously growing population due to its versatile advantages as a transportation fuel. Hythane®, was a trademark by Hydrogen Component Inc. (HCI) in their patent wherein they used a mixture of H2 and CH4 in internal combustion engines, and

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claimed its burning to reduce the emissions of NOx with equivalent energy of CNG (Bolzonella et al. 2018). The H2 content in hythane is 10–25% by volume. By combining the advantages H2 and CH4, hythane is considered as one of the important fuels involved in achieving the transition from a fossil fuel-based society to a renewable energy-based society (Fulton et al. 2010, Buitron et al. 2014, Mishra et al. 2017). Biohythane consisting of H2 and CH4 via AD of waste biomass is probably an alternative to the fossil based hythane (Fig. 4.5). The production of biohythane is basically accomplished by two methods. One method is to add clean H2 to natural gas. Two separate gas channels with quantitative control of the gas flow rates would be required to produce hythane with the desired H2/CH4 ratio. Another method is to biologically produce H2 and CH4 simultaneously in one system via twostage fermentation (Liu et al. 2012). If the system could reach the desired H2/CH4 ratio suitable for hythane, a sustainable route for the generation of biohythane can be achieved. The biological method is more promising as it maintains the H2/CH4 ratio easily by adjusting the conditions of the microbial fermentations. Therefore, biohythane production from organic matter via two-stage fermentation could be a new innovation, as it provides an output of both H2 and CH4 (Fulton et al. 2010). The conversion of waste into biohythane could resolve the problems of both environmental pollution and energy crisis. Chemical production of hythane is costly, energy intensive process and releases toxic end products which are harmful to the environment. On the contrary, industrial wastewater for the biochemical conversion into hythane has a potential to use all the end products as a substrate for methanogenesis (Kothari et al. 2012, Arizzi et al. 2016). The most efficient method for biological process is DF. However, it produces metabolic end-products such as acetate, butyrate,

Fig. 4.5. Steps of biohythane production by two-stage AD.

Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy 73

propionate, ethanol and lactic acid which remain unused (Das and Veziroglu 2001). Different organic wastes, viz., potato wastes (Zhu et al. 2008), ethanol stillage (Luo et al. 2011), food waste (Lee et al. 2010), wheat straw hydrolysate (Kongjan et al. 2011) and garbage slurry and paper waste have been used for the two-stage biohythane production under mesophilic conditions. Khongkliang et al. (2015) reported a high H2 (81.5 l/kg COD) and CH4 yield (310.5 l/kg COD) using thermophilic consortium. Among different waste substrates, organic wastewater including distillery effluent has high organic load which is detrimental for the environment (Lin et al. 2012). According to Central Pollution Control Board (CPCB 2010), India, distilleries are ranked among the top 17 heavily polluting industries of the country. Annually, 40 billion litres of effluent are disposed from distilleries. The distillery wastewater, owing to its huge organic load, is an ideal substrate for the biohythane production. It comprises of high COD (60,000–120,000 mg/l) and BOD (45,000–60,000 mg/l) (Krishnamoorthy et al. 2017). 3.3.1 Principle of Biohythane Production Two-stage anaerobic digestion (AD) is the most viable process for the combined production of two valuable biofuels; H2 and CH4, in the form of biohythane. In the first stage, the substrate is fermented to H2, CO2, VFAs, lactic acid and alcohols, and second stage is the production of CH4 and CO2. The fermentation products from H2 production process are very important for the entire biohythane system performance because they can affect the loading rate, degradation efficiency and operating stability of the methanogenesis. The conversion rate from VFAs to acetic acid will affect the methanogenic quantity, and subsequently affect the degradation rate of acetic acid and CH4 yield. The basic principle of a two-stage process is shown in Fig. 4.5 (Fulton et al. 2010). The first stage includes hydrolysis, acidogenesis and acetogenesis where hydrolytic and fermentative bacteria excrete enzymes to break down the complex organic compounds (carbohydrate, protein and lipids) into simple molecules (mono sugar, amino acids and long chain fatty acids and/or glycerol). The acidogenesis, acidogenic bacteria convert the hydrolysis products into CO2, H2, VFAs, lactic acid and alcohols. High H2 production was achieved by fermentative bacteria via acidogenesis process under pH range of 5–6 and at HRT of 1–3 days. Under optimum conditions, acidogenic bacteria could convert carbohydrate to H2 and CO2 via the acetate and butyrate pathways. In acetogenesis, acetogenic bacteria convert all VFAs into acetic acid and more H2. The acetogenic bacteria could produce acetic acid along with additional H2 and CO2 from butyric acid, propionic acid and lactic acid. In the second stage, the acetic acid in the H2 eluent is anaerobically converted to CH4 and CO2 by acetolastic methanogens. These reactions occur under an optimal pH range of 7–8 and HRT of 10–15 days. The two-stage anaerobic fermentation process is also characterized by a significantly reduced fermentation time with overall fermentation time of 13–18 days. Following the first report on CH4 and H2 coproduction from waste water sludge (Wang et al. 2003), further investigation on the effects of methods of pre-treating sludge substrate were carried on the coproduction of H2 and CH4 using Clostridium sp. to enhance H2 production (Ting and Lee 2007). The original and pre-treated sludge (acidified, basified and freeze/thawed) were tested. The acidified sludge with Clostridium sp. significantly enhanced the H2 production, whereas the original, basified and freeze/thawed sludge with Clostridium sp. 1 had similar CH4 production. The highest gas yields were 16 l H2/kg TS and 182 l CH4/kg TS, respectively. Ueno et al. (2007) established a thermophilic two-stage system fed with organic wastes consisting of artificial garbage and milled paper. The hydrogenic operation was more efficient than solubilizing with a shortened retention time, resulting in an H2 yield of 2 mol/mol hexose and a CH4 yield of 129 l/kg COD. Biohythane has proved as a better alternative of CNG due to the addition of H2 which improves the low inflammability range of CH4, reduces the burning time of engine without NOx emissions into atmosphere. Thus, biohythane is an economical as well as eco-friendly energy source.

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4. Advantages for Anaerobic Fermentation of Wastewater There are normally two methods used at industrial scale for treatment of wastewater; the anaerobic and aerobic process. The anaerobic fermentation process converts organic matters to biogas with good features of storage and transportation, which increases its commercial value as a bioenergy source. Various by-products are produced along with CH4 such as H2, VFAs, CO2 and alcohol during anaerobic fermentation. The heat values for CH4 and H2 are 35.8 kJ/l-CH4 and 10.8 kJ/l-H2, respectively (Neumann et al. 2016, Yang and Wang 2017). Furthermore, most of the value-added products such as enzymes, bio-plastics, biofuels, bio-fertilizer, bio-flocculants and bio-pesticides, are produced by pre-treatment followed by fermentation process as the main steps with wastewater and sewage sludge as raw materials. Furthermore, heavy metals in the digested sludge have lower biodegradability and crop-absorption characteristics than raw sewage sludge because of the formation of stable metal-humus complexes in the digested sludge, so it is suitable to be used as a soil fertilizer in the agricultural field.

5. Key Challenges and Possible Solutions for Anaerobic Fermentation of Wastewater In spite of a number of advantages of anaerobic fermentation approach for wastewater treatment, there are many challenges linked with these processes, which need to be addressed for successful application of these strategies for accomplishments of the targets. Some of the key challenges with possible solutions are discussed below.

5.1 Composition of Waste Water/Sewage Sludge Industrial waste water is composed of various toxic compounds such as phenol or higher concentration of various ions, which poses a threat to the reactors (Rajeshwari et al. 2000). Moreover, the sewage sludge has low C/N ratio, which goes against the nutrition balance of microorganisms, therefore the co-digestion of sewage sludge with carbon-rich substrate is widely applied for nutrition adjustment (Liu et al. 2013). Further, the flocculating structure is not easy to break, and the components of activated sludge is hard to degrade, thus the release of the organics from sludge has been restrained, so the fermentation efficiency is low and long retention time is required. Further, it was reported that the sewage sludge pre-treatment technologies had good effects on organic matters release and hydrolysis.

5.2 Biomass Washout Biomass washout is another common issue faced in the anaerobic treatment plants of wastewater, which could be resolved by using membranes or immobilized cells along with the digester for retention of biomass (Ross and Strogwald 1994, Leenen et al. 1996).

5.3 Design of Reactors A number of reactors termed as the “second generation reactors” or “high-rate digesters” carrying the high COD loading rate and up-flow velocity of 24 kg/m3 day and 2 ± 3 m/h, respectively with low HRT, have been developed for the wastewater treatment by anaerobic fermentation (Lettinga 1995). Nevertheless, these reactors are sensitive to various process factors such as composition of substrate, e.g., concentration of ions and toxic compounds (e.g., phenol) present in the wastewater, temperature, pH, etc. (Rajeshwari et al. 2000). Furthermore, designs of reactors also depend on various other factors, e.g., requirement of pre-treatment, control of operating conditions, dilution of substrate, etc. For instance, slaughterhouse wastewater treatment reactor does not need pre-

Fermentative Approaches in Wastewater Treatment for Harnessing Renewable Energy 75

treatment, on the other hand, high-rate digesters like Upflow Anaerobic Sludge Blanket reactor (UASB) requires a pre-treatment step for removing suspended solids and fats before treatment (Rajeshwari 2000). Hence, more attention should be paid towards selecting the appropriate design of a reactor according to the various requirements or improving the existing design of reactors along with some advanced operating techniques. It has been reported that a plug flow reactor, e.g., Upflow Staged Sludge Bed reactor (USSB) is a better choice than any other typically used reactor due to the reduced generation of VFAs in the effluent, high retention of sludge and steadiness. However, considering the cost, operation and maintenance in addition to the high loading rate, COD reduction, biomass retention, UASB along with fixed film configurations is an excellent choice for anaerobic fermentation of wastewater (Rajeshwari 2000).

5.4 Insufficient Reduction in BOD/COD Generally, a single process is not sufficient to treat wastewater due to a variety of contaminants for instance, DF during wastewater treatment generally generates a maximum of 4 moles of H2 per moles of glucose leading to only 33% of COD removal causing 70–80% degradable material left for the secondary or tertiary treatments (Sivagurunathan et al. 2017). Therefore, anaerobic treatments can be integrated with other anaerobic fermentation processes such as AD and photo fermentation for complete degradation of organic matter, maximizing the energy recovery and to increase the valuable byproduct generation.

6. Conclusions Anaerobic fermentation is one of the best options to treat wastewater in terms of the efficient energy recovery by producing valuable products after fermentation such as CH4 and H2. The challenging features of anaerobic fermentation, e.g., composition of waste water/sewage sludge, hard to degrade components of sewage sludge with reverse effect on the AD, biomass washout, problems with reactors design and insufficient reduction in BOD/COD requires the use of co-digestion of multiple feedstocks, use of the membranes or immobilized cells, selection of the appropriate reactor design with/or improvements and combinations of one or more anaerobic processes for the improved treatment efficiency of wastewater. The fermentation efficiency of sewage sludge could be further improved by pre-treatment, two-stage fermentation and high solid fermentation. These processes could be further investigated at a large scale to evaluate the economics, environmental feasibility and stability of the process. Furthermore, optimization of specific process should also be based on the availability of raw materials and local requirements for biogas and fertilizers.

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Patil, V.S. and H.V. Deshmukh. 2015. Anaerobic digestion of vegetable waste for biogas generation: A review. Int. Res. J. Environ. Sci. 4(6): 80–83. Prasertsan, P., S.O. Thong and N.K. Birkeland. 2009. Optimization and microbial community analysis for production of bio-hydrogen from palm oil mill effluent by thermophilic fermentative process. Int. J. Hydrogen Energy 34: 7448–7459. Rai, P.K, S.P. Singh and R.K. Asthana. 2012. Biohydrogen production from cheese whey wastewater in a two step anaerobic process. Applied Biochem. Biotechnol. 167(6): 1540–1549. Rajeshwari, K.V., M. Balakrishnan, A. Kansal, L. Kusum and V.V.N. Kishore. 2000. State-of-the-art of anaerobic digestion technology for industrial wastewater treatment. Renew. Sust. Energ. Rev. 4: 135–156. Redwood, M.D. and L.E. Macaskie. 2006. A two stage, two organism process for biohydrogen from glucose. Int. J. Hydrogen Energy 31: 1514–1521. Ren, N.Q., H. Chua, S.Y. Chan, Y.F. Tsang, Y.J. Wang and N. Sin. 2007. Assessing optimal fermentation type for biohydrogen production in continuous flow acidogenic reactors. Biores. Technol. 98: 1774–1780. Ross, B. and H. Strogwald. 1994. Membranes add edge to old technology. Water Qual. Int. 4: 18–20. Roy, S. and D. Das. 2016. Biohythane production from organic wastes: present state of art. Environ. Sci. Pollut. Res. 23: 9361–9410. Roy, S., M. Vishnuvardhan and D. Das. 2014. Improvement of hydrogen production by newly isolated Thermoanaerobacterium thermosaccharolyticum IIT BT-ST1. Int. J. Hydrogen Energy 39: 7541–7552. Sarma, S.J. and J.H. Tay. 2018. Aerobic granulation for future wastewater treatment technology: Challenges ahead. Environ. Sci.: Water Res. Technol. 4: 9–15. Sen, B., J. Arvind, P. Kanmani and C.H. Lay. 2016. State of the art and future concept of food waste fermentation to bioenergy. Renew. Sustain. Energy Rev. 53: 547–557. Shi, X.Y. and H.Q. Yu. 2006. Continuous production of hydrogen from mixed volatile fatty acids with Rhodopseudomonas capsulate Int. J. Hydrogen Energy 31: 1641–1647. Shin, H.S., J.H. Youn and S.H. Kim. 2004. Hydrogen production from food waste in anaerobic mesophilic and thermophilic acidogenesis. Int. J. Hydrogen Energy 29: 1355–1363. Siddiqui, Z., N.J. Horan and M. Salter. 2011. Energy optimisation from co-digested waste using a two-phase process to generate hydrogen and methane. Int. J. Hydrogen Energy 36: 4792–4799. Singh, R. and S. Kumar. 2019. A review on biomethane potential of paddy straw and diverse prospects to enhance its biodigestibility. J. Clean. Prod. 217: 295–307. Singh, R., M. Hans, S. Kumar and Y.K. Yadav. 2021. Potential feedstock for sustainable biogas production and its supply chain management. pp. 147–165. In: Balagurusamy, N. and A.K. Chandel (eds.). Biogas Production. Springer, Cham. Singh, S., A.K. Sudhakaran, P.M. Sarma, S. Subudhi, A.K. Mandal, G. Gandham and B. Lal. 2010. Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments. Int. J. Hydrogen Energy 35: 10645–10654. Sinha, P. and A. Pandey. 2011. An evaluative report and challenges for fermentative bio-hydrogen production. Int. J. Hydrogen Energy 36: 7460–7478. Sivagurunathan, P., G. Kumar, A. Pugazhendhi, G. Zhen, T. Kobayashi and K. Xu. 2017. Biohydrogen production from wastewaters. pp. 197–210. In: Farooq, R. and Z. Ahmad (eds.). Biological Wastewater Treatment and Resource Recovery. Intech Open, U.K. Srikanth, S., S.V. Mohan, M.P. Devi, D. Peri and P.N. Sarma. 2009. Acetate and butyrate as substrates for hydrogen production through photo fermentation: Process optimization and combined performance evaluation. Int. J. Hydrogen Energy 34: 7513–7522. Tang, G., J. Huang, Z. Sun, Q. Tang, C. Yan and G. Liu. 2008. Biohydrogen production from cattle wastewater by enriched anaerobic mixed consortia: Influence of fermentation temperature and pH. J. Biosci. Bioengg. 106: 80–87. Tilley, E., L. Ulrich, C. Lüthi, P. Reymond and C. Zurbrügg. 2016. Compendium of Sanitation Systems and Technologies. Swiss Federal Institute of Aquatic Science and Technology (Eawag), Duebendorf, Switzerland. p. 175. ISBN 978-3-906484-57-0. Ting, C. and D. Lee. 2007. Production of hydrogen and methane from wastewater sludge using anaerobic fermentation. Int. J. Hydrogen Energy 32: 677–682. Van Ginkel, S.W., S.E. Oh and B.E. Logan. 2005. Biohydrogen gas production from food processing and domestic wastewater. Int. J. Hydrogen Energy 30: 1535–1542. Vazquez, G.D., C.B.C. Navarro, L.M.R. Colunga, A.D. Rodriguez and E.A. Flores. 2009. Continuous biohydrogen product using cheese whey: Improving the hydrogen production rate. Int. J. Hydrogen Energy 34: 4296–42304.

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5 Potential of Extremophilic Algae for the Synthesis of Value-added Products Aishwarya Atakkatan,1,# Sandra Innesent,1,# Shreya Padmesh Prajapat,1,# Soumya Pandit 2 and Namita Khanna1,3,*

1. Introduction Life on Earth can be witnessed both within and beyond the human scope of vision and terrain. What may seem as inhabitable, such as the extremely high temperature of the Mushroom Spring at the Yellowstone National Park or the hyper-saline conditions of the Dead Sea, has shown signs of life in remarkable ways. The research surrounding the existence of life in such extreme environments not only focuses on the scope of biotechnological applications but also, extends towards astrobiologythe field of science which explores the origin, evolution, distribution and the future possibility of life on extraterrestrial realms (Capece et al. 2013). The organisms which thrive in such extreme environments are described as “extremophiles”. Studies have found that extremophiles are not just restricted to one domain of life such as Archaea, but span over all the three domains of life, namely; Archaea, Bacteria and Eukarya. In general, extremities tolerated by extremophiles include- (i) temperature (ii) pH (iii) salinity (iv) heavy metal concentration (v) radiation and (vi) CO2 levels. Extremophilic microalgae and cyanobacteria have evolved both morphologically and metabolically to acclimate and adapt to various environmental stress conditions (Varshney et al. 2015). To better understand and characterize these metabolic adaptations and responses, several modern techniques have been employed such as transcriptomics, proteomics, lipidomic and genomic analyses. Extremophilic microalgae have been comprehensively studied for the production of a number of high-value products, such as, biofuels (e.g., bioethanol, biohydrogen production), natural pigments (e.g., carotenoids, phycobilins) (Varshney et al. 2015) secondary metabolites (e.g., vitamins, exopolysaccharides), nutrient supplements (Berthold et al. 2020) (e.g., polyunsaturated fatty acids such as omega-3 and omega-6), bioactive compounds that exhibit anti-bacterial, antifungals, anticancer properties (proteins, extremozymes, extremolytes), bioplastics (e.g., PHB synthesis) (Rahman and Miller 2017).

Department of Biotechnology, Birla Institute of Technology and Science, Pilani, Dubai Campus, Dubai, UAE. Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida-201306, India. 3 Nuclera, 137 Cabridge Science Park, Cambridge CB4 0GD, UK. * Corresponding author: [email protected] # Equal contribution 1 2

Potential of Extremophilic Algae for the Synthesis of Value-added Products 81

Additionally, extremophiles that tolerate more than one stress factor are called “polyextremophiles”. In light of polyextremophilic microalgae, a team of researchers in Brazil proposed Galdieria sulphuraria as a suitable candidate for deriving high-value products such as anti-oxidants, phycocyanins (a phycobiliprotein of significant antimicrobial, antitumor, antiinflammatory and neuroprotective activity) and biofuels. Its capacity to thrive in environments with high heavy metal concentration, low light intensity, high temperatures and acidic environments can be utilized to produce a wide array of bioactive compounds by altering cultivation methods to influence certain pathways (Sydney et al. 2019). Several research groups are focusing on “synthetic polyextremophiles”, wherein, microalgae are genetically engineered to withstand multiple stress factors and/or develop tolerance. Therefore, exploring the diverse population of extremophilic microalgae may help find answers to some of the questions surrounding the definition of life on Earth. It may also foster innovative and efficient ways to tap into the natural resources for procuring various products of commercial importance. Next some of the different stress conditions tolerated by extremophilic microalgae, followed by the adaptation mechanisms adopted to combat various stress factors will be explored. The phylogeny of extremophilic microalgae species and reviewing notable methods of extremophile cultivation will also be analyzed. Lastly, the value-added products obtained from extremophilic microalgae and, a brief insight on synthetic extremophiles and their potential applications will be described.

2. Extremophilic Environment and Metabolic Adaptations Prokaryotic cyanobacteria and eukaryotic extremophilic algae have the ability to thrive under harsh environmental conditions and tolerate various abiotic stresses (Fig. 5.1) such as temperature, pH, light, salt, heavy metal and carbon dioxide. Some of the abiotic stress tolerance mechanisms include—transcriptional regulators and DNA protection/repair, involvement of stress related proteins such as Heat Shock Proteins (HSP), antioxidative enzymes, cellular lipid and carbohydrate accumulation and carotenoid production (Babele et al. 2019). Table 5.1 lists a few examples of extremophilic microalgae and cyanobacteria with respective value-added products obtained upon cultivation.

2.1 Temperature Temperature is one of the vital forces that influence life on Earth. The majority of terrestrial life forms cannot withstand high heat as most structural proteins and enzymes denature at high temperatures while low temperatures render most biological activity stagnant. The temperature extremes on Earth are notably the coldest regions in Antarctica, hot springs, deserts such as the Lut Desert, and the deep-sea hydrothermal vents in the oceans. Quite remarkably, organisms (in our context, microalgae) which tolerate these extremities of temperature have been isolated and are broadly classified as— (i) hyperthermophiles, which perform optimally at temperatures above 80°C; (ii) thermophiles, grow at temperatures above 50°C and (iii) psychrophiles thrive at temperatures 15°C or lower. Although most thermophiles grow between 70–80°C, eukaryotic alga have an upper limit of about 62°C. This still holds true for applications concerning the extraction of value-added products, as cultivation of extremophiles exclude the possibility of contamination by other competitive organisms (Varshney et al. 2015). To combat temperature stress, microalgal species have evolved and developed strategies by altering their pathways and undergoing changes in their structure. 2.1.1 Psychrophiles Psychrophilic microalgae comprise of snow and glacial algae species with most belonging to the phyla-Chlorophyta (order-Chlamydomonadales) and Streptophyta (order-Zygnematales),

82 Extremophiles: Wastewater and Algal Biorefinery

Fig. 5.1. Diagrammatic representation of adaptations employed by eukaryotic extremophilic algae in response to abiotic stress conditions.

Potential of Extremophilic Algae for the Synthesis of Value-added Products 83 Table 5.1. A comprehensive table listing notable examples of extremophilic microalgae and cyanobacteria and the value-added products obtained. Organism Classification

Organism

Growth Condition

Product

Product Yield

Reference

TEMPERATURE STRESS Thermophile

Coelastrella sp. FI69

Temperature: 45–50°C, Mode of nutrition: Photoautotrophic Illumination: 1500–500 µE s−1 m−2

Presence of SFA, MUFA, PUFA and C18:1 (precursor for biodiesel production)

31.43% of C18:1%

(Narayanan et al. 2018)

Psychrophile

Chlamydocapsa spp.

GREEN PHASE Temperature: 10°C, Nutrition: Bold’s basal medium (with 9 mM nitrate), pH: 5.5, Illumination: continuous PAR of 120–220 µmol photons m–2 s–1 RED PHASE Temperature: 10°C, Nutrition: Errötungs medium (with 0.08 mM nitrate), pH: 5.5, Illumination: PAR at 150–300 µmol photons m–2 s–1 (for 24 hours), then, at 800–960 photons m–2 s–1

α–tocopherol (a type of vitamin E)

883 µg/g of freezedried biomass (green phase)

(Leya et al. 2009)

Raphidonema spp.

417 µg/g of freezedried biomass (red phase)

SALINITY STRESS Halotolerant

Scenedesmus sp. IITRIND2

Nutrition: Seawater media and NaCl supplemented BBM growth media, Salinity: Four levels of salinity (sea water or NaCl supplemented)-no salt, 8.75 g/L (quarter strength), 17.5 g/L (half strength), 35 g/L (full strength) Temperature: 25°C, Illumination: 70 μmol photons m−2 s−1 (white fluorescent lamps) Sparging: 2% (v/v) CO2

Biofuel (biodiesel) production

Seawater (17.5 g/L): 31.8% Lipids 35 g/L seawater: 26.9% Lipids

(Arora et al. 2019)

Table 5.1 contd. ...

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...Table 5.1 contd. Organism Classification

Organism

Growth Condition

Product

Product Yield

Reference

Halophile

Spirulina platensis

Nutrition: Zarrouk’s media, Temperature: 25°C

Methylcobalamin

38.5 ± 2 µg (microbiological assay) and 35.7 ± 2 µg (chemiluminescence assay) per 100 g of dried biomass

(Kumudha et al. 2010)

Dunaliella salina CCAP19/18

Nutrition: Artificial Seawater medium, Salinity: 2 M NaCl Illumination: 150 µmol/m²s

Carotenoids

Total carotenoid content (21 days): 8.6 mg L–1, or, 19.11 pg/cell

(Fazeli et al. 2006)

Dunaliella salina SA 134

Temperature: 25 ± 1°C, Salinity: 2 M NaCl Illumination: 12:12 light-dark photoperiod, light intensity of 42.4 µmol m−2 s−1

Lipids

248.33 mg L−1

(Ahmed et al. 2017)

Haematococcus pluvialis

Temperature: 20°C, pH: 6.4, Illumination: 150 µE/m2/s

6.5 mg L–1

(Park and Lee 2001)

20 mg/g dry weight

(Sloth et al. 2006)

38% of the algal biomass

(Chowdhury et al. 2019)

LIGHT STRESS Light stress tolerant

Astaxanthin

pH STRESS Acidophile

Galdieria sulphuraria strain 074 G

Temperature: 42°C, pH: 2, Batch Culture Nutrition: 5:1 Carbon source (glycerol): Nitrogen source (NH4)2SO4 Illumination: 79 µmol photons m−2 s−1

Alkaliphile

Chlorella vulgaris

pH: 9.6–9.8 Lipids periodic sparging of 99.99% pure CO2 Illumination: Low light intensity (60–90 μmol/m2/s) Higher light intensity (240–280 μmol/m2/s) Reactor: 3.5 L Stirred tank reactor

Phycocyanin

Table 5.1 contd. ...

Potential of Extremophilic Algae for the Synthesis of Value-added Products 85 ...Table 5.1 contd. Organism Classification

Organism

Growth Condition

Metallotolerant Acidophile

Desmodesmus sp. MAS1

Nutrition: modified Bold’s basal medium (BBM), originally composed of heavy metals such as Cu, Fe, Mn and Zn at 0.02, 1.0, 0.50 and 0.11 mg L–1, respectively Temperature: 23 ± 1°C, pH: 3.5, Illumination: Continuous (60 μmol m–2 s–1)

Product

Product Yield

Reference

Presence of Fe: 12.8–25.6% dry wt. Presence of Mn: 25.5–35 % dry wt.

(Abinandan et al. 2019)

HEAVY METAL TOLERANCE

Heterochlorella sp. MAS3

Fatty Acid Methyl Esters (FAME) (Biodiesel)

Presence of Fe: 12.8–25.6% dry wt. Presence of Mn: 22.9–32.9 % dry wt.

POLYEXTREMOPHILES Thermophile CO2 tolerant

Asterarcys quadricellulare

Nutrition: Bold’s Basal medium, Temperature: 37°C, Illumination: Light intensity of 250 μmol photons m−2 s−1; 14:10 light-dark cycle, Flue gas concentration: 5% CO2 + 80 ppm NO

Lipids

44.3% of dry cell weight

(Varshney et al. 2018)

Thermophile Light stress tolerant

Anabaena sp. ATCC 33047

Batch Culture -Temperature: 40–45°C* CO2 concentration: 6% (v/v) Illumination: 460 µE–2s–1 -Temperature: 40°C CO2 concentration: 6% (v/v) Illumination: 920–1840 µE–2s–1

Exopolysaccharides

12–15 g L–1*

(Moreno et al. 1998)

10–12 g L–1*

* Approximate data deduced from the findings of the research paper.

respectively. They are known to flourish in the liquid water film between ice crystals and melted snow. Based on the pigment composition and population density, the discoloration of snow and ice occur. The distribution of these algal species is limited, depending largely on snow and ice cover, ecological and climatic conditions (Hoham and Remias 2020). Two examples of notable psychrophilic eukaryotic microalga are—Chlamydomonas nivalis, a snow alga that flourishes in the liquid water film associated with melted snow and Mesotaenium berggrenii, a glacial alga known to dominate the Akken Glacier ice cover in the Russian Altai Mountains and other glacial areas in the Northern Hemisphere (Hoham and Remias 2020, Malavasi et al. 2020).

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The ability of psychrophiles to survive at low temperatures and tolerate the increased water viscosity can be attributed to specific adaptations such as enzymes having superior catalytic efficiency at low temperatures and, higher accumulation of polyunsaturated fatty acids (PUFAs) in their membrane lipids to maintain membrane fluidity (Varshney et al. 2015). Transcriptomic data from psychrophilic algae in the Antarctica reveal complex adaptive modifications in photosynthesis, marked by a stable photosynthetic apparatus, multiple antioxidant strategies as well as improved membrane fluidity (Zhang et al. 2019). Similarly, some algae such as the Arctic alga Chlorella-Arc and Tetraselmis sp., acclimate to the cold stress by the regulation of carbon between sugars and lipids (Shin et al. 2016, Song et al. 2020). The genes for PUFA synthesis enzymes, transport proteins and other chaperon proteins of the Antarctic ice alga Chlamydomonas sp. ICE-L, were found to have great similarity with those of the Antarctic bacteria. Hence, suggesting the occurrence of horizontal gene transfer between the algae and its symbiotic microbes (Liu et al. 2016). 2.1.2 Thermophiles Thermophilic microalgae are native to habitats with elevated temperatures such as hot springs, deep-sea hydrothermal vents, geysers and volcanic environments, to name a few. Amongst these, the Yellowstone National Park (Wyoming, USA) incorporates the major and extensively studied hot springs, with phototrophic microbiota flourishing at temperatures around 74°C. Thermophilic microalgae and cyanobacteria possess incredible properties that enable them to grow at high temperatures while concomitantly utilizing sunlight and CO2. Thus, the phototrophic nature of microalgae can be effectively utilized to capture CO2 and convert it to useful algal biomass and lipids for biofuel or bio-electricity production (Patel et al. 2019). Under heat stress, some microalgae adjust membrane fluidity via changing the fatty acid saturation level by the integration of de novo-synthesized Saturated Fatty Acids (SFA’s) into the membrane lipids (Légeret et al. 2016). The structure of fatty acids is controlled with the help of enzymes primarily belonging to the family of desaturases and elongases. Heat stress leads to the denaturation and aggregation of unfolded proteins, which has been shown to lead to the expression and upregulation of heat shock proteins to re-establish the protein homeostasis in algae (Barati et al. 2019). A microarray study on the red alga, Cyanidioschyzon merolae native to acidic hot springs, revealed that its resistance to heat shock up to 63°C was due to the upregulation of two small Heat Shock Proteins (sHSP) during the stress (Kobayashi et al. 2014). In addition to the upregulation of genes encoding heat shock proteins, heat stress can also induce expression of genes related to redox homeostasis to overcome toxic reactive oxygen species. A study on the halophilic green alga Dunaliella bardawil subjected to heat stress showed that the cells tended to shift from aerobic to glycolytic metabolism for energy production to increase survival chances under heat stress (Liang et al. 2020). Inhibition of photosynthesis and other non-essential metabolic processes are some additional mechanisms employed by Pyropia haitanensis to high-temperature stress (Xu et al. 2014). Therefore, primarily, in algae, temperature stress induces modifications in the fatty acid composition of their membrane to maintain homeostasis. Further, regulation of carbohydrate metabolism, production of antioxidants, maintenance of photosynthetic apparatus and expression of heat shock proteins are some of the strategies adopted by the algae to withstand and overcome the temperature stress.

2.2 pH Tolerance Organisms that grow well at the extremes of pH are categorized as—(i) acidophiles, which thrive optimally below pH 3, and (ii) alkaliphiles, those flourishing well at pH greater than 9. Acidophiles and alkaliphiles studied so far represent all domains of life. Further, organisms that grow optimally

Potential of Extremophilic Algae for the Synthesis of Value-added Products 87

at neutral pH, called neutrophiles, have exhibited coping mechanisms that allow sub-optimal growth at the extremes of pH- these species are typically termed as “acido-tolerant” or “alkali-tolerant”, respectively (Capece et al. 2013). 2.2.1 Acidophiles Highly acidic environments are a consequence of both natural and anthropogenic activities and, are havens to a diverse range of acidophiles including microalgae. Usually, in regions of acid drainage, many conditions co-occur, such as high temperatures, heavy metal concentrations and salinity. Thus, most acidophilic microorganisms are adapted to multiple stresses (Malavasi et al. 2020). A notable acidophilic microalga, Coccomyxa onubensis, which was first isolated from the acidic waters of a river in Spain (Tinto River), showed considerable lipid accumulation, anti-oxidant activity (RuizDomínguez et al. 2015) and anti-microbial activity (Navarro et al. 2017). Acidophilic microalgae such as, Chlamydomonas acidophila LAFIC-004, have also been studied for efficient nutrient removal from secondary effluents of wastewater plants at high CO2 concentrations (Neves et al. 2019). Possible mechanisms employed by neutrophilic algae to evolve and adapt themselves to acidic environments, suggest the upregulation of the genes encoding for heat shock proteins and plasma membrane proton pump (H+-ATPase). Certain key enzymes such as malate synthase and isocitrate of the glyoxylate cycle which are involved in the conversion of acetyl-CoA to succinate were lost. Thereby indicating that, fermentation pathways which contribute to the acidification of the cytosol with the production of acetate and succinate were inhibited. These mechanisms were studied by performing a comparative genome and transcriptome analysis between the acidophilic green alga C. eustigma and neutrophilic alga C. reinhardtii (Hirooka et al. 2017). Another study on polar lipids of the extremophile red alga, Galdieria sulphuraria, found in hot and cold sulfur springs with pH values ranging between 0–4, led to the identification of about 14 classes of lipids including regioisomers. The low pH prevented the dissociation of phosphoric acid and led to phosphorous starvation. This condition was overcome by the algae replacing the cellular phospholipids with betaine lipids (Vítová et al. 2016). When algae are subjected to heavy metal stress as a consequence of acidic environments, such as the extremophilic green algae Coccomyxa melkonianii SCCA 048, the accumulation of lipids was employed to cope with acidic stress. The strain even exhibited phenotypic plasticity (Soru et al. 2019). 2.2.2 Alkaliphiles Alkaliphiles have been isolated not only from naturally occurring alkaline environments such as soda deserts, saline soda soils and soda lakes such as the Soap Lake in Washington, USA, but also from neutral environments, alkali-thermal hot springs, sewage, oceanic bodies and alkaline hydrothermal systems. There are also numerous highly alkaline environments which are a consequence of anthropogenic activities such as drainages, ponds and lakes around steel, soda and bauxite processing sites. These areas of high alkalinity also include wide-ranging temperatures and usually have moderate to high concentrations of dissolved salts (Capece et al. 2013, Mamo and Mattiasson 2020). Often, alkaline environments contain high concentrations of bicarbonate ion. Given the existing knowledge of “Carbon Concentrating Mechanisms” (CCMs), in microalgae, it is quite evident that soda lakes are photosynthetically productive. Diatoms are exclusively suited for survival in alkaline environments due to their unique C4 metabolism which allows them to convert bicarbonate to CO2 via a C4 organic acid. The CO2 thus generated is utilized by RuBisCO in the carbon-fixation cycle (Beardall and Raven 2020). An alkaliphilic diatom, Fistulifera sp. 154-3, isolated from Lake Okeechobee in South Florida, was found capable of producing high-value lipid compounds, such as ω-3, ω-7 and palmitic acid (Berthold et al. 2020).

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Similarly, Chlorella vulgaris, isolated from a saline water body, has demonstrated promising potential for fuel production from saline wastewater (Chowdhury et al. 2019). Quite recently, an alkaliphilic chlorophyta, Picocystis sp., isolated from alkaline residual water from a sewage pond, showed high tolerance to, and high removal efficiency of Bisphenol A (BPA) via biotransformation/ biodegradation. This aspect provides a promising potential in the purification of water from BPA contaminants using algal-based systems (Ouada et al. 2018). High pH is also known to induce maximum accumulation of lipids and changes in structure of microalgae such as, C. melkonianii. Extreme pH conditions cause accumulation of different lipids. The FAME analysis has led to the identification of different compounds which can then be exploited for several biotechnological applications in various industries (Soru et al. 2019).

2.3 Salinity Extremophiles that flourish optimally in hypersaline environments are called halophiles. Halophiles may be classified further as—(i) Slight halophiles, which tolerate up to 5% (w/v) NaCl in environments; (ii) Moderate halophiles, which thrive in environments containing up to 20% salt; and, (iii) Extreme halophiles, which require saturated environments of about 20–30% NaCl for their survival. Meanwhile, halotolerant species typically survive in environments with only minute amounts of salt, with an upper limit of 5% (w/v) NaCl. However, the average ocean salinity which is around 3.5% NaCl, is much lower than these concentrations. Both prokaryotic and eukaryotic halophiles have been identified and isolated from a range of diverse environments such as seawater brine solutions, ancient rock salts, saline marshes and salt lakes including, the Great Salt Lake (USA), the Dead Sea and Lake Magadi (Kenya) (Capece et al. 2013). Dunaliella salina, first isolated from evaporation ponds in Australia, is a notable halophilic microalgal species that has been extensively studied for various biotechnological applications such as biofuels, algal biomass, heavy metal remediation from contaminated water, production of bio-active compounds (anti-microbials) and essential value-added compounds such as β-carotene (Krishnakumar et al. 2013, Ranjbar et al. 2015, Solovchenko et al. 2015). These algal species often overcome salinity stress by accumulation of antioxidants and increased levels of protective enzymes which help in detoxifying and eliminating the reactive oxygen species. This can also be further mediated through the intracellular accumulation of glycerol and is proportional to the extracellular salt concentration (Tammam et al. 2011, Farghl et al. 2015). Glycerol acts as an important osmoregulatory solute which is inert in nature and produced by several algae under salt stress such as Chlamydomonas sp., Dunaliella sp., Chlorella sp. (Shetty et al. 2019). Studies indicate that algae as well as cyanobacteria overcome salt stress by enhancing the total lipid content, production of carotenoids, sugars and other monosaccharides as well as other cell proteins (Farghl et al. 2015, Pietryczuk et al. 2014, Tammam et al. 2011, Verma et al. 2019). Accumulation of solutes, such as, glucosyl glycerol, trehalose and glycine betaine also acts as a basic mechanism for salinity adaptation in the blue-green algae (Rezayian et al. 2019). Similarly, it was also observed that prokaryotic cyanobacteria accumulated phenylpropanoid which helped in enhancing the antioxidative property and provided tolerance to the varying salt concentrations (Singh et al. 2014). Transcriptome analyses show the role of genes under salinity stress and the way they affect the functioning of different pathways in microalgae. Under salt stress, the number of upregulated genes were much higher as compared to those which were downregulated in the chlorophyte C. vulgaris. It was observed that the calcium signaling pathway in the cytoplasm which is responsible for regulation of homeostasis was highly upregulated. On the other hand, the genes involved in the photosystem I of light harvesting complex were downregulated (Abdellaoui et al. 2019). Salt stress can also upregulate the expression of key genes involved in photosynthesis and glycerol metabolism which help in the survival of halophiles such as Dunaliella salina in hypersaline environments (Li et al. 2019).

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Varying habitat and salt concentration can produce different levels of stress response markers as suggested by a study involving freshwater and marine Chlorella sp., C. salina and C. vulgaris, respectively (Farghl et al. 2015). Other mechanisms to overcome salt stress include enhancement of glycolysis metabolism to reduce carbohydrate accumulation, use of acetate, as an alternative source of energy, to overcome the photosynthetic impairment and maintenance of lipid homeostasis by regulating phosphatidic acid (Wang et al. 2018). Proteomics and qRT-PCR analysis of Arthrospira plantensis-YZ under salt stress identified the expression of proteins involved in about 31 metabolic pathways such as photosynthesis, metabolism of glucose, cysteine and methionine, fatty acids, glutathione, etc. Stress Response Proteins (SRPs), heat shock proteins and transporter proteins were also identified which may be responsible for salt stress acclimation (Wang et al. 2013).

2.4 Heavy Metal Tolerance Excessive amounts of hazardous substances is leached into surface waters daily as a consequence of rapid industrialization and urbanization. Amongst these pollutants, heavy metals impose the most dangerous risk to many life-forms due to their toxic nature, resistance to biodegradation, persistence and biomagnification across the food chain. Despite the negative consequences heavy metals impose on the environment, various manufacturing industries such as textiles, smelting, plating, mining, plastics, etc., continue to use them in numerous technological processes. Therefore, elimination of heavy metals from wastewater is highly essential to reduce their negative effects on the environment (Bulgariu and Gavrilescu 2015). Algal-based systems provide a cost-efficient and environment-friendly alternative to conventional techniques owing to their photosynthetic capability and fast growth rates under extreme conditions such as nutrient deprivation, temperature stress, high heavy metal concentration and others (Leong and Chang 2020). Microalgae are known to take up metals by the process of bio-accumulation and bio-sorption. Dried algal biomass of C. vulgaris and Scenedesmus sp. for instance, have been studied for the remediation of the toxic hexavalent Chromium, Cr (VI), via biosorption. The advantage of using non-living algal biomass for metal remediation is the possibility of reusing the algal biomass after HM desorption, thereby eliminating the need for additional growth media (Sibi 2016, Pradhan et al. 2019, Pavithra et al. 2020). Different molecular mechanisms have been adapted by microalgae to cope with heavy metal stress. Generally, these include the production of antioxidants such as superoxide dismutase (SOD), peroxidase, catalase and glutathione reductase in the algae to maintain oxidative stress and metal homeostasis. Chlorella vulgaris has been shown to adapt to cadmium by changing the activity of these enzymes and thereby overcoming the stress (Cheng et al. 2016). Metal stress is also known to induce the expression of enzymes such as, phytochelatin synthase enzyme (involved in the production of phytochelatins glutathione and cysteine), which help in binding, chelating and ultimately, the detoxification of heavy metals such as lead. Phytochelatin synthase enzyme expression was increased in Acutodesmus obliquus cells in addition to the enhancement of the xanthophyll cycle as well as, certain changes in the hormonal homeostasis when subjected to lead stress in varying concentrations (Piotrowska-Niczyporuk et al. 2017). The oxidative stress caused by a mixture of heavy metals such as Cu, Cd and Zn, is often overcome by strategies that include- increased expression of metallothionein, lipid content, lipid peroxidation as well as, peroxidase activity (Jamers et al. 2013). It has been observed that metal solubility is greatly enhanced in highly acidic environments. As described by Sydney et al. 2019, the polyextremophilic microalgae, Galdieria sulphuraria, is of great interest in heavy metal removal from wastewaters along with the production of other highvalue products (Varshney et al. 2015).

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2.5 Solar and Ionizing Radiation Tolerance Our planet’s ozone layer and magnetic field protect the terrestrial life-forms from enormous amounts of cosmic radiation. Ultraviolet (UV) and ionizing radiations pose risks to terrestrial life, especially the short-wavelength UVB and UVC that cause DNA damage by dimerization of pyrimidines (Capece et al. 2013). Microorganisms thriving in regions with strong irradiation have developed a range of physiological strategies to tolerate stressful growth conditions including ROS scavenging, repairing damage due to excessive UVB and PAR (Photosynthetically Active Radiation), and many others. (Varshney et al. 2015). These organisms occupy a broad range of environmental niches where UV radiations are high, such as higher altitudes (mountain ranges) and open fields (Gabani and Singh 2013). Rivas et al. (2016) discussed the photosynthetic efficiency of Chlorella sp., isolated from a microalgal community, thriving on snow, in Antarctica. The organism exhibited resistance to solar radiation when exposed to higher temperatures, along with the recovery of photosynthetic activity after inhibition by UV light. In addition to growing well under the extremes of PAR and UVB, certain algae and cyanobacteria might also be reliable sources of antioxidants and photo-protective compounds, which are usually by-products of various stress adaptation mechanisms. These adaptation mechanisms have been, in general, associated with PS II and assessed with the help of Non-Photochemical Quenching (NPQ). The NPQ study on different green microalga revealed differences which may be due to variations in their molecular machineries and the organism’s response to stress (Finazzi and Minagawa 2014). Under high light conditions, in algae, such as Haemotococcus pluvialis, in addition to synthesis of astaxanthin other morphological changes occur, including differentiation of the cell into non-motile red cyst, falling of the flagella and formation of thick secondary cell wall. It was also seen that initially the high light stress inhibited the photosynthetic activities of the cell, however gradually, the activity was regained in concurrence with the accumulation of astaxanthin (Hu et al. 2019). Under high light conditions, genes encoding the production of antioxidants are upregulated to scavenge the ROS from the chloroplasts (Pospíšil 2016, Erickson et al. 2015, Xiong et al. 2015, Hu et al. 2019). Two genes encoding for enzymes in photorespiratory pathway, glycolate dehydrogenase and malate synthase were also seen to be highly upregulated in response to the high light stress. These pathways are known to contribute to metabolites which are involved in nitrogen assimilation which may contribute in photo-acclimation to the stress (Davis et al. 2013). Some microalgae species such as C. reinhardtii also exhibit negative chemotaxis and move away from high light conditions. When chemotaxis is not possible, the alga switches to regulating the Light Harvesting Complexes (LHC) and rearranging the antenna in a short time scale. This leads to alterations in the electron transport, which involves dissipation and balancing of the excitation with the one that is manageable by the cell’s capacity. High light conditions cause the upregulation and expression of a Light Harvesting Complex Stress Related (LHCSR) in Chlamydomonas sp. ICE-L which is known to play an important role in photo-protection (Mou et al. 2012). In case of longer duration of exposure to high light, changes in PS II and gene expression occur so as to help the alga acclimate to the stress (Erickson et al. 2015). Certain DNA-binding proteins (Dps) such as those found in the cyanobacterium, Nostoc punctiform, help in acclimating to high light intensities via osmotic stress protection and maintenance of iron homeostasis (Li et al. 2018, Moparthi et al. 2016). In addition, there has been an increased generation of radioactive wastes in the environment owing to the extensive use of radioactive elements and compounds for nuclear energy, in medicine, research and industry. Nuclear power plant disasters in the past such as the 1986 Chernobyl disaster and the 2011 Fukushima Daiichi disaster, have also contributed to the presence of radionuclides and radioisotopes in the environment (Gabani and Singh 2013).

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A novel extremophilic microalgal species, Coccomyxa actinabiotis sp., isolated from spent nuclear fuels in the cooling pool of a nuclear reactor in France, showed resistance to the radiative, nutritive and metallic stress continuously prevalent there. Laboratory controlled irradiation studies revealed that the species could tolerate about 2000 times the dose of intense gamma-radiation lethal to humans via accumulation of radionuclides; making it an ideal choice for remediation of radioactively-contaminated environments and their decontamination (Rivasseau et al. 2016). Similarly, cyanobacteria such as Nostoc commune, Scytonema javanicum and Stigonema ocellatum, have also been studied to show significant removal of radioactive iodine, 125I (Fukuda et al. 2014).

2.6 CO2 2.6.1 Limited CO2 and CCMs The oxygenation of the biosphere has been increasing for the past 2.4 billion years while, CO2 levels have seen a downward trend, having a minimal at glacial regions. Almost all autotrophs, including cyanobacteria and eukaryotic microalgae, depend on the Calvin Cycle and the enzyme, RuBisCO (Ribulose-1,5-bisphopshate carboxylase oxygenase) for the conversion of inorganic carbon (CI ) to organic matter. The following gradual drop in CO2 levels and rise in oxygen has resulted in O2/CO2 competition, restricting net carbon fixation. Moreover, RuBisCO evolved at a time when the CO2 levels in the atmosphere were much higher than it is today and hence, autotrophic organisms possess different forms of the enzyme. The different forms of RuBisCO have varying degrees of affinity towards CO2 as, cyanobacterial versions possess low affinity while those present in green algae have comparatively higher affinity for CO2. Further, an inverse-relation between CCMs and CO2 specificity has been observed-low CO2 usually perpetuate higher CCMs (Beardall and Raven 2020). Therefore, to better concentrate CO2 at the active sites of RuBisCO, cyanobacteria and algae such as, Chlamydomonas reinhardtii, maintain their photosynthetic activity with the help of photorespiration or by adopting CO2—Concentrating Mechanisms (CCMs). Photorespiration involves recycling of phosphoglycolate that is formed as a result of the oxygenase reaction, catalyzed by RuBisCO (Moroney et al. 2013). The CCM helps in compensating for the decreased activity of the RuBisCO enzyme in the Calvin cycle (Beardall and Raven 2020). This is mediated with the help of active transport of CO2 to ensure sufficient internal concentration of carbon dioxide (Ci) in the organism (Fan et al. 2016, Wang et al. 2016). Carbon concentrating mechanism involves different routes to overcome CO2 limitation. A study on the eukaryotic green alga C. reinhardtii revealed that this involves the use of inorganic carbon transporters to increase the Ci within the cells. The pyrenoid structure, located in the chloroplast is rich in enzyme RuBisCO and is analogous to the carboxysome present in cyanobacteria. It acts as a site of increased Ci concentration. An important enzyme, carbonic anhydrase helps in the conversion of the accumulated bicarbonate to CO2 (Jungnick et al. 2014). The bicarbonate transporters help in accumulation of HCO3– within the cytoplasm, which is then converted to CO2 (Jungnick et al. 2014, Moroney et al. 2013, Raven et al. 2020). Low CO2 concentration also induces the production of saturated fatty acids (Fan et al. 2016). CO2 concentrations vary greatly within saline environments due to biological activity such as a diatom bloom. CO2 levels in the surface water of the Western Antarctic Peninsula dropped from 480 to 80 µatm during a large diatom spring bloom. CCM regulation by the dominant phytoplankton species was observed in response to CO2 concentrations during and after the bloom, which maintained near saturation of RuBisCO by CO2 throughout the season (Kranz et al. 2015). The green alga Dunaliella salina exhibited an enhanced ability to utilize low levels of CO2 via an extracellular carbonic anhydrase whose activity was heightened in hyper-saline waters, indicating the possibility of a CCM (Booth and Beardall 1991).

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2.6.2 Elevated CO2 Levels Owing to anthropogenic activities such as industrialization and urbanization, atmospheric CO2 levels have been on a steady rise, impacting global climate. To sequester the large amounts of CO2, many algal-based systems have been proposed. Duarte et al. (2017) described the CO2 biofixation capacity of the microalga Chlorella fusca LEB 111 and, the cyanobacteria Spirulina sp. LEB 18 from coal flue gas, isolated from coal power plants in Brazil. The microalgal strain showed a higher CO2 biofixation rate as compared to cyanobacteria, Sprirulina sp. thereby, implying its potential as an alternative to reduce flue gas emission along with the generation of useful algal biomass. A study in India investigated the CO2 tolerance of the microalga Scenedesmus dimorphus, isolated from freshwater bodies, which showed potential for simultaneous CO2 sequestration and PUFA-rich lipid production with tolerances to a wide range of pH and salinity levels (Vidyashankar et al. 2013). In Japan, two Micractinium sp. microalgal strains, isolated from freshwater reservoirs, were found to be able to grow at 30–80% CO2 (China and Fujii 2018). Under CO2-rich conditions, CO2 tolerance in microalgae such as Desmodesmus sp., is achieved with the help of pH homeostasis, rapid acclimation of the photosynthetic apparatus and the shutdown of the CCM mechanism (Solovchenko et al. 2015). Some algal species also exhibit an increase in the overall biomass, carbon, carotenoid as well as lipid content. Studies also record an increase in polyunsaturated fatty acid (PUFA) content with palmitic and oleic acids constituting a major proportion of PUFAs (Fan et al. 2016, Swarnalatha et al. 2015). An increase in the availability of free CO2 resulted in decreased activity of carbonic anhydrase (Swarnalatha et al. 2015). Increasing CO2 also affected the characteristics of the extracellular polymeric substance and induced the production of humic substances in the green alga Scenedesmus acuminatus (Li et al. 2016). High concentrations of CO2 also induce other regulatory mechanisms that include-regulation of the transporters responsible for carbon uptake, induction of photosynthesis, carbon fixation and glycolysis. An upregulation of photosynthetic machinery such as photosystem reaction centers, cytochromes, phycobilisomes and downregulation of proteins involved in photo-protection and redox maintenance (Mehta et al. 2019).

3. Phylogenetic Analysis of Extremophilic Microalgae Here the evolutionary relationships between Bacteria, Archaea and Algae, employing a phylogenetic tree (Fig. 5.2) derived from the Maximum Likelihood approach were analyzed. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version X (Kumar et al 2018). The tree was constructed from the alignment of 16S rDNA sequences of prokaryotic organisms and 16S rDNA plastid sequences of eukaryotic organisms obtained from the GenBank. The sequences were aligned using MUSCLE algorithm and the resulting alignment gaps were autofilled. Neighborjoin and BioNJ algorithms were applied to a matrix of pairwise distances, estimated by Tamurai Nei Model. Finally, the initial tree for heuristic search was selected by considering the topology with the highest log likelihood value. Microalgae are a diverse group of unicellular, photosynthetic microorganisms, found in a variety of habitats from freezing polar deserts to marine water bodies. The evolutionary history of microalgae has been modulated by three phenomenal forces: endosymbiosis, vertical and horizontal gene transfer (Hopes and Mock 2015). These factors lay the underlying basis for the spectrum of characteristics portrayed by microalgae. The extremophilic characteristics in some microalgae trace their evolutionary journey along the direction of the Bacterial and Archaean kingdoms. The microalgae act as a bridge between the prokaryotic and eukaryotic kingdoms. Hence, their evolutionary history speculates the interest of many. Phylogenetic trees using specific conserved molecular markers can help identify the deviatory paths taken by organisms to reach their current taxonomical destination. The most commonly used

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Fig. 5.2. Phylogenetic relationship of extremophiles.

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molecular marker for determining phylogenetic relationships between organisms is the small subunit rDNA sequences. The small subunit rDNA sequences are conserved enough to enable sequence alignment of varied species yet variable enough to resolve closely related species (Bhattacharya 1997). Bacteria are the oldest living forms present on Earth. They have witnessed all kinds of conditions throughout the dramatic development of Earth. Bacteria are known to have ascended directly from the last common ancestor. Adaptation to different environments has diversified the organisms under this group. However, existing extremophilic bacteria can point out evolutionary pathways for several species. Archaea are the leading group of organisms that display extremophilic traits. These organisms are known to inhabit a wide variety of habitats from hyperthermophilic environments to glaciers. These organisms share characteristics with both Eukarya and Bacteria. Analysis of universal rooted trees have demonstrated Archaea and Eukarya as sister lineages with a recent last common ancestor (Gribaldo and Brochier-Armanet 2006). Thus, the development of extremophilic characteristics in algae may have connections to Archaean origins. Prokaryotic algae or cyanobacteria are one of the oldest living forms on Earth, predicted to have originated around 2700–3500 million years ago (Varshney et al. 2015). The formation of an oxygenic atmosphere is credited to the photosynthetic activity of cyanobacteria (Shestakov and Karbysheva 2017). Cyanobacteria have been argued to have originated from either local Archean settlements or shallow warm water bodies during the Archean era. The Early Archean atmosphere consisted of high amounts of gases such as H2, CO2, N2 and methane (Shestakov and Karbysheva 2017) and held high-temperature environments (Sleep 2010) suitable to sustain thermophiles. Cyanobacteria in shallow warm water bodies survived under anoxygenic conditions and eventually replaced methane-dominant environment with oxygen (Shestakov and Karbysheva 2017). This replacement lowered the prevailing greenhouse effect and paved the way for cooler glaciations in the early Proterozoic era. The resultant climatic changes led to the development of new adaptation characteristics in cyanobacteria and other organisms. The development of extremophilic microalgae can be understood better by tracing the origins of algae. Evolutionary derivation of algae stems from polyphyletic origins (Bhattacharya 1997). The incipient occurrence of algae is determined to be in the early Proterozoic era (2500 million years ago). The first common ancestor of Archaeplastida (red algae, green algae, glaucophytes and land plants) existed around 1.9 billion years ago (Sánchez-Baracaldo et al. 2017). The early Proterozoic environment was characterized by cold glaciations and steadily increasing oxygen content in the atmosphere. One of the major forces responsible for the inclusion of a diverse variety of organisms within microalgae is endosymbiosis. Endosymbiosis is a form of symbiotic survival mechanism wherein one organism resides within another. This evolutionary force is responsible for the presence of plastid in all photosynthetic eukaryotic organisms. The position of plastid is either taken up by cyanobacterium in primary endosymbionts or an alga in secondary symbionts (Heimann and Huerlimann 2015). Also, Horizontal Gene Transfer (HGT) from diverse species played an essential role in the evolutionary development of algae. Gene transfer from the endosymbiont and horizontal gene transfer to the host genome has been proven to shape algal metabolic processes (Hopes and Mock 2015). For example, diatoms are proved to have metabolic processes shaped from bacterial, green and red algae and heterotrophic host origins (Hopes and Mock 2015). In addition, the red alga, Galdiera sulphuraria, has shown the presence of bacterial and archaean genes within its genome (Hopes and Mock 2015). Such evolutionary mechanisms may be responsible for the development of extremophilic characteristics in microalgae. In the presented phylogenetic tree (Fig. 5.2), the 16S rDNA sequences of extremophilic algal have been represented along with extremophilic bacteria and archaea. Extremophilic traits have been demonstrated in green and red microalgae. Phylogenetic analyses depict red algae and

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green algae, formed from a first endosymbiotic event involving cyanobacteria, as sister groups. Extremophilic algal groups with traces of secondary endosymbiotic events, Euglenoidea and Heterokonts (diatoms), have also been analyzed to deduce relationships. Euglenoids are derived from the secondary endosymbiosis of chlorophytes (Heimann and Huerlimann 2015). As depicted in the phylogenetic tree, extremophilic Euglenoid representatives, Euglena mutabilis, and Euglena gracilis are linked to the green algal class, Trebouxiphyceae. Heterokonts, excluding some diatom species, have not depicted special extremophilic representatives (Varshney et al. 2015). Heterokontophytes show traces of secondary endosymbiosis involving rhodophytes (red algae) (Heimann and Huerlimann 2015). The phylogenetic tree shows the linkage between diatoms and the red algae, Cyanidiales. A recurring trend is observed in the emergence of extremophilic traits in the phylogenetic tree. Organisms with extremophilic traits such as thermophilicity, metal tolerance and CO2 tolerance emerged before other extremophilic organisms. This evolutionary development is at par with the development of climatic changes in the Early Earth from extreme anoxygenic, thermophilic conditions in the Archean era to the cold psychrophilic conditions of glaciers in the Proterozoic era. This argument is also supported by the fact that a thermophilic organism is considered to be the last common ancestor of life (Chakravorty et al. 2012). However, photosynthetic organisms have not shown survival at temperatures over 75°C owing to chlorophyll instability (Varshney et al. 2015). On the other hand, cold biomes are often additionally exposed to high ultraviolet radiation, low nutrient conditions and high salt concentrations in water bodies. Marine psychrophilic organisms are often additionally equipped with halophilic and light stress-tolerant traits (Hoover and Pikuta 2009). In short, microalgae show polyphyletic origins and one of the oldest eukaryotes to inhabit Earth. The presence of extremophilic traits in microalgae is a result of adaptation and genetic material inherited from several organisms.

4. Cultivation of Microalgae Extremophilic algae are perceived as the future platform for manufacturing a wide array of valueadded products. An essential technology required to sustain the growth of the microalgae industry is their economical large-scale cultivation of microalgae. The cultivation of microalgae depends on numerous factors such as, (i) nutrient availability (carbon, nitrogen and phosphorus) (ii) pH (iii) light intensity and supply (iv) temperature (v) salinity and (vi) agitation control. An optimal supply of the above-mentioned requirements ensures successful cultivation of microalgae for biotechnological applications. The ability of extremophiles to overcome harsh culture conditions such as high temperature, extreme pH and high light intensity, is advantageous for large-scale cultivation as these stress conditions not only induce the production of a variety of value-added products, but also, eliminate concerns of contamination from mesophilic species. The polyextremophilic microalga, Galdieria sulphuraria and the red alga, Cyanidioschyzon merolae, which exhibit growth at pH 1.0–2.0, and the alkaliphilic microalga, Chlorella sorokiniana, that grows at pH 11.0–12.0, are notable examples for the cultivation of extremophiles in obtaining value-added products. The extremophilic species can be cultured in any mode of cultivation- autotrophic, mixotrophic or heterotrophic (Rashid et al. 2019). To achieve commercially and economically viable production of algal biomass and high-value products, suitable and innovative operating designs of microalgae cultivation system are necessary (Yen et al. 2019). In general, large-scale microalgae cultivation are carried out in either open systems such as, raceways, tanks and ponds or closed systems such as photobioreactors (PBRs). Sometimes, a combination of a closed and an open system, known as a hybrid system, may be applied to accomplish high biomass production and nutrient removal (e.g., from wastewater) (Razzak et al. 2017).

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4.1 Open Systems Open pond systems are the most commonly used extremophile cultivation systems for commercialscale applications. Characteristically, the open pond cultivation technique is used for cultivation of all variants of extremophiles except temperature. Being an open-ended system, the maintenance of extreme high or low temperatures is challenging. Open pond systems usually comprise of shallow, circular ponds, tanks and raceway ponds. The positive aspect of open pond systems is the ease of construction and operation when compared to closed systems. Open ponds and raceways, though light-limited at greater depths, are exposed to high light intensity, including UV radiation at the surface. Thus, microalgae acclimated to high light stress are suitable for cultivation in open systems as they often produce value-added products such as astaxanthin and other anti-oxidants which help overcome light stress (Davis et al. 2013, Hu et al. 2019). Simple open ponds have a large rotating mixer installed at the center which ensures the algal biomass remains in suspension along with the provision of a constant supply of nutrition and aeration. Whereas, raceway ponds consist of a racetrack-like set-up driven by paddle wheels (Fig. 5.3), which provide the driving force for liquid flow, keeping the microalgae in a suspended state, and near the surface for ample exposure to sunlight or other light sources. Most raceway ponds are operated at a water depth of 15–20 cm in a continuous mode with continual CO2 and nutrient feed supplied to the system, while the microalgae culture is removed for harvesting at the end of the raceway (Yen et al. 2019). Further, open systems are ideally suitable for bio-sequestration of CO2 especially when the algal species is adapted to high light conditions and fixation of high concentrations of CO2 through CCMs. Quite often, inorganic carbon can get depleted quickly in dense cultures, which limits photosynthesis. CCMs, thus, help most microalgae and cyanobacteria to overcome the limitation of carbon assimilation (Varshney et al. 2015). Ramanan et al. (2010) demonstrated that miniraceway pond cultivation of the microalgae, Chlorella sp. and the alkaliphilic cyanobacterium, Spirulina platensis, increased CO2 fixation by 46 and 39% mean fixation efficiency, respectively, under 10% (v/v) CO2 input. However, the productivity and generation of algal biomass is limited largely due to the poor utility of light, evaporative losses, low gas transfer rates due to insufficient mixing, CO2 losses to the atmosphere and the requirement of large land areas (Yen et al. 2019). Since, outdoor cultivation of microalgae is often limited by the high CO2 demand, high alkaline culture conditions (pH > 10) have been studied to facilitate greater scavenging of CO2 from the atmosphere by alkaliphilic microalgae that have adapted to sustain carbon fixation through CCMs. This not only provides an economically feasible option for cultivation of microalgae, but also, greatly reduces the possibility of mesophilic contamination due to the employment of extreme culture (here, alkalinity) conditions. An alkaliphilic algal strain, Chlorella sorokiniana SLA-04, showed comparable biomass and lipid productivities as neutrophilic algal species when cultured indoors and outdoors under the same conditions (Vadlamani et al. 2017). Similarly, Spirulina sp. is mass cultivated in raceway ponds operated with manual mixing for the production of nutraceuticals by non-profit organizations such as, Antenna Nutritech Foundation in Madurai, India. The alkaliphilic species is cultivated at pH greater than 10 to not only maximize nutraceutical production but also, reduce the chances of contamination, making the process efficient and economically feasible (Kumar et al. 2015). The relatively simple operation and easy scale-up process make open systems a viable option for microalgal cultivation, especially via the photoautotrophic mode, pertaining to industrial applications. Moreover, open systems are also applied when wastewater is used as the source of nutrients, integrated with CO2 provided by flue gas (McGinn et al. 2011, Yen et al. 2019).

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Fig. 5.3. Schematic diagram of an open raceway pond for microalgal cultivation.

4.2 Closed Systems In closed cultivation systems, often known as photobioreactors, there is no direct exchange of gases between the outside environment and inside of the photobioreactor. Necessary gas exchanges such as those involving air and/or CO2, are done by passing the air stream through a sterile gas filter thus, virtually eliminating contamination of the algal cultures. Moreover, there is relatively efficient process-control over culture conditions such as temperature, CO2 concentrations, light, pH, etc., in closed systems when compared to open systems. There are several kinds of closed cultivation systems such as tubular (vertical and horizontal) photobioreactors, flat-plate photobioreactors, filtration photobioreactor and bag photobioreactors, to name a few. In fact, extremophilic microalgal species belonging to the genera, Chlorella, Dunaliella and Scenedesmus, have been cultivated in closed photobioreactors and have achieved high productivities (Cheah et al. 2015). A vertical column photobioreactor consists of a transparent vertical tubing (either glass or acrylic) that permits the penetration of light for photoautotrophic microalgal cultivation. At the bottom of the reactor, a gas sparger system is installed which converts the inlet gas into small bubbles. These bubbles serve as the driving force for mass transfer of CO2, ensure proper mixing of the nutrients and also remove the O2 produced during photosynthesis. Usually, a separate agitation system is not implemented while designing a vertical column photobioreactor. Based on the flow patterns inside the bioreactor, vertical column photobioreactors include a bubble column and airlift reactors (Fig. 5.4). While bubble column photobioreactors use a single sparger system, airlift reactors are equipped with two internal tubes called the “riser” and the “downcomer”, in which the medium circulates and required mixing and gas exchange occur (Yen et al. 2019). The sparging of CO2 inside the photobioreactors ensures greater process control and supply to the microalgal cells resulting in higher productivities. Cultivation of the acidophilic microalga Chlamydomonas acidophila LAFIC-004, in photobioreactors injected with 5 and 10%

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Fig. 5.4. Schematic diagrams of vertical column photobioreactors—(a) Bubble column photobioreactor, (b) Airlift photobioreactor.

CO2, showed considerable potential in CO2 fixation and nutrient removal from treated wastewater (Neves et al. 2019). Flat-plate photobioreactors are cost-effective and excellent for biomass productivity as they possess a large surface area to volume ratio which allows sufficient illumination of the vertically semi-transparent flat plates on both sides. Agitation and mixing are achieved by the generation of gas bubbles as in tubular photobioreactors and can be enhanced by the addition of baffles for aeration along the light gradient. A flat-plate PBR can be up-scaled, with an accommodation capacity of 1000–2000 L (Cheah et al. 2015, Yen et al. 2019). The potential of mass cultivation of the halophilic green alga, Dunaliella salina, in vertical flat-plate photobioreactors under semi-continuous mode resulted in higher biomass and essential lipids accumulation (Khadim et al. 2018). Thus, closed system cultivation can provide sufficient control over culture conditions. In addition, the evaporative and CO2 losses are reduced, leading to higher productivity in biomass and target products such as biofuels and bio-active compounds. The only negative aspect of photobioreactors is the high equipment cost, which is however, often compensated by the numerous advantages associated with this system of cultivation (Cheah et al. 2015).

5. Value-added Products Obtained from Extremophiles Microalgae and cyanobacteria have immense potential to satisfy the energy demands and supply bioactive compounds with high commercial value. Microalgae with extremophilic characteristics have several advantages over mesophilic, industrial microorganisms. First, extremophilic microalgae produce and accumulate products of commercial value as a response mechanism to stress conditions. Second, the ability to proliferate in harsh conditions enable them to adapt to common unfavorable outdoor cultivation conditions such as high temperature and light intensity (Varshney et al. 2015). Lastly, extremophilic culture conditions prevent contamination from competing pathogens and other contaminating mesophilic organisms (Malavasi et al. 2020). Microorganisms with such characteristics are much in demand in the sphere of White Biotechnology. Here examples of some of these commercially valuable extremophilic microalgae and the value-added products derived from them will be discussed.

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5.1 Biotechnology Tools: Enzymes Extremophilic enzymes are one of the most industrially–relevant biotechnological products. Several industrial chemical reactions prefer enzymes as biological catalysts over their chemical counterparts, owing to their high substrate specificity, fewer by-products, controllable kinetics and environmentally friendly nature. However, enzymes are susceptible to environmental influences. Industrial processes often bear unfavorable environmental conditions, thus, affecting enzyme stability and action (Varshney et al. 2015). Temperature is one of the critical parameters influencing enzyme kinetics. Enzymes obtained from mesophilic organisms are unable to demonstrate optimum activity at too low or high temperatures. Therefore, enzymes obtained from thermophilic and psychrophilic microalgae can be more efficient in industrial settings than their mesophilic counterparts. Thermostable enzymes from thermophilic microalgae serve as valuable products for several industries. Industrial processing carried at high temperatures requires thermostable enzymes. Higher temperatures are especially favorable as it results in faster reaction rates, better mixing rates and higher mass transfer rates (Zamost et al. 1991). Bio-reactions carried at higher temperatures also provide the added advantage of lower pathogenic and microbial contamination (Zamost et al. 1991). Furthermore, thermostable enzymes can be exploited in the biotechnological sector for genetic manipulation and cloning experiments in other microorganisms or higher plants. Phormidium is a genus of non-nitrogen-fixing cyanobacterium. Some species of Phormidium thrive well at higher extremes of temperature. This high-temperature tolerance of the organism has been studied extensively and considered for industrial exploitation because of its biotechnological products. For example, Phormidium laminosum samples, obtained from Hyemen Terrace Spring at Yellowstone National Park, showed the optimum activity of lipases at 48°C. Similarly, the thermostable glutamine synthetase was extracted and purified from Phormidium lapideum samples from Matsue hot springs, Japan. This glutamine synthetase enzyme showed at an optimum activity at a temperature of 45°C (Sawa et al. 1988). A study by Ogbaga et al. (2018) analyzed the engineering potential of thermostable Rubisco Activase (RCA), from thermophilic cyanobacterium, to confer resistance to higher plants from heat stress. The activity of Rubisco is dependent on the state of RCA. Heat–tolerant Rubisco Activase can prevent subsequent inactivation of Rubisco and decline in crop productivity under high-temperature conditions. The cyanobacterium Dactylococcopsis salina sp. nov. demonstrates another example of thermostable enzymes (Laue et al. 1991). This halophilic cyanobacterium possesses class II restriction endonucleases Dsa I–VI, which shows optimal activity in the temperature range of 30–60°C (Laue et al. 1991). While thermostable enzymes have a high demand for industrial and genetic engineering applications, enzymes tolerant to low temperatures are also useful. For instance, reaching optimum temperatures for enzymes in some industrial processes can be considered energy consuming (Cavicchioli et al. 2011). In such situations, enzymes acquired from psychrophilic organisms can be beneficial. Enzymes tolerant to low temperatures compensate the low reaction rates with increased flexibility, which improves molecular motions (Feller 2013). Some common snow species such as Chloromonas, Chlamydomonas, Chlorella, and Scenedesmus can be employed to extract cold-tolerant enzymes (Varshney et al. 2015) which can be used for the production of heat-sensitive products (Cavicchioli et al. 2011). These enzymatic characteristics find application in food, feed and biotechnological industries (Cavicchioli et al. 2011). Enzymes such as lipases and proteases are essential in animal feed and detergent products. Additionally, enzymes such as β-galactosidases, lactases and α-amylases are essential in dairy, wine and bakery industries (Cavicchioli et al. 2011). Furthermore, the genes conferring such extremophilic characteristics have immense value for cloning experiments. These organisms can serve as gene assets for genetic manipulation experiments.

100 Extremophiles: Wastewater and Algal Biorefinery Such experiments grant other organisms traits to increase tolerance to an extremophilic environment (Varshney et al. 2015).

5.2 Value Added Metabolites and Compounds Microalgae produce a diverse range of bioactive compounds and metabolites that are of human interest. These highly valuable products have applications in food, cosmetics, pharmaceutical and medical industries. Bioactive metabolites such as proteins, fatty acids, vitamins, amino acids and organic compounds are synthesized from the metabolic pathways of these organisms. In extremophilic organisms, some compounds and metabolites are synthesized to support the organism’s intracellular environment in response to the stress. These environmental influences can be adopted in industrial microbial cultivation to enhance the accumulation of these compounds. Some of these valuable products obtained from microalgae include lipids, vitamins, amino acids and organic chemicals. Microalgae are promising assets for the commercial extraction of fatty acids. Fatty acids are organic compounds with high status with applications in pharmaceuticals, nutraceuticals, cosmetics, fuels and fine chemicals. Essential fatty acids, especially polyunsaturated fatty acids (PUFAs), have salubrious benefits and are sold as supplements. Microalgae possess abilities to produce large amounts of polyunsaturated fatty acids. Essential fatty acids such as γ-linolenic acid (GLA), Arachidonic Acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can be extracted from microalgae (Sathasivam et al. 2019). At lower temperatures, microalgae tend to produce fatty acids with much higher unsaturation as a coping mechanism. Such psychrophilic microalgae can be utilized to establish a commercial level of production setup for PUFAs. For example, in a study conducted by (Steinrücken et al. 2017), psychrophilic microalgal isolates were collected from the North Atlantic and analyzed for their long chained PUFA content. These isolates belonged to Diatoms, Chlorophytes and Cyanobacteria (Steinrücken et al. 2017). The study recorded Total Fatty Acid (TFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content as high as 42.9 ± 1.13%, 24.1 ± 0.15% and 6.60 ± 0.18% of the dry weight at the end of stationary phase (Steinrücken et al. 2017). Microalgae also serve as rich sources of vitamins. Psychrophilic organisms such as Chloromonas nivalis, Chlorococcum spp., Chlamydocapsa spp. and Raphidonema spp. have showcased the ability to produce α-tocopherol (Vitamin E) during both actively proliferating green phase and resting red phase. Chlamydocapsa spp. produced the highest amount of α-tocopherol during the green phase (883 µg per g of freeze-dried biomass) (Leya et al. 2009). Meanwhile, during the red phase, Raphidonema spp. produced the highest amount of α-tocopherol (417 µg per g of freeze-dried biomass) (Leya et al. 2009). Additionally, the halophilic cyanobacteria, Spirulina sp., synthesize high amounts of Cobalamine (Vitamin B12). For instance, Spirulina platensis is one such species that has been studied and analyzed for its methylcobalamin (a form of Vitamin B12) content. Study results showed values of 38.5 ± 2 µg and 35.7 ± 2 µg of methylcobalamin per 100 g of dried biomass from two different assays (Kumudha et al. 2010). Vitamin C, an essential organic compound with antioxidant and immunomodulatory characteristics, can also be extracted from microalgae (Sathasivam et al. 2019). Additionally, halophilic Dunaliella tertiolecta (Barbosa et al. 2005) and acidophilic Auxenochlorella protothecoides (Running et al. 2002) have showcased potential for commercial production of ascorbic acid (Vitamin C). While metabolites, as mentioned earlier, have demonstrated diverse applications, certain amino acids in cyanobacteria and microalgae are specifically employed in cosmeceuticals as natural sunscreens. Sunscreens are especially sought - after cosmetic products to protect the skin from the harsh UV rays of the Sun. Sunscreens require frequent application on the skin for appropriate protection and hence preferably have to be as skin-friendly as possible. Biologically derived UV screening compounds can achieve this required quality. Biological compounds from some marine algae and cyanobacteria have been identified for their UV screening properties. Moreover, some

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cyanobacteria possess pigments that have UV screening properties. One of these compounds with “sunscreen – like” activity are Mycosporine – like amino acids. Mycosporine – like amino acids (MAAs) come under a family of secondary metabolites. They show maximum absorption in the range of 310–365 nm, which corresponds to the UV region of the spectrum. They possess maximum absorption capacity for UV – A and UV – B (Browne et al. 2014). This compound shows ubiquitous presence in marine organisms that thrive in shallow water environments, thus, indicating the vital role of this compound throughout the evolutionary history. A study by Elsayed et al. (2015) analyzed the ability of the terrestrial cyanobacteria, Nostoc commune (pH 3–10) (Varshney et al. 2015), to produce MAAs under different stress factors. The production and accumulation of MAAs were significantly amplified with an increase in the duration of exposure to UV – A and UV – B radiation (Elsayed et al. 2015). In addition to the myriad of value-added metabolites, microalgae have commercial importance as sources of industrially relevant organic compounds. For instance, some halophilic microalgae produce glycerol to tolerate high saline environments. Glycerol is a valuable organic chemical with applications in the pharmaceutical, cosmetic, food and chemical industries. Dunaliella, a green algal genus under the phylum Chlorophyta, consists of species that have adapted to halophilic environments. The production of glycerol imparts the tolerance to high osmotic stress in halophilic conditions. Glycerol prevents the inactivation and inhibition of enzymes in high salinity conditions. Accumulation of intracellular glycerol can reach up to 50% of its dry weight under osmotic stress conditions in halophilic environments (Hosseini Tafreshi and Shariati 2009). Another halotolerant green alga, Asteromonas gracilis, also displays glycerol accumulation in response to osmotic stress (Ben-Amotz and Grunwald 1981). The amount of accumulated glycerol is directly proportional to the medium’s salinity, with a maximum glycerol amount of 400 picograms/cell obtained at 4.5 M NaCl (Ben-Amotz and Grunwald 1981). Here, glycerol leakage into the outside medium was observed at salinities above 3.5 M NaCl and temperatures above 40°C. The leakage capacity increased with a further increase in temperature (Ben-Amotz and Grunwald 1981). Thus, the costs involved in extraction and downstream processing can be reduced in this mode of production. Currently, the demand for glycerol is met through petrochemical sources (Hosseini Tafreshi and Shariati 2009). A shift to algal sources can increase production with a reduction in processing costs. Extremophilic microalgal based production have booming commercial potential but is still in embryonic stages of development. Innovations are a necessity for the proper establishment of this industry. Improvements in biomass productivity, selection of algal strains and advancements in genetic engineering can further the growth of the microalgal industry (Varshney et al. 2015).

5.3 Pigments Pigments in photosynthetic organisms are essential components for light absorption and harvest. However, the colorful characters of these pigments make them exceptional candidates for use as natural colorants. Moreover, these pigments have been proven to display salubrious benefits when consumed in the diet. Thus, the commercial exploitation of these pigments has been long considered. Microalgae can be employed for commercial extraction of these pigments owing to their simple culture requirements and photoautotrophic traits. Some of these popular pigments with potential for commercial exploitation from microalgae and cyanobacteria are described below. 5.3.1 Carotenoids Carotenoids are a class of isoprenoid pigments, present in all photosynthetic organisms. Carotenoids can be classified either based on the function of the pigment or the chemical structure. Based on the function, carotenoids are categorized into primary and secondary carotenoids. Primary carotenoids

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are essential components of the photosynthetic apparatus. Meanwhile, the production of secondary carotenoids is induced when exposed to certain environmental stimuli. Carotenoids have significant commercial value as coloring agents in food products, pharmaceuticals and cosmetics (Qaisar et al. 2019). The most popular candidate for carotenoid production is the photosynthetic green algae, Dunaliella. The carotenoids produced by Dunaliella include β-carotene, α-carotene, zeaxanthin, lutein, neoxanthin and cryptoxanthin (Ye et al. 2008). These halotolerant microalgae can thrive in a diverse range of salinities. Dunaliella accumulates β-carotene to prevent chlorophyll photodamage caused by high irradiances (Hosseini Tafreshi and Shariati 2009). β-carotene is produced in the form of two isomers: 9-cis and all-trans. The ratio of the isomers produced depends on the intensity of light, exposed during the cell division cycle (Ye et al. 2008). (Orset and Young 2000) observed that this ratio can be promoted at lower irradiances in the range of 20 to 50 μmol photons m–2 s−1. β-carotene production can be maximized by controlling stress conditions producing up to 14% of the dry weight (Malavasi et al. 2020). Stressful culture conditions, such as high light intensity, high salinity, low temperature and nutrient limitation (N2 ), can help increase carotenoid production (Raja et al. 2007). For example, in Dunaliella salina, the collaborative effect of high light intensity and salinity increased carotenoid production (Fazeli et al. 2006). Another suitable candidate for the commercial extraction of carotenoid pigments is the red alga, Cyanidioschyzon merolae, a dweller of the acidic, hot springs (Cunningham et al. 2007). Carotenoid pigments with β-rings such as β-carotene and zeaxanthin can be extracted from this group of unicellular red algae (Cunningham et al. 2007). Carotenoid pigment production capabilities have also been demonstrated by certain microalgal species residing in low-temperature conditions, called, snow algae. Snow algae are a group of freshwater algae that can thrive biting cold conditions of snow deserts. These organisms are exposed to dehydration, osmotic stress and high light intensity conditions (Leya et al. 2009). For example, species belonging to the class Chlorophyceae such as Chloromonas nivalis, Chlorococcum spp. and Chlamydocapsa spp. were forced into the stage of dormancy (red phase) due to nitrogen-deficient and high light intensity conditions. During this red phase, the strains displayed secondary carotenoid (echinenone, canthaxanthin, astaxanthin and adonixanthin) synthesis and accumulation (Leya et al. 2009). Light stress damages photosynthetic apparatus and is counteracted by activation of the carotenoid biosynthesis pathway. Unlike primary carotenoids which are accumulated within the plastid, secondary carotenoids are exported to the cytoplasm. Thus, the accumulation of secondary carotenoids around the chloroplast protects high irradiance (Leya et al. 2009). The highest secondary carotenoid accumulation was observed in Chlamydocapsa 101-99/ R2 strain, which produced 921 µg of canthaxanthin per g of freeze-dried dry mass (Leya et al. 2009). The strains mentioned in (Leya et al. 2009) can also be utilized for the extraction of primary pigments and xanthophyll pigments during the green phase, which is characterized by low light conditions. Astaxanthin is a secondary carotenoid with antioxidant, anti-cancer and immunomodulatory properties. This carotenoid is an essential component of marine animal feed and is also exploited as a coloring agent, natural preservative and a super antioxidant (Park and Lee 2001). Nutraceutical industries extract astaxanthin from its common microalgal source, Haematococcus pluvialis. This pigment is produced in the organism on exposure to high light intensity conditions. The accumulation capabilities in Haematococcus pluvialis are also influenced by the wavelength of the exposed light (Katsuda et al. 2004). Other microalgal species that have astaxanthin accumulation capabilities are Chlorella, Dunaliella (Guedes et al. 2011), Chlamydomonas nivalis (Remias et al. 2005) and Chloromonas nivalis (Remias et al. 2010). Astaxanthin production is induced to counteract the oxidative stress encountered as a result of high irradiance. However, the effect of

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irradiance is also affected by other operating culture conditions. Nitrogen deprivation also helps induce increased astaxanthin production (Guedes et al. 2011). 5.3.2 Phycocyanin Phycobiliproteins are water-soluble pigments – proteins found mainly in cyanobacteria and algae such as Rhodophyta, Glaucocystophytes and Cryptomonads (Hu 2019). Phycocyanin is a type of phycobiliprotein identified as a blue colored protein - pigment complex. Phycocyanin has applications as a fluorescent marker in histochemistry and dye in food and cosmetics. Microalgae such as Galdiera sulphuraria and cyanobacteria Arthrospira (Spirulina) platensis are commercially exploited to extract phycocyanin. The amount of phycocyanin produced by Galdiera sulphuraria is lower than Arthrospira (Spirulina) platensis. However, the biomass productivity of Galdiera sulphuraria is higher. A study by (Sloth et al. 2006) analyzes the effect of different culture conditions on phycocyanin accumulation in the algae Galdiera sulphuraria 074G. Nitrogen-rich conditions were found to favor phycocyanin accumulation. The phycobiliproteins act as nitrogen storage compounds and are mobilized in the face of nitrogen-limited conditions. Hence, the amount of phycocyanin content decreases as a result of nitrogen starvation. Additionally, carbon deficient conditions and low light intensity conditions favor phycocyanin accumulation. 5.3.3 Scytonemin Scytonemin is a yellowish-brown pigment present in cyanobacteria. Scytonemin pigments are endowed with UV-screening, anti-inflammatory and anti-proliferative properties. The pigment shows maximum absorption at 384 nm (Browne et al. 2014). The biosynthesis of Scytonemin is triggered under specific environmental influences such as high photon fluence rate, high light intensity, high temperature, exposure to UV – A radiation, deficiency of elements (Fe, Mn, N2) and desiccation (Browne et al. 2014). Cyanobacterial species exposed to high solar irradiances are potential candidates for commercial production of scytonemin. Nostoc, Calothrix, Rivuleria, Scytonema, Lyngbya are examples of some cyanobacteria that synthesize scytonemin pigment. Commercial exploitation of algal pigments is welcomed for their salubrious characteristics; however, this industry is still in the infancy stages of development. Commercially viable algal strains and technologies are pre-requisites for the development of this industry. Advances in genetic engineering and research on genetic and metabolic factors of production will build up the future of this industry.

5.4 Biofuels The idea of replacement of fossil fuels with biofuels is gaining popularity in the present century, as can be witnessed, degrading environmental conditions and depleting fossil fuel reserves. Biofuels provide an environmentally friendly solution to the dangerous by-products and the increasing energy demands. Microbial organisms serve as the reserves for the production of these biofuels, with particular consideration for microalgae. Microalgae satisfy all the criteria required for an environmentally friendly and cost-efficient fuel production. Low nutritional requirements, high photosynthetic capabilities and faster growth rate than plants prove them to be excellent candidates for the purpose. Microalgae have shown potential for the production of different kinds of biofuels. Biodiesel, bioethanol and biohydrogen are some of the biofuels that can be extracted from microalgae. 5.4.1 Biodiesel Diesel derived from crude oil is the dominant global energy supplier since its discovery in the 19th century. However, the prolonged dependence on crude oil has depleted its reserves to an alarming extent. This crisis can be resolved by accepting new sources of oil. Algal biomass is highly

104 Extremophiles: Wastewater and Algal Biorefinery recommended as a biodiesel reserve owing to its high biomass productivity and high lipid content. Extremophilic characteristics provide an added advantage for the industrial-scale production of biodiesel. The halophilic microalgae, Dunaliella salina, demonstrates high lipid production and accumulation. The intracellular lipid content falls in the range of 6–25%. Ahmed et al. (2017) analyzed the effect of salinity (0.5 M–2.5 M) on lipid production and accumulation. The results demonstrated an increased lipid accumulation at higher salinities. This phenomenon is an adaptation response to the stress condition. While the maximum lipid per biomass percentage was obtained at the highest salinity (2.5 M), the biomass productivity decreased. However, high biomass concentration (1231.66 ± 1.26 mg L−1) and lipid content (248.33 mg L−1) was observed in the case of salinity of 2 M (Ahmed et al. 2017). Furthermore, two polyextremophiles, Asterarcys quadricellulare and Chlorella sorokiniana have also demonstrated high lipid accumulation abilities (Varshney et al. 2018). These green algae are tolerant to high temperatures of up to 40–43°C, high light intensity and the presence of flue gases such as, CO2 and NO. Lipid accumulation as high as 44.3, and 46.4% of the dry cell weight was observed in Asterarcys quadricellulare and Chlorella sorokiniana, respectively (Varshney et al. 2018). Biomass from these lipid rich microalgae is often converted to biodiesel by means of various thermochemical and biochemical technologies. 5.4.2 Bioethanol The liquid fuel, bioethanol, is another promising alternative for fossil fuels. Bioethanol is produced by microbial fermentation of raw materials rich in sugar or starch, such as crop residues (sugar cane, wheat, corn), lignocellulosic biomass (wood, grass) or microalgal biomass. Oxygen constitutes 35% of the fuel (Balat et al. 2008). This high oxygen content encourages the complete combustion of the fuel and reduces carbon monoxide, hydrocarbon and particulate emission. Recently, microalgae have been preferred over other raw materials for bioethanol production. Qualities such as high growth rate, high biomass productivity and high carbohydrate content prove that microalgae are ideal bioethanol fuel reserves (Lam and Lee 2015). Microalgal species such as Chlorella sp. (19%), Chlorella vulgaris (12–17%), halophilic Spirogyra sp. (33–64%), Nannochloropsis oculata (8%) and Chlamydomonas reinhardtii (17%) have high carbohydrate content. The carbohydrate and sugar-rich biomass derived from microalgal species are fermented to produce bioethanol (Lam and Lee 2015). 5.4.3 Biohydrogen Biohydrogen is a clean, environment-friendly and energy-efficient biofuel. Considered the “fuel of the future,” this propitious energy form is predicted to replace all forms of fossil fuel in the future. It is a zero-emission fuel and produces mainly water when combusted with oxygen. Photosynthesis in some cyanobacteria and microalgae species have hydrogen production and evolution mechanisms. These metabolic pathways can be manipulated by genetic and metabolic engineering to enhance hydrogen production and convert cyanobacterial and microalgal cells into “hydrogen biofactories”. Enhanced biohydrogen production can be achieved under specific extreme environmental settings such as high irradiance, high CO2 and high-temperature conditions (BayroKaiser and Nelson 2017). Extremophilic organisms suited to these conditions can be exploited for commercial-scale biohydrogen production. Extremophilic organisms can be genetically engineered to enhance hydrogen production. For instance, Chlamydomonas reinhardtii is the model organism for biohydrogen production. However, it is cultured at moderate environmental conditions and thus, poses a barrier for industrial-scale production (Bayro-Kaiser and Nelson 2017). However, the hydrogenase genes from this organism can be engineered into an extremophile, amenable to genetic manipulation, for large scale biohydrogen production. Biofuel production from algae is portrayed as ideal replacements for fossil fuels. However, the commercial viability of the process is yet to be improved for complete adoption of these biofuels.

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Extremophilic algal strains possess characteristics for commercial exploitation but can be further improvised by genetic manipulation to achieve the purpose.

5.5 Bioremediation The mark of industrialization in the 18th century was responsible for the development of modern economy and society. Urbanization and increased economic growth have paved the way for our present lifestyle. However, the negative outcome of these activities has been reflected on environmental health. Realizing the harsh impact of this modern revolution, we have come to embrace the development of a green economy which favors sustainable development. The first step to pave the way for this green economy is realizing the need for environmental remediation. Physical and chemical methods of remediation cause further harm and disruption in the process of cleanup. Biological remediation serves as an excellent solution to preserve the state of the natural state of the habitat. Bioremediation is the technology of utilizing biological organisms or their products to treat a polluted environment. For this purpose, microalgae, which can multiply in such contaminated conditions, have been assessed to degrade pollutants. The qualities of microalgae, such as short generation time, fast growth rate and autotrophic nature, are favorable for bioremediation (Devi et al. 2014). This section analyses some microalgae with different extremophilic characteristics that play a role in bioremediation. Microalgae capable of thriving in the presence of high concentrations of gases such as CO2, CO, oxides of nitrogen and sulfur, classified as pollutants, are especially valuable for the treatment of flue gases. Treatment of exhaust gases from industries and other facilities due to the combustion of fossil fuels is a critical concern for the maintenance of environmental health. Biosequestration strategies employed by microalgae can be employed to treat greenhouse gases and other harmful emissions in industrial exhausts. For example, Chlorella pyrenoidosa XQ-20044 is shown to have tolerance to a high concentration of SO32– and NO2– valued at 20 mmol L–1 and 8 mmol L–1, respectively (Du et al. 2019). This microalga was used to clean up flue gas from CO2, SO2, and NO with the simultaneous production of lipids. The simulated flue gas consisting of pollutants 15% CO2, 0.03% SO2, 0.03% NO with 85% N2 was removed with an efficiency of 95.9, 100, and 84.2%, respectively, by Chlorella pyrenoidosa XQ-20044 (Du et al. 2019). The study proved that CO2, SO2 and NO are assimilated by the organism and contribute to biomass production and lipid accumulation. The commercial exploitation of the lipids as biofuel can alleviate the costs associated with the microalgae cultivation and treatment. Another category of extremophilic algae capable of participating in bioremediation is the metal - tolerant strain. Microalgae that can proliferate in heavy metal-laden environments find applications in the treatment of industrial effluents, waste from mining activities, leaded oil spills, fertilizer–treated areas and municipal wastes. Geothermal environments are often characterized by the presence of the hazardous element arsenic. However, indigenous organisms of this environment can carry out redox reactions on arsenic to produce fewer toxic compounds. For example, the study conducted by Qin et al. (2009) described the arsenic detoxification ability of the red algae, Cyanidioschyzon sp. isolate 5508. The red alga has acidophilic as well as moderate thermophilic characteristics. The organism conducts redox reactions on the available species, As (III), and transports it to the cytosol. As (III) is methylated by the enzyme, As (III)-S-adenosylmethionine methyltransferase (T opt = 60–70°C) to produce the readily dispersible, volatile gas trimethylarsine [TMAs (III)] (Qin et al. 2009). Through this mechanism, Cyanidioschyzon sp. isolate 5508 prevents the intracellular accumulation of toxic As (III) entities. Abinandan et al. (2019), studied the heavy metal removal capacity of two acid-tolerant microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3. The strains showed

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simultaneous removal of about 40–80% and 40–60% removal of Fe and Mn, respectively as well as increased biodiesel production when cultivated at pH 3.5. Thus, indicating suitability for sustainable biofuel production in locations like acid mine drainages rich in metals. Recently, exopolysaccharide-producing cyanobacterial strains such as, Cyanothece sp. CCY 0110, have attracted attention in the bioremediation of heavy metals from water as, the large number of negative charges on the external cell layers serve as chelating agents for removing the positively charged heavy metal ions from water solutions (Mota et al. 2013). Another concern in environmental remediation is the treatment of soil and land excessively tampered by the use of chemical pesticides. The use of chemical insecticides and herbicides in agriculture has plummeted soil quality and negatively affected the soil ecosystem. Despite the toxic quality of soil, certain microalgae are capable of sustaining in the presence of these chemical compounds. For example, the cyanobacterium Phormidium valderianum BDU 20041 is capable of tolerating and metabolizing the insecticide, chlorpyrifos (Palanisami et al. 2009). Enzymes such as peroxidases, cytochrome P450s, polyphenol oxidases, glutathione S-transferases and class A esterases are responsible for the detoxification of the insecticide compound. Additionally, the presence of chlorpyrifos generates reactive oxygen species that are counteracted by enzymes such as catalase and superoxide dismutase (Palanisami et al. 2009). In addition, wastewater treatment using polyextremophilic microalgae can be more beneficial than current treatment systems. Such microalgae are required to possess tolerance to high amounts of CO2 gas, heavy metals and high temperature. Besides, these microalgae can generate productive and useful biomass by capturing CO2. Microalgae can also be utilized to produce biofuels and value-added products, proving to be more economically viable. An acidophilic (pH 1.0–4.0) red algae, Galdieria sulphuraria CCMEE 5587.1, was used to treat primary – settled wastewater. The low pH survival characteristic of the organism is especially desirable to cease the contamination by competing pathogens. At the end of the third day, the BOD was reduced by more than 64%. Additionally, the nitrogen and phosphorus contents were reduced by more than 86 and 85%, respectively (Henkanatte-Gedera et al. 2017). Use of extremophilic microalgae in bioremediation techniques serves as a good alternative to conventional remediation technologies. In addition to the exemplary microalgal traits, some microalgae belonging to the class of Cyanophyceae are capable of tolerating high doses of ionizing radiation (Kraus 1969). Such microalgae can be exploited to open up new techniques of dealing with biological media contaminated with radiation. Furthermore, bioremediation clustered with the extraction of microalgal derived products allows opportunities for sustainable and cost-efficient means of industrial production. Such advancements in algal technologies can revolutionize the future of the global economy.

6. The Biotechnological Potential of Synthetic Extremophilic Microalgae Genetic and metabolic engineering approaches may be applied to enhance the value-added products from extremophilic algae. In this context, in particular, studies were conducted to augment the biofuel and carotenoid production potential of algal extremophiles. Presently, most of these studies are limited to a few select model strains like Dunaliella and Haematococcus sp. A study carried out on the oleaginous diatom Fistulifera solaris, revealed overexpression of glucose-6-phospahte dehydrogenase and phosphogluconate dehydrogenase, which are NADPHproducing enzymes in the pentose phosphate pathway, played a significant role in enhancing the lipid production under nutrient depletion stress condition (Osada et al. 2017). A 12% increase in the total long chain poly unsaturated fatty acid content of D. salina was obtained on the introduction of Acetyl CoA carboxylase and Malic enzyme in pGH. This proves to be of great help in large scale biodiesel production (Talebi et al. 2014). The halophilic marine microalgae Dunaliella salina is known for producing a high quantity of β-carotene. Metabolic engineering using Agrobacterium-mediated transformation for carotenoid

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synthesis was carried out in Dunaliella by introduction of the β-carotene ketolase (bkt) gene from H. pluvialis. In parallel, the chloroplast was also targeted for the production of ketocarotenoids. The success of this engineering was evidenced by the production of canthaxanthin and astaxanthin (Anila et al. 2016). Similarly, a three and two fold increase in violaxanthin and zeaxanthin respectively, was observed on the introduction of β-carotene hydroxylase from C. reinhardtii into D. salina, using Agrobacterium vector under conditions of high irradiation and nitrogen starvation (Simon et al. 2016). A dual genetic engineering strategy applied in Dunaliella tertiolecta helped in the production and accumulation of Medium-Chain-length Fatty Acids (MCFA). Expression of plant lauric acid-biased thioesterase and ketoacyl-Acyl Carrier Protein (ACP) led to the accumulation of lauric acid by seven times and myristic acid by four times as compared to the native strain. This study carries great potential for improving the production of biodiesel using MCFA based approach (Lin and Lee 2017). Chloroplast transformation in D. tertiolecta via particle bombardment and selection using erythromycin antibiotic, caused the production of significant quantities of recombinant enzymes such as xylanase, phytase, α-galactosidase and phosphate anhydrase (Georgianna et al. 2013). Overexpression of phytoene desaturase (PDS), a rate-limiting enzyme in the carotenoid biosynthesis pathway was successfully brought about using chloroplast genetic engineering in Haematococcus pluvialis. The biolistic transformation and expression led to 67% increase in the astaxanthin accumulation than in the wild type, which were induced with exposure to high light intensity and nitrogen depletion (Galarza et al. 2018). Therefore, an enormous scope and potential lies in engineering extremophilic algae for enhanced production of biofuels and other value-added products. Further research in this field would provide a significant breakthrough in the food, industry, environmental, health and pharmaceutical sector.

7. Conclusion The unique ability of microalgae to tolerate a wide array of extremities has opened up a plethora of prospects ranging from obtaining high value-added products and bioremediation to production of biofuels. The various metabolic mechanisms employed to withstand extreme conditions lays the foundation for the metabolic engineering of microalgae to further enhance the production of value-added products. Further, a deeper understanding of the nature of extremophilic microalgae can help devise better strategies to optimize cultivation methods. Coming to the origins of microalgae, these organisms are polyphyletic. The evolution of microalgae has been affected by forces such as endosymbiosis, vertical and horizontal gene transfer, which may influence the inheritance of extremophilic characteristics. The phylogenetic tree derived from 16S rDNA sequences of Bacteria, Archaea and Algae, shows a pattern in the development of extremophilic characters over the evolutionary period and demonstrates connections between the organisms. Nevertheless, over the years, extremophilic microalgae have proved to be effective and sustainable tools in providing revolutionary solutions to certain problems faced by the environmental, food and health sectors.

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6 Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes Dhanshree Mone1 and Nitin Trivedi 1,2,*

1. Introduction The oceanic environment is a natural reservoir for several living organisms with unique and important functions. These organisms play a crucial role in maintaining the ecosystem and are also examined by human beings for large industrial applications. Among them, marine microorganisms and algae, mainly seaweeds have been extensively probed for their potential human applications. Marine microorganisms can survive in different conditions of the sea like pH, temperature, pressure, etc., and have emerged as a source of several value-added biological compounds, mainly enzymes. To date, several enzymes like carbohydrase, lipases, proteases, algal hydrolyzing enzymes have been reported from normal and extreme (extremophiles) habitats of the marine environment (Dalmaso et al. 2015). These enzymes are gaining industrial importance due to their high activity and stability in different harsh conditions in several industrial applications. So far enzymes from marine microbes (extremophiles) have been widely used in dairy, pharma, cosmetics, textiles, detergents, food and beverages, biorefinery, leather, bioremediation, bioenergy and other applications (Kohli et al. 2020). In 2019, the world-wide enzyme market was valued at USD 9.9 billion and is to rise with a CAGR of 7.1% from 2020 to 2027. The global enzyme market is dominated by microbial enzymes followed by animals and plant enzymes. The major players in enzyme production are Novozymes, DuPont Danisco and DSM with a global market share of more than 75% (https://www.grandviewresearch. com/industry/catalysts-and-enzymes). Similarly, another valuable product of the marine environment is seaweeds which have demonstrated applications as a source of food, feed, fertilizers, biochemicals, phycocolloids, bioactive compounds, bioenergy, biorefinery, etc. The global seaweed production in 2018 was 32.38 million tons of fresh weigh biomass with an estimated market value of USD 11.17. Most of the seaweed production is carried out by South East Asian countries like China, Indonesia, Korea and the Philipinnes (FAO 2020). These seaweeds contain different polysaccharides mainly

DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai, Maharashtra – 400019, India. Department of Marine Biotechnology, Gujarat Biotechnology University Gandhinagar-382355, Gujarat, India. * Corresponding author: [email protected] 1 2

116 Extremophiles: Wastewater and Algal Biorefinery Sulphated Polysaccharides (SPs) which comprise up to 30% of seaweed biomass and 60–70% of total seaweed carbohydrates. These SPs like agar, carrageenan, ulvan, alginate, porphyran, etc., can be hydrolyzed by the chemical or enzymatic route. The enzymatic hydrolysis of these SPs can be achieved using enzymes like agarase, alginate lyase, ulvan lysae, carageenase, porphyranase, etc. So far, several microorganisms mainly from a marine origin have been known to produce these enzymes (Garcia-Ruiz et al. 2016). However, very few seaweed hydrolyzing enzymes are produced commercially compared to other enzymes. In recent times, marine extremophiles have also been explored to produce these seaweed degrading enzymes due to their high activity and stability. In the coming years, these extremozymes would play a game-changing role in seaweed biotechnological industries.

2. Marine Extremophiles The marine ecosystem consists of a large array of microorganisms. Some of these organisms can withstand extreme environmental conditions of temperature, pH, pressure and several other physicochemical conditions, which are known as extremophiles (Donato et al. 2019). Extremophiles include thermophiles (high temperature), psychrophiles (low temperature), acidophile (low pH), alkaliphile (high pH), anaerobic (absence of oxygen), piezophile (high pressure), halophile (high salt concentration) and several microbes living in extreme conditions (Poli et al. 2017). For example, microorganisms living at pH below 3 are called acidophiles, and those living at pH more than 10 are called alkaliphiles (Dalmaso et al. 2015). Polyextremophiles are microorganisms that can survive more than one extreme condition. For example, bacteria that can resist higher temperatures and acidic pH conditions are known as thermoacidophilic bacteria. Altogether the three domains, i.e., Eukarya, Bacteria and Archaea consist of extremophiles. Bacteria and Archaea hold a large diversity of the marine extremophiles which belong to Alphaproteobacteria, Actinobacteria, Acidobacteria, Cyanobacteria, Deltaproteobacteria, Gammaproteobacteria and Flavobacteria phyla (Donato et al. 2019). Adaptation to the extreme environment is driven by the survival pressure against extreme habitats of extreme temperature, pressure, pH, saline conditions, oceanic depth and hydrothermal vents. These adaptation constraints give rise to the production of beneficial components like polymers, osmolytes and enzymes (extremozymes) which are also useful for the biotechnological aspects as a promising biocatalyst. Therefore, in the last few decades, marine extremophiles have gained recognition for its extremozymes. Extremozymes help to improve industrial and biotech processes, making them economical and eco-friendly. Extremozymes produced by the marine extremophiles include lipases, proteases, glycosidases that could be employed in the pharmaceutical, food, medical, cosmetics, environmental, textile, agricultural, chemical fields along with other industrial and biotechnological purposes (Poli et al. 2017). To obtain an extremozyme, the extremophiles could be cultivated in the bioreactor by providing optimal growth conditions of pH, temperature, aeration, nutrients and other parameters (Dalmaso et al. 2015). The extremophiles are categorized into different types depending on their habitats. A few important examples of extremophiles are discussed here.

2.1 Thermophiles Microorganisms living at extreme temperatures are called thermophiles. Microorganisms that can resist 60°C to 80°C are generally thermophiles, and which grow above 80°C are known as hyperthermophiles (Donato et al. 2019). The highest optimal temperature for the majority of the heterotrophic species is under 105°C. Different thermophiles can survive different temperatures, for instance, Pyrodictium, Pyrolobus fumarii, Desulfurococcales strain 121, Methanopyrus can survive 110°C, 113°C, 121°C, and 122°C, respectively. Thermophiles generally belong to the Archea mainly

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Euryarchaeota and Crenarchaeota. Species belonging to Euryarchaeota are thermophiles, aerobic, methane-producing (methanogens), sulphate, iron, nitrate sulphur reducers whereas Crenarchaeota are thermophilic, hyperthermophilic and heterotrophic. Methane-producing Archaea belongs to Methanosarcinales grows by producing a biofuel that shares boundaries with the hydrothermal flow and able to survive at 80°C. Species belonging to genera Thermotoga and Desulfurobacterium grow in 60–80°C whereas Thermus and Bacillus grow in 60–75°C (Poli et al. 2017). Thermophilic microorganisms also include Actinobacteria sp., Bacillus sp., Clostridium sp., Desulfotomaculum sp., Thermus sp., Thiobacillus sp., fermenting bacteria, spirochetes and phototrophic bacteria such as cyanobacteria, green and purple bacteria (Dalmaso et al. 2015). Hyperthermophiles survive up to the highest temperature of about 120°C hence, to survive such extreme temperatures, microorganisms show adaptations in proteins and other structures. Peptides, amino acids and ATP could tolerate above 250°C, shows the same normal three-dimensional structure, but the different amino acid composition with several charged residues on the surface of proteins which result in the easy formation of the crystallized structure to achieve stability and resist unfolding. Proteins present in thermophiles also contain numbers of disulphide bonds to increase the stability of the quaternary structure. They help to make industrial processes faster, increase the solubility of the substrate, miscibility of solvent, decreases the risk of contamination and viscosity of solutions. Therefore, extremozymes from thermostable microorganisms are used in biofuel, paper, bleaching and biorefinery industries. Taq polymerase isolated from Thermus aquaticus is a well-known example of a thermozyme utilized in a Polymerase Chain Reaction (PCR) in the field of molecular and recombinant DNA technology (Dalmaso et al. 2015).

2.2 Psychrophiles Microorganisms that grow at a temperature of 15°C are called psychrophiles. The maximum and minimum growth temperature for psychrophiles is 20°C and 0°C, respectively. Also, a microorganism that can tolerate up to 25°C with optimum growth is called psychrotolerant (Hamdan 2018). Microorganisms such as Colwellia psychrerythraea 34H (a γ-proteobacterium) (Carillo et al. 2015), Euplotes focardii (Pischedda et al. 2018), Psychrobacter namhaensis SO89 (Makled et al. 2017), Psychroserpens jangbogonensis (Baek et al. 2015), Psychrobacter oceani (Matsuyama et al. 2015) include psychrophiles. Survival at lower temperature integrates an elevated quantity of fatty acids having a low melting point, such as mono and polyunsaturated fatty acids along with branched-chain fatty acids to provide the necessary fluidity of the membrane (Hassan et al. 2020). When mesophilic microorganisms are kept at a low temperature, they produce Cold Shock Proteins (CSPs). Later reduction in CSPs production has been observed after adaptation to the low-temperature conditions. Also, antifreeze proteins are produced by some bacteria to prevent crystallization and osmotic imbalance. Certain solutes such as mannitol and sucrose help in lowering the freezing point for the cytoplasm which results in prevention from freezing and desiccation. Denaturation of proteins and free radical scavenging is prevented by trehalose in species such as in Listeria monocytogenes, Burkholderia pseudomallei (Kumar et al. 2020).

2.3 Halophiles Salt-loving microorganisms, which are capable of growing at 2.5 M (15% w/v) are called halophiles present in all domains of life, i.e., Eukaryota, Archaea and Bacteria (Edbeib et al. 2016). Depending on the salt concentration required (w/v) for growth, halophiles are categorized as extreme (15 to 30%), moderate (3 to 15%), and slightly halophiles (1 to 3%) (Zhang et al. 2018). The habitats for halophiles include artificially prepared solar salterns, salt mines, coastal and submarine pools sea brines, saltwater lakes, etc. (Singh et al. 2019). Halophiles include Haloferax mucosum (López-Ortega et al. 2020), Halococcus agarilyticus (Gaonkar and Furtado 2020), Halomonas sp.

118 Extremophiles: Wastewater and Algal Biorefinery (Thomas et al. 2019), Acinetobacter sp. (Mohanta et al. 2020), Dunaliella salina (Abomohra et al. 2020). To survive in such an extreme saline environment, microorganisms adopt strategies to avoid excessive loss of water and to maintain osmolarity with the external environment. Microorganism shows adaptations such as synthesis of osmoprotectants or building up high salt concentration in the cytoplasm the same as that of the outer environment (Edbeib et al. 2016). The protein shows an increase in the concentration of acidic amino acids, i.e., glutamic and aspartic acid, and a decrease in the amount of lysine and hydrophobic amino acids (Zhang and Yi 2013). The negative charge present on these acidic amino acids interact with cations from water, increasing the solubility of the proteins and preventing its precipitation, aggregation and denaturation (Karan et al. 2012, DasSarma and Arora 2001).

2.4 pH Extremes (Acidophile, Alkaliphile) Microbes have different pH requirements for their growth. Microorganisms that prosper at or below pH 3 are called acidophiles (Mirete et al. 2017) and above pH 9 are called alkaliphiles (TiquiaArashiro and Rodrigues 2016). Habitats of acidophiles include hydrothermal vents, mine drainages and mines. It shows the production of amylases, glucoamylases, proteases, cellulase, oxidase enzymes. Alkaliphiles live in coastal regions and soda lakes. Alkaliphiles produces enzymes like proteases, cellulase, amylases, lipases, cyclodextrinases (Rekadwad and Khobragade 2017). Some example of acidophilus includes Ferrimicrobium acidiphilum, Sulphobacillus thermosulfidooxidans, Leptospirillum ferriphilum (Rivera-Araya et al. 2020), Chlamydomonas acidophila (Hirooka et al. 2017) and that of alkaliphiles includes Bacillus circulans, Paenibacillus tezpurensis AS-S24-II, Stenotrophomonas maltophilia MTCC 7528 (Sarethy et al. 2011), Neochloris oleoabundans (Chowdhury et al. 2019). To maintain the cytoplasmic pH at such a high proton gradient, acidophiles show low membrane permeability for protons (Konings et al. 2002) and mechanisms to repay protons out of the cell decide the survival of the cell. Certain bacteria have ω-alicyclic fatty acids in their membrane to increase acid resistance (Chang and Kang 2004). In red algae, C. Caldarium discharge of protons is carried out by ATPase present on the plasma membrane (Enami et al. 2010). Most of the alkaliphiles show low pmf (proton motive force) which affects ATP synthesis based on oxidative phosphorylation. Cardiolipin and cytochrome C retains the proton gradient pump by the respiratory complex enhancing pmf and that of ATP production efficiency (Mamo 2019).

2.5 Piezophiles Microorganisms that grow under 0.1–10 MPa pressure are called piezotolerant. Piezophiles grow under 10–70 MPa pressure. Hyperpiezophiles grow under more than 70 MPa pressure, these microorganisms do not grow below pressure 50 MPa. A few piezophilic microorganisms include Moritella japonica DSK1, Shewanella benthica F1A, Photobacterium profundum DSJ4, Psychromonas profunda, Colwellia piezophila (Fang and Kato 2016). The habitat piezophile mainly includes hydrothermal vents present in the deep-sea (Charlesworth and Burns 2016), subsurface region, deep oceans (Peoples et al. 2020). To maintain the fluidity at such high-pressure piezophiles contain an enhanced quantity of unsaturated and polyunsaturated fatty acids, pressure control operons and heat shock proteins, strong DNA repair system is also present (Rekadwad and Khobragade 2017).

2.6 Radiophiles The microorganisms living in high radiations are known as radiophiles or radioresistant. High radiation could lead to damage to the DNA, i.e., ionizing radiation cause double-strand breaks.

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To survive such DNA damage caused by radiation, these organisms show proficient DNA repair systems to reorganize the fragmented DNA in a short period. Its application includes bioremediation of the radionuclide polluted sites (Rekadwad and Khobragade 2017). Deinococcus radiodurans was the first reported radiophile. Other reported species of Deinococcus are Deinococcus radiophilus, Deinococcus proteolyticus, Deinococcus radiopugnans and Deinobacter grandis (Shukla et al. 2020).

2.7 Polyextremophiles The microorganisms which can resist a variety of environmental conditions are called polyextremophiles (Maizel et al. 2019). Microorganisms able to tolerate high temperature and pressure are called thermopiezophilic organisms found at deep-sea hydrothermal vents. A rise in temperature increases the volume of the cell which is counterbalanced by the enlarged pressure maintaining the structural integrity. The example of thermopiezophilic organisms is Marinitoga piezophila. Microorganisms resistant to high temperature and acidic environments are called thermoacidophiles. Membrane permeability grows with an increase in temperature, which causes a rise in acidification in the cytoplasm, which has been resolved by the presence of glycerol ether lipids in the archeal membrane and ω-alicyclic fatty acids in the bacterial membrane. The stability of RNA codon is achieved by an increase in purine concentration but at this extreme acidic pH, glycosidic bonds are prone to acid hydrolysis. This issue was resolved in Picrophilus torridus by the inclusion of fewer purines in short open reading frames and more purine in longer open reading frames (Capece et al. 2013). Some examples of polyextremophiles include Bacillus halodurans TSEV1, a thermoalkaliphile, is tolerant of extreme temperature and alkaline pH. Similarly, B. halodurans PPKS-2, a haloalkaliphile, is tolerant of pressure and alkaline pH (Dalmaso et al. 2015). Different types of extremophiles and their habitats are provided in Fig. 6.1.

Fig. 6.1. Various types of extremophiles and their habitats.

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Extremophiles: Wastewater and Algal Biorefinery

3. Seaweeds and their Potential Applications Seaweeds (macroalgae) are photosynthetic, multicellular, macroscopic, benthic marine algae. Seaweed sequester CO2 by the process of photosynthesis generating 90% of the Earth’s oxygen. Seaweeds, a source of food and shelter for other marine life, are generally not differentiated into the root, stem, vascular tissue like in higher plants. It typically contains a thallus body which includes (a) lamina/blade, (b) floats, (c) stripes and (d) holdfast. The blade is a leaf-like flattened structure responsible for photosynthesis. The float is attached to the blade which lifts the seaweed to reach towards the surface of the water for sunlight. Stipe is a stem-like structure in the seaweed that provides support and is involved in transporting sugars to the rest of the plant from the blades. The blade and stripe portion together are called the frond. Seaweed is attached to the sea bottom by a root-like structure called a holdfast, whose sole purpose is physical attachment and not nutrient extraction (Sudhakar et al. 2018). Some seaweed species are freely floating, such as Sargassum, and forms Sargasso Sea (Wang et al. 2019). Seaweeds are mainly categorized into three divisions: Rhodophyta (red seaweeds), Phaeophyta (brown seaweeds) and Chlorophyta (green seaweeds). Red seaweeds have approximately 6500 species worldwide, and mainly contain phycobiliproteins such as phycoerythrin and phycocyanin which hides chlorophyll and other photosynthetic pigments and gives the red colour to the seaweed (Kılınç et al. 2013). It contains a high amount of polysaccharides mainly floridean starch, sulphated galactans such as carrageenan, agar, porphyrin, etc., along with proteins, minerals and bioactive compounds. Red seaweeds have several applications in the food, pharmaceutical, cosmeceuticals industries. A major application of red seaweed includes the production of hydrocolloids such as agar and carrageenans. These hydrocolloids are used for gelling and thickening purposes (Yoon et al. 2017, Torres et al. 2019a). Brown seaweed (Phaeophyta) comprises about 1750 species (Kılınç et al. 2013). It contains 50–60% carbohydrates, which include cellulose, fucoidan, laminarin and alginates as well as 1–3% lipids, 7–38% minerals, vitamins such as B12, C, D, E, and K and essential amino acids (Vijay et al. 2017). In addition to this, it contains bioactive compounds like fucoxanthin which is effective against obesity and diabetes (Miyashita et al. 2011). Brown seaweeds are used as food, feed, fertilizer, have anti-cancer, antioxidant, antimicrobial, anti-inflammatory properties and several other pharmaceuticals, nutraceutical and cosmeceutical applications (Milledge et al. 2015, Wijesinghe et al. 2012). Green seaweed (Chlorophyta) comprises up to 1500 species (Kılınç et al. 2013). The most common species include Ulva lactuca also known as sea lettuce. It contains sulphated polysaccharide ulvan, which along with other cell wall polysaccharide cellulose, xyloglucan and glucuronan contribute up to 45% of the dry weight (Kidgell et al. 2019). Green seaweeds also contain macroelements (Ca, K, Mg, Na), microelements (Al, B, Ba, Co, Cr, Cu, Fe, Mn, Ni, Zn) (Berik et al. 2019). It is used as a food source (Tabarsa et al. 2012) and posseses several biological activities like immunomodulating, antioxidants, anticancer, anticoagulant, antihyperlipidemic, antiviral and plant defense (Kidgell et al. 2019). Some potential applications of seaweeds are discussed here.

3.1 Source of Food and Feed Seaweed is an important food resource in Asian countries like China, Japan and Korea where it is used in soups and salads. Seaweeds contain vitamins, minerals and omega-3 polyunsaturated fatty acids (ω-3 PUFA) and it helps in the reduction of omega-6 and omega-3 polyunsaturated fatty acid ratio (ω-6/ω-3 PUFA) (Kılınç et al. 2013). Carbohydrate content in the seaweeds ranges from 4 to 76% on the DW basis. The major polysaccharides in all the three seaweeds are ulvan, agar, alginate, carrageenan, fucoidan, laminarin along with minimal quantities of cellulose and starch. Macroalgal phycocolloids like agar, carrageenan are used for gelling, stabilizing and emulsifying agents in food industries. The protein content of seaweeds ranges from 12–27% on the DW basis and is used to

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produce bioactive compounds that show antimicrobial, antitumour and antiviral activities. Bioactive compounds have a neutraceutical value which offers several health benefits and is effective against certain health conditions. For example, blood coagulant peptides from seaweed show similar effects as heparin and are non-cytotoxic (Lafarga et al. 2020). To meet the increase in protein demands for animal feed, seaweed would be the preferable option because of its high protein contents. The benefits of using seaweed as animal feed include less land use, lower freshwater requirement and higher biomass productivity (Bikker et al. 2016). The integrated multi-trophic farming system is also an innovative approach in which fish and shrimp cultivation is integrated with oysters, scallops, mussels along with seaweed production. In this approach, the waste and the byproducts produced by the fish, and shrimps will be recycled by filtering species such as seaweeds, oysters, scallops and mussels. For example, N and P produced by fish will be used by seaweeds as food and energy source with the production of O2 and biomass (Nardelli et al. 2018).

3.2 Pharmaceutical Potential Seaweed sulphated polysaccharides like carrageenans and agar, fucoidans, ulvans extracted from red, brown and green seaweed, respectively exhibit antibacterial, antiviral, antitumour, immunomodulatory potential. Polysaccharides like alginate are used as bio-adhesive for wound dressing and drug delivery owing to non-toxicity. Alginate also exhibits anticoagulant and antitumour activities. Agar serves as an inactive medium for drug delivery such as capsule case for oral solid dosage. Fucoidans show anti-coagulant, anticancer, anti-inflammatory, antithrombotic and anti-proliferative activity. Ecklonia cavaextract, phlorotannin extracts from Ascophyllum nodosum show antioxidant and antidiabetic properties, respectively. Polyphenols, phlorotannins present in brown algae protect from photo-carcinogesis and harmful UVB rays (Leandro et al. 2020). Drug delivery application is exhibited by sulphated polysaccharides such as carrageenan (Cunha and Grenha 2016). The issue of drug-resistant bacterial infections could be resolved with natural approaches such as seaweed bioactive compounds like phlorotannins, polysaccharides and peptides. A mixture of different seaweed extracts or different groups of seaweed extracts exhibits several medicinal properties. For example, type 2 diabetes (T2D) could be treated with a mix of brown (Fucus vesiculosus) and green seaweed (Cladophora sp., Monostroma sp., Ulva compressa) and along with other seaweed species (Daniels 2007).

3.3 Nutraceutical Potential Worldwide, seaweed is used as a food source due to its immense nutritional health benefits. Seaweed sulphated polysaccharides such as carrageenan and agar (red seaweed), fucoidan, alginate, laminarin (brown seaweed), ulvan (green seaweed) hold many beneficial qualities such as anticoagulant, anti-inflammatory, antioxidant, anticarcinogenic and antiviral activities (Tanna and Mishra 2019). Seaweed also contains vitamins B1, B2, B12, C, E, β-carotene, which fulfills the vitamin requirements of the body. Seaweeds could be consumed in the form of dietary supplements or nutraceuticals. The health benefits of vitamins include reduction of blood pressure (vitamin C), cancer (vitamin C, E, carotenoid) and cardiovascular diseases (b-carotene) (Škrovánková 2011). Seaweed contains omega-3 and omega-6 polyunsaturated fatty acids (ω-3 and ω-6 PUFAs) which successfully bring down the chances of cancer, diabetes, osteoporosis and cardiovascular diseases (Mišurcová et al. 2011). Seaweeds are known for their nutritional value as they contain macronutrients (Na, Ca, Mg, K, Cl, S, P) and micronutrients (I, Fe, Zn, Cu, Se, Mo, F, Mn, B, Ni, Co) (FAO 2018).

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A large number of minerals such as iron, iodine are present in seaweeds which act as nutraceuticals (Mišurcová et al. 2011). Seaweeds could be an inexpensive source of protein for the treatment of protein deficiency. The protein content of the seaweed ranges between 5 to 47% on a DW basis. Among all seaweeds; red seaweed holds higher protein content of 47%. The protein profile of seaweed resembles that of the egg protein. It contains 50% of essential amino acids (Černá 2011).

3.4 Cosmeceutical Potential Many bioactive compounds of seaweeds have been investigated for their cosmeceutical potentials. Seaweed bioactive components such as sulphated polysaccharides, phlorotannins, proteins (amino acids), fatty acids show cosmeceuticals properties such as reduction of hyperpigmentation, UV protection, antiageing, antiacne, anticellulite, moisturizing, antimicrobial, antioxidant, antiinflammatory agents, emulsion, stabilizers, antiallergenic, chelating agents, anti-wrinkling effect, colloids, gelling, immunostimulating agents, stimulation of collagen synthesis and improves hair growth (Jesumani et al. 2019, Pereira 2018, Jahan et al. 2017).

3.5 Biofilm/Bioplastic To reduce plastic pollution, one of the promising biodegradable alternatives could be seaweed bioplastic, which is completely natural and biodegradable. Seaweed can be grown in large quantities and become an inexpensive source for the production of seaweed bioplastic. Seaweed contains approximately 50% polysaccharides. These polysaccharides along with other natural products (mainly polysaccharides from various seeds of avocado, jackfruit, and durian) could be used to prepare bioplastic with superior mechanical and physicochemical properties (Rajendran et al. 2012, Hii et al. 2016, Yusmaniar et al. 2019).

3.6 Biofuel & Biorefinery Seaweeds contain a higher proportion of carbohydrates, and a lower percentage of lignin than that of terrestrial plants, therefore they are a suitable feedstock to produce different biofuels using several biochemical and thermochemical methods. Different biofuels like biodiesel, bioethanol, biobutanol, biomethane, bio-oils are produced from several seaweed species such as Ulva, Cladophora, Laminaria, Chaetomorpha species, Laminaria saccharina Linnaeus. The processes such as combustion, pyrolysis, gasification, transesterification, hydrothermal liquefaction, fermentation, anaerobic digestion have been used to produce biofuel from seaweed biomass (Jiang et al. 2016, Milledge et al. 2014, Torres et al. 2019b, Gao et al. 2020). A biorefinery is the efficient utilization of biomass, which should lead to a zero-waste approach. In the past few years, seaweed biorefinery has gained substantial importance due to the extraction of multiple bioproducts such as polysaccharides, protein, lipids, pigments and biofuels in a sequential step. This cascading approach will make seaweed industries more viable and economical (Torres et al. 2019b, Balina et al. 2017).

3.7 Agricultural Potential Plant growth and yield are enhanced by the bioactive components present in seaweed-like auxins, cytokinins, gibberellins, betaines along with macronutrients like calcium, potassium, phosphorus and micronutrients like iron, copper, zinc, boron, manganese, cobalt and molybdenum. These bioactive components improve the yield, germination and tolerance to biotic and abiotic stress. Therefore, seaweed extract or seaweed sap is used in agriculture for its beneficial qualities for crop plants (Zodape et al. 2011, Begum et al. 2018).

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4. Types of Seaweed Polysaccharides 4.1 Fucoidans Fucoidan is categorized under sulphated polysaccharides present in brown seaweeds. It has a different degree of sulphation along with different chain lengths and branching. The major component of fucoidan is fucose accompanied by other monomer residues such as galactose, mannose, glucuronic acid and xylose (Lim et al. 2016, Ale et al. 2011). Two different types of fucoidan are present: the first type consists of a polymer of L-fucopyranose linked with α-1,3 glycosidic linkages, and the second type consists of a branched polymer of L-fucopyranose with alternate α-1,3 and α-1,4 glycosidic linkages (Jiao et al. 2011). Sulphate and/or acetate of the α-Lfucopyranose is often situated in C-2, C-4 position, sometimes in C-3 or distributed in C-2, C-4 and C-2, C-3 positions (Ale and Meyer 2013). Its molecular weight ranges from around 43 to 1600 Kilodalton (kDa) (Fletcher et al. 2017). Only 10–20% of Fucoidan is present in the total dry weight of seaweed, a maximum of 20% is recorded in Fucus vesiculosus (Bilal and Iqbal 2020). The yield of fucoidan varies with the type of algae and season of collection. Autumn contains a higher percentage of fucoidan than any other season. It is primarily involved in the maintenance of water and ion, and protects from osmotic stress and drying up during low tides. Extraction of fucoidan could be carried out by subjecting the sample to ultrasounds (Ultrasound-Assisted Extraction: UAE), microwaves (Microwave-Assisted Extraction: MAE), enzymes (enzyme assisted extraction), supercritical fluids (Supercritical Fluid Extraction: SFE) conventional extraction methods such as hot water, acid (HCl) extraction from brown seaweeds such as Fucus evanescens, Ascophyllum nodosum, Sargassum oligocystum, N. Zanardinii, P. Tetrastromatica, Padina tetrastromatica, etc. (Jönsson et al. 2020). These polysaccharides showed a broad range of biological properties, namely antitumour, anticancer, antidiabetic, anti-inflammatory, anticoagulant, antioxidant, immunomodulatory, antibacterial, antiproliferative and antithrombotic (Hentati et al. 2020).

4.2 Alginate Alginate is a linear structure that consists of D-mannuronic acid linked with β-1,4 glycosidic linkages (M) and L-guluronic acid linked with α-1,4 glycosidic linkages (G) which are consecutively arranged M residues or G residues or alternating M and G residues, depending on the type of seaweed (Bilal and Iqbal 2020). Physical, mechanical properties and biocompatibility of the compound depend on the M/G ratio, copolymer size, G block properties (Zhao et al. 2012). Salt of alginic acid is known as alginate and the cell wall of brown seaweed contains 17–47% of the alginate on a dry weight basis. Alginate is extracted from the seaweed in the form of its sodium or calcium salts, predominantly from brown seaweed species such as Ascophyllum, Durvillaea, Lessonia, Ecklonia, Laminaria, Macrocystis, Turbinaria and Sargassum (Bilal and Iqbal 2020). Alginate is primarily not soluble and forms a colloidal solution with water. The calcium salt of alginic acid forms water-insoluble gels whereas, sodium alginic acid salts and magnesium alginic acid salts form water-soluble due to interaction between monovalent sodium and the carboxyl group of alginic acid. The addition of divalent ions of alkali metal, such as magnesium (Mg+2), calcium (Ca+2), barium (Ba+2) and strontium (Sr+2) increases the gelation property of alginate at the same temperature. Alginate is a non-toxic, bioactive, biocompatible, biodegradable polymer and contains stabilizing, emulsifying and gelling properties (Bilal and Iqbal 2020). Due to these properties, alginates are used in medicine, cosmetics, agriculture, paper, textile, food industries and tissue engineering (Jönsson et al. 2020).

4.3 Carrageenan Carrageenan is an anionic sulphated polysaccharide obtained from red seaweed along with protein, lipids, polysaccharides and pigments. It has a high molecular weight with alternating

124 Extremophiles: Wastewater and Algal Biorefinery α-1,3-D-galactopyranosyl and β-1,4-D-galactopyranosyl groups and 3,6-anhydrogalactose residues (Jönsson et al. 2020). Depending on the quantity, place of sulphate ester groups and 3,6-anhydrogalactose numbers, carrageenan is categorized into λ, θ, ι, κ, µ, ν namely lambda, theta, iota, kappa, mu and nu. Lambda, kappa, along with iota forms a remarkable and commercially important structure of carrageenans (Iqbal et al. 2018). Lambda, kappa and iota sequentially contains 32–39%, 25–30%, 28–30% sulphate ester, also kappa, iota contains 28–35%, 25–30% anhydrogalactose, respectively (Jönsson et al. 2020). Various red seaweed species comprise of different carrageenan, for example, Kappaphycus alvarezii mostly contains κ-carrageenan, Eucheuma dendiculatum contains ι-carrageenan also Chondrus crispus and Sarcothalia crispata contains κ- and λ-carrageenan together (Jönsson et al. 2020). Biologically, iota, kappa, lambda produce nu, mu, theta forms of carrageenan. The water solubility, viscosity, gelation property depends on the sulphate ester and cations (K+ and Na+) equilibrium. An increase in the quantity of sulphate ester decrease solubility temperature and gel strength in terms of affecting carrageenan applications. All forms of carrageenans are hydrophilic and insoluble within organic solutions. The viscosity of carrageenan increases with a rise in the concentration of crosslinkers like gum, salt and organic compounds (Bilal and Iqbal 2020).

4.4 Agar Agar is a polysaccharide of red seaweeds extracted mainly from Gelidium and Gracilaria species (Lebbar et al. 2018). Agar is comprised of agarose and agaropectin. Agarose is a high molecular weight subunit and contributes a large portion of approximately 70% of agar whereas agaropectin is a lower molecular weight subunit and contributes only 30% of agar. Agarose is a linear polymer of disaccharide agarobiose having (β-1,3) D-galactose linked with (α-1,4) 3,6-anhydro-L-galactose (Pérez et al. 2016). It constitutes a double helical structure, and shows water holding capacity. Agarose and agaropectin share similar linear structures, the only difference is that in agaropectin a large quantity of sulphate esters and different proportions of pyruvic and D-glucuronic acid is present. It alters its gelling properties (Bilal and Iqbal 2020).

4.5 Ulvan Ulvan is a sulphated polysaccharide obtained from green seaweed species such as Ulva and Monostroma. It consists of rhamnose, xylose, glucuronic acid, iduronic acid, i.e., Rha, Xyl, GlcA, IdoA and sulphate groups. In Ulva species, ulvan is composed of repeating disaccharides like uronic acid-rhamnose where uronic acid could be either glucuronic acid or iduronic acid. In Ulva pertusa, repeated groups rhamnose-glucose, rhamnose-xylose are present (Hentati et al. 2020). The average molecular mass of this uronic acid-rich polysaccharide, i.e., ulvan is ranged from 189 kDa to 8200 kDa (Bilal and Iqbal 2020). The extraction of ulvan is carried out using hot water and its purity could be increased by ethanol precipitation and subsequent freeze-drying. The quantity and constitution of ulvan are dependent on seaweed species, environmental conditions, season and the extraction method. Groups like iduronic acid and sulphated rhamnose in an ulvan form important compounds. Iduronic acid is used for the synthesis of heparin equivalent with antithrombotic activities (Bilal and Iqbal 2020).

4.6 Laminarin Laminarin is predominantly a storage glucan of all brown seaweeds like that of starch in terrestrial plants (Graiff et al. 2016, Bilal and Iqbal 2020). Laminarin is a linear molecule of β-1,3-D-glucopyranose with branching at β-1,6-D-glucopyranose (Kraan 2012). It is a short polymer of 20–25 monomeric units having D-glucose (50–69%) and D-mannitol (1–3%). It has a molecular mass of 2.9 kDa to 3.3 kDa. The quantity of β-1,3-D-glucopyranose to β-1,6-D-glucopyranose

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125

differs in various algal species. The polymerization degree, duration of extraction and solvent used for extraction affect the molecular mass. Molecular mass expands with an increase in the duration of extraction similarly, HCl extraction gives higher molecular mass laminarin as compared to H2SO4 (Saccharina and Laminaria species) (Bilal and Iqbal 2020). The water solubility of laminarin is related to β-1,6 branching. With a rise in branching, water solubility also increases (Graiff et al. 2016). The amount of laminarin varies because of environmental factors such as season, habitat and recorded up to 32% of the total mass (Bilal and Iqbal 2020). Two types of laminarins have been investigated G-chain and M-chain depending on differences in their reducing end. G-chain consists of D-glucopyranose and M-chain consists of D-mannitol residue at the reducing end (Kadam et al. 2014). The appearance of the percentage of M-chains decides the solubility of laminarin. Laminarin having 75% M-chains are generally soluble, and the one that is less than or equal to 45% is found to be insoluble (Hentati et al. 2020). Laminarin and its derived oligosaccharides show promising health benefits such as prebiotic, antioxidant, anticoagulant, hypocholesterolemic, antimutagenic, anti-inflammatory and anti-cancer activity (Jönsson et al. 2020).

4.7 Other Polysaccharides (Porphyran, Cellulose & Starch) Apart from SPs, seaweeds also contain polysaccharides like cellulose and starch. The cellulose is present in all three seaweeds and ranges from 2–20% on a DW basis. The highest yield of cellulose is reported from green seaweeds as they have high growth and photosynthetic rates. Cellulose has been reported from different seaweed genera like Ulva, Chaetomorpha, Gracilaria, Gelidium, Kappaphycus, Champia, Sarconema, Sargassum, Dictyota, etc. (Siddhanta et al. 2009, Trivedi et al. 2013a, Baghel et al. 2015). Further, seaweed cellulose has also been explored to produce bioethanol, biomethane, paper, biodegradable films by several researchers across the globe. Another polysaccharide reported from seaweeds is starch. Though very few reports are available on seaweedderived starch, most are reported from green seaweeds. The starch content in green seaweed sp. Ulva has been reported up to 32% on a DW basis (Prabhu et al. 2019, Korzen et al. 2015). Starch has proven applications in textile, food, pharma, fermentation, cosmetic and biopolymer industries (Santana et al. 2014). The schematic representation of seaweed polysaccharides, their respective degrading enzymes and possible applications have been shown in Fig. 6.2.

Fig. 6.2. Schematic presentation of seaweed polysaccharides, potential seaweed polysaccharide degrading enzymes and their possible applications.

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5. Seaweed Polysaccharide Degrading Marine Extremophilic Enzymes and Applications The marine habitat is composed of a huge variety of living microorganisms that can sustain extreme environmental conditions such as extreme temperature, pressure, salt concentration, etc., and give rise to the extremophilic enzymes to confirm their existence. These extremozymes produced by extremophiles exhibiting abilities such as thermostability, halostability could be used for the development and enhancement of the processes and product development in the biotechnological sector (Jahromi and Barzkar 2018). Therefore, the major part of this chapter mainly focuses on the marine extremozymes and its application. The enzymes produced by extremophiles include various seaweed polysaccharides degrading enzymes such as agarase, carrageenase, alginate lyase, ulvan lyase, cellulases, porphyranase, laminarase. Marine bacterial species Saccharophagus degradans strain 2–40 isolated from salt marsh cordgrass in the Chesapeake Bay watershed. It can degrade several polysaccharides which involve agar, cellulose, alginic acid, pectin, β-glucan, chitin, laminarin, xylan, pullulan and starch using it as the only carbon source (Fraiberg et al. 2011). To identify the particular enzyme-producing bacteria 16S rRNA sequencing method was implemented. In this technique, the 16S rRNA sequence is amplified using PCR with specific primers. Further, the PCR product is purified and sequenced. The sequence is compared with the NCBI database by the BLAST algorithm, the closest match will be the isolated species (Fu et al. 2008). Similarly, fungal species identification is carried out by the amplification of ITS (Internal Transcribed Spacer) from the fungal DNA using ITS primers (Furbino et al. 2018).

5.1 Agarase Red seaweed genera such as Gracilaria, Gelidium mainly produce agar. The degradation of agar is carried out by the agarase enzyme. The agarase producing microbes include Vibrio, Pseudomonas, Alteromonas, Microbulbifer, Thalassomonas, Salegentibacter, Zobellia, Agarivorans, Pseudoalteromonas, Paenibacillus, Bacillus, Acinetobacter, etc. (Jahromi and Barzkar 2018). Several agarase-producing microorganisms were isolated from marine sediments, seawater, marine algae, marine mollusks capable of producing agarase conversely only a few freshwater microorganisms have been reported having agarase producing ability (Fu and Kim 2010). The enzyme responsible for the hydrolysis of α-1, 3 linkages, β-1, 4 glycosidic linkages of agar is known as α-agarase, β-agarase respectively (Jahromi and Barzkar 2018). Lysis of agar converts it into agaro-oligosaccharides and neoagaro-oligosaccharides by α- and β-agarases respectively. The agaro-oligosaccharides hold anti-cancer as well as antioxidant properties while neoagarooligosaccharides hold antioxidant, antitumour, macrophage stimulating, skin moisturizing and probiotic properties (Kim et al. 2011a, Ohta et al. 2005a). Marine microorganisms can survive several extreme environmental conditions and are shown to secrete thermostable and halostable enzymes (Barzkar et al. 2018). Agarase is also used for its stability in a wide range of temperature, and pH, its high substrate affinity, high agarose degrading activity, because of this it shows promising application in food, cosmetics and medical industries (Freitas et al. 2012). 5.1.1 Applications of Agarase (a) Agarses are used in the production of oligosaccharides which is beneficial for human health, as oligosaccharides comprise key biological and physiological functions (Fu and Kim 2010). (b) Agarase enzyme has been used for the degradation of agar and eventually the cell wall of seaweed species to extract several bioactive substances. These substances show antioxidant

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(Wang et al. 2004a), anti-inflammatory (Yun et al. 2013), antibacterial properties. Therefore, agarase has attracted the interest of several biotechnological industry applications. (c) Agarase is used for protoplast isolation from seaweeds (Fu and Kim 2010). (d) One of the applications of the agarase is to retrieve DNA from agarose gel (Fu and Kim 2010). (e) It helps to study the structure of polysaccharides of the seaweed cell wall (Fu and Kim 2010). 5.1.2 Qualitative Assay of Agarase To determine the presence of the extracellular agarase producing bacterial colonies on the agar Petri plate, Lugol’s iodine is used. The dark stained portion indicates the presence of the polysaccharides in the sugar and the absence of the stain around the colonies is observed in the case of agarase producing microorganisms, which indicates that the polysaccharides are broken down into the monosaccharides and oligosaccharides (Kim et al. 2011). SDS-PAGE (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is carried out by the method described by Laemmli and gel can be stained with the Coomassie Brilliant Blue for the protein identification. Also, molecular weight determination was done by comparing with molecular mass standards. Removal of SDS is carrying out by dunking the gel, three times in 50 mM MOPS for 30 minutes total, followed by overspreading the SDS gel on 1.5% agar and 50 mM MOPS sheet at 37°C for 30 minutes. This agar sheet was stained with iodine to visualize the agarase (Ohta et al. 2004). 5.1.3 Quantitative Assay of Agarase Agarase degrades agar which will lead to a rise in the concentration of the reducing sugars and it is estimated spectrophotometrically by Nelson method with D-galactose as a standard (Fu et al. 2008) or with DNS method with D-glucose as standard (Hu et al. 2009). One unit of enzyme activity (U/mL), i.e., agarase activity is the quantity of enzymes required to liberate 1 µmol of reducing sugar (D-glucose or D-galactose) from agar per minute (Hu et al. 2009). 5.1.4 Purification of Agarase Numerous methods have been used together for agarase purification, such as ammonium sulphate precipitation and subsequent anion exchange chromatography, gel filtration, acetone precipitation, hydroxyapatite and hydrophobic chromatography (Jahromi and Barzkar 2018). The α-agarases have low activity and productivity than β-agarases (Seok et al. 2012). This will result in complications in isolations of α-agarases, which could be resolved by optimization of fermentation conditions. This problem of low activity and productivity could also be resolved by using a recombinant technology to obtain α-agarase (Jahromi and Barzkar 2018). A number of articles have been reported for the isolation of marine extremozymes such as agarase, carrageenase, alginate lyase, ulvan lyase, etc. A few examples of agarase isolated from marine extremophiles include the bacterial species Microbulbifer sp. SD-1 isolated from mud, seaweed and algae samples in Busan, Korea. It is capable of hydrolyzing agar which is confirmed by the zone surrounding the bacterial colonies when grown on a 1.5% agar Petri plate (Kim et al. 2011a). Also, a further study reported the isolation of agar degrading bacteria called Thalassomonas sp. JAMB-A33 produces endo-type α–agarase which can degrade not only agarose, but also neoagarohexaose, agarohexaose and porphyran (Ohta et al. 2005a). Fungi such as Penicillium chrysogenum, Penicillium sp., Beauveria bassiana, Pseudogymnoascus sp., Doratomyces sp. isolated from cold conditions of the Antarctica shows the production of agarase and carrageenase (Furbino et al. 2018). Some of agarase producing marine extremophiles have been shown in Table 6.1.

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Table 6.1. Different agarase producing marine extremophiles. Species Name

Category

Extremophile

Optimum pH

Optimum Temperature

Marine Extremozyme

Characteristic of Extremozyme

Isolated From

Reference

1.

Microbulbifer sp. SD-1

Bacteria

Thermophile 60°C (68% of maximum growth rate)

6 upto pH 9.0

30°C upto 60°C

Extracellular Agarase

Degrade agar into a monosaccharide and oligosaccharide

Busan, Korea from mud, sea water, seaweed samples

Kim et al. 2011a

2.

Thalassomonas sp. JAMB-A33

Degrade agarose into agarotetraose also capable of degrading agarohexaose, neoagarohexaose, and porphyran

Sediment off Noma Point, Japan, at a depth of 230 m

Ohta et al. 2005a

Bacteria

Piezophiles

8.5

45°C

Endo-type α-agarase (agarasea33)

Fungi

Psychrophiles

–

15°C

Carrageenase and agarases

Carrageenolytic and agarolytic activities

Macroalgae from maritime Antarctica

Furbino et al. 2018

Penicillium chrysogenum Penicillium sp. 3.

Beauveria bassiana Pseudogymnoascus sp. Doratomyces sp.

4.

Bacillus sp. BI-3

Bacteria

Thermophile

6.4

70°C

Thermostable Agarase

Degrade agar to form neoagarobiose

Hot spring on the coast of Kalianda Island, Indonesia

Li et al. 2014

5.

Flammeovirga sp. OC4

Bacteria

Piezophiles

–

–

Thermostable and ph-Stable β-Agarase

Degrade agar

Deep-Sea South China Sea

Liu et al. 2015, Chen et al. 2016

Extremophiles: Wastewater and Algal Biorefinery

Sr. No.

Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes 129

5.1.5 Future Direction: Agarase The recombinant techniques could be the solution to enhance the activity, productivity and stability of the enzymes for industrial purposes. The common host will be E. coli for the production of recombinant enzymes also, Bacillus subtilis is used in some cases such as Microbulbifer (Ohta et al. 2004, Jahromi and Barzkar 2018) (Table 6.2). As agar starts gel formation at a temperature around 42–45°C depending on the concentration of agar. Due to this, the isolation of DNA from agar gel agarase should have thermostability. The engineered bacterial species that produce agarase having thermostability and pH-stability have been reported previously (Chen et al. 2016). Hence, according to the requirement of the industrial and biotechnological process, microorganisms could be modified by various processes such as genetic engineering to produce specific enzymes such as thermostable agarase, to improve the production quantity, which could be further used in the cosmetic, pharmaceutical and food industries (Table 6.2).

5.2 Carrageenase Carrageenan is linear sulphated polysaccharides present in the extracellular matrix of the Rhodophyta, i.e., Red algae (Campo et al. 2009). Depending on the sulphate substituents attached to disaccharide units, carrageenans are categorized as kappa (κ), iota (ι) and lambda (λ) carrageenan and contains one, two and three sulphate substituents, respectively (Ghanbarzadeh et al. 2018, Chauhan and Saxena 2016). Carrageenase is the enzyme that degrades carrageenans into small molecular weight oligosaccharides which have pharmacological and therapeutic applications. It forms water-soluble colloids. Structurally, it contains sulphate half-esters attached to the linear polysaccharide chain of sugar. Carrageenase cleaves β-(1,4)-linkage forming evennumbered oligosaccharides as all the carrageenase are generally endolytic enzymes (Zhu et al. 2018). The carrageenase is mainly produced by Proteobacteria and Bacteroidetes. Mostly Gramnegative bacteria (Cellulophaga, Pseudomonas, Cytophaga, Pseudoalteromonas, Tamlana, Vibrio, Catenovulum, Microbulbifer, Zobellia, Alteromonas) are employed for the degradation of red seaweed. However, some Gram-positive bacteria such as Bacillus sp. and Cellulosimicrobium also show the degradation of red seaweed (Chauhan and Saxena 2016). Many studies have been reported for the isolation of the carrageenase producing microorganisms from the marine environment (Zhu et al. 2018). Pseudoalteromonas porphyrae LL1 strain was isolated from the decayed seaweed from the Yellow Sea, China (Liu et al. 2011). Further, its genes for the carrageenase activity is cloned and expressed in Brevibacillus choshinensis. κ-carrageenase could be produced from the given cloned microorganism, which can degrade κ-carrageenan into κ-carrageenan oligosaccharides (Zhao et al. 2018). Extracellular κ-carrageenase producing Pseudoalteromonas sp. QY203 is also isolated from decayed red algae collected from the coast of Qingdao, China (Li et al. 2013). Cellulophaga lytica strain N5-2 isolated from the sediments (Yao et al. 2013). Cellulophaga sp. QY3 from red algae named Grateloupia livida was reported from Qingdao coastal water Yellow Sea, China (Ma et al. 2013). Similarly, carrageenan degrading Pseudoalteromonas sp. AJ5-13, Pseudomonas elongate has been isolated from holothurian Apostichopus japonicas, marine algae from a holothurian farm and sea area in Dalian, China, and red algae collected from the west coast of India, respectively (Ma et al. 2010, Khambhaty et al. 2007). Marine extremophile Pseudoalteromonas bacterium present in the deep-sea sediments also shows the production of the carrageenase (Ohta and Hatada 2006) (Table 6.3).

130

Table 6.2. Genetically engineered marine extremophiles producing agarase. Type of Agarase

Source

Extremophile

Isolated From

Expression Strain

Name of the Gene

Number of Amino Acid Produced

Optimum pH

Optimum Temperature

Improvement

Reference

1.

β-agarase

Flammeovirga pacifica WPAGA1

Piezophiles

Deep-sea sediment

E. coli BL21 (DE3)

aga4383

965 amino acids

9

50°C

Thermostable and pH-Stable

Hou et al. 2015

2.

β-Agarase

Flammeovirga sp. OC4

Piezophiles

Deep-sea water of the South China Sea

Escherichia coli BL21 (DE3)

aga4436

456 amino acids

6.5

50−55°C

Thermostable and pH-Stable

Chen et al. 2016

3.

β-Agarase

Microbulbifer sp. JAMB-A7

Piezophiles

Sediment samples in Sagami Bay at a depth of 1,174 m

Bacillus subtilis cells (RagaA7)

agaA7

441 amino acids

7

50°C

Thermostable and resistant to various chemical reagents

Ohta et al. 2004

4.

extracellular α-agarase

Thalassomonas sp. strain JAMB-A33

Piezophiles

Sediment in Kagoshima Bay, Japan, at a depth of 230 m

Bacillus subtilis

AgaA33

1463 amino acid

8.5

45°C

Improvement of its antioxidant activity with regard to freeradical-scavenging capacity and superoxide radical anion scavenging activity

5.

β-Agarase

Agarivorans sp. JAMB-A11

Piezophiles

South side of the Kuril Trench at a depth of 4152 m

Bacillus subtilis

AgaA11

995 amino acids

7.5–8.0

40°C

30-fold greater production

Hatada et al. 2006

Ohta et al. 2005b

Extremophiles: Wastewater and Algal Biorefinery

Sr. No.

Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes 131 Table 6.3. Different carrageenase producing marine extremophiles. Sr. No.

Species Name

1.

Pseudoalteromonas bacterium

Extremophile

Marine Extremozyme

Characteristic of Extremozyme

Isolated From

Reference

Piezophiles

endo-type λ-carrageenan

Degrade Carrageenase Gives final obtained is tetrasaccharide

A sediment sample from Suruga Bay, Japan, at a depth of 2409 m

Ohta and Hatada 2006

5.2.1 Application of Carrageenase (a) Carrageenan, a polysaccharide when degraded by the carrageenase enzyme released an even number of oligosaccharides. These oligosaccharides possess health benefits such as antitumour, anti-inflammatory, antithrombosis and anticoagulation effect (Zhu et al. 2018). (b) Carrageenase enzyme also helps to isolate and form algal protoplast (Khambhaty et al. 2007). (c) It is used to study the detailed structure of carrageenan and also the metabolism of the bacteria because carrageenase is specific for carrageenan (Anastyuk et al. 2011). (d) Abundant carrageenan polysaccharide waste has been generated by the seaweed processing exercises. This waste could be converted into fermentable sugars to save the marine environment. These resultant sugars have the potential to generate bioethanol (Kang and Kim 2015). (e) Carrageenase is used in the textile industry to withdraw excess printing paste left after textile printing. The common practice is to wash these printing pastes with the help of water. But, for this the water requirement is large because of the more viscosity of the printing paste. This issue could be resolved by the use of carrageenase along with other polysaccharides, which helps to lower down the processing time, energy and water requirement (Pedersen et al. 1995). (f) One of the applications of the carrageenans is in dairy products such as jams, ice-creams, yogurt. Due to the high affinity towards cellulose fibres, carrageenan is responsible for laundry stains. Carrageenase can hydrolyze these polysaccharides efficiently. Along with laundry stains, carrageenase is used in dish wash, contact lens and oral cleaners (Chauhan and Saxena 2016). (g) Carrageenase produced by the marine bacteria helps to prevent the red algal bloom. It is also used to prevent biofouling of the submerged surface like pipes (Chauhan and Saxena 2016). 5.2.2 Qualitative Assay of Carrageenase The presence of the carrageenase producing the bacterial colonies was identified by pouring 10% cetylpyridinium chloride on a solid media plate. A clear zone is observed around the carrageenase producing colonies. Another method for the identification of the enzyme carrageenase, SDS-PAGE is performed by the method described by Laemmli (Laemmli 1970). The gel, then is soaked in 50 mM MOPS having 0.4 M NaCl three times for the total period of 30 minutes. It is overspread on gel containing 0.5% ι-carrageenan, agarose (1%) and 50 mM MOPS (pH 7.0) and incubated at 25°C for 3 hours. The gel is further stained with 10% cetylpyridinium chloride, a clear zone indicates the presence of carrageenase (Ohta and Hatada 2006). Similarly, the carrageenase is visualized by using alcian blue. The SDS-PAGE is overspread on a gel containing polysaccharides, which will degrade by the carrageenase from SDS-PAGE, oligosaccharides are washed out and polysaccharides will be stained with the alcian blue. Hence, the presence of the carrageenase is represented by the presence of a clear zone (Smith et al. 2005). Molecular weight determination of carrageenase is carried out by staining SDS-PAGE with Coomassie brilliant blue and comparing with the standards (Ohta and Hatada 2006).

132

Extremophiles: Wastewater and Algal Biorefinery

5.2.3 Quantitative Assay of Carrageenase The quantitative analysis for the estimation of the carrageenase is detected by measuring the concentration of the reducing sugar after hydrolysis of carrageenan (Yao et al. 2013, Kidby and Davidson 1973). One unit of enzyme activity (U/mL), i.e., the carrageenase activity is the quantity of enzymes required to liberate 1 µmol of reducing sugar, i.e., D-galactose per minute (Ohta and Hatada 2006). The dinitrosalicylic method could also be used for determining the increasing concentration of reducing sugar spectrometrically for the quantitative estimation of the carrageenase (Miller 1959). 5.2.4 Purification of Carrageenase After fermentation, initial purification of carrageenase is commonly carried out by the ammonium sulphate fractionation (Li et al. 2013). Whereas, ion-exchange chromatography, gel filtration chromatography, affinity chromatography could be used for the further purification (Liu et al. 2011, Ohta and Hatada 2006). An example of the purification κ-carrageenase is reported by Yao et al (2013). It started with ammonium sulphate fractionation, followed by gel filtration by SephadexG-200 and SephadexG-75. This purified enzyme is further confirmed by the SDS-PAGE (Yao et al. 2013). 5.2.5 Future Direction: Carrageenase To scale up the production, recombinant carrageenases producing strain could be used. A cloned strain helps in increasing the production of the carrageenase than the wild strain (Liu et al. 2014). Cloning could not only improve the production of the carrageenase and tolerance to extreme conditions but also will help to improve the enzyme activity (Yu et al. 2017, Li et al. 2017) (Table 6.4).

5.3 Alginate Lyase Alginate is present in brown seaweeds and made up of a different arrangement of β-D-mannuronate (M block) and α-L-guluronate (G block) or alternate β-D-mannuronate and α-L-guluronate (MG block). In the process of hydrolysis, oligosaccharides are formed when alginate lyase targets glycosidic linkage between poly M, G, MG block called poly M lyase, poly G lyase, poly MG lyase respectively (Xue et al. 2019). Also, alginate lyase is classified as endo and exo which degrade alginate by β-elimination mechanism. Exo type alginate lyase forms monosaccharides on the degradation of alginate and its oligosaccharides while endotype of alginate lyase forms unsaturated disaccharides, trisaccharides and tetrasaccharides on alginate degradation (Li et al. 2016, Zhu and Yin 2015). In the case of exo alginate lyase, reducing sugar formation rate is more and slower viscosity reduction is seen because of the terminal cleavage site. In the case of endo alginate lyase reducing sugar, the formation rate is slow, and rapid viscosity reduction is seen because of the intermediate cleavage site (Xue et al. 2019). Further, alginate lyase is categorized based on the hydrophobic cluster in their primary structure into seven major families, i.e., PL (polysaccharide lyase)-5, 6, 7, 14, 15, 17, 18 (Zhou et al. 2020). Exo-alginate lyase belongs to PL-15, 17 and endo alginate lyase belongs to PL-5, 7 families. Also, it can be differentiated based on their molecular weight, as small, medium, large alginate lyase, which have 20~35 kDa, 40 kDa, 60 kDa of molecular weight, respectively (Xue et al. 2019). To date, many alginate lyases has been isolated from brown seaweeds, marine micro-organisms, fungi, molluscs, terrestrial bacteria and viruses (Wong et al. 2000, Chen et al. 2018). Bacterial species that secrete alginate lyase comprises Bacillus, Vibrio, Pseudomonas, Microbulbifer, Flavobacterium, Klebsiella, Nitrogen-fixing bacteria, Bacillus, Enterobacter, Streptomyces. Certain marine organisms also possess alginate lyase in their hepatopancreas (Xue et al. 2019).

Table 6.4. Genetically engineered marine extremophiles producing carrageenase. Isolated From

Expression Strain

Name of the Gene

Number of Amino Acid Produced

Optimum pH

Optimum Temperature

Improvement

Reference

ι-carrageenase

Flavobacterium sp. YS-80-122

Psychrophile

Yellow sea sediment

E. coli strains DH5α and BL21 (DE3)

cgiF

484 amino acid

7.6

30°C

New coldadapted and thermo-tolerant

Li et al. 2017

2.

ι-carrageenase

Microbulbifer thermotolerans JAMB-A94

Piezophiles

Deep-sea Bacterium

Bacillus subtilis

cgiA

569 amino acids

7.5

50°C

improved production

Hatada et al. 2011

3.

κ-carrageenase

Pseudoalteromonas tetraodonis JAM-K142

Piezophiles

deep-sea bacterium

E. coli DH5α

Cgk-K142

397 amino acids

8

55°C

–

Kobayashi et al. 2012

Type of Carrageenase

Source

1.

Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes 133

Extremophile

Sr. No.

134 Extremophiles: Wastewater and Algal Biorefinery Some of the examples of alginate lyase producing marine microorganisms include Vibrio sp. SY08 isolated from the marine environment which have the ability to degrade alginate producing Unsaturated Alginate Disaccharides (UAD). This UAD has an industrial application in the production of antioxidants (Li et al. 2017). Another microorganism Flammeovirga sp. NJ-04 having alginate lyase ability has been isolated from the South China Sea (Li et al. 2019). Alginate lyase AlyL isolated from cloned marine bacteria Agarivorans sp. L11 shows the cold-adapted and surfactant-stable alginate lyase properties (Li et al. 2015). Extracellular alginate lyase producing marine bacteria Microbulbifer sp. have been isolated from rotten brown algae (Zhu et al. 2016). Microorganisms living in extreme conditions such as high temperature, salt conditions have also shown the production of the alginate lyase. Defluviitalea phaphyphila Alg1 bacteria has the optimum temperature of 55–60°C isolated from coastal sediment of an amphioxus breeding zone in Qingdao, China shows alginate lyase production (Ji et al. 2016). Halophilic bacteria, such as Isoptericola halotolerans CGMCC5336, Pseudomonas sp. W7 showed alginate lyase production (Dou et al. 2013, Kong et al. 1995) (Table 6.5). 5.3.1 Application of Alginate Lyase (a) Alginate lyase is used for the production of algal protoplast which could be adopted for the production of industrial material and in the food industries (Wong et al. 2000). (b) Alginate lyase is also used in the substrate specification studies. When polysaccharides are degraded by the poly M specific alginate lyase, which could be further used for the determination of the substrate specificity of the other alginate lyase (Wong et al. 2000). (c) Alginate oligosaccharides contain several biological functions such as prebiotic properties. It helps to increase the beneficial bacteria, i.e., Lactobacillus, Bifidobacterium in rats when fed with unsaturated alginate oligosaccharides (Kim et al. 2011b). (d) Alginate oligosaccharides also promote the growth of the plants such as banana plantlets (Kim et al. 2011b). (e) Alginate oligosaccharides possess therapeutic applications like the stimulation of cytotoxic cytokine secretion in human macrophages, increase in granulocyte colony secreting factor, tumour necrosis factor-a (TNF-a), inhibit the production of ROS from immune cells (Kim et al. 2011b). (f) Alginate monosaccharides are used in the production of biofuel such as bioethanol, biohydrogen, biodiesel (Kim et al. 2011b). (g) The alginate lyase is used for the degradation of seaweed waste (Tang et al. 2009). (h) It is also used for the treatment of cystic fibrosis (Islan et al. 2014) and against pathogenic bacteria (Lamppa and Griswold 2013). 5.3.2 Qualitative Assay of Alginate Lyase Bacterial colonies producing alginate lyase degrade the alginate present in the media. The absence of the alginate gives a clear zone when stained with cetyl pyridinium chloride or ruthenium red when treated for 30 minutes (Gacesa and Wusteman 1990). Also, Gram’s iodine is used to stain the alginate, a clear zone around the colonies represents the production of alginate lyase (Sawant et al. 2015). 5.3.3 Quantitative Assay of Alginate Lyase The quantity of alginate lyase is measured by the amount of reducing sugar produced by a variety of methods such as DNS method, Somogyi-Nelson method and UV absorption method.

Table 6.5. Different alginate lyase producing marine extremophile. Species Name

1.

Psychrobacter, Winogradskyella, Psychromonas and Polaribacter, Pseudoalteromonas

Bacteria

Psychrophile

7

10–30°C

2.

Defluviitalea phaphyphila Alg1

Bacteria

Thermophile

7 and 8

55–60°C

Category

Extremophile

Optimum pH

Optimum Temperature

Marine Extremozyme

Characteristic of Extremozyme

Isolated From

Reference

Alginate lyase

Cold condition adapted alginate lyase

Intertidal zone of the Kings Bay in Mar. Drugs 2012, 10 2483 Ny-Ålesund, Svaldbard during the Chinese Arctic Yellow River Station

Dong et al. 2012

Exotype alginate lyase

Degrade brown algae

Coastal sediment of an amphioxus breeding zone in Qingdao, China

Ji et al. 2016

Rotten seaweed samples from an industrial production area of sodium alginate in Lianyungang City, China

Dou et al. 2013

3.

Isoptericola halotolerans CGMCC5336

Bacteria

Halophile

7

50°C

Alginate lyase

Bifunctional substrate specificity for polyguluronate and polymannuronate used for alginate oligosaccharides production with low polymerisation degrees

4.

Pseudomonas sp. W7

Bacteria

Halophile

–

25°C

Alginate lyase

–

Laver in the southern sea of Korea

Kong et al. 1995

5.

Psychromonas sp. C-3

Bacteria

Psychrophile

8

20°C

Alginate lyase

First dimeric endo-alginate lyase structure. Salt activated alginate lyase

The Arctic brown alga Laminaria

Xu et al. 2020

Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes 135

Sr. No.

136

Extremophiles: Wastewater and Algal Biorefinery

In the DNS method, absorbance is measured spectrophotometrically at 540 nm, when alginate lyase is reacted with sodium alginate gives reddish-brown coloured complex. In the Somogyi-Nelson method, absorbance is measured at 520 nm, when copper oxide is mixed with the arsenomolybdate. In the UV absorption method, increased absorbance is measured spectrophotometrically at 235 nm (Dharani et al. 2020). 5.3.4 Purification of Alginate Lyase Purification of the alginate lyase starts with the ultrafiltration of the supernatant followed by the ion-exchange chromatography. The fraction obtained from ion-exchange chromatography is further applied to the gel filtration chromatography. Protein is routinely monitored spectrophotometrically at 280 nm. Molecular weight determination of the fraction containing alginate lyase is done by the SDS-PAGE (Hu et al. 2006). Other methods such as affinity chromatography, size exclusion chromatography, gel-permeation chromatography could be explored for the purification of the alginate lyase (Dharani et al. 2020). 5.3.5 Future Direction: Alginate Lyase To enhance the quality, production of the extremozymes techniques such as cloning, overexpression, purification are used. The increase heat stability, production of the heat stable oligosaccharides can be achieved by the cloning (Inoue et al. 2016, Zhu et al. 2017) (Table 6.6).

5.4 Ulvan Lyase Ulvan is a polyanionic heteropolysaccharide produced by green algae and mainly consists of rhamnose, glucuronic acid, iduronic acid, xylose monosaccharides. The enzyme which degrades ulvan by the β-elimination reaction into its oligosaccharides is known as ulvan lyase. Most of the ulvan lyases are endo-lytic enzymes that hydrolyze β-1,4 glycosidic bonds and form different oligosaccharide units. The ulvan lyase is predominantly isolated from the marine bacteria and the bacteria present in the intestine of the marine animals (Li et al. 2020). Ulvan lyase is categorized into five polysaccharide lyase families, i.e., PL-24, 25, 28, 37 and 40 based on their protein sequence. The majority of the ulvan lyase shows an ideal temperature range from 30°C to 50°C. For example, ulvan lyase such as NLR42, ALT3935 obtained from Nonlabens ulvanivorans NLR42, Alteromonas sp. A321, respectively showed the optimal temperature at 50°C. It shows the optimum pH ranges from 7.0 to 8.0 but, NLR42 enzyme shows the ideal pH of 9.0 (Li et al. 2020). The sources of ulvan lyase includes bacteria, such as Alteromonas (Foran et al. 2017), Pseudoalteromonas, Nonlabens ulvanivorans (Ulaganathan et al. 2017), Formosa agariphila (Reisky et al. 2018). Ulvan lyase from Formosa agariphila KMM 3901T shows the adaptation to the temperature at which it is being isolated, turning over a number of the enzyme and ulvan affinity is affected by high NaCl concentration. Divalent metal ions like Ca2+ act as a cofactor for the ulvan lyase (Reisky et al. 2018). Alteromonas portus HB161718T isolated from coastal sand from Tanmen Port in Hainan, PR China have also shown ulvan lyase production (Huang et al. 2020). Ulvan degrading pathway has been observed in F. agariphila KMM 3901T 88 isolated from Japanese Sea green algae (Reisky et al. 2019, Reisky et al. 2018). Ulvan lyase producing Alteromonas sp. KUL17 isolated from faeces of small marine Molluscs and shrimps having high ulvan degrading ability extracted from the Ulva ohnoi (He et al. 2017). Cloning in Escherichia coli BL21 (DE3) increases the thermostability in ulvan lyase gene, ALT3695 which was isolated from Alteromonas sp. A321 mainly produces disaccharides and tetrasaccharides after degrading ulvan (Gao et al. 2019). Salt tolerant marine extremophile Alteromonas sp. (AsPL) also shows the ability of ulvan degradation (Qin et al. 2020) (Table 6.7).

Table 6.6. Genetically engineered marine extremophiles producing alginate lyase. Sr. No.

Endotype alginate lyase

Source

Flammeovirga sp. NJ-04

2.

Endotype alginate lyase

Agarivorans sp.

3.

Alginate lyases

Nitratiruptor sp. SB155-2

Expression Strain

Name of the Gene

Number of Amino Acid Produced

Optimum Temperature

Improvement

Reference

7

50°C

Heat stable with produce oligosaccharites with degree of polymerization (DP) of 2–5

Zhu et al. 2017

289 amino acids

9

30°C

High alkaline, salt activated alginate lyase

Kobayashi et al. 2009

243 amino acids

6

70 °C

Heat stability

Inoue et al. 2016

Extremophile

Isolated From

Piezophiles

Deep seawater of South China Sea (118°01′E, 21°21′N) at the depth of 5219 m

E. coli BL21 (DE3)

FsAlgA

550 amino acid

Piezophiles

Deep-sea sediments were collected off Cape Nomamisaki, at the southwestern tip of Kyushu Island, Japan, at a depth of 254 m

E. coli TOP10

A1m

Piezophiles

Deep-sea hydrothermal vents at a water depth of 1,000 m

Escherichia coli

NIS_0185

Optimum pH

Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes 137

1.

Type of Alginate Lyase

138

Extremophiles: Wastewater and Algal Biorefinery Table 6.7. Different ulvan lyase producing marine extremophile.

Sr. No. 1.

Species Name

Category Extremophile

Alteromonas Bacteria sp. (AsPL)

Halophile

Optimum Optimum Marine pH Temperature Extremozyme 7.5 to 9.5

30–50°C

Ulvan lyase

Characteristic Isolated of Reference From Extremozyme Salt tolerance

Marine Qin et al. origin 2020

5.4.1 Applications of Ulvan Lyase (a) Ulvan lyase is used in the preparation of the ulvan oligosaccharides from the ulvan. These oligosaccharides comprise the anti-thrombus and antioxidant properties (Li et al. 2020). (b) It is used in the determination of the structure of the ulvan (Chi et al. 2020). (c) The problem of a huge amount of macroalgae from green tides could be resolved with the help of ulvan lyase. This resultant macroalgal biomass could be used for biorefinery purposes (Reisky et al. 2018). 5.4.2 Enzyme Assay of Ulvan Lyase The determination of the ulvan lyase is carried out by observing the reducing end formation by the ferricyanide method by Lane and Lawen (2008). The reducing end formation is observed spectrophotometrically at 415 nm. Ulvan lyase is also determined by the estimation of double bond formation at 235 nm (Kopel et al. 2016, Lane and Lawen 2008, Reisky et al. 2018). 5.4.3 Purification of Ulvan Lyase The ulvan lyase protein is purified by affinity chromatography using immobilized Ni2+ metal column followed by the size exclusion chromatography. After size exclusion chromatography single peak is observed. Protein profiling is further observed using gel (polyacrylamide) electrophoresis (Konasani et al. 2018). The quantity of the recombinant proteins is observed by using Nanodrop (Kopel et al. 2016).

5.5 Cellulase Cellulases are the most common and widely studied enzymes used for the saccharification of cellulose. Cellulases have been used for several applications in textiles, paper and pulp, biofuels and the production of platform chemicals. Cellulose is abundantly present in both terrestrial and marine plants. Marine biomass mainly seaweeds are found to have ~ 20% (DW) of cellulose and are a source of the production of biofuels like bioethanol, biobutanol and cellulose derived platform chemicals. These days, emphasis is given to cellulase produced by the bacterial species because of its robust nature, stability, tolerance to harsh conditions and the presence of the multi-enzyme complex (Acharya et al. 2012). Cellulose from several seaweed genera like Ulva, Gracilaria, Geliedella has been explored to produce bioethanol using cellulases from both isolated microbes and commercial origin (Trivedi et al. 2015, Baghel et al. 2015, Trivedi et al. 2016). Many marine bacteria have been isolated, having the ability to cellulolytic properties. Cladosporium sphaerospermu, Pseudoalteromonas sp. isolated from the marine environment with cellulase producing ability (Trivedi et al. 2015, Trivedi et al. 2013b). Bacteria present in the marine extreme environment also show the cellulytic ability along with the tolerance to the extreme environment. Bacillus flexus, Bacillus aquimaris are isolated from the Ulva, having a cellulytic ability (Trivedi et al. 2011a, Trivedi et al. 2011b). Bacteria, such as Clostridium thermocellum, Thermosipho affectus sp. Ik275mart, Bacillus sp. SR22 isolated from the extreme environments anaerobic, thermophilic, pH extreme conditions shows the cellulose production (Ji et al. 2012, Podosokorskaya et al. 2011, Dos Santos et al. 2018) (Table 6.8).

Table 6.8. Different cellulose-degrading marine extremophile. Species Name

Category

Extremophile

Optimum pH

Optimum Temperature

Marine Extremozyme

Characteristic of Extremozyme

Isolated From

Reference

1.

Bacillus flexus

Bacteria

Alkalihalotolerant

10

45°C

Cellulase

The enzyme showed 70% activity at 15% nacl

Degraded macroalgae Ulva lactuca

Trivedi et al. 2011a

2.

Bacillus aquimaris

Bacteria

Solvent tolerant

11

45°C

Cellulase

The enzyme was functionally stable with 85% residual activity at pH 12 and 95% residual activity at 75ºC

Degraded macroalgae Ulva

Trivedi et al. 2011b

3.

Clostridium thermocellum

Bacteria

Anaerobic and thermophilic conditions

7

60°C

Cellulase

A potential biofuel production and in lignocellulose conversion

Coastal marine sediment

Ji et al. 2012

4.

Thermosipho affectus sp. Ik275mart

Bacteria

Thermophilic

5.6–8.2

37–75°C

Cellulolytic bacterium

Ferment starch, cellulose and cellulose derivatives

Mid-Atlantic Ridge deep-sea hydrothermal vent

Podosokorskaya et al. 2011

5.

Bacillus sp. SR22

Bacteria

Temperature and pH tolerant

Cellulase

Salt-tolerant thermostable cellulase

From aseptically collected tissues of different Siderastrea stellata colonies at Cabo Branco coral reefs, Paraiba State, Brazil

Dos Santos et al. 2018

6.5

60°C

Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes 139

Sr. No.

140

Extremophiles: Wastewater and Algal Biorefinery

5.5.1 Applications of Cellulase Enzyme (Kuhad et al. 2011) (a) Food industry: Cellulase helps to improve the quality of food products, such as texture, flavour, processing, aroma, volatile properties. Along with this, it improves the starch and protein extraction and release of antioxidants from fruits and vegetables. (b) Agriculture: Cellulase helps to control pathogens and diseases, enhances soil quality, seed germination, root quality. It also helps to generate the plant and fungal protoplast. (c) Textile: Cellulase improves the softening of textile, quality, absorbance and the stability of the cellulosic fabrics. It also helps to remove the excess dye from the textiles. (d) Detergents: Cellulose acts as a better detergent because of the brightening and dirt removing capability without damaging the fabrics. (e) Paper and pulp industry: It is used for the production of biodegradable paper towels, cardboard, and sanitary papers. It helps to improve the strength, brightness of the fibers along with the reduction in energy requirement. (f) Bioconversion: It helps in the conversion of the cellulosic biomass to fermentable sugars, which further helps in the production of ethanol. 5.5.2 Qualitative Assay of Cellulase Isolated microorganisms grown on the CMC agar plates overnight is further treated with the Congo red (0.1%) for 15 minutes and washed with the 1 M NaCl solution. The cellulase-producing microorganisms show the clear zone because cellulase has been degraded into simple sugars (Ponnambalam et al. 2011). Similarly, CMC agar plates with microorganisms have been filled with the Gram’s iodine for 3 to 5 minutes. The Gram’s iodine reacts with the cellulose giving a bluishblack complex, but not with degraded cellulose, hence microorganisms secreting cellulose show the clear zone because of the cellulose degradation (Kasana et al. 2008). 5.5.3 Quantitative Assay of Cellulase For the quantitative estimation of the cellulolytic enzyme, it is shown to react with the Carboxy Methyl Cellulose (CMC) (1%) in phosphate buffer pH 5.8 for 20 minutes at 40ºC. The reducing sugars released is measured spectrophotometrically at 540 nm. The amount of sugar released per ml of enzyme per minute defines the enzyme activity (Ponnambalam et al. 2011, Saha et al. 2006). 5.5.4 Purification of Cellulase Partial purification of the cellulase is initialized with the ammonium sulphate precipitation, followed by dialysis. The molecular weight determination of the purified enzyme can be carried out by the SDS-PAGE (Shanmugapriya et al. 2012).

5.6 Other Seaweed Degrading Enzymes Porphyranase, a glycoside hydrolase, is an enzyme that hydrolyzes sulphated polysaccharide porphyran present in red seaweeds. Most of the porphyranase reported are of β type which cleaves the beta-1,4 linkage between beta-D-galactopyranose and alpha-L-galactopyranose6-sulfate (Hehemann et al. 2010, Zhang et al. 2019). Several microorganisms like Wenyingzhuangia fucanilytica, Pseudoalteromonas atlantica, Zobellia galactanivorans have been reported for producing porphyranase enzyme (Zhang et al. 2019, Przybylski et al. 2015, Correc et al. 2011).

Marine Extremophiles as a Source of Seaweed Polysaccharide Hydrolyzing Enzymes 141

Laminarase is another glycoside hydrolases which hydrolyzes laminarin. This enzyme specifically cleaves the β-1,3- and β-1,6-glycosidic linkages of laminarin (Davies and Henrissat 1995). Several microbial strains and genera like Aspergillus nidulans, Penicillium brevicompactum, Trichoderma paraviridescens, Rhizopus oryzae, Pseudoalteromonas citrea, Emericellopsis spcs., Acremonium spcs., Cytophaga, Alteromonas/Pseudoalteromonas, etc., have displayed laminarase activity (Patyshakuliyeva et al. 2020, Bakunina et al. 2000, Descamps et al. 2006). Hyperthermophilic Pyrococcus furiosus produce laminarase. The extracted laminarinase have also shown the adaptation at extreme temperatures (Gueguen et al. 1997, Ilari et al. 2009). Similarly, fucoidanase is the enzyme that hydrolyze the fucoidan (sulphated polysaccharide) mainly present in brown seaweeds. Fucoidanase has been produced by several microorganisms such as Mucor sp. 3P, Fusarium sp. (LD8), Vibrio sp. N-5, Bacillus sp. H-TP2, Pseudoalteromonas citrea (Rodriguez-Jasso et al. 2013, Qianqian et al. 2011, Sakai et al. 2003, Furukawa et al. 1992, Bakunina et al. 2002, Wang et al. 2004b). Enzymes produced by microbes living in extreme conditions have gained interest because of their industrial and biotechnological applications. Further to improve the quality, production and to achieve the specific requirement these extremophiles could be cloned, overexpressed and purified. The marine environment is the richest source for such microorganisms having a fascinating range of extremozymes producing microbes. Hence, it is crucial to study marine extremophiles producing extremozymes for diverse industrial applications.

6. Future Perspectives Marine extremozymes are naturally customized enzymes of aquatic extremophiles, having multifaceted applications in food, pharmaceutical, cosmeceuticals, paper, textiles, etc. Further, the stability of the extremozymes under harsh conditions could be explored for countless applications. The pertinence of these extremozymes has been increasingly examined in recent years, still, there is a need to investigate the application of these extremozymes on the bioengineering and molecular biological grounds. It is essential to apply research on a large scale, i.e., industrial level. To scale up the applications of the marine extremophiles, there is a requirement of optimization of culture conditions, and efficient handling procedure. Further, delving into the marine extremophiles on a metagenomics and proteomics basis could help to design customized microorganisms that hold genes for marine extremozymes by the process of recombinant DNA technology which are likely to be used for industrial applications. Marine extremozymes have been widely used to explore terrestrial resources. However, they have limited use in marine resources mainly algae. Marine macroalgae have proven applications due to their higher carbohydrate composition. If researchers can isolate unique extremophilic microorganism which can produce seaweed degrading extremozymes, it would boost the seaweed industry to produce food, feed, biofuels, bioactive compounds, platform chemicals and many other industrially important products. Very limited companies are producing seaweed degrading enzymes and there is a huge scope for the same if novel extremozymes can be obtained from marine microorganisms and extremophiles. By comprehensively investigating marine extremophiles based on their metabolism, proteomics, genetics; it could be used in a circular approach increasing reusability. All these will assist to pursue the eco-friendly, sustainable, economical, least waste generating, innovative industrial, environmental applications of marine extremozymes.

Acknowledgments The authors would like to acknowledge the Department of Science and Technology (DST), New Delhi, India for the DST INSPIRE Faculty grant. DM and NT have equally contributed to this book chapter.

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Yu, Y., Z. Liu, M. Yang, M. Chen, Z. Wei, L. Shi, L. Li and H. Mou. 2017. Characterization of full-length and truncated recombinant κ-carrageenase expressed in Pichia pastoris. Front. Microbiol. 8: 1544. Yun, E.J., S. Lee, J.H. Kim, B.B. Kim, H.T. Kim, S.H. Lee, J.G. Pelton, N.J. Kang, I. Choi and K.H. Kim. 2013. Enzymatic production of 3, 6-anhydro-Lgalactose from agarose and its purification and in vitro skin whitening and anti-inflammatory activities. Appl. Microbiol. Biotechnol. 97: 2961–2970. Yusmaniar, Y., D.I. Syafei, M. Arum, E. Handoko, C. Kurniawan and M.R. Asali. 2019. Preparation and characterization of seaweed based bioplastic blended with polysaccharides derived from various seeds of Avocado, Jackfruit and Durian. J. Phys. Conf. Ser. 1402: 055097. Zhang, G. and L. Yi. 2013. Stability of halophilic proteins: From dipeptide attributes to discrimination classifier. Int. J. Biol. Macromol. 53: 1–6. Zhang, X., Y. Lin and G.Q. Chen. 2018. Halophiles as chassis for bioproduction. Adv. Biosyst. 2: 1800088. Zhang, Y., Y. Chang, J. Shen and C. Xue. 2019. Expression and characterization of a novel β-porphyranase from marine bacterium Wenyingzhuangia fucanilytica: A biotechnological tool for degrading porphyran. J. Agric. Food Chem. 67: 9307–9313. Zhao, X., B. Li, C. Xue and L. Sun. 2012. Effect of molecular weight on the antioxidant property of low molecular weight alginate from Laminaria japonica. J. Appl. Phycol. 24: 295–300. Zhao, Y., Z. Chi, Y. Xu, N. Shi, Z. Chi and G. Liu. 2018. High-level extracellular expression of κ-carrageenase in Brevibacillus choshinensis for the production of a series of κ-carrageenan oligosaccharides. Process Biochem. 64: 83–92. Zhou, H.X., S.S. Xu, X.J. Yin, F.L. Wang and Y. Li. 2020. Characterization of a new bifunctional and cold-adapted polysaccharide lyase (PL) family 7 alginate lyase from Flavobacterium sp. Mar. Drugs 18: 388. Zhu, B. and H. Yin. 2015. Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications. Bioengineered 6: 125–131. Zhu, Y., L. Wu, Y. Chen, H. Ni, A. Xiao and H. Cai. 2016. Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates. Microbiol. Res. 182: 49–58. Zhu, B., F. Ni, Y. Sun and Z. Yao. 2017. Expression and characterization of a new heat-stable endo-type alginate lyase from deep-sea bacterium Flammeovirga sp. NJ-04. Extremophiles 21: 1027–1036. Zhu, B., F. Ni, Y. Sun, X. Zhu, H. Yin, Z. Yao and Y. Du. 2018. Insight into carrageenases: Major review of sources, category, property, purification method, structure, and applications. Crit. Rev. Biotechnol. 38: 1261–1276. Zodape, S.T., A. Gupta, S.C. Bhandari, U.S. Rawat, D.R. Chaudhary, K. Eswaran and J. Chikara. 2011. Foliar application of seaweed sap as biostimulant for enhancement of yieldand quality of tomato (Lycopersicon esculentum Mill.). J. Sci. Ind. Res. 70: 215–219.

7 Extremophilic Microalgae The Potential Source for Wastewater Treatments and Biofuel Productions Sourav Kumar Bagchi,1,3,* Reeza Patnaik,2 Navneet Sharma3 and Ramasare Prasad 1

1. Introduction Extremophiles are a diverse group of microorganisms that have the potential to survive and grow in environmental conditions that are considered to be most extreme and challenging to sustain any kind of carbon-based living organisms. These extremophilic organisms are mostly unrevealed or unexplored that have the credibility to bloom in various extreme environmental conditions. This group has different anthropogenic considerations in contrary with the other carbon-based organisms that could live in some moderate environmental conditions and thus termed as neutrophiles or mesophiles. The word ‘extremophile’ derived with a combination of two Latin and Greek words named ‘extremus’ and ‘philia’ means ‘extreme’ and ‘love’, respectively (Rothschild and Mancinelli 2001). These groups of microorganisms have the unique genetic adaptability to thrive in diverse hostile environmental conditions and they have very primordial life forms gradually evaluated for billions of years on this blue planet (Arora and Panosyan 2019). Extremophiles are not only important for its ecological perspective but they have also proved to be the potential source of industrial biotechnology researches, viz., wastewater bioremediations, biofuel productions, biopolymer productions and extractions of bioactive industrially important proteins, fatty acids, pigments, polysaccharide, etc. Halophilic organisms or halophiles possess halotolerant proteins and cell membranes which can resist the elevated concentrations of inorganic salts, ions and various other solutes. Likewise, thermophilic or thermotolerant microorganisms have the potential to grow in high temperatures with proteolysis resistance, while barophilics have cell wall stability at high pressure. Acidophilic and alkaliphilic organisms have pH resistivity in high acidity or high alkaline conditions with excess ion solutes’ pump out process. These organisms can sustain their membrane stability under acute conditions and have the ability to protect their genetic traits in extreme environmental conditions (Arora and Panosyan 2019). Often some microorganisms

Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Haridwar District, Uttarakhand-247667, India. 2 DBT IOC Centre for Advanced Bioenergy Research, Indian Oil Corporation R&D Centre, Sector-13, Faridabad-121007, Haryana, India. 3 School of Life Science & Technology, IIMT University Meerut, Ganga Nagar, Meerut, Uttar Pradesh-250001, India. * Corresponding author: [email protected] 1

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Fig. 7.1. Industrial biotechnology applications of extremophilic microalgae.

acquire additional qualities to survive with very high levels of CO2 or they can grow in the presence of a high concentration of metallic ions and can also bloom in combinations of different stresses which are often called ‘polyextremophiles’. Some other organisms even have a noteworthy potential to grow under elevated levels of ionizing radiations (Rivasseau et al. 2013). These organisms are specifically utilized in industrial biotechnology domains. It has been depicted that the extremophiles acquire unique genetic modules to multiply under intense ecological conditions. Therefore, these extremophilic bacteria, fungi and algal species are distinctive and are used exclusively for industrially important compounds under the adverse pH, temperature, pressure even in the presence of other organisms and pollutants. Although prokaryotic extremophilic organisms have been of unquestionable importance in the biotechnology realm, this chapter is focused on the potential application of microalgae in the industrial biotechnology sector (Fig. 7.1).

2. Microalgae Extremophilic microorganisms play a very important role in attaining the goal of biobased sustainability (Malavasi et al. 2020). They are potentially used for the commercial productions of enzymes, can be used for bioremediation of metal ions in urban or heavy industrial wastewater bodies and also serve as the credible source for bioenergy and biofuels productions exclusively by the tint organism ‘microalgae’ which are often termed as ‘Green-gold’. Microalgae, also known as phytoplanktons or microphytes, are very small plant-like organisms that do not have roots or leaves and are generally a few micrometres in diameter. The microalgae are typically found in aquatic environments both in freshwater and saline water and can survive in both the water column and sediments (Thurman 1997). They are unicellular or multi-cellular species that can exist either individually or in groups or chains. They are capable of performing photosynthesis and can generate half of the oxygen required to survive other living organisms on earth. There are nearly 2,00,000 to 8,00,000 different microalgal species available on this earth, but only a few hundred are described in the literature until now (Thrush et al. 2006). Algae may be prokaryotic in nature, viz., cyanobacteria (Chloroxybacteria) or maybe eukaryotic in nature like red algae (Rhodophyta), brown algae (Phaeophyta), diatoms (Bacillariophyta), green algae (Chlorophyta) and many more. But the most recent widely accepted algae classification of inland waters was reported by Krienitz (2009) based on the phylogenetic analysis, morphological characterization and advanced RNA sequencing. According to this study, algae are mainly divided into two groups, Prokaryota and Eukaryota. Prokaryota has two major divisions, Cyanophyta (Cyanobacteria)

Extremophilic Microalgae: The Potential Source for Wastewater Treatments and Biofuel Productions 153 Table 7.1. Microalgal cultivation with elevated lipid production under challenging environmental conditions of high light intensity and salt stress. Name of the Microalga

Stress Condition

Dunaliella sp. Haematococcus pluvialis Nannochloropsis sp.

Reference

NaCl stress

71

Takagi et al. (2006)

High light intensity

35

Damiani et al. (2010)

15% CO2 under N-starvation + high light intensity + wastewater

60

Jiang et al. (2011)

Chlorella vulgaris

NaCl stress

Scenedesmus obliquus Chlorella sp.

Lipid Content (% dcw)

40 39

Gorain et al. (2013)

Light intensity of 10,000 lux

32

Han et al. (2015)

Chlorella pyrenoidosa

NaCl stress

44

Bajwa and Bishnoi (2015)

Scenedesmus obliquus (Turpin) Kützing

Elevated level of 15% CO2 supplementation in photobioreactor

44

Bagchi and Mallick (2016)

*dcw means dry cell weight of lipid yield.

and Prochlorophyta. Eukaryota is also classified into eight major divisions, viz., Rhodophyta, Chlorophyta, Heterokontophyta, Haptophyta, Cryophyta, Dinophyta, Euglenophyta and Charophyta. These divisions are also classified into several other classes and sub-classes (Krienitz 2009). Hence, there are immense possibilities to explore many new and indigenous isolated microalgal species which may be useful for different kinds of human benefits starting from food, healthcare to advanced biofuel production. Since the last decade, microalgae, more specifically, green microalgal feedstocks, have been getting immense importance in the field of biodiesel production due to their rapid growth rate coupled with high lipid contents. However, biodiesel production from microalgae is primarily dependent upon the biomass yield and cellular lipid accumulation potential of the selected species. Moreover, microalgae can be cultivated in waste or sea water and an elevated oil content of 50–60% of dry cell weight (dcw) can be achieved as compared with some best terrestrial oil crops of only 10–15% oil content (Li et al. 2008) (Table 7.1). It is noteworthy that microalgae can bioremediate wastewater by removing NH4+, NO3– and 3– PO4 from a number of wastewater sources. Researchers reported that microalgae could grow in various wastewaters, and the wastewater sources proved to be a potential source of low-cost lipids for biodiesel production (Woertz et al. 2009). In another report, Mandal and Mallick (2011) showed that the green microalga Scenedesmus obliquus was grown successfully in three kinds of waste discharges, viz., the secondary settling tank discharge of municipal wastewater, fish pond and poultry litter discharges. The lipid accumulation reached up to 1.0 g L–1 with an increased level of saturated fatty acid content.

2.1 Microalgal Growth Under Challenging Environmental Stresses Various microalgal species were able to grow, and their lipid yields were boosted under different extreme environmental conditions applied in the laboratories during the experiments. Takagi et al. (2006) studied the effect of NaCl stress on the accumulation of lipids and triacylglycerides in the marine microalga Dunaliella sp. An increase in initial NaCl concentration from 0.5 M (marine water) to 1.0 M resulted in a higher lipid accumulation of 71% (dcw). Damiani et al. (2010) examined the effects of high light intensity (300 µmol photons m–2 s–1) on lipid accumulation in Haematococcus pluvialis. A lipid content of 35% was recorded under the high light intensity as compared to 15% in control. Gorain et al. (2013) demonstrated that the microalgae Chlorella vulgaris and Scenedesmus obliquus could grow under a high salinity environment, and this NaCl-induced osmotic stress showed lipid accumulation of 40% (dcw) for both the species. In

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another report (Ren et al. 2014), the microalga Scenedesmus sp. showed lipid content of 47% (dcw) against 10% control, under a low dosage of calcium (Table 7.1). An elevated lipid content of 32% (dcw) was noticed under the 10,000 lux light intensity for the test alga Chlorella sp. (Han et al. 2015). In contrast, in another study, it was depicted that the lipid accumulation was boosted significantly for the microalga Chlorella pyrenoidosa under high saline (NaCl) cultivation (Bajwa and Bishnoi 2015).

2.2 Microalgal Outdoor Mass Cultivation For a couple of decades, researchers are focusing on the isolation and growth accumulation for various microalgal species which can thrive in some extreme environmental growth conditions like elevated or extreme cold temperature, high light or low light conditions, cope with seasonal variations and contamination by other microorganisms. These aspects are largely imperative for microalgal large-scale cultivations in outdoor raceway ponds and photobioreactors (PBRs) due to shared environmental challenges (Vonshak and Richmond 1988, Bagchi et al. 2018). At this time, the most acclaimed strategies for large-scale productions of microalgae are algal growth in raceway ponds which are so termed because of its raceway shape. However, scientists have strongly addressed that the biggest bottleneck for the microalgal cultivations in the open raceway ponds is the high costs of the chemical-grade growth medium (Bhattacharya et al. 2016). The feasible commercial-scale production of microalgae is large photobioreactors and raceway ponds (Chisti 2007). The most cost-effective mass cultivation system for the commercial production of biodiesel from microalgae is to grow the microalgae in an outdoor raceway pond (Koley et al. 2019). Compared to the photobioreactors, raceway ponds are generally preferable for largescale algal biomass production due to lesser capital investment and lower operational expenditure, utilization of wasteland or barren lands and easy maintenance (Chisti 2008, Brennan and Owende 2010). In raceway ponds, the cultures’ temperature is not controllable and usually fluctuates depending on seasonal variation (Gross 2013). Temperature and sunshine hours therefore, have profound effects on biomass and lipid accumulation in microalgae grown in outdoor raceway ponds (Bhattacharya et al. 2016). Notably, in tropical and sub-tropical areas, the growing season’s timespan strongly influences the average biomass productivity of microalgae (Chisti 2012). Our recent study has shown that the biomass yield for one locally isolated microalga Scenedesmus obliquus (Turpin) Kützing was considerably higher during the winter season (1.15 g L–1) followed during the summer (0.97 g L–1) and minimum during the rainy (0.88 g L–1) season. This organism has proved to be the extremophilic microalga with the capacity to grow in temperatures ranging from a minimum 16–18°C to a maximum of 42–45°C. It was also recorded that the alga was able to tolerate the pH level of 10.5–11.5, which was found to be incompatible for many other invading microorganisms during the large-scale experiments with 40,000 L polyhouse covered raceway ponds constructed in IIT Kharagpur, West Bengal, India (Bagchi et al. 2019, Bagchi et al. 2022). Interestingly, the newly isolated Scenedesmus obliquus (Turpin) Kützing seems to be a better CO2 biofixer that could tolerate the elevated CO2 levels of 15% (v/v) under the mixotrophic growth condition with the CO2 biofixation rate of 0.77 g L–1 day–1, as compared to Chlorella sp. MTF-7 (0.674g L–1 day–1) (Chiu et al. 2011), Chlorella vulgaris (0.612 g L–1 day–1) and Botryococcus terribilis (0.614 g L–1 day–1) (Nascimento et al. 2015), the CO2 biofixers of higher-order reported till date Table 7.2. In this context, it should be mentioned that the 250 L capacity receway ponds are constructed in the Department of Biosciences and Bioengineering, IIT Roorkee where the microalga Tetradesmus obliquus was able to thrive in the extremely high pH of 10–11 under the industrial wasterwater cultivation medium (Fig. 7.2). The maximum biomass was found to be 2.01 g/ L under this high pH alkaline medium growth. Hence, it can be concluded that the microalga was found to be a potential organism to grow under large-scale conditions. Various studies have already shown the efficiency of microalgal bio-

Extremophilic Microalgae: The Potential Source for Wastewater Treatments and Biofuel Productions 155 Table 7.2. Microalgal cultivations using for wastewater treatments under high pH (high alkaline) and variations in temperature under pilot-scale explorations. Name of the Microalga Microalgal consortium

Chlorella vulgaris A consortium of microalgal isolates Parachlorella sp. JD076 Mixed microalgal consortium

Growth Medium

Areal Lipid Productivity

Reference

Wastewater treatment Area21 m2 (outdoor condition with pH/Temperature variations)

3.7 g m−2 day−1

Lee et al. (2014)

Alga cultivation using wastewaters

3.75 g m−2 day−1

Rogers et al. (2014)

Wastewaters from dairy farms

Annual productivity 25.9 t ha–1 year–1

Hena et al. (2015)

Municipal wastewater

3.5 g m−2 day−1

Yun et al. (2018)

Community wastewater utilized

(Not reported)

Buchanan et al. (2018)

Fig. 7.2. Alkaliphilic chlorophycean microalgal cultivation (Tetradesmus obliquus) with paper pulp derived industrial wastewater as its growth medium under pH 10–11 in the semi-large scale Raceway Ponds of 250 L capacity developed in the Department of Biosciences & Bioengineering, Indian Institute of Technology Roorkee (IIT Roorkee), Uttarakhand, India.

filters for the consumption of nitrogen, phosphorous and other nutrients to clean water bodies. It should be practised under mass-scale cultivations with a defective target to convert the algal biomass to biofuels and other industrially essential bioproducts. In this way, extremophilic microalgae may offer several advantages in industrial biotechnological applications.

2.3 Microalgal Tolerance for Wastewaters Under High pH and Abrupt Temperature At the present, it is also highly necessary to investigate the feasibility of culturing some oleaginous as well as halophilic or halotolerant and acidophilic microalgal strains by utilizing heavy industrial wastewaters that may offer a reasonable and unconventional alternate to traditional and conventional technologies in the treatment of heavy metals like arsenic, copper, cadmium, chromium and lead, etc., that are generally present in industrial wastewater samples and can cause diabetes, cancer, anemia, osteomalacia and many neurotic or nephrotic syndromes (Uberoi 2003, Lefebvre et al. 2006). However, the execution is not as easy as it sounds. Researchers reported that heavy industrial wastewaters were well characterized by their high alkalinity resulting in the pH value of ~ 8.0 due to the heavy chemicals used in the technological processes. They have also recorded that the Total Dissolved Solids (TDS) concentrations of industrial wastewaters are up to the elevated level of 37.0 g L–1. In contrast, the suspended solid concentrations were measured as 5.3 g L–1 (Leta et al. 2004).

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The solution for wastewater treatment is to cultivate some alkaliphilic microalgae in the wastewater bodies. Alkaliphiles are a class of extremophilic microbes competent of continued existence in alkaline (pH approximately 8–11) surroundings, growing optimally more or less a pH of 10–11. Hence, to pragmatically comprehend and perform these experiments are major critical tasks by researchers for evaluations and commercialization aspects (Kongjao et al. 2008). Moreover, when it is evaluated from the industrial and commercial point of view, nowadays it is undoubtedly most indispensable to follow some more efficient techniques by which the wastewater grown wet algal biomass can directly be converted into bio-crude oil without adopting or involving the numbers of cost-intensive and time-consuming processes essential for biodiesel production such as dewatering, drying, lipid extraction with solvents, followed by the transesterifications. On a serious note, it can be commented that these lengthy conventional techniques are the real stumbling blocks for biodiesel production on commercial scales. It is also a fact that microalgal biodiesel production is practically an energy and cost-intensive approach owing to these lavish and time-consuming harvesting, drying and solvent-mediated lipid production techniques (Mathimani and Mallick 2018). From the above analysis and with concern of the major non-cost-effectiveness associated with the biodiesel production till date, it seems that there is an indispensable need to produce the bio-crude oil directly from wet microalgal slurries and utilization of the ‘bio-crude’ as crude oil in the petroleum refinery named ‘petro-crude’. Tanning is one of the oldest industries in the world. During ancient times, tanning activities were organized to meet the local demands of leather footwear, drums and musical instruments. With the growth of population, the increasing requirement for leather and its products led to the establishment of large commercial tanneries. Tanneries are typically characterized as pollutionintensive industrial complexes that generate widely varying, high-strength wastewaters. Tannery effluent is among one of the hazardous pollutants of industry. Major problems are due to wastewater containing heavy metals, toxic chemicals, chloride, lime with high dissolved and suspended salts and other contaminants (Uberoi 2003). Tannery wastewaters are characterized by great alkalinity with very high salinity, resulting pH value of > 8.0 due to various chemicals used in leather technological processes. The total dissolved solids (TDS) concentrations of the tannery wastewaters are reported up to the level of ~ 35.0 g L–1 (Kongjao et al. 2008). Hence, this chapter intends to discuss different research works done in the last decade and recent years, while focusing on exploring the prospect of utilizing the extremophilic microalgal species particularly some halophilic or acidophilic strains bioremediate the toxic metal ions and harmful nutrients from the industrial or urban wastewater samples. Various laboratory-scale as well as large-scale trials are discussed in this context with reference to different published articles for wastewater bioremediation using halotolerant and acidophilic microalgae. The chapter is also extended to focus on the possibilities of the algal refinery approach with spent algal biomass for biochar production after the lipid extraction process for biodiesel production as well as bioethanol production from microalgae. Biochar is a charcoal-like substance produced from agriculture and plant wastes that may contain more than 70% carbon component. Algal biochar could be used as the enhancer of soil to increase the soil fertility and prevent soil degradation with carbon sequestering in soil. Finally, the microalgae-based bio-remediated wastewaters can be reused.

3. Background 3.1 Thermophile Microalgae Thermophilic microorganisms are a class of extremophiles that are capable of thriving with an optimum temperature of 40–60°C and can even tolerate the maximum temperature of 80–90°C. These organisms have one particular class of thermostable enzymes. There are various strains of freshwater or saline water microalgae that can grow in these challenging environmental conditions with the temperature ranges ≥ 45°C. These polyextremophilic microalgal strains have some potential

Extremophilic Microalgae: The Potential Source for Wastewater Treatments and Biofuel Productions 157

industrial biotechnological interests for extracting various enzymes, lipids, pigments and other coproducts. In a research work, α-Tocopherol and β-carotene were extracted from the thermophilic microalga Raphidonema (Leya et al. 2009) whereas; the microalga Chloromonas nivalis was used for the production of commercial class of astaxanthin (Remias et al. 2010). Blue-green algae, the novel thermophilic algal strain, Thermosynechococcus elongatus isolated from the hot springs of Taiwan, depicted elevated biomass productivity in the temperature condition of ~ 50°C grown with 20% CO2 supplementation. The pigment phycocyanin was also produced, which has well-defined industrial applications (Leu et al. 2013).

3.2 Applications Alkaliphilic and Halophilic Microalgae for Wastewater Treatments For extremophilic microalgal biomass to have a significant and meaningful impact on meeting the present energy demand in the world; there is an urgent need for large-scale cultivation and adaptations of various processing techniques associated with the production of biodiesel in some low-cost, energetically favourable manner (Sydney et al. 2019). In comparison with physicochemical treatment technology for industrial wastewaters, microalgae-based treatments are more efficient and safe, besides removing toxicants, including heavy metals, in a cost-effective manner. In this context, extremophile microalgal species are to bioremediate the industrial wastewaters that would offer a promising alternative to traditional technologies in treating heavy metals and nutrients present in the industrial wastewater samples. Wisely scheming and regulating the experiments is as important as the execution of the investigation itself. It is noteworthy that microalgae can bioremediate wastewater by removing NH4+, NO3– and 3– PO4 from a number of wastewater sources. Researchers reported that microalgae could grow in various wastewaters, and the wastewater sources proved to be a potential source of low-cost lipids for biodiesel production (Woertz et al. 2009). The increasing energy demand and the massive level of water consumption by humans have caused an alarm for the imperative need for energy security and efficient wastewater treatment and its reuse. Conventional wastewater treatment systems do not seem to be the definitive solution to pollution and eutrophication problems. Secondary sewage treatment plants are specifically designed to control the quantity of organic compounds in wastewaters. However, pollutants, mainly nitrogen, phosphorus, sulphur, etc., are only slightly affected by this type of treatment (Mallick et al. 2016). It is highly striking that the tiny organism ‘microalgae’ has considerable potential to bioremediate wastewaters by removing NH4+, NO3– and PO43– from a number of wastewater sources (Bagchi et al. 2018). Researchers reported that microalgae could grow in various wastewaters, and the wastewater sources proved to be a potential source of low-cost lipids for biodiesel production (Woertz et al. 2009, Arora et al. 2016). Hence, microalgae cultivation using industrial wastewater has been studied as an alternative approach for conventional wastewater treatment. In Algeria, the green microalga Chlorella pyrenoidosa was successfully cultivated in a desert area at a domestic wastewater treatment plant site. In this experiment, the maximum areal biomass productivity was recorded as > 35 g m−2 day−1 (Dahmani et al. 2016). In another study, the locally green isolated microalga Chlorella sp. was cultivated in a wetland raceway pond using piggery wastewaters. Various significant parameters for this wetland cultivation were investigated, such as the aeration rate, nutrient removal by the alga from wastewaters, biomass yield and the Fatty Acid Methyl Esters (FAMEs) compositions. The maximum biomass productivity was recorded as 79.2 mg L−1 d−1 and the total nitrogen, phosphorus removal efficiencies were found to be 80.9 and 99.2%, which was much higher than the overall chemical oxygen demand value recorded as 74.5% (Lee and Chen 2016). One recent report has depicted areal biomass productivity of 6.16 g m−2 day−1, for the microalgal consortium comprised with Chlorella sp., Scenedesmus sp., and Stigeoclonium sp. (CSS) and

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cultivated using the municipal wastewater in the 0.4 ton capacity of High Rated Algal Ponds (HRAPs) at an optimized culture depth of 20 cm. The algal consortium was also found to be quite sufficient to bioremediate 82.5 and 89.7% of nitrogen and phosphorous, respectively, from the wastewater medium (Kim et al. 2018). In contrary to this, researchers evoked that the microalga Parachlorella sp. JD076 showed relatively higher biomass productivity of ~ 20.0 g m−2 day−1 (Yun et al. 2018) when cultivated using municipal wastewater in outdoor tanks.

3.3 The Harnessing of Biofuels from Alkaliphilic Microalgae Bioenergy is a kind of renewable energy derived from various biological sources to generate heat or to produce liquid biofuels for transportation. The current scenario is the most widely used renewable energy that provides 10% of the global primary energy supply. Moreover, it was also reported that a gross 370 TWh of electricity was generated in 2012 from the renewable bioenergy resources which were equivalent to 1.5% of the global electricity production in 2012. The target has been set to raise the value up to 560 TWh electricity generation from bioenergy sources at the end of 2018, which will be at least 3000 TWh by 2050 (IEA 2013). It should be noted that the term bioenergy is often coined as ‘biofuels’ in a more specified sense. Its’ primary rationale is that the biomass itself is a store of fuel, and the energy contained in the fuel is termed as bioenergy. In general, the term ‘biofuels’ are defined as gaseous or liquid fuels derived from biomass and are used as a replacement of conventional fossil fuels like diesel or petrol for transportation and other portable applications. At this time, most of the renewable energy resources like solar, tidal, wind or geothermal energy are targeted for the production of electricity only, whereas significant global energy consumption is based on the production of liquid fuels (Campbell 2008). The last decade had already witnessed a tremendous impetus on biofuel research due to the irreversible depletion of fossil fuels and the escalating emissions of greenhouse gases in the atmosphere. Biofuels are subdivided into two primary liquid fuels, viz., bioethanol and biodiesel. The lignocellulosic crops like sugar cane, corn, etc., are fermented to produce bioethanol. In contrast, biodiesel, the other transportation fuel, is produced from various sources starting from many food products like vegetable oils to tiny microorganisms. Bioethanol is generally used in combination with gasoline, and the biodiesel is blended usually with diesel to open an option of alternative fuel utilization in transport sectors. However, nowadays, the main concern for a biofuel product’s suitability depends on the two major factors, i.e., land use and appropriate biomass to fuel energy conversion (Kosinkova et al. 2015). The global biofuel production has rapidly growing in the last 10 years, from 15 billion litres in 2000 to 110 billion litres in 2013. It has captured a share of 3.5% of the world’s total transportation fuel (REN21 Report, 2015). It is will be hopeful when we find that biofuels meet nearly one-fourth of the road transport fuel requirements in Brazil. The global biofuel production was projected to around 140 billion litres by the end of 2018. This report showed that global biofuel production had already reached 123.7 billion litres at the end of 2014. Thus, it is not unanticipated that thermophiles and halophiles microalgae have a notable ability to endure variations in temperature, pH and other environmental challenges. This quality brings forward a comprehensible benefit in the progress of a commercially attainable process. This is a quality that is predominantly imperative for the production of third-generation biofuels from algae. The challenging environmental conditions whether it is high or low temperature or alkaline or acid conditions for microalgal growth under outdoor raceway ponds, generally these stresses also can induce the production of lipids, carotenoids, fatty acids, astaxanthin and other industrial important co-products from algae. Thus, algae have the huge potential to thrive in environmental stresses and can accumulate high lipid bodies and carotenoids in their cellular components (Mallick et al. 2016, Patnaik and Mallick 2019). The algae under these elevated temperatures and pH have the potential to bioremediate toxic ammonia, nitrate, phosphate and most of the metal ions from the

Extremophilic Microalgae: The Potential Source for Wastewater Treatments and Biofuel Productions 159

different wastewater samples, viz., municipal, agricultural, rural and industrial wastewater bodies. This chapter presents a critical evaluation of extremophilic microalgae’s current standings and their impending applications for wastewater treatments vis-à-vis biofuel harnessing. The chapter discusses the large-scale microalgal cultivation stratagems essential for commercial applications of microalgae.

4. Current Developments 4.1 Blueprint for Wastewater Treatment to Lipid Harnessing Followed by Biodiesel Production In the present world scenario of rapid urbanization, waste discharges from metropolitan urban city sewages, industrial farms, heavy industrial discharges, agriculture, etc., are the primary sources of water pollution. It is reiterated that the conventional unadventurous wastewater treatment plants do not seem to be the best solution for pollution and eutrophication problems. They suffer mainly from two drawbacks, viz., cost-effectiveness and nutrient non-recycling. Secondary sewage treatment plants are purposely designed to minimize only the quantity of organic compounds in wastewaters. It is remarkable that some microalgal strains with their potent capacity to tolerate the adverse environmental (halotolerant and acidophilic/besophilic nature) conditions can bioremediate the waste samples and wastewaters with the removal of NH4+, NO3– and PO4– as well as diminish the heavy metal ions like Hg, As, Cu, Cd, Fe, Zn, Mn, etc., from a number of waste materials and wastewater bodies. Various researchers reported that some extremophilic microalgae that can be found in wastewaters have proved to be the best potential source of cost-effective biofuel production (Woertz et al. 2009). Since the inception of algae to biofuels, there are verities of extremophilic algae strains being isolated, which can tolerate the high/low temperature, pH and can thrive under other environmental challenges. It was mentioned earlier, that there is a large number of microalgal strains such as Scenedesmus, Dunaliella, Chlorella, etc., that can act as extremophiles which tolerate both high temperatures and low/high alkaline pH, having elevated growth rates at 40–42°C and pH above 10 (Koley et al. 2019, Patnaik et al. 2019). The advantages of using extremophilic microalgae would be to minimize the contamination by providing a high alkaline or low pH under the outdoor largescale raceway pond systems or even in the photobioreactor (PBR) cultures. However, despite the prospective for using algae for biodiesel harnessing, the cost of microalgal biodiesel is a hindrance due to the cultivation cost associated with the medium and the high cost associated with the harvesting as well as following the conventional drying techniques (Bagchi et al. 2015). The cost-effective biodiesel production aims to utilize wastewaters and use various waste materials like poultry litter, dairy manures, fish-pond discharges and municipal discharges, etc., for algal growth and rapid proliferations. Thereby various scientific reports of microalgal cultivation and lipid followed by biodiesel production using wastewaters and various waste products as the cost-effective growth-medium ingredients are a requisite to be experimented thoroughly with the isolation of locally available thermophilic, alkaliphilic or any acidophilic algal strains in the large-scale raceway ponds. In this context, it needs to be mentioned that ammonium is the primary nitrogen source in any wastewaters. Controlling its high level of toxicity is the primary challenge for the microalgae to thrive and proliferate with the decisive goal of elevated lipid productivities (Ribeiro et al. 2020). The chlorophycean microalga Scenedesmus obliquus has shown an elevated biomass and lipid yield by using a mixture of poultry litter and municipal secondary settling tank discharges in the amount of 15 g L–1. The lipid content was also boosted significantly up to 53.0% (dcw) by using these waste-disposal mixtures in its specified quantities (Mandal and Mallick 2011). The digestive swine manure wastewater was successfully utilized for the cultivation of 97 microalgae obtained from algae-bank and 50 other algal strains isolated from the local waterbodies in Minnesota,

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United States of America. The maximum biomass yield and lipid content were achieved upto 2.03 g L–1 and 23% (dcw) for the locally isolated microalgal strain UMN 271 (Zhou et al. 2012). In one report, it was observed that the mixed microalgal consortium was cultivated in two phases comprising of initial growth phase under mixotrophic mode using domestic sewage wastewater followed by temperature stressed starvation phase for enhanced lipid induction. The maximum biomass yield and lipid content were recorded as high enough in this biodiesel production process (Venkata Subhash et al. 2014). Another report also demonstrated that the microalga Chlorococcum sp. was grown in sea-water based saline medium supplemented with waste glycerol available from the biodiesel industries with a maximum biomass yield and lipid content were 0.85 g L–1 and 39.0% (dcw), respectively (Beevi et al. 2015). The green microalga Chlorella vulgaris was grown under ammonia-rich wastewater cultivations as poultry litter uses in which the lipid content of ~ 50% (dcw), respectively (Markou 2015). The utilization of wastewaters was also found to be quite effective for algae cultivation as per the research work carried out in our laboratory where the microalga Chlamydomonas debaryana IITRIND3 was successfully cultivated in different wastewaters as obtained from sources of domestic, sewage, paper mills and dairy wastewaters, respectively. The maximum biomass yield was depicted as 3.66 g L–1 in dairy and 3.56 g L–1 in domestic wastewater, whereas the lipid content was recorded as 45.0% (dcw) in dairy, followed by ~ 42% (dcw) in domestic sewage, respectively (Arora et al. 2016). These biomass and lipid yield found in this process by using wastewaters was quite productive, and the yield values were significantly higher than many other reports published till date. The ability of the microalga for bioremediation, various parameters like chemical oxygen demand (COD), NH4 –N, and TP were measured in the course of the cultivation periods and their average removal efficiencies were reported as 78, 95, and 81%, respectively (Dahmani et al. 2016). In another study, the locally isolated microalga Chlorella sp. was cultivated in a fabricated outdoor wetland under the mixotrophic cultivation techniques using piggery wastewaters. Various significant parameters for this wetland cultivation were investigated, such as the aeration rate, nutrient removal by the alga from wastewaters, biomass yield and the fatty acid methyl esters (FAMEs) compositions. The maximum biomass productivity was recorded as 79.2 mg L−1 d−1 and the Total Nitrogen (TN), phosphorus (TP) removal efficiencies were found to be 80.9 and 99.2% (Lee and Chen 2016), which was much higher than the overall Chemical Oxygen Demand (COD) value depicted as 74.5%. One current report has also demonstrated that the microalga Chlorella sp. was successfully cultivated by utilizing the aerated seafood processing wastewater for higher biomass accumulation, lipid production as well as the major nutrients’ removal from the wastewater. The study also has shown that the total nitrogen (TN) and total phosphorous (TN) contents in the wastewater were constantly decreased during the end of the cultivation period of the microalgae. The total nitrogen concentration was reduced to a very low level of 4.11 mg L−1, which was only 3.4% of the initial concentration. Further calculations have also indicated that ~ 93 and ~ 50% of the eliminated nitrogen and phosphorous were assimilated by the alga during the end of the course of the investigation, showing that the tiny organisms ‘microalgae’ are effectually the potential sources to use for removing the nitrogen and phosphorous from the wastewater bodies (Gao et al. 2018).

4.2 Stratagem for Cost-effective Biofuel Harnessing with Wastewater Utilization as the Growth Medium for Extremophilic Microalgae Cost-effective cultivation is the main bottleneck for the extremophilic microalgal biodiesel production in a large-scale scenario. A considerable upstream energy cost in terms of material energy expenditures for microalgae cultivation is using chemical graded AR or LR medium. Scientists are now focused on successfully utilizing various wastewater resources for microalgal growth that may aid markedly in addressing the challenges of lowering the material costs for upstream processing for biofuel production from algae (Ekendahl et al. 2018). A detailed study conducted by Sipaúba-

Extremophilic Microalgae: The Potential Source for Wastewater Treatments and Biofuel Productions 161

Tavares et al. (2017) illustrated and projected that the total medium cost for producing 3,722 tonnes of dried algal biomass was USD 22,200 use of the chemical graded medium. In continuation, Ribeiro et al. (2020) estimated a 1000 L agricultural grade fertilizer growth medium composed of 510 mg L–1 of Urea and 35 mg L–1 of Monoammonium Phosphate costs USD 10 which is significantly low-cost compared to the conventional chemical LR grade medium. However, the upstream processing of algal biomass to biofuel needs more cost reduction in terms of growth medium for its up-scaling. Therefore, it is a fact that the inorganic chemical medium cost is substantially higher, which in turn affects the overall production cost of lipids, thereby increasing the biodiesel price and puts a big question mark for the commercial applicability of biodiesel. Hence, locally available wastewater as a cultivation medium for the extremophilic microalgal growth seems to be the only preeminent solution in this aspect. Several studies have already been carried out for the cost-effective microalgal growth, lipid production followed by the harnessing of biofuels with the use of the cheap source of growth medium i.e., domestic, agricultural, urban or industrial wastewaters under open raceway pond cultivations. In a study, the microalga Chlorella zofingiensis could survive in wastewater with elevated lipid productivity of 70 mg L–1 day–1. Moreover, the total N (nitrate + nitrite + ammonium) and P removal efficiencies were recorded as 83 and 98%, respectively (Zhu et al. 2013) (Table 7.3). A recent report published by Kim et al. (2018), also evoked that the mixed microalgal consortium including Chlorella sp., Scenedesmus sp., and Stigeoclonium sp. (CSS) obtained from various wastewater sources were able to thrive in urban municipal wastewaters, which are generally highly toxic with the presence of elevated quantities of ammonium and other inorganic ions. Hence, the microalgal consortium proved to be the potent alkali/acid and halotolerant with higher lipid productivity of 30.80 mg L–1 day–1 with the inorganic total nitrogen and phosphate removal efficiencies of 82 and 90%, respectively (Table 7.3). However, there are still some significant hurdles and research gaps. Therefore, some more efforts should be continued to suggest some novel stratagems that will minimize the cultivation costs and improve biofuel production sustainability (Balat and Balat 2010). The cultivation technique must attach with the application of the inexpensive carbon dioxide sources such as flue gas generated from various industries which are detrimental for mankind (Shirvani et al. 2011). Moreover, coupled with flue gas, wastewaters that are rich with nutrients must diminish the requirement of additional Table 7.3. Comparative assessment of various study reports with lipid productivity, nutrient removal efficiencies for different alkaliphilic and/or acidophilic microalgal species grown with wastewaters. Microalgae

Wastewater Specification

Chlorella sp.

Urban wastewater

8.6

89

81

Li et al. (2011)

Chlorella sp.

Sewage wastewater

8.0

41

100

Han et al. (2014)

Chlorella zofingiensis

Piggery based wastewater

70.0

83

98

Zhu et al. (2013)

Chlorella debaryana

Piggery based wastewater

~ 11.5

88

54

Hasan (2014)

Seafood processing wastewater

Total lipid yield –1.55 g L–1

93

~ 50

Gao et al. (2018)

Municipal wastewater

30.80

82

90

Kim et al. (2018)

Chlorella sp.

Mixed microalgal consortium made up with Chlorella sp., Scenedesmus sp., and Stigeoclonium sp. (CSS)

Lipid Productivity (mg L–1 day–1)

(%) Total N Removal Efficiency

(%) Total P Removal Efficiency

Reference

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nitrogen and phosphorous. The microalgae treated wastewaters will also be reused for various purposes in future endeavours. These wastewater and waste product utilization techniques will minimize the costs of raw materials for algal cultivations and counterbalance and utilize the waste products to contribute to microalgal biofuels’ sustainability. Indigenous isolated extremophilic microalgae obtained from the wastewater discharge sites could prove to be the best biological substitute for inorganic nutrient removals from various wastewater bodies. Microalgae are expedient for this tender owing to their capability to use nutrients into biomass that again could be valorized in bioenergy, raw chemicals or other products just as a perfect example of ‘waste to wealth’ applications.

4.3 Possibility of Use Extremophilic Microalgae for Bioactive Co-products Harnessing These days, the neccessity of pharmaceutically and health important products’ generations are gaining significant attention worldwide amidst the sudden and rapid SARS COVID-19 outbreaks. When one is protecting oneself from the virus and bacteria from outside, it is imperative to protect oneslf within our body by strengthening our healthy immune system. The tiny organism ‘microalgae’ is one of the emerging sources of sustainable energy producers as well as its impending potential for health benefits such as pigments, polysaccharides, carotenoids and Omega-3, Omega-6-fatty acids generations. Researchers have decisively commented that these thermophilic, halophilic, alkaliphilic and acidophilic microalgal species, with their diverse wide range of inhabiting environments, have a significant prospective for biotechnological applications with bioactive co-products generations (Patel et al. 2019, Malavasi et al. 2020). The wide ranges of extremophilic algal species as the promising sources of industrial biotechnology and bioactive co-products generations have just started to be explored (Sydney et al. 2019) however more work needs to be made in this field. In this proposed project work, the target is set to investigate the feasibility of culturing of some extremophilic Chlorophyceae microalgae and cyanobacteria for the cost-effective biodiesel harnessing and the subsequent utilization of the extremophilic algal biomass for further exploitation of various human health important co-products like pigments, omega-3-fatty acids, omega-6-fatty acids, β-carotenes (precursor of Vitamin A), polysaccharides, etc., harnessing (Patel et al. 2019). The extremophilic microalgal research direction is also set for higher CO2 levels to accumulate lipids and other important health bioactive co-products’ harnessing, such as omega-3 omega-6fatty acids and β-carotenes. The microalgal polyunsaturated fatty acids are very essential for body metabolism in humans also. The microalgal extracts show antimicrobial activities accredited to the presence of various fatty acids and pigments (Khozin-Goldberg et al. 2011). Researchers have suggested that omega-3 and omega-6-fatty acids can reduce the systolic and diastolic blood pressures in people with hyper tension and these two fatty acids have many other pivotal roles for immunity-boosting in humans (Gutiérrez et al. 2019).

5. Future Prospects From the above discussions, it is exemplified, unlike the other extremophilic organisms which require some specific culture conditions in the laboratory, microalgae can be cultivated in freshwater, wastewater and in outdoor culture conditions, limited freshwater and nutrients used for conventional agriculture. Extremophilic microalgal biomass has a meaningful impact on meeting the current energy demand in the world. The primary restricted access for the commercialization of algal biomass to biofuel is the higher cost of the cultivation medium, energy-expensive drying of biomass and the high volumes of the solvent utilization for biomass biodiesel production purposes (Malavasi et al. 2020). Describing the recent studies mentioned above, it is indeed an unaccountable fact that microalgae-based wastewater treatments are highly efficient and safe, besides removing toxicants including heavy metals in the cost-effective manner, when compared with different

Extremophilic Microalgae: The Potential Source for Wastewater Treatments and Biofuel Productions 163

conventional physicochemical treatment technologies adopted till date. Thus, the future research work’s primary target can be set as an integrated effort to study the CO2 bio-fixation and costeffective biodiesel production for some oleaginous thermotolerant and alkaliphilic microalgal strain cultivations under large scale explorations by combining it with the industrial wastewater treatments.

6. Conclusions Studies of extremophilic microalgae have revealed a substantial industrial biological potential for their growth at high temperatures of ~ 50°C and under extremely high or low pH culture conditions. Verities of microalgal strains in attendance at elevated temperature can be subjugated to harness numerous products like biodiesel and enzymes and high-value co-products as like astaxanthin, betacarotene, omega-3, omega-6 fatty acids, phycocyanins from cyanobacteria, etc. In this chapter, the effort is was made to discuss various reports published based on microalgal growth under challenging environmental stresses, mainly focusing on its potential for wastewater treatments under high or low temperature and elevated pH conditions. These capabilities to thrive its growth under these adverse conditions with the potential for wastewater treatment vis-à-vis biodiesel productions have proved to be a potential extremophile with numerous advantages. Moreover, being omnipresent with its photoautotrophic growth, microalgae can also be easily maintained in the laboratory as well as under the large-scale outdoor conditions compared to the extremophilic bacteria, fungi, etc., which require more precise culture conditions and maintenance in the laboratory.

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Takagi, M., Karseno and T. Yoshida. 2006. Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J. Biosci. Bioeng. 101: 223–226. Thrush, S.F., J.E. Hewitt, M. Gibbs, C. Lundquist and A. Norkko. 2006. Functional role of large organisms in intertidal communities: Community effects and ecosystem function. Ecosystems 9: 1029–1040. Thurman, H.V. 1997. Introductory Oceanography, Prentice Hall College, New Jersey,USA, ISBN 0-13-262072-3. Uberoi, N.K. 2003. Environmental Management, Excel Books Publisher, New Delhi, p. 269. Venkata Subhash, G., M.V. Rohit, M. Prathima Devi, Y.V. Swamy and S. Venkata Mohan. 2014. Temperature induced stress influence on biodiesel productivity during mixotrophic microalgae cultivation with wastewater. Bioresour. Technol. 169: 789–793. Vonshak, A. and A. Richmond. 1988. Mass production of the blue-green Spirulina: An overview. Biomass 15: 233–247. Woertz, I.C., L. Futon and T.J. Lundquist. 2009. Nutrient removal and greenhouse gas abatement with CO2supplemented algal high rate ponds. p. 13. WEFTEC Annual Conference, Water Environment Federation, October 12–14, USA. Yun, J.-H., D.-H. Cho, S. Lee, J. Heo, Q.-G. Tran and Y.K. Changa. 2018. Hybrid operation of photobioreactor and wastewater-fed open raceway ponds enhances the dominance of target algal species and algal biomass production. Algal Res. 29: 319–329. Zhou, W., B. Hu, Y. Li, M. Min, M. Mohr and Z. Du. 2012. Mass cultivation of microalgae on animal wastewater: a sequential two-stage cultivation process for energy crop and omega-3-rich animal feed production. Appl. Biochem. Biotechnol. 168: 348–363. Zhu, L., Z. Wang, Q. Shu, J. Takala, E. Hiltunen and P. Fenga et al. 2013. Nutrient removal and biodiesel production by integration of freshwater algae cultivation with piggery wastewater treatment. Water Res. 47: 4294–4302.

8 Extremophilic Microalgae as a Potential Source of High-Value Bioproducts Meenakshi Singh,1 Nitin Trivedi,2 Navonil Mal 2 and Sanjeet Mehariya3,*

1. Introduction Microalgae, including cyanobacteria, are highly diverse microscopic single-celled lifeforms inhabiting the earth since the origin of life. They are capable of oxygenic photosynthesis in the extreme atmospheric and physiological conditions. They are found in all aquatic environments and can survive in highly stressed conditions considered unfit to survive like icebergs, permafrost soil, mining sites, geothermal springs, volcanic rocks, dead sea and marine deep waters. The extensive studies on adverse environments by (Mohamed 2008, Raddadi et al. 2015, Schmidt et al. 2018, Zechman et al. 2010) suggested that microalgae along with prokaryotes are well-suited to harsh conditions and act as the potential source of biologically active compounds required for normal growth and development of microorganisms such as primary and secondary metabolites, organic acids, pigments, proteins, coenzymes and extremolytes. These are therapeutically explored by human beings in treating various diseases caused by bacteria, fungus, larvae, helminths, etc. (Moore 1996). In limiting conditions of salinity, light, pH, temperature, osmotic gradients and key nutrients, only a few microalgae can sustain, by releasing more by-products to protect cell membranous structures against free radical attack. To survive, in unfavorable conditions microalgae lose their flagella, become spherical and store reserve food material. Such ‘stress-thriving’ microorganisms can stabilize and remain catalytically active to colonize challenging environmental conditions. In this context, the short generation time of unicellular microalgae and adapted biomolecules have broad and versatile biochemical metabolic pathways coupled with physiological capacities widely useful in biotechnological and industrial applications to generate value-added products (Varshney et al. 2015). Besides this, the potential utility of extremozymes in food, cosmetic, medical applications, dairy industries, as well in the production of biofuel, bioelectricity generation and piggery-waste treatment processes act as an efficient and cost-effective solution (Elleuche et al. 2014, Coker 2016, Dopson et al. 2016, Chandrasekhar and Ahn 2017, Enamala et al. 2020, Mehariya et al. 2020a, Goswami et al. 2021).

Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat – 390002, INDIA. 2 Department of Marine Biotechnology, Gujarat Biotechnology University, Gandhinagar-382355, Gujarat, India. 3 Department of Engineering, University of Campania “Luigi Vanvitelli”, Real Casa dell’Annunziata, Via Roma 29, Aversa (CE), 81031– ITALY. * Corresponding author: [email protected] 1

168 Extremophiles: Wastewater and Algal Biorefinery

1.1 Definition of Extremophilic Microorganisms The earth has a great variation in its demography and climatic conditions that keep changing with time. Since, the origin of life, the earth has witnessed a lot of changes in the evolutionary diversity of lifeforms, withstanding changes in atmospheric, land and water conditions further accelerated by anthropogenic activities and climate change. There are certain typical environments, which are considered unsuitable for the normal growth and reproduction of most of the microorganisms. In such a case, the organisms that thrive well in harsh habitats of acidic and alkaline, cold and hot stress, hypersaline and pressure gradient environments are known as extremophilic microorganisms. The extremophiles possess special characteristics to cope with stressed and limiting resources available to adapt and acclimatize rapidly to the exposed high amount of greenhouse gases, low amount of oxygen, toxic level of metal ions, long duration of ultraviolet radiations and other factors (Stockwell et al. 2009).

1.2 Morphological Diversity of Extremophilic Microalgae in a Stressed Habitat The world of extremophiles is uncovered with groundbreaking discoveries and recent microbiological advancements that clearly depict the clues to the diversity and versatility of tiny microscopic entities. Based on morphological diversity in extreme microhabitats either permanent or ephemeral, extremophilic microalgae can be broadly classified as—(i) Microalgae which require one or more harsh conditions to grow, and (ii) Microalgae which can sustain complex physiological parameters while growing in standard conditions (Rampelotto 2013). Though extremophilic microorganisms include all three domains of life, i.e., Prokaryotes (Bacteria and Archaea) and Eukaryotes (Algae); but here, we will be focusing only on eukaryotic microalgae including cyanobacteria (blue-green algae). The microalgae can be considered as thermophilic (lovers of high temperature, 60–80oC), hyperthermophilic (optimum growth above 80oC), psychrophilic or cryophilic (cold-loving organisms, 15oC or lower), halophilic (salineloving organisms), acidophilic (low pH, ranging between 1–5), alkaliphilic (high pH, more than 9), radiation-resistant phototrophs resisting UV radiation, barophilic (thriving high hydrostatic pressure), oligotrophic (growing in limited nutrient environments); endolithic (growing in/under a rock or stones) and xerophilic (growing in arid conditions). Sometimes, they can acclimatize and amplify in multiple stress conditions, then they are known as ‘polyextremophiles’ (Seckbach and Oren 2007). All of them show a complex life cycle, alternating with dormant a spore stage, vegetative and sexual cycles. To sustain in extreme and stressful conditions, some kind of adaptive mechanism is employed at the biochemical as well at the genetic level (Giddings and Newman 2015). The adaptive response of extremophilic microalgae is persistent throughout their short lifecycle and growth strategy irrespective of fluctuations in the harsh habitats (Rossoni and Weber 2019). Although past studies, considered such habitats as lifeless for many years but recent studies proved it wrong, as the findings showed the presence of highly adaptive species (Duarte et al. 2012). In the case of municipal waters or wastewaters, the presence of a significant amount of ammonical nitrogen, phosphate and other metallic ion makes concentration and low pH gradient creating it difficult to reuse for agricultural or human consumption (Mehariya et al. 2018, Goswami et al. 2020a, 2020b). A recent study by Čížková et al. 2020 experimented on acidophilic microalgae, Galdieria sulphuraria which successfully biosorbed precious metals (gold and palladium) from metal-containing wastewaters. However, certain limitations of nutrients, physical and geochemical factors play an important role in developing resistance to drastic conditions. A detailed account of the diversified habitats of different microalgae are given in Table 8.1.

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Table 8.1. The diversified stressed habitats of extremophilic microalgae. S. No.

Extremophilic Microalgae

Stressed Habitat

References

1.

Acutodesmus sp.

Wastewater

Varshney et al. 2016

2.

Arthrospira platensis

Alkaline waters

Berry et al. 2003

3.

Anabaena sp.

Alkaline lakes, Salt tolerant aquifers

Gimmler and Degenhard 2001

4.

Chlamydomonas acidophila

Acidic water

Garbayo et al. 2008

5.

Chlamydomonas nivalis

Arid region

Duval et al. 2000, McKay et al. 2003, D’Amico et al. 2006

6.

Chlamydomonas botryopara

Acidic water

Novis and Harding 2007

7.

Chlorella ohadii

Deserted soils

Treves et al. 2013

8.

Chlorella sorokiniana

High ionic concentration and thermophilic

Varshney et al. 2018

9.

Desmodesmus sp. F2

Thermophilic and high metal concentration

Bhattacharya and Pal 2011

10.

Desertella, Eremochloris, and Xerochlorella

Desert soil crusts

Fučíková et al. 2014

11.

Dunaliella salina

Hypersaline lakes

Vincent 2000, Varshney et al. 2015

12.

Dunaliella viridis

High salt concentration

Oren 2014

13.

Galderia sulphuraria and Pseudochlorella sp. YKT1

Acidic lakes

Hirooka and Miyagishima 2016

14.

Gloeocapsa sp.

Sandstone habitat

Sokoloff et al. 2016, D’Alessandro and Antoniosi Filho 2016

15.

Graesiella sp.

Geothermal springs

Mezhoud et al. 2014

16.

Haematococcus pluvialis

Zinc enriched hot springs

Pentecost 2011

17.

Mesotaenium berggrenii

Glaciers

Remias et al. 2009

18.

Melosira sp.

Antarctica soil

Seckbach and Oren 2007

19.

Microchloropsis gaditana CCMP526

Hypersaline lakes

Karthikaichamy et al. 2018

20.

Nitzschia westii

Antarctica soil

Seckbach and Oren 2007

21.

Navicula sp.

Antarctica soil

Seckbach and Oren 2007

22.

Nostoc commune

Antarctica soil

Maria Grilli Caiola 2007

23.

Pinnularia acoricola

Acidic water

Novis and Harding 2007

24.

Pinnularia cymatopleura

Antarctica soil

Seckbach and Oren 2007

Oscillatoria sp., Mastigocladus sp., Phormidium sp., Calothrix sp.

Geothermal habitat

Ward and Castenholz 2006

25.

Raphidonema sp.

Polar ice caps

Leya et al. 2009

26.

Synechococcus vulcanus

Thermal springs

Yu et al. 2015

27.

Synechococcus elongatus PCC 11801

Thermophilic

Jaiswal et al. 2018

28.

Trebouxia sp.

Sandstone habitat

D’Elia et al. 2018

2. Growth Engineering Systems Microalgae can be cultivated easily in extreme conditions of municipal wastewaters, seawater, agricultural runoff, organic effluent from dairy and food industries for the viable alternative technologies for metabolite productions (Mehariya et al. 2021a, 2021b, 2021c, 2021d). In this way,

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an extremophilic microalgae is the most suitable organism to adapt well to inhabitable conditions and resist contamination. They utilize the available nitrogen and phosphorus and remove toxic metals from the water and produce high biomass. This provides the additional economic benefit of sustainable wastewater bioremediation. There are certain strains extremophilic microalgae considered for upscale operations, like Chlorella sp., Arthrospira platensis, Dunaliella salina, Chlorococcum sp., Scenedesmus sp., Haematococcus pluvialis, etc. These microalgae are well-suited to stressed environmental conditions and can be cultivated using natural resources, like sunlight, CO2, alkalinity and salinity. The mass cultivation generates viable biomass, used for various value-added products. Presently, two types of mass-cultivation systems are employed worldwide: (I) Outdoor cultivation, and (II) Photobioreactors (PBRs).

2.1 Outdoor Cultivation The outdoor farming of autotrophic microalgae is either perfomed in an open system, closed system or a hybrid of both (Olivieri et al. 2014). This is the economical way of producing large scale biomass for biofuels, animal feed, nutraceuticals and high-value bioproducts (Borowitzka and Vonshak 2017). It requires a basic low-cost setup of shallow raceway pond well equipped with a paddle wheel for the flocculation to remove the aggregates and cost-effective harvesting methods. However, the outdoor pilot/large-scale microalgal cultivation suffers from various limitations like increased salinity, high light irradiance, low or high temperature, nutrient starvation under stressed conditions (Borowitzka and Vonshak 2017). Table 8.2 specifies the recent study reports of outdoor cultivations for different types of extremophilic microalgae, growth system, total volume of cultures, biomass productivity and high value components produced. Table 8.2. High value component production by extremophilic microalgae in outdoor culture systems. Extremophilic Microalgae

Outdoor Culture System

Total Volume

Biomass Productivity

High Value Component

Reference

Chlorella vulgaris

Mod. Kuhl med., Temp. 25 ± 2°C, pH - NA, LI - 140–200 μmol·m2·s−1

1000 L

31.71 mg/L/d

Lipd

El-Sheekh et al. 2019

Coccomyxa melkonianii SCCA 048

Mod. BBM med., Temp. 25 ± 2°C, pH - 6.8, LI - 80–100 μmol·m2·s−1

1.8 L

10 mg/L/d

Lipid

Soru et al. 2019

Coelastrella sp. F169

BG11 med., Temp. 25 ± 2°C, LI - μmol·m2·s−1 pH - NA

4.5 L

380 mg/L/d

Lipid

Narayanan et al. 2018

Scenedesmus species novo

ST med., LI - 6524–7360 μmol·m2·s−1 Temp. ±40°C, pH - NA

NA

10.41 × 106 cells/ml

Lipids and carotenoids

Durvasula et al. 2015

Coccomyxa onubensis ACCV1

NPK med., Temp. 10–25°C, pH 2.5–3.0, LI - NA

800 L

140 mg/L/d

Lutein

Fuentes et al. 2020

Graesiella sp.

BBM med., Temp. 30 ± 1°C, LI - 120 μmol·m2·s−1 pH - NA

100 L

500 mg/L/d

Extracellular polymeric substances

Gongi et al. 2020

Nitzschia frustula

F med., Temp. 23 ± 1°C, LI - 250 μmol·m2·s−1 pH - NA

40 L

1.4 × 106 cells/ml

Lipids and amino acids

Subba Rao et al. 2005

Temp. – Temperature, LI – Light intensity, NA – not available, Mod. – modified media.

2.2 Photobioreactors (PBRs) This technique is efficiently using microalgae in heterotrophic and mixotrophic conditions to cultivate in a closed and controlled environment. It requires high operational and maintenance

Extremophilic Microalgae as a Potential Source of High-Value Bioproducts 171

costs but offers better control over undesirable contamination and physiological conditions. It also yields high biomass, superior in quality (high cell densities) that is used for generating bioactive compounds (De Morais et al. 2015). Given the increasing demand for natural products with bioactive nutraceutical properties, the interest in the development of efficient and scalable processes to obtain such products is increasing. Outdoor PBR processes represent an attractive alternative to synthetic procedures in this regard (Ruiz-Domínguez et al. 2015). However, algal harvesting is a difficult task as culturing and harvesting methods differ according to species-to species, so there is no ‘one-size-fits-all’ solution. Some other factors such as growth conditions, geographical constraints, availability of natural resources or waste streams. Therefore, to design an effective and efficient algal cultivation system, it should be engineered to generate zero waste and provide numerous benefits. Microalgae can be preferred over other plant sources as it can readily adjust to physico-chemical variations and does not comprise on its nutrient uptake ability for growth and reproduction (Enamala et al. 2018).

3. High-Value Bioproducts Microalgae offer a great range of high-value bioproducts, and more than 4000 experimental studies were published between 1926 and 2016 on bioactive compounds of plants (Michalak et al. 2017). However, only limited studies have been done so far on the high-value bioproducts extracted from extremophilic microalgae, including cyanobacteria. The conventional extraction techniques of high-value metabolites from algae mainly depend on the used solvents (organic and residual) and the end-product may compromise on quality standards. Thus, emerging algaltechnologies, like supercritical fluid extraction, enzymatic hydrolysis, mechanical disruption and many more are employed to obtain various bioproducts with the best quality (Di Sanzo et al. 2018, Leone et al. 2019, Mehariya et al. 2019, 2020b, Molino et al. 2018a, 2018b, 2018c, 2019a, 2019b, Saini et al. 2021). They are mostly used in food supplements, cosmeceuticals, animal feed and pharmaceutical industries which are discussed in detail next. A schematic diagram representing the potential commercial applications of valuable bioproducts from extremophilic microalgae as shown in Fig. 8.1.

Fig. 8.1. The commercial applications of extremophilic microalgae and their high-value bioproducts.

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3.1 Pigments The commercial production of algal pigments, from freshwater and marine waters, has been practised for many years. The major pigments are β-carotene and Astaxanthin synthesized by Dunaliella salina and Haematococcus pluvialis, respectively. Lutein pigment is synthesized from Scenedesmus sp., Muriellopsis sp., and Chlorella sorokiniana is good for the health of skin and delays the aging process (Stoyneva-Gärtner et al. 2020). Pigments like phycocyanin and phycoerythrin, are mainly used as food colorants and also used to manufacture cosmetics (Liang et al. 2004). They also show many anti-inflammatory properties and boost immunity in human beings, as they are a good source of antioxidants (Ciccone et al. 2013). Various natural pigments and their derivatives are effective against cancerous cells as chemo-preventive agents. Tiwari and Tiwari 2020 have reviewed the emerging drug technologies as “cyanotherapeutics” and concluded that the “cytotoxic assay of the pigment for anticancer activity depicted an increased activity under nutrient deprived conditions, confirming the chemotherapy potential of pigments”.

3.2 Bioactive Compounds Microalgae from cold stressed environments evolve an exclusive membrane assembly to protect the cell from freezing damage, and to maintain their fluidity for transporting proteins. Whereas, in case of thermal springs, some potential enzymes help to regulate the tolerance towards rising temperatures in the exposed habitat. So, there are different types of bioactive metabolites present in algae from the untapped extreme environments, such as Carotenoids (β-carotene, astaxanthin, lutein, etc.); PUFAs; peptides; phenolic compounds, etc. (Molino et al. 2020a, 2020b). However, for mass-cultivation of potential strains, standardization of screening protocols and optimized research methods are required to produce several medical diagnostic agents (Jacob-Lopes et al. 2019).

3.3 Protein-rich Biomass The protein-rich biomass obtained from algae is reported to excel in protein concentration in quality as well as quantity. On a dry weight basis of whole biomass, the protein content may range between 46 to 71% in blue-green algae, Spirulina platensis (Zhu et al. 2014), and up to 58% for green algae, Chlorella sp. (Borowitzka and Moheimani 2013). Besides this, microalgae have a well balanced essential amino acid profile, equivalent to plants and other non-vegetarian protein sources, such as beta-lactoglobulin in animal milk, egg white and soybeans. But algal protein-based diets in food applications are still limited, due to the cellulosic cell wall which makes it hard to digest for humans as well as the unsavoury taste and lack of flavor. However, algal proteins isolated from Dunaliella salina are used in bakery products, without any digestion problems, for past many decades (Finney et al. 1984).

3.4 Lipids Algal lipids have more fluidity and mobility than plant lipids, they tend to change their composition according to stress factors like temperature, light intensity, pH, CO2 levels and nutrient deprivation. In cold-thriving conditions, the algae swaps the lipid content as per the fluctuation in temperature to thrive the freezing stress and maintain the fluidity in the cell membranes. The thickness of the membrane varies according to the saturation and length of the fatty acid chains. Longer fatty acids are harder to move than shorter fatty acids. The recent work of Martelli et al. 2020, on blue green algae, has reported that these “glycolipids or lectins can be used to develop certain formulations, and fermented products that can destroy the development of the disease-causing processes, even in cases where antibiotics are ineffective”.

Extremophilic Microalgae as a Potential Source of High-Value Bioproducts 173

3.5 Others Some of the potential and interesting applications of extremophilic microalgae are as follows: (i) Antibiotics – In recent years, bioprospecting of microalgae from harsh environments and the screening of secondary metabolites, produced by them have shown antibiotic activity. The algal extracts from wastewaters can inhibit bacterial growth more effectively than algae growing in clean waters (Lustigman 1988). The experimental study done by Ghazala et al. 2004 on “Tetraspora cylindrica, collected from wastewater and thermal effluents showed antibacterial activity against Corynebacterium diphtheria, Klebsiella pneumoniae and Shigella boydii, and many more”. (ii) Glycerol – It is excreted by halotolerant algal strains, during excessive saline conditions. Its production in polar environments can be constrained if exposed to high solar radiations. As per Ahmad and Hellebust 1986, “the osmoregulatory role of glycerol and other inorganic ions are confirmed in microalgae, Chlamydomonas pulsatilla in the salty marsh habitats”. Similarly, Dunaliella salina flourish well in salt stress conditions and release more glycerol, besides carotenoids (Hadi et al. 2008). An experimental study by Raymond 2014 investigated green algae in cold stressed habitat, “Chloromonas brevispina, can produce glycerol or ice-nucleating proteins that not only prevent the cells from freezing temperatures but also bind to ice crystals to prevent recrystallization”. (iii) Bio-manure – During algal cultivation, the residues obtained from microalgal biomass, after aerobic digestion can be used as a fertilizer. This bio-manure is safe for agricultural use with zero discharge of pollutants Renuka et al. 2015. (iv) Livestock feed – The poultry industry including pigs, hens (laying and feeding hens) successfully use defatted algae-based animal fodder to enhance health and production. According to Madeira et al. 2017, the growth results in farm animals (pigs) were found using more algal ingredients, as they contain a high amount of polysaccharide degenarating enzymes combined with protease, supporting the growth and development metabolism. A great potential of algae as a protein source was evident by the studies of Fredriksson et al. 2006 on laying hens, which showed “enhanced resistance to disease, improved color of the yolk, and reduced fat as compared to traditional feeding formulas”. (v) Functional foods – These are the innovative healthy foods incorporating the bioactive compounds to meet the nutritional demand without compromising on health benefits and taste (Borowitzka and Moheimani 2013). Microalgae from stressed environments have a lot of potential as a functional food, with adequate nutritional effects, satisfactorily being included in human diets. For example, the marine alga, Isochrysis galbana biomass is also present with other food ingredients to prepare biscuits that are naturally loaded with polyunsaturated fatty acids, PUFAs (Gouveia et al. 2010). A recent experimental studies by Vieira et al. 2020 confirmed the application of “3D printed functional cookies fortified with Arthrospira platensis, which adds the natural ink/colorant to the innovative functional food with antioxidant properties”.

4. Medical Applications of Bioactive Compounds Nowadays, microalgae are used in several therapeutic applications as a major candidate in drug development programs, after the discovery of several bioactive compounds. This has led to their tremendous medical utility to treat several fatal illnesses, practically tested and proven by the clinical scientists across the world. According to Patel et al. 2019 extremophilic microalgae have “various kinds of biomolecules (active compounds), which include different types of

174 Extremophiles: Wastewater and Algal Biorefinery vitamins, minerals, fibers, sterol, polyphenols, polysaccharides”. They are well adapted in harsh conditions of desiccation, high temperatures, extreme pH, cold, osmosis, salt, light, nitrogen and high salinity (Singh 2018) and therefore, best suitable as diagnostic agents in the medical world because of their antibacterial, antifungal, antiplasmodial, antiviral, anticarcinogenic properties and immunomodulatory actions.

4.1 Anticarcinogenic Several algal extracts have the potential to induce neoplastic cell death (apoptosis) by caspasemediated cell death or pre-apoptosis either by caspase-dependent or caspase-independent pathways and its medicinal properties are often explored to avoid the side-effects of chemo-medication and X-ray radiation therapy to study various cancer diseases by conventional ways (Majumder et al. 2015). For example, methanolic extract of Rhizoclonium riparium (Roth) Harvey, collected from different blocks of the Sundarbans mangrove ecosystem located in India shows cytotoxicity on HeLa cells with IC50 value 506.081 ± 3.714 μg/ml (Paul and Kundu 2013). The pigment c-phycocyanin, isolated from Spirulina platensis have been reported to trigger structural alterations at the genetic level. Li et al. 2006 reported that “pigment extracts from Spirulina platensis binds with dioxyribonucleic acid fragmentation, increased Fas expression, ICAM expression, decreased Bcl-2 expression and activation of caspase 2, 3, 4, 6, 8, 9, 10 in HeLa cell line”. A similar kind of study using extracts of Aphanizomenon flos-aquae and Haematococcus pluvialis has been reported to impose antiproliferative activity in HL-60 and MV-4-11 cell lines (Bechelli et al. 2011).

4.2 Immunostimulants Most of the polysaccharides derived from algae are referred to as sulfated polysaccharides and have proved to show unique immunomodulatory functions. The basic mechanism relies upon the stimulation of macrophages and the acceptance of Reactive Oxygen Species (ROS), nitric oxide and various other types of cytokines/chemokines (Schepetkin and Quinn 2006). Sulfated polysaccharides have been known to suppress several viral infections, such as Encephalomyocarditis virus (EMC) in animals, Herpes simplex virus types 1 and 2 (HSV1 and HSV2), Human Immunodeficiency Virus (HIV), swine flu virus and others (Smelcerovic et al. 2008). Another scientific study by De Jesus Raposo et al. 2015 on carrageenan, a sulfated polysaccharide, can attach “directly to human papillomavirus (HPV) to inhibit not only the viral adsorption process but also the input and the subsequent process of the uncoating of the virus”.

4.3 Antioxidants Microalgae from extreme habitats are mainly exposed to severe oxidative stress and develop efficient protective systems against ROS and free-radicals to protect their bioactive molecules. The supercritical fluid (SCF) extraction method including generally recognized as safe (GRAS) – certified CO2 as extracting solvent could be used for getting a cancer prevention agent of Phormidium concentrates (Chatterjee et al. 2014). The presence of large amounts of antioxidants in β-carotene and lutein pigment found in the acidophilic green alga, Coccomyxa acidophila when grown in urea decrease the risk of skin cancer (Casal et al. 2011). A few cryophilic species of green algae, Chlamydocapsa sp. and Raphidonema sempervirens can produce α-tocopherol (vitamin E) that reinforce antioxidants production same as carotenoids (Hassan et al. 2013). Scytonemin, a phenolic molecule, having UV radiation protection characteristic is obtained from thermophilic algal genera, like Scytonema, Stigonema, Nostoc, Calothrix, Lyngbya, Rivularia, Chlorogloeopsis and Hyella (Garcia‐Pichel et al. 1992). Mycosporine-like Amino Acids (MAAs) were considered to be potent antioxidants with ROS scavenging activity. The presence of MAAs in significant amounts was found among snow algae Chlamydomonas nivalis and Scotiella nivalis that protects from solar ultraviolet-radiations (Sinha et al. 2001).

Extremophilic Microalgae as a Potential Source of High-Value Bioproducts 175

4.4 Anti-inflammatory There are many algal derived metabolites that can suppress chronic inflammation by regulating cellular enzymatic activities (Robertson et al. 2015). The thermophilic alga, Mastigocladus laminosus has proved to reduce the expression of pro-inflammatory genes (anti-inflammatory effect) by inducing exopolysaccharides antioxidants (Stoyneva-Gärtner et al. 2020). This can be linked to the production of various proinflammatory mediators, including the antioxidant properties of microalgal oils. The studies of Yates et al. 2014 supported that the microalgal oil composition may differ in natural habitats due anthropogenic activities and other multi-scale drivers.

5. Commercial Use of Extremophilic Microalgae Extremophilic microalgae play a significant role in the isolation of biochemicals and their derivatives of industrial applications; in the field of bioenergy, drug manufacturing, natural skin and hair products, biofertilizers, innovative food drinks, cakes, cookies and other health supplements.

5.1 Biotechnology The biotechnological studies on the functioning of heat-stable enzymatic activity by Greco et al. 2013 confirmed “the identification of thermostable site-specific endonucleases in thermophilic blue-green algae, Dactylococcopsis salina as a series of class-II restriction endonucleases (5′-C↓CRYGG-3′ (DsaI); 5′-GG↓CC-3′ (DsaII); 5′-R↓GATCY-3′ (DsaIII); 5′-G↓GWCC-3′ (DsaIV); 5′-↓CCNGG-3′ (DsaV); and 5′-GTMKAC3′(DsaVI)”. Some cyanophyte strains of Phormidium also contain thermostable enzymes working efficiently at high temperatures ranging from 65°C to 80°C (Piechula et al. 2001).

5.2 Pharmaceuticals Pharma valuable products from extremophilic microalgae and its industrial commercialization can be seen as a gateway to a multibillion-dollar industry in the present and coming future (Bhattacharjee 2016). Algae from extreme habitats exhibit oxidative stress, have defensive mechanisms to overcome stressed conditions and contain active chemical constituents of immense medicinal importance. For instance, change in a healthy lifestyle has a positive impact on natural pigments and contributes a great share of the market in the pharmaceutical industry. Dunaliella salina contain β-carotene almost 14% of total dry weight have natural anti-inflammatory properties (Metting 1996) and Haematococcus pluvialis, contain astaxanthin which is 1.5–3% of dry weight (Todd and Gerald 2000). Their market price may range from US$ 300 to US$ 3000/kg and are often consumed as major food supplements in developed countries (Hejazi and Wijffels 2004). A eukaryotic acidophilic microalgae, Coccomyxa onubensis extracts shows good antimicrobial activity, against Gram-negative and Gram-positive bacteria and fungus, Candida albicans (Navarro et al. 2017). Some cold-adapted strains of green microalgae can produce antifreeze proteins (AFPs) that demonstrates their antifungal activities (Elena and Carlos 2018). However, more studies on the biochemical and molecular aspect in the future are needed to characterize and purify several other novel bioactive molecules from extremophilic microalgae for assessing their potential as pharmaceutical sources.

5.3 Nutraceuticals A nutraceutical can be defined as a product either isolated or purified from foods and is normally available in therapeutics, which are not generally linked with foods, possesses demonstrable physiological benefits and provides protection against several chronic diseases. Algal-sourced protein are generally recognized as safe (GRAS) for human consumption, having favorable protein

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and amino acid profile equivalent to soy-proteins and animal proteins (Spolaore et al. 2006, Kent et al. 2015). Some of the certified GRAS commercially available species include several extremophilic green algae like Haematococcus sp., Arthrospira sp., Dunaliella sp. and Chlorella sp. (Koyande et al. 2019). According to (Jacobsen et al. 2013) a dinoflagellate microalgae, Crypthecodinium cohnii, accumulates 60% of essential docosahexaenoic acid (DHA), an important component of polyunsaturated fatty acids, crucial in early child development and growth.

5.4 Bioremediation of Wastewater With the advent of the industrial revolution, harmful chemical discharge into natural water bodies and rivers via streams/canals has greatly impacted aquatic vegetation and biodiversity. This has caused predominance of blue green algae, bacteria and toxins in the waterbodies, disturbing aquatic life and even causes death of fishes in some cases. Thus, to save the ecological balance and convert toxic waterbodies back to normal, algal-technologies are implemented. It is a well-established biosorption technology to remove excessive nutrients and even heavy metals from the water using low investments and operational costs (Craggs et al. 2012). A novel approach to treat complex and hazardous wastewater by emerging hybrid technology of bioelectrochemical cells (BEC) using algal-microbial interaction. It delivers promising aspects of generating bioenergy with concurrent waste electro-remediation (Chandrasekhar et al. 2020a). The nitrogen fixating thermophilic algae are very effective at removing nutrients, such as nitrogen and phosphorus, from nuclear reactor thermal effluents (Radway 1992, 1994). In a study conducted by Sawayama et al. 1998 one thermophilic cyanobacterium, “Phormidium laminosum was immobilized at 43°C on hollow fibres in a photobioreactor to improve the removal of nitrate and phosphate ions from water”. Moreover, a research by de-Bashan et al. 2008a on the green microalga, “Chlorella sorokiniana has shown its ability to remove ammonium ions from the wastewater at temperatures of 40–42°C and light intensity of 2500 μmol·m2·s−1 for 5 h in a day”.

5.5 Biofuels The rapid decline of non-renewable energy sources around the globe has increased the demand for alternative biofuels feedstock (algal biomass) for a circular bioeconomy (Chandrasekhar et al. 2020b). This can be achieved by developing more renewable biologically produced fuels (bioethanol and biodiesel) for energy consumption (Borowitzka and Moheimani 2013). To increase the lipid synthesis, extremophilic microalgae are one of the most promising agents because they have adapted well to the mechanism to combat with nutrient limitations and can produce more fatty acids into glycerols in the cytosol during nutrient starvation. Many of the steps in biofuel production deal with high temperatures and extremes of pH; therefore, extremophiles serve as ideal candidates for biofuel feedstock (Satpati and Pal 2018). A recent experiment on a thermophilic alga, Micractinium sp. conducted by Abu-Ghosh et al. 2020 to check “microbial lipids synthesis under nitrogen starvation conditions at different light regimes (time-integrated photon dose of 1000 µmol photons m−2·s−1 ). The results exhibited lipid content (22.7%), and lipid productivity (0.04 g·L−1·d−1)” at elevated temperatures up to 50°C. The effect of salinity gradients on lipid productivity extensively studied on Dunaliella sp. also showed a high amount of C16 and C18 fatty acids production (Sharma et al. 2012).

5.6 Cosmeceuticals Chemical free personal care products are a new trend in people’s lifestyles. One of the recent review by Stoyneva-Gärtner et al. 2020 suggested that “nowadays people are more concerned about natural and chemical/preservative-free products”. For many decades, microalgae and seaweeds have been used to prepare cosmetic products due to their skincare and haircare potential. To meet the soaring

Extremophilic Microalgae as a Potential Source of High-Value Bioproducts 177 Table 8.3. Bioactive compounds found in extremophilic microalgae and their cosmetic applications. Extremophilic Microalgae

Bioactive Compounds

Cosmetic Applications

Reference

Anabaena vaginicola

Lycopene

Skin protection and rejuvenation

Hashtroudi et al. 2013

Stichococcus bacillaris

α-tocopherol (Vitamin E)

Skin and hair care products

Hassan et al. 2013

Laurencia obtusa

Phycoerythrin

Natural colorants

Azeem et al. 2019

Chlamydomonas nivalis

Astaxanthin

Anti-aging and UV protectant moisturizer

Remias et al. 2010

Coccomyxa acidophila

Lutein

Skin protection

Casal et al. 2011

Nannochloropsis sp.

Bioctive compounds

Anti-aging products

Zanella and Vianello 2020

Chlorella vulgaris

β-carotene

Hair conditioners and shampoos

León et al. 2003

Scotiellopsis rubescens

Sporopollenin polymer

Sunscreen moisturizer

Sharma et al. 2016

Nostoc commune

Mycosporine-like amino acids (MAAs)

Sun care and skin nourishment products

Bohm et al. 1995

demand, untapped natural resources of extremophilic algae are explored to extract biomolecules used in cosmetics as listed in Table 8.3. All novel biomolecules in cosmetic applications undergo standard safety trials before being marketed for human use and applications.

5.7 Aquafeed Microalgae are considered as an ideal meal in the fishery and hatchery cultivation. It has a short lifecycle with good nutritional property, leaving residual biomass after harvesting, rich in lipid, protein and polysaccharides contents (Hemaiswarya et al. 2011). In the aquaculture production units, with the use of emerging algal cultivation techniques, the biomass is a good source of polyunsaturated fatty acids (PUFAs), linoleic acid and α-linoleic acid which are key nutrients for aquaculture. These algal pellets make fish healthy and bigger in size, hence turning fish farming into a profitable business in a short period. According to Kiron et al. 2012 “Nannochloropsis sp. has great potential as aquafeed for Atlantic salmon, common carp and white leg shrimp”. Further, Catarina and Xavier 2012, suggested “diatom Chaetoceros sp., to be ideal for the rearing of bivalves, under standard conditions because they are rich in secondary metabolites like PUFAs, EPA (eicosapentaenoic acid), and DHA (docosahexaenoic acid)”. Thus, gives good yield and market value.

6. Recent Developments for Enhanced EPA and DHA Production The emerging trends target the manipulation of the precise molecular scenario and the nutrient regime, which are the two major players behind the synthesis of several value-added products from extremophilic microalgae. With the advent of several omics-technologies, it is now possible to presume several precise clear-cut blueprints to enhance the production of high-value components. Microalgae are a sustainable reservoir of polyunsaturated fatty acids, but interestingly their extremophilic counterparts are usually very rich in these components like PUFAs, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Nonetheless, marine algae as primary producers have the potent ability to store EPA and DHA (Omega 3-PUFAs) and can reduce the excess demand from fish stock. Patel et al. 2020 developed a mixotrophic cultivation system that can valorize the growth rate and gives better yield of high value chemicals and which can be further scaled up for the industrial stage. The successful isolation results of extremophilic microalga Galdieria sp. USBA-GBX-832, from acidic hot springs of Los Nevados, Colombia by Lopez et al. 2019 showed their promising metabolic versatility. It can be cultivated in both autotrophic,

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heterotrophic as well as mixotrophic cultivation system and comparitive studies confirm the dynamics of their lipid profile, clearly pointing out that heterotrophy and mixotrophy provides better PUFA accumulation than autotrophy. The research study by Fuentes et al. 2020 on chlorophycean extremophile Coccomyxa onubensis, showed that they can tolerate pH extreme (upto pH 2.5 and even lower) making it suitable for outdoor cultivation and reduces the chance of contamination. The cells grown under nutrient-limited conditions contain high a level of polyunsaturated fatty acids (Ruiz-Domínguez et al. 2014). Moreover, molecular approaches have been taken into considerations, through genetic manipulations and metabolic engineering in which the key enzymes elongases and desaturases can synthesize PUFA. Thiyagarajan et al. 2019, focused on such two fatty acid biosynthetic genes, Δ6-elongase from Isochrysis sp. and Δ5-desaturase from Pavlova sp. involved in EPA and DHA production. The findings suggested that under nitrogen-starved growth conditions, expression of these two genes increased by 1.7 fold which further improves the lipid production. Also, these stress conditions can be generated through the manipulation of cultivation systems. For example, in a salinity-gradient strategy, halotolerant strain Dunaliella salina has been grown in salt extremes, which is contemporary to its lipid accumulation. As, an addition of salt is a simple technique to enhance lipid productivity, it can enhance the optimal lipid productivity to 223.2 mg L–1 d–1 and lipid content up to 59.4% (Ho et al. 2014). The lipid contents of Isochrysis galbana increased from 24.0 to 47.0% on using 10 practical salinity units for 2 days during the second stage cultivation system (Ra et al. 2015). Whereas, on stressed exposure of Light-Emitting Diode (LED) for a continuous period of 2 days resulted in Nannochloropsis salina, Nannochloropsis oceanica and Nannochloropsis oculata reaching the maximum lipid content 52.0, 53.0 and 56.0% (w/w) at 100 µmol m–2s–1 of blue and green LED respectively, in the two-phase culture system for biomass production and lipid accumulation (Ra et al. 2016).

7. Conclusion The present work studied the untapped potential of extremophilic microalgae found in different stressed conditions. The different algal cultivation technologies, with emphasis on extremophilic microalgae growth systems to isolate and upscale the high-value products is described. The bioproducts obtained from extremophilic microalgae such as proteinaceous biomass, lipids, pigments and other bioactive compounds have multiple applications in the medicinal, pharmaceutical and nutritional value as anticarcinogenic, immunostimulants, antioxidants and anti-inflammatory agents. Additionally, their role as biotechnological therapeutics by creating a gene pool of thermostable enzymes and as antifreeze proteins, suitable for cosmetic products. The presence of high amount of EPA and DHA not only enhance fish productivity and market value, but also improves the nutritional value of food, essential for human growth and development. The emerging technology of hybrid electro-remediation led to the removal of toxic ions from wastewater using algal resources, present in the stressed environment. Also, their well-adapted mechanism of lipid synthesis makes them an ideal candidate for biofuel generation. All these approaches will develop a positive impact on extremophilic microalgal farming, using innovative techniques to generate a profitable income in a short span using natural resources.

Acknowledgement We thank Dr. G.P. Singh, Algal Biotechology Laboratory, University of Rajasthan, Jaipur, Rajasthan (INDIA) for the valuable corrections and proof-reading.

Conflicts of Interest The authors declare no conflict of interest.

Extremophilic Microalgae as a Potential Source of High-Value Bioproducts 179

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9 Value-Added Products and Biofuels from Extremophilic Microalgae Biomass Usharani K.,1,* Lakshmanaperumalsamy P.2 and Jayesh M.S.3

1. Introduction Algae can mineralize nutrients, metals available in wastewater and are capable of their biotransformation for their growth and development. Algae biomass is the quantity of algae in a water body at a given time. Algae biomass fuel is among the most recent forms of biomass fuels and can reduce carbon dioxide (CO2) emissions from smokestacks throughout its development process, while also producing cleaner fuel. Photosynthetic algae comprise a broad range of photosynthetically energetic green, red and brown algae in addition to cyanobacteria. Since they are able to fix CO2 using light as the sole source of energy, they are important microbial cell factories to produce bio‐ based energy carriers and value-added products. Besides photoautotrophy, numerous microalgal species are competent to make chemoheterotrophic or mixotrophic metabolism that promotes the management of industrial wastewater holding an elevated organic load (Judd 2016). Microalgae in general, conventional and extremophile can play an important role in a circular bio-economy by providing high-quality products that include proteins, lipids and colorants inside the biomass produced by the wastewater treatment (WWT) cleaning process. The usage of microalgae in WWT plants has two main aims: (1) The direct utilization or conversion of water contaminants, and (2) Progressing the decontamination of bacterial systems (microalgae-bacteria aggregates) by providing additional oxygen from photosynthesis (symbiotic co-cultures), and thus dropping the total energy costs of direct (gassing performance) or indirect (stirring performance) oxygen supply.

Department of Environmental Science, PSG College of Arts and Science, TN, India. Department of Environmental Sciences, Bharathiar University, TN, India. 3 Department of Chemistry, Laval University, Quebec, Canada. * Corresponding author: [email protected] 1 2

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2. Types of Extremophiles The major types of extremophilic microorganisms and their activities concerning life at high temperatures, extremes of pH, salt concentration, pressures is shown in Fig. 9.1, and their examples are given in Tables (Table. 9.1a and 9.1b). • • • • • • •

Thermophiles (Temperature lovers), Psychrophiles (extreme cold lovers), Acidophiles (acid lovers), Alkaliphiles, Halophiles (salt lovers), Barophiles or Piezophiles Radiophiles (radiation lovers) - Radiation resistant microorganisms.

2.1. Thermophiles are organisms that can thrive at temperatures between 60ºC and 85ºC humidity (Dumorne et al. 2017), many of the thermophiles inhabit volcanoes, hot springs, geysers and deepsea hydrothermal vents, making these places as popular hunting grounds for the search for new types of thermophilic microorganisms. 2.2. Psychrophiles are organisms having a growth temperature optimum of 10ºC or lower, and a maximum temperature of 20ºC (Dumorne et al. 2017), are generally distinct as such organisms that have their optimum temperature below 15–20oC (Cavicchioli et al. 2011, Irwin and Baird 2004). 2.3. Acidophiles are organisms with a pH optimum for growth at or below 3–4 (Dumorne et al. 2017). They are connected with acidic environments that are often associated with volcanic activity, hot sulfur springs, mud pots, etc. Another type of low pH environment is caused by microbial activity. These microorganisms maintain their cytoplasm at near-neutral pH values through powerful proton pumps in their cytoplasmic membrane, which keeps a proton concentration gradient of four, five or even more orders of magnitude (Oren 2018, Irwin and Baird 2004).

Fig. 9.1. Major classification of Extremophiles.

Value-Added Products and Biofuels from Extremophilic Microalgae Biomass 189 Table 9.1a. Types of Extremophiles with their growth conditions and examples. Environmental Factor

Type

Growth Conditions

Examples

References

Temperature

Hyper thermophile

Maximum growth > 80oC

Pyrolobus fumarii (113oC)

Goswami et al. 2016 Anderson et al. 2011 Irwin and Baird 2004 Rothschild and Mancinelli 2001 Blöchl et al. 1997 Blamey et al. 1993 Zillig et al. 1983

Crenarchaeota-nitrate reducing Thermococcus celer Thermophile

Maximum growth 60–80oC

Synechococcus lividis Thermus aquaticus (> 70oC) Deinococcus–Thermus group Bacillus stearothermophilus

Goswami et al. 2016 Irwin and Baird 2004 Rothschild and Mancinelli 2001 Kullberg 1981 Brock et al. 1969

Mesophile

Maximum growth 15–60oC

Homo sapiens Escherichia coli

Goswami et al. 2016 Rothschild and Mancinelli 2001

Psychrophile

Maximum growth < 15oC

Pscychrobacter sp. Alteromonas haloplanctis Polaromonas vacuolata

Goswami et al. 2016 Rothschild and Mancinelli 2001

Acidophile

Low pH affectionate

Sulfolobus acidocaldarius (pH 3) Cyanidium caldarium, Ferroplasma sp. (pH 0)

Chen et al. 2005 Dopson et al. 2004 Irwin and Baird 2004 Rothschild and Mancinelli 2001 Doemel et al. 1971

Alkaliphile

pH > 9

Natronobacterium, Bacillus firmus OF4, Spirulina spp. (all pH 10.5) Natronomonas pharaonis

Goswami et al.,2016 Rothschild and Mancinelli 2001 Falb et al., 2005

Salinity

Halophile

Salt-loving (2–5 M NaCl)

Halobacteriaceae, Dunaliella salina Natronomonas pharaonis

Goswami et al. 2016 Irwin and Baird 2004 Rothschild and Mancinelli 2001

Radiation

Radiophile

Radiation resistant microbes

Deinococcus radiodurans Dunaliella radioduransis, Dunaliella bardawil, Rubrobacter sp.

Goswami et al. 2016 Irwin and Baird 2004 Rothschild and Mancinelli 2001

Pressure

Barophile Piezophile

Weight-loving Pressure-loving For

Unknown microbe, 130 MPa

Goswami et al. 2016

Gravity

Hypergravity Hypogravity

> 1 g None known < 1 g None known

None known None known

Goswami et al. 2016 Rothschild and Mancinelli 2001

Vacuum

Vacuumphile

Tolerates vacuum (space devoid of matter)

Tardigrades, insects, microbes, seeds

Goswami et al. 2016 Rothschild and Mancinelli 2001

Desiccation

Xerophile

Anhydrobiotic

Artemia salina; nematodes, microbes, fungi, lichens

Goswami et al. 2016 Rothschild and Mancinelli 2001

pH (Acidic/ Alkaline)

Table 9.1a contd. ...

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...Table 9.1a contd. Environmental Factor

Type

Growth Conditions

Examples

References

Oxygen tension

Anaerobe

Cannot tolerate O2 tension

Methanococcus jannaschii

Irwin and Baird 2004 Rothschild and Mancinelli 2001

Micro aerophile

Tolerates some O2

Clostridium

Irwin and Baird 2004 Rothschild and Mancinelli 2001

Aerobe

Requires O2

H. sapiens

Goswami et al. 2016 Rothschild and Mancinelli 2001

Gases Metals

Can tolerate high concentrations of metal

C. caldarium (pure CO2) Ferroplasma acidarmanus (Cu, As, Cd, Zn); Ralstonia sp. CH34 strain, (metalotolerant) (Zn, Co, Cd, Hg, Pb)

Irwin and Baird 2004 Anton et al. 1999 Rothschild and Mancinelli 2001

Chemical Extremes

Table 9.1b. Major extremophiles and its extremozymes with applications. S. No.

Extremophiles

Extremozymes

Applications

1.

Psychrophiles

Alkaline Phosphatase Proteases Lipases Cellulases Amylases Dehydrogenase

Bioremediation, Biosensors, Methanogens-methane synthesis Detergent and bakery Feed, Textiles

2.

Thermophiles

DNA Polymerase, DNA Ligase, Alkaline Phosphatase, Proteases Xilanases

Molecular Biology, Surfactant, Oil degradation, Waste Treatment, Methane Production Paper Bleaching

3.

Halophiles

Polyhydroxyalkanoates, Bacteriorhodopsin, Rheological Polymers, Liposome Dehydrogenase Proteases

Bioelectrodes Bioelectronoics Oil recovery Drug delivery Biocatalysis in organic Peptide synthesis

4.

Alkaliphiles

Proteases, Cellulases, Xylanases Pullulanases Pectinases

Detergents, Food industry Paper industry Waste Treatment

5.

Acidophiles

Amylase, glucoamylase Proteases, cellulases Oxidases

Starch processing Feed component Desulfurization of coal

2.4. Alkaliphilies are organisms with optimal growth at pH values above 10 (Dumorne et al. 2017), and these microorganisms are widespread in nature. Bacteria such as, Bacillus sp. which grow at pH 9–10, while not being capable of growing at neutral pH can simply be isolated from soils (Irwin and Baird 2004). 2.5. Halophiles are organisms requiring at least 1 M salt for growth (Dumorne et al. 2017), and linked with hypersaline environments are easily generated or produced when seawater dries up in

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coastal lagoons and salt marshes and in manmade evaporation ponds of saltern systems built to produce common salt by evaporation of seawater (Irwin and Baird 2004). 2.6. Barophiles or Piezophiles are organisms that live optimally at hydrostatic pressures of 40 MPa or higher (Dumorne et al. 2017), these organisms facilitate the requirements with a highpressure environment in order to grow. They can withstand a high-pressure habitat, which is the deep-sea environment including ocean floors and deep lakes where the pressure can exceed 380 atm (Irwin and Baird 2004). 2.7. Radiophiles are organisms resistant to high levels of ionizing radiation (Dumorne et al. 2017) and are radiation resistant microorganisms found on terrestrial surfaces, air-borne microbes, microbes living in the upper layers of the sea and other aquatic environments exposed to direct sunlight, including a considerable amount of potentially harmful ultraviolet radiation. Those microorganisms have to protect themselves against radiation damage and the protection mechanisms comprise repair mechanisms for damaged deoxyribo nucleic acid (DNA) (Lynn Rothschild and Rocco Mancinelli 2001, Fred Rainey and Ahran Oren 2006, Irwin and Baird 2004, Goswami et al. 2016). Examples of radiophile include, Dunaliella radioduransis, Dunaliella bardawil, Rubrobacter species. The bacterium D. radiodurans is well-known for its capability to resist ionizing radiation (up to 20 kGy of gamma radiation) and UV radiation (doses up to 1,000 J m12), but this extraordinary resistance is considered to be a by-product of resistance to extreme desiccation (Battista 1997). The other organisms to facilitate which can place high levels of radiation are two, Rubrobacter species (Ferreira) and the green alga Dunaliella bardawil (Ben-Amotz and Avron 1990) (Table 9.1). The examples of extremophiles include Picrophilus torridus (a thermoacidophile adapted to hot, acidic conditions), Antarctic krill (a psychrophile) and the Pompeii worm (a thermophile).

3. Growth of Extremophiles and Importance of Extremozymes “Extremozymes” are specialized enzymes that are highly stable and can tolerate extremes of temperature, pH, salinity that would inactivate other enzymes which are all industrially important. They are produced from extremophiles that are valuable in industrial production procedures and research applications due to their capability to remain active under the harsh conditions (e.g., high temperature, pressure and pH) characteristically engaged in these processes (Gerday and Glansdorff 2007). They are also of research importance in the field of astrobiology. Biocatalysts isolated by these extremophiles are termed extremozymes and they possess extraordinary properties of salt allowance, thermostability and cold adaptivity. They are very resistant to extreme conditions due to their great hardness and create novel opportunities for biocatalysis and biotransformation and the expansion of the economy and innovative research through their application. Thermophilic proteins, piezophilic proteins, acidophilic proteins and halophilic proteins have been studied during the preceding years. Amylases, proteases, lipases, pullulanases, cellulases, chitinases, xylanases, pectinases, isomerases, esterases and dehydrogenases have huge potential application for biotechnology field such as in agricultural, chemical, biomedical (Kelly Dumorne et al. 2017) and biotechnological processes, a particular application in biofuel production (bioethanol, biodiesel, biobutanol and biogas, relying on the use of substrates such as sugars, starch and oil crops, agricultural and animal wastes and lignocellulosic biomass) (Desire Barnard et al. 2010. The focus of the study on extremozymes and their main applications have come into prominence in recent years (Seckbach et al. 1970, Beardall and Entwisle 1984, Pick 1994, Isken and de Bont 1998, Seki and Toyoshima 1998, Nies 2000, Desire Barnard et al. 2010, Kelly Dumorne et al. 2017, Kour et al. 2019). The extremozymes from extremophilic microbes have been in high demand in the various commercial applications, due to their potential applications in different processes. Extremophiles such as acidophile, alkaliphile, halophile, metallotolerant, piezophile, psychrophile, psychrotolerant, radioresistant, thermophile, hyperthermophile, toxitolerant, xerophile and their extremozymes produced, include amylase, lipase, pectinase, protease, β-galactosidase, β-glucosidase, cellulase,

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laccase, chitinase and xylanase are essential for various commercial applications (agriculture, medicine and the food industry). The potential applications in areas such as agriculture, bioconversion of hemicellulose, biodegradation, bioethanol production, biorefinery, chemical industry, composting, dairy industry, detergent industry, feed supplement, food industry, feed industry, leather industry, paper and pulp industry, peptide synthesis, pharmaceutical industry, molecular biological work and therapeutic agent (Table 9.1b). This potential use in health-related and food related areas is sufficient to research for novel and more efficient extremozyme (Rasuk et al. 2016, Kour et al. 2019). Dunaliella, green unicellular microalgae isolated from high salinity aquatic habitat with sodium chloride (NaCl) concentrations higher than 3 M (Borowitzka and Huisman 1993) and the other is Spirulina. The genus Dunaliella, notably D. salina and D. viridis, are found all over the world in salty lakes and saltern evaporation and crystallizer ponds and brines at salt concentrations up to NaCl saturation. Dunaliella strains are used for the commercial production of β-carotene, often under optimized conditions Both green D. viridis and β-carotene-rich D. salina play a significant role in the salt-lake ecosystem (Hamburger 1905, Oren 2014). Extremophiles have developed special mechanisms that allow the cell to grow and thrive under extreme conditions. The well-known accumulation of glycerol as an osmo-regulant in Dunaliella or the accumulation of beta-carotene as a protective agent against excess light is the basis for the development of the mass culturing of Haematococcus pluvialis for the extraction of astaxanthin (Fan et al. 1998, Varshney et al, 2014). Spirulina, is a filamentous blue green microalga and an excellent source of plant protein and could replace expensive animal derived proteins in fish feed. The uses of spirulina as a fish meal for different fish species were commercially applied. Spirulina are prebiotic obtained from dried biomass of the cyanobacterium, Arthrospira platensis and are a rich source of proteins, vitamins, essential amino acids and other phytochemicals. The spirulinaincorporated diets produced better Specific Growth Rate (SGR) and Feed Conversion Ratio (FCR) than probiotic diets, the PER indicates that supplementing diets with spirulina, followed by yeast and bacteria, significantly improves carp performance (Ramakrishnan et al. 2008, Feng et al. 2020). Extremophilic biomass production is incredibly important to provide sufficient material for enzyme and biomolecule isolation and characterization, ultimately enlightening particular features of industrial significance. The production of biomass, associated enzymes and biomolecules from extremophile resources, particularly center on the application of new fermentation approaches. The enzymes from extremophiles known as ‘extremozymes’ have a potential in multiple areas, either by using the enzymes themselves or by using them as sources of information to modify mesophile-derived enzymes (Hough and Danson 1999). In most cases the reaction medium is aqueous. However, results have indicated that aqueous or organic and nonaqueous media allow modifying of reaction equilibria and enzyme specificity, creating pathways for synthesizing novel compounds 120. The fastidious growth conditions for extremophiles means that it is often economically advantageous to express the gene in a more tractable host organism such as E. coli. The growth rate of the bacterium (example, E. coli) reached maximum at optimum temperature (optimal growth temperature) and low at minimum and maximum temperature (Fig. 9.2). Extremophiles flourish in antagonistic environments such that they are utilizing an ecological niche for which they are extremely well-adapted, and characteristically small or no antagonism within it. Natronomonas pharaonis is an archaeon adapted to two extreme conditions which include high salt concentration and alkaline pH, when it grows aerobically on acetate, it uses carbon to oxygen consumption ratio that is hypothetically near-optimal for growth and energy production. The most often used nutritional selection method in extreme thermophiles is supported on the uracil prototrophic selection from anauxotrophic parental strain. Synthesis of uracil engages enzymes encoded by the pyrE (orotate phosphoribosyl transferase) and pyrF (orotidine-5′-phosphate decarboxylase) genes (Yamagishi et al. 1996).

Value-Added Products and Biofuels from Extremophilic Microalgae Biomass 193

Fig. 9.2. Growth curve (E. coli) showing growth rate and its optimum peak.

Their role in uracil production further converted and changed the synthetic chemical 5-fluorooroticacid (5-FOA) in to the cytotoxic fluorode oxyuridine (Jund and Lacroute 1970, Krooth et al. 1979). Hence the growth of strains with functional uracil pathways on media containing 5-FOA selects for natural mutants with disruptions in pyrE or pyrF (Krooth et al. 1979, Worsham and Goldman 1988).

3.1 Biocatalysts-Extremozymes Isolated from Extremophiles Extremozymes are a source of novel enzymes due to their stable nature and potential to exist under extreme conditions. In an exacting manner, thermophilic enzymes have a huge ability for biotechnological applications because of their elevated resistance under extreme temperature, chemicals, organic solvents and pH. Extremozymes are economically potential applications in various fields which include agriculture, food beverages, pharmaceutical, detergent, textile, leather, pulp and paper, bioleaching and biomining industries. The expansion of new industrial processes based on extremozymes and the increasing demand of biotech industries for novel biocatalysts are of great interest for extremophile research (Egorova and Antranikian 2005, Ferrer et al. 2007, Kelly Dumorné et al. 2017). By definition, biofuels are the fuel products obtained from biomass (including sugar cane, corn, beets, wheat, sorghum, rapeseed, sunflower, soybean, palm, coconut and jatropha) as well as the biodegradable component of industrial and municipal wastes (Dufey 2006). Although chemical and thermo-chemical processes are current technologies for biofuel production (Luque et al. 2008), the biological conversion of biomass to biofuel by microorganisms is more cost-effective and has gained great momentum over the last several years. Furthermore, extremophiles’ potential application and their robust enzymes in this process have recently been explored (Desire Barnard et al. 2010). Researchers are constantly determined to advance the various features of such existing technologies in view of the number of substrates available for biofuel production jointly with the limitations expressed in present manufacture customs. Numerous studies have accounted the application of enzymes obtained from microorganisms, mainly those from extremophiles (Desire Barnard et al. 2010).

3.2 Functions of Microalgae in Biotechnology Certain microalgal strains that have reached the stage of being a business-related traded product for example, Dunaliella, a green unicellular micro-algae isolated from high salinity water bodies with NaCl concentrations exceeding 3 M (Borowitzka and Huisman 1993). Spirulina, a filamentous cyanobacterium that blooms in alkaline lakes with high pH in the range of 9–11 (Silli et al. 2012). Dunaliella is used as a natural source of β-Carotene, while Spirulina has a market as a food and feed additive in human and animal nutrition. A key factor in the two species’ commercial success is their ability to grow under specific extreme conditions that help reduce the contamination by other algal species (Avron and Ben-Amotz 1992). Under mass culturing conditions, these species are reported to grow at 10–60 g/m2/day1.

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In view of this, extremophilic microalgae could offer the following benefits in biotechnological applications. • Capability to grow under local climatic conditions and exclude potential contaminants • High-value products from extremophilic micro-algae • Sources of genes that yield products of interest

4. Advantages of Microalgal Technology It includes the following advantages along with economic and ecologic assets:Ø Wastewater Remediation, biotreatment process of liquid wastes, zero waste discharge. Ø Bioenergy: Biofuel, Biodiesel, Bioethanol, Biochar, Biorefinery, Microbial fuel cells and Bioelectricity production. Ø High value-added products (biomass as food and feed for supplements, cosmetics and pharmaceuticals, etc.). Ø CO2 sequestration and Reduced greenhouse gas emissions, reduce global warming.

4.1 Algenol Biofuels It uses crossbreed blue-green algae and specially designed photobioreactors as a maintainable, correct platform for making its products from carbon dioxide, sunlight and saltwater. These technologies knock biodiesel industries to refine algal oil from its carbon capture and biofuels production facility next to a power megawatt (MW) coal-fired power station. Judd (2016) stated that about 35–86% price decline is on account of combined harnessing of CO2 and nutrients from waste sources. This evaluates with 12–27% for preventing fertilizers obtained through utilizing wastewater nutrient source or reprocessing the liquor from the removed algal biomass waste and 19–39% for CO2 fixation from a chimney gas feed.

4.2. Algal Biomass Exploitation for Resource Recovery and Limitations for Large Scale Application Ø Ø Ø Ø Ø Ø

Algae as carbon neutral energy Industrial applications of microalgal bio-products Sustainability and environmental challenges Zero-waste algal biorefinery for bioenergy and biochar The cost-benefit ratio of algal biorefinery for bioenergy and biochar Possible solutions and future directions

4.3 Bio-products Production from Wastewater Treatment by Microalgae Technology Ø Ø Ø Ø Ø Ø Ø

Biogas (methane and CO2, Biomethane, Biohydrogen) Biodiesel, Biocrude Acetone, Butanol, Ethanol Feed (protein for aquaculture and agriculture) Phycocyanin products (pigments, antioxidants) Biofertilizer Bioplastics

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5. Value-Added Products from Wastes using Extremophiles 5.1 Microalgal Bioactive Compounds Production and Applications in Pharmaceutical, Cosmetics, Food Industries Extremozymes are extremely tolerant to extreme conditions owing to their immense strength, they create innovative prospects for biocatalysis and biotransformation processes. The proteins from these organisms, including thermophilic proteins, piezophilic proteins, acidophilic proteins and halophilic proteins, are important for different biotechnological applications. Enzymes such as proteases, amylases, lipases, chitinases, pectinases, isomerases, esterases, dehydrogenases, pullulanase, xylanases and cellulases are resourceful applications in various activities including agricultural, chemical, biomedical and biotechnological processes. The diversity and outstanding properties of extremozymes including reproducibility, high performance and fiscal viability, include increased biotechnological application for different industrial processes (Gurung et al. 2013). The cellulolytic enzymes recognized the immense biotechnological potential in industries associated with food which include agriculture, brewing and wine, biomass refining, paper and pulp, textile and laundry, while cellulases, protease and lipase are used in the detergents industry and are capable of altering cellulose to increase the color intensity, feel and dirt elimination from cottonblend clothes (Singh et al. 2007, Staley and Konopka 1985, Young 1997). Moreover, marine algae are measured as valuable sources of structurally diverse bioactive compounds such as hydrocolloids alginate, agar and carrageenan. And some of these marine algae are also rich in Sulfated Polysaccharides (SPs) such as carrageenans in redalgae (Rhodophyta), fucoidans in brown algae (Phaeophyta) and ulvans in green algae (Chlorophyta) (Ngo and Kim 2013). These SPs demonstrate numerous health beneficial nutraceutical effects such as antioxidant, antiallergic, anti-human immuno deficiency virus, anticancer and anticoagulant activities (Borowitzka 2013, Lin 2013, Xin Zhang et al. 2014). 5.1.1 Microalgal Probiotics in Nutritional and Therapeutics benefits Probiotics are live microbes that provide health benefits to their host organisms by maintaining or improving the beneficial microbial balance to the intestinal medium (Fuller 1989, Gismondo et al. 1999, Holzapfel and Schillinger 2002, Shah 2001, Bhowmik et al. 2009, Beheshtipour et al. 2013, Patel et al. 2019). The health benefits of probiotics when added with selected food products include, antimicrobial, antiviral activity, improve lactose metabolism, reduce serum cholesterol level and blood pressure, improve mineral absorption, stabilization of the gut, urogenital infections, atopic diseases, immune system stimulation, anti-mutagenic, anti-carcinogenic properties, antidiarrheal, anti-constipation properties, improve inflammatory of bowel disease and suppression of Helicobacter pylori infection (Nakasawa and Hosono 1992, Nighswonger et al. 1996, Kailasapathy and Rybka 1997, Riordan and Fitzgerald 1998, Salminen and Wright 1998, Sanders 2012, Beheshtipour et al. 2013). In the food-dairy industry, the viability of probiotic bacteria during the production and storage of fermented milks is the most important and the addition of microalgae into fermented milk products in order to augment the sustainability of probiotics. Spirulina and Chlorella are the most significant and widely renowned microalgae used for fermented milks (Beheshtipour et al. 2013). Spirulina species, a photoautotrophic blue green microalga, is widely recognized as a food and dietary supplement owing to its excellent nutritive value which act as a prebiotic obtained from dried biomass of the blue green algae (cyanobacterium), Arthrospira platensis and as a rich source of proteins, vitamins, essential amino acids and phytochemicals. Spirulina acts as a noble dietary nutritional supplement and the three groups of probiotic combinations, Lactic Acid Bacteria (LAB), Bacillus strains and their mixture, are commonly used for commercial applications in the food industry (Bhowmik et al. 2009, Beheshtipour et al. 2013, Patel et al. 2019). It contains about

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63% protein, 18% carbohydrate and 4% fats. Spirulina is also a rich source of carotenoids as it contains (on dry w/w basis) about 0.5% total carotenoids including about 0.2% b-carotene, the pro-vitamin A (Gershwin and Belay 2007, Patel et al. 2019). The Spirulina, combination with yogurt benefits and enhanced carotenoid/b-carotene content to combat diarrheal and vitamin A deficiency. The Spirulina biomass on microbiological viability and growth of lactic acid and probiotic bacteria in fermented milk and yogurt with beneficial effects has been reported in several studies (De Caire et al. 2000, Varga et al. 2002, Beheshtipour et al. 2012). The studied report suggested that the addition of dry biomass of S. platensis at various concentrations of 1 mg, 5 mg, 10 mg/ml promoted growth of Lactobacillus acidophilus up to 171.67 and 185.84%, respectively at pH 6.2, and there was simultaneous antibacterial activity of S. platensis against three grams negative and three grams positive bacteria (Bhowmik et al. 2009). The nutritional and therapeutic benefits of microalgae are as follows, I. Nutritional benefits • Antioxidants, fat-soluble (carotenoids) and water-soluble • Vitamins (B2, B6, B8, B12, A, E, K) • Minerals such as Fe and Ca • High level of valuable amino acids and proteins • Carbohydrates • Fibers • Saturated and unsaturated fatty acids II. • • • • • • • • • •

Therapeutics benefits Decrease in blood cholesterol Reducing blood sugar and controlling diabetic patients Reducing hyperlipidemia Reducing heart failure Reducing anemia Reducing hypertension Anti-tumoral effects, Anti-viral effects, Anti-allergic effects Increase immune level Stress-controlling effects Protecting effect against chemical materials

5.2 Petroleum, Agriculture and Environmental Applications Enzymes from extremophilic microorganisms give a diverse biotechnological view for biocatalysis and biotransformations, owing to their permanence at high and low temperatures, range of pH, ionic strengths and salinities and also their capability of usefulness in organic solvents that would denature the majority of other enzymes (Adrio and Demain 2014, Karmakar and Ray 2011). The enzymes are used in many commercial products and industrial processes (Adrio and Demain 2014). More than 3,000 enzymes are identified, and nearly 65% are used in the detergent, textile, pulp, paper and starch industries and 25% are used for food processing (Birgisson et al. 2003). The enzyme xylanases have accessible huge applications in biotechnology, the industry in the bioleaching of paper and pulp, thus lesser environmental pollution by halogens (Mohammed and Pramod 2009, Yumoto 2002). Extremozymes such as proteases, lipases, cellulases and amylases are commercial enzymes that have been used in industry, especially in detergents (Chang et al. 2004, Das and Singh 2004, Hashim et al. 2004, Von Solingen et al. 2001).

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5.2.1 Extremophiles used in the Production of Biofuels Considering the number of substrates available for biofuel production together with the limitations faced in current production practices, researchers are continually driven to improve the various aspects of such existing technologies. Countless studies have reported the application of enzymes derived from microorganisms, particularly those from extremophiles, each specific with regards to its intended purpose biofuels (Desire Barnard et al. 2010). Given the number of substrates available for biofuel production jointly with the limitations expressed in present manufacture customs, researchers are repeatedly determined to progress the various features of such existing technologies. Numerous studies have accounted for the application of enzymes obtained from microorganisms, mainly those from extremophiles (Desire Barnard et al. 2010). There are several advantages of using extremophiles in industrial applications, particularly in the production of biofuels. Extremophiles are vigorous organisms producing stable enzymes. They are frequently able to withstand and tolerate changes in environmental conditions such as pH and temperature. When considering the use of extremophiles in biofuel production, it has become obvious that the majority are of thermophilic source. Since thermophiles have a notable ability to tolerate fluctuations in pH, temperature and environmental change (Gerday and Glansdorff 2007), an attribute which offers a clear advantage in the development of a commercially viable process (Dien et al. 2003, Zaldivar et al. 2001). Thermophiles readily ferment pentose and/or hexose sugars from biomass and, in some cases, even structurally complex carbohydrates, a quality which is particularly important for the production of second-generation biofuels (Zaldivar et al. 2001, Sommer et al. 2004). Besides, thermophilic industrial fermentations are less prone to microbial contamination and require lower energy inputs due to the reduced cooling steps needed between the fermentation steps. Despite the dominance of thermophiles in biofuels, other extremophile groups have also been applied in this field, including methanogens (typically thermophilic, anaerobic archaea) and psychrophiles. Methanogens contribute a vital role in biogas production, whereas psychrophiles are being exploited for their cold-adapted lipases for use in biodiesel. The application of these extremophilic organisms and their respective enzymes in the production of biofuels, mainly for bioethanol, and to a lesser degree in the production of other biofuels (Desire Barnard et al. 2010). Geobacillus are thermophilic bacilli with high catabolic flexibility and possible metabolic engineering (Taylor et al. 2008, Liao et al. 1986). A few specific species can ferment sugars such as D-glucose, D-xylose and L-arabinose at temperatures between 55°C and 70°C to produce a mixture of lactate, formate, acetate and ethanol from glucose (San Martin et al. 1992). More complex carbohydrates that include xylan are also degraded by specific Geobacillus strains due to the presence of xylanase (Wu et al. 2006). Geobacillus stearothermophilus can produce ethanol at 70°C at yields comparable to those of S. cerevisiae (Hartley and Payton 1983). A Geobacillus thermoglucosidasius strain has been isolated which can tolerate ethanol as high as 10% (v/v), although without growth (Fong et al. 2006). For these reasons, there is an immense deal of interest in these organisms for industrial bioethanol production. Saccharomyces cerevisiae and Zymomonas mobilis produce ethanol through the pyruvate decarboxylase (pdc) gene and the alcohol dehydrogenase (adh) gene (Dawes et al. 1966, Witt and Heilmeyer 1966). These two enzymes are sufficient to convert intracellular pools of pyruvate and NADH to ethanol. The acetaldehyde generated by the pyruvate decarboxylase from pyruvate is then converted to ethanol by alcohol dehydrogenase (Desire Bernard et al. 2010). Thermoanaerobacter species are thermophilic anaerobes which are extremely similar to thermophilic clostridia. Some were originally classified as Clostridium species (Collins et al. 1994) and the main products from Thermoanaerobacter fermentations are lactic acid and ethanol (Lamed and Zeikus, 1980) through lactate dehydrogenase and alcohol dehydrogenase activities (Lamed and Zeikus 1980). Thermoanaerobacter ethanolicus can ferment both D-glucose and D-xylose (Lacis and Lawford 1991) to form ethanol, though their ethanol tolerance is low (Burdette et al. 2002). The T. ethanolicus strain was adapted to tolerate up to only 4% (v/w) of ethanol (Lovitt et al. 1988).

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Ethanol tolerance in T. ethanolicus seems to be linked to the function of alcohol dehydrogenase because T. ethanolicus mutant gene was tolerant to 8% ETOH (ethanol) (v/v) (Burdette et al. 2002). Other species that have been evaluated for ethanol production are T. thermohydrosulphuricus and T. brockii (Lamed and Zeikus 1980, Mori and Inaba 1990, Desire Bernard et al. 2010). Thermoanaerobacterium is a hemicellulolytic thermophilic anaerobe (Garrity et al. 2005). It is capable of utilizing pentose sugars such as xylose to produce ethanol, as well as organic acids (acetic acid is formed by pyruvate: ferredoxin oxidoreductase (POR), phosphate acetyltransferase (Pta) and acetate kinase (Ack), while lactic acid is formed by L-lactate dehydrogenase (Ldh) (Shaw et al. 2008). To increase ethanol yields, metabolic engineering of end product metabolism has been carried out to generate a single knockout mutant for lactate dehydrogenase in Thermoanaerobacterium saccharolyticum (Desai et al. 2008). This mutant had reduced levels of lactate production and a four-fold increase in ethanol yields. Thermophilic clostridia are fermentative anaerobes with an optimal growth between 60 and 65°C (Lamed and Zeikus 1980). They are capable of degrading lignin-containing materials, such as lignocellulosic waste, because of the existence of multiple cellulases and hemicellulases frequently contained within the cellulosome (Demain et al. 2005). The cellulosome is a multienzyme complex to be found on the outside of the cell membrane and is involved in the enzymatic degradation of cellulosic substances, including crystalline cellulose (Demain et al. 2005). The enzymes in this complex include endo-β-glucanases, exoglucanases, β-glucosidases, cellodextrin phosphorylases, cellobiose phosphorylases, xylanases, lichenases, laminarinases, pectin lyases, polygalacturonate hydrolases, pectin methylesterase, β-xylosidases, β−galacosidases and β−mannosidases (Demain 2009, Demain et al. 2005). The cellulosome of Clostridium thermocellum allows for cellulose degradation to cellobiose and cellodextrins, and hemicellulose to xylose, xylobiose and other pentose sugars. Cellobiose and cellodextrins are taken into the cell, where C. thermocellum is able to ferment them to ethanol, acetate, lactate, H2 and CO2 (Lamed and Zeikus 1980). Clostridium thermocellum, is a superior candidate for ethanol fermentation from cellulosic biomass. Galdieria sulphuraria, also known as Cyanidium caldarium, is one of the most interesting microalgae with extremophilic growth properties. The strains of this Rhodophyta (red algae) species are capable of growing mixotrophically and heterotrophically on 27 different sugars and sugar alcohols (Gross and Schnarrenberger 1995, Schmidt et al. 2005). Galdieria sulphuraria is able to grow not only in neutral environments but also in extremely acidic environments, down to pH 1.8 (Merola et al. 1981). The strain of G. sulphuraria is capable of acidifying its environment by an active proton efflux, thus reducing the costs of pH control and the risk of contamination (Enami and Kura-Hotta 1984, Delanka-Pedige et al. 2019, Wollmann et al. 2019). Besides its acidophilic nature, G. sulphuraria shows thermophilic growth activities up to 56ºC (Selvaratnam et al. 2014). G. sulphuraria is improved by elevated levels of the phycobili protein phycocyanin, which is progressively being accepted as a natural colorant/nutraceutical in the food industry (FernandezRojas et al. 2014, Greque et al. 2018), cosmetics industry (Couteau and Coiffard 2018), and as a fluorescence marker in molecular biology (Pagels et al. 2019). The metabolic flexibility coupled with the ability to produce value added phycocyanin, makes G. sulphuraria a very promising candidate for treating high chemical oxygen demand-loaded, acidic or high-temperature wastewater (Wan et al. 2013). The potential of growing G. sulphuraria 074G, heterotrophically on hydrolysates of food waste from restaurants and bakeries (Sloth et al. 2017). In a first field study, Lammers and co-workers (Henkanatte-Gedera et al. 2017) reported that G. sulphuraria was able to grow well in primary-settled wastewater while significantly reducing levels of organic carbon (46–72%), ammonium (NH4-N) (63–89%), and phosphate (PO4) (71–95%). Additional promising acidophilic microalgal strains can be found within the Chlorophyta (green algae). Chlamydomonas acidophila have been isolated from an acidic river in a mining area, with pH values ranging between 1.7 and 3.1 (Cuaresma et al. 2011). It has been shown that C. acidophila can grow mixotrophically without CO2 by using different carbon sources, especially glucose, glycerol and starch, at pH 2.5 (Cuaresma

Value-Added Products and Biofuels from Extremophilic Microalgae Biomass 199

et al. 2011), and its capacity to remove NH4 (Escudero et al. 2014). The added value of C. acidophila biomass from waste sources is its ability to accumulate high concentrations of antioxidants such as the carotenoid lutein (Garbayo et al. 2008, Wollmann et al. 2019). Chlorella protothecoides var. acidicola has been isolated from acidic (pH 2.5–2.6) mine water and has shown good heterotrophic growth on glycolic acid (Nancucheo et al. 2012), which is part of the wastewater load of fruit and vegetable processing industries. Chlorella sorokiniana, a well-studied thermophilic green microalgae has revealed high photoautotrophic growth rates up to 43ºC (Varshney et al. 2018). The efficient P and N removal rates in heterotrophically grown C. sorokiniana cultures, which is an essential precondition for many WWT processes. The superior removal performance for heterotrophic C. sorokiniana cultures, compared with photo- and mixotrophic cultures (Kim et al. 2013 a,b). Cells of C. sorokiniana can accumulate high levels of valuable bioproducts, e.g., lutein (Chen et al. 2018), fatty acids (Chen et al. 1991, Leon-Vaz et al. 2019) and proteins (Blackburn and Volkman 2012), making the sustainably produced biomass a good source for animal feed or biofuel production. The co-immobilization with the microalgae growth promoting bacterium Azospirillum brasilense considerably enhanced the P-removal efficiency of C. sorokiniana (Hernandez et al. 2006, Hernandez et al. 2013). Another challenge is the energy-efficient treatment of low-temperature wastewater. Psychrophilic species such as Koliella antarctica have temperature optima below10ºC (Andreoli et al. 1998), making them an interesting potential biological system for treating wastewater from fresh fruit processing industries. Koliella antarctica has also been shown to produce high levels of EPA, DHA, astaxanthin and lutein (Fogliano et al. 2010, Wollmann et al. 2019). 5.2.2 Biodiesel Production and Pollution Control by Microalgae Cultivation Microalgae are being used as the superior raw material for biodiesel production and for all the stages of microalgae biodiesel process chain. Policy measures promote biofuel derived from microalgae and its manufacture is growing all over the world (Xin Zhang et al. 2014). The capital and operating costs of algae-based biodiesel are still higher than petrol diesel, which makes it difficult to develop microalgae biodiesel technology on a commercial scale (Singh and Olsen 2011, Singh et al. 2011a). Recently, some research has suggested that the dual use of microalgae cultivation for environmental pollution control (especially wastewater treatment) coupled with biofuel generation is an attractive option in terms of reducing the energy cost, CO2 emissions, nutrient and fresh water resource costs (Sun et al. 2013b). The earlier study expanded an effective heteroautotrophic mode for enhanced wastewater nutrient removal, wastewater recycling and superior algal lipid accumulation with Auxeno chlorella protothecoides UMN280. It was noted that the maximal biomass concentration and lipids content reached 1.16 g/L and 33.22% dry weight (Zhou et al. 2012). Besides biodiesel, different high-value chemical compounds such as pigments, antioxidants, b-carotenes, polysaccharides, vitamins and biomass can be extracted from microalgae, and they are largely used as bulk commodities in diverse industrial sectors (e.g., pharmaceuticals, cosmetics, nutraceuticals, functional foods) (Mata et al. 2010). The b-carotene, a vitamin-A precursor in health food was the first high-value product commercially manufactured from Dunaliella bardawil. The biomass of microalgae as a sun-dried or spray-dried powder or in a compressed form as pastilles, which are available in the human health food market. It is the predominant product in microalgal biotechnology. The final product of biomass production was used both in aquaculture and as well as in animal husbandry as animal nutrition feed stock (Pulz and Gross 2004, Xin Zhang et al. 2014). Biodiesel production is a grown technology used in compression-ignition (diesel) engines. The plant raw materials’ expenditure averages 70% of the total production cost (Behzad and Farid 2007), which engages processing of vegetable oils by transesterification into monoalkyl esters of the plant fatty acids. If biodiesel is to turn into an economically feasible resource and for further efficient novel sources of oil, such as microalgae as well as from extremophilic organisms, need to be investigated. Photosynthetic microalgae biodiesel microalgae production are eukaryotic

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photosynthetic microorganisms that convert sunlight, water and CO2 to algal biomass. Under optimal growth conditions, these organisms produce fatty acids for esterification in to glycerolbased membrane lipids which can amount to 5–20% of their dry cell weight (Hu et al. 2008). Under stressful environmental conditions some microalgae, such as Botryococcus braunii, can produce very long-chain hydrocarbons (C23 to C40), similar to those in petroleum, which can exceed 80% of their dry cell weight (Banerjee et al. 2002, Metzger and Largeau 2005). There are several advantages for using microalgae for the production of lipids for conversion into biodiesel (Desire Bernard et al. 2010). Ø they accumulate lipids and oils in large amounts; Ø they grow rapidly, often doubling biomass within 24 hours (Chisti 2008); Ø they are able to grow in saline waters and wastewaters without the need for fresh water (Rhodes 2009); Ø photobioreactors for the growth of microalgae can be located in arid or semi-arid areas that are not suitable for agriculture; Ø the nutrients needed for growth can be provided from waste sources such as agricultural runoff, industrial or municipal wastewater and animal feeds (Hu et al. 2008); Ø they remove CO2 emitted from burning fossil fuels; Ø unlike crops, their growth is not seasonal; Ø a large number of microalgae produce valuable by-products, such as biopolymers, pigments and polysaccharides, which can be harvested; Ø after lipid extraction, the algal biomass can be anaerobically converted into biogas, which can provide more energy than the energy produced from the lipids (Lantz et al. 2006, Sialve et al. 2009). There are a large number of extremophilic microalgae, such as Cyanidium caldarium and Galdieria sulphuraria, which tolerate both high temperatures and low pH, having high growth rates at 50ºC and pH 1 (Luca et al. 1981, Pulz and Gross 2004). The advantage of using extremophilic microalgae would be to minimize contamination within the photobioreactors, which tends to be problematic in outdoor cultures. Lipid content in certain microalgae, such as Ochromonas danica and Nannochloropsis salina, has been shown to grow with increasing temperature (Hu et al. 2008). Biobutanol is greater potential alternative fuel, the extremophiles are used in the production of butanol, most microorganisms are unable to grow at butanol concentrations above 2% (Knoshaug and Zhang 2009). However, certain organisms, such as certain species of Bacillus, can tolerate butanol concentrations as high as 2.5–7% (Sardessai and Bhosle 2002). Higher tolerance has been shown for organisms belonging to the Pseudomonas genus. Pseudomonas achieve high solvent tolerance by removing solvent using efflux pumps and physico-chemical changes of their membrane lipids (Segura et al. 2004, Ramos et al. 2002). The P. putida S12 has inherent moderate tolerance to butanol (de Carvalho et al. 2004), while other P. putida strains have been evolved to tolerate 6% w/v butanol (Rühl et al. 2009). This fact and the recent engineering of P. putida to produce butanol (Nielsen et al. 2009) has opened a new field of research to produce butanol from solvent-tolerant organisms. The UK-based company Green Biologics (www.greenbiologics.com) uses a mixture of thermophiles as well as thermostable enzymes for the production of butanol from waste biomass. The thermophilic fermentations are conducted with genetically modified microbial strains optimized to produce butanol which is commercially sold as in the name of Butafuel™. 5.2.3 Wastewater Treatment, Nutrient Removal by Microalgae Cultivation In the pilot-scale of microalgae culture, utilization of vast amounts of potable water is a serious problem, which would conflict with land crops and human activities. Large amounts of nitrogen and

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phosphorus can be recovered from wastewater, but their cost in pure chemical form is high and their sustainability is low (Lardon et al. 2009). As a result, the microalgae production using wastewater as the nutrient source is a potential method, which offers added environmental advantages. Microalgae production is effective in removing nitrogen, phosphorus and toxic metals from an extensive range of wastewaters, producing cleaner effluents with high concentrations of dissolved oxygen (Gomez et al. 2013). On the other hand, it cannot be ignored that there are many endogenous bacteria in real wastewater systems that affect the growth of microalgae. The use of C. vulgaris for nitrogen and phosphorus removal from municipal wastewater with the highest removal rates of 9.8(N) and 3.0(P) mg/L/day (Cabanelas et al. 2013). Other microalgae broadly used for nutrient removal from different wastewater streams are the Chlorella sp. (Gonzales et al. 1997, Cabanelas et al. 2013), Scenedesmus sp, (Martınez et al. 2000) and B. braunii (Álvarez-Díaz et al. 2013). Cai et al. (2013) reported that due to wastewater’s multifaceted characteristics the tests of growing algae in wastewater are typically at the laboratory scale. Pilot-scale algae cultivation continues to face several issues including contamination, in consistent wastewater components and unstable biomass production (Xin Zhang et al. 2014). 5.2.4 CO2 Biofixation from Flue Gases and NOx Removal by Microalgae Cultivation The concern of global warming is one of the key challenges and CO2 acts as a principal GHG effect to contribute to global warming. About 75% of the total anthropogenic CO2 emissions were derived from fossil fuel burning (Nakanishi et al. 2014). So far, several technologies have been used to obtain CO2, i.e., physical and chemical adsorption, cryogenic distillation and membranes separation (Abu-Khader 2006). The acquired CO2 is then transported and accumulates in geological formations. These procedures must only be considered as short-term solutions because they are energy consuming and the obtained CO2 needs to be disposed. Additionally, the CO2 biofixation by microalgae has drawn much attention as an environmentally friendly CO2 mitigation strategy. Carbon is a chief resource for successful microalgae production as it is the major element of microalgae (36–65% of the dry matter). The diffusion of CO2 from the atmosphere in to microalgae culture is inadequate to support the rapid growth of microalgae (growth velocity less than 5% of its prospective power) (Benemann 1993). Therefore, by using flue gases, the microalgae culture can reduce GHG emissions, while addressing the microalgae culture carbon supply and leading the production of biomass energy through photosynthesis. The flue gases contain numerous different compounds which include H2O, O2, N2, nitrogenoxides (NOx), sulfuroxides (SOx), unburned carbohydrates (CxHy), CO, heavymetals, halogenacids and PM. Several chemical compounds (SOx, heavy metals, etc.) have shown to be toxic to some microalgae (Lee et al. 2000), but well tolerated by others (Xin Zhang et al. 2014). Moreover, flue gases also contain different NOx species restricted by legislation and considered necessary to remove additional gas treatment steps such as chemical reduction and adsorption. NO is the main NOx species present, comprising about 90–95% (Fritz and Pitchon 1997). As nitrogen is one of most vital nutrients for algal production, it should be noted that NOx can serve as a nitrogen source for microalgae cultivation and can be metabolized by microalgae compared to the requirement of expensive catalysts or adsorbents (Cant and Liu 2000). Consequently, microalgae’s biological denox (bio-denox) method may be a commendable method of promotion for flue gas treatment to reduce NOx emissions and merits further studies (Xin Zhang et al. 2014).

6. Microalgae Based Wastewater Treatment Process The microalgae-based wastewater treatment (WWT) approaches include. • Conventional microalgae • Extremophile microalgae • Photobioreactor systems

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• Suspended WWT systems • Immobilized WWT systems It uses an enclosed modular high-yield algae growth manufacturing method/system to manufacture sustainable and renewable oil for fuel. The enclosed photo-reactor system provides cost, scale and yield compensation over the open pond method. This technology is designed to capture carbon dioxide waste (Carbon Capture and Storage or CCS) from power plants and manufacturing facilities that feeds into the algae growth system. In the global development stage, the industrial biotechnology companies make biofuels and high-value industrial chemicals cost-effective from abundant, renewable resources (Fig. 9.3). In this technology, the integrated process significantly improved the energy balance and economics of biofuel production and also the wastewater treatment plant (WWTP). They monitored the fiscal analysis and verified that higher biomass production along with technology development that was required to attain operational feasibility and profitability of the current microalgae-based bio-oil production. Carbon capture, storage and utilization by microalgal biomass and bio-products production from wastewater using microalgae culture technology are shown in Figs. 9.3 and 9.4 and Table 9.2. The microalgae play an important role in transforming elements in the aquatic environments and have gained more attention in recent years as they can biotransform (absorb and detoxify) the heavy metals. In this technology, the design and size of the process equipment were achieved to gain a realistic assessment and evaluation of possible manufacturing cost. Nugroho et al. (2018) confirmed that nutrient savings was achieved by wastewater and CO2 utilization to the polyculture or mixed culture of native microalgae. The process model provided a predictable CO2 sequestration of 82.77 to 140.58 tons ha−1year−1 with 63 to 107 tons ha−1year−1 of prospective biomass manufacture. The data proved that the integrated process significantly improved the energy balance and economics of biofuel production and the wastewater treatment plant (WWTP). They observed the fiscal analysis and verified that elevated biomass production and technology development were required to achieve operational feasibility and profitability of the current microalgae-based bio-oil production.

Fig. 9.3. Microalgal technology for bioproducts production from wastewater.

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Fig. 9.4. Flow chart showing microalgal technology at different stages using various systems.

7. Scale-up Bioprocess and Execution of Microalgae Technology Bioprocess engineering of microalgae are able to produce a diversity of bio-products. In sight of the enormous biodiversity of microalgae and the current advancements in genetic and metabolic engineering, these microalgae are the major agreed source for the innovative products production and applications. With the advancement of thorough culture and screening techniques, microalgal biotechnology can congregate the high demands of food, energy and pharmaceutical industries. The bioprocess engineering of microalgae technology, cultivators is a unique high surface area, biofilmbased approach to enhance light penetration, productivity, harvest density and gas transfer. Microalgae harvesting is one of the major constraints in microalgae biotechnology development at an industrial scale (Gonzalez and Ballesteros 2013 and Pragya et al. 2013) due to the considerable costs and energy concerned in the step. Ground-breaking progresses and breakthroughs in bioprospecting novel strains advance in culture strategies and process optimization are encouragingly us to be optimistic about the expectations of microalgal biorefinery. Heterotrophic procedures are a put into practice to rise above the long and costly autotrophic scale-up development process for large-scale microalgae production. Preliminary scale-up using heterotrophically grown microalgae culture technology up to a 5-L fermenter (heterotrophic route). This process was compared to the average scale-up process whose inoculum corresponded to microalgae cells cultivated photoautotrophically in flasks with the highest volume of 5 L (autotrophic route). Either inoculum was used to seed 1 m3 Fat Panel (FP) photobioreactors operated outdoors under photoautotrophic conditions. The culture volumes (liters) and the duration (days) of each scale-up step are indicated. Industrial scale-up using heterotrophically grown Chlorella vulgaris up to a 5000-L fermenter (heterotrophic route). This process facilitated the direct inoculants (seeding) of eight 100-m3 industrial photobioreactors, instead of the average scale-up process using photoautotrophically grown inocula (autotrophic route), declining scale-up time and the area of the production plant committed to the scale-up process (Figs. 9.4 and 9.5). The whole metabolic pathways for microalgal biomass to biofuel bio-hydrogen conversion are shown in (Figs. 9.6a, b and c) (Kit et al. 2017, US DOE 2010).

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Extremophiles: Wastewater and Algal Biorefinery Table 9.2. Wastewater treatment by microalgae as bioinoculant and their biomass.

Bioinoculant (Microalgae)

Type of Wastewater

Biomass (Dry Weight) (mg/L/day)

Nutrient Removal

References

Chlorella sp.

Dairy

59

NH4+-N = 96% P = 99% in 12 days

Woertz et al. 2009

Chlorella vulgaris

Textile

0.0019

77% (dye) 69.9% (COD)

El-Kassas and Mohamed 2014

Chlorella vulgaris Autotrophic

Textile

730

N = 9.8 mg/L/day P = 3 mg/L/day

Zhang et al. 2014

Chlorella vulgaris JSC-6

Swine wastewater

1960

COD = 60–70% NH4+-N = 40–90%

Wang et al. 2015

Chlorella vulgaris

Landfill leachate

0.24

P-70% N- < 50% CO2 biofixation rate of 0.46 g CO2/L/d

Chang et al. 2018

Chlorella vulgaris

Activated Sludge

98% (N removal)

Leong et al. 2018

Chlorella pyrenoidosa

Textile

Chloride (61%), nitrate (74.43%), phosphate (70.79%)

Brar et al. 2019

Chlorella pyrenoidosa with bacteria (Kluyvera sp.)

Municipal

350

NH3–N = 91%

Zhou et al. 2020

Botryococcus brauni

Municipal

345.6

N = 91%

Orpez et al. 2009

Botryococcus brauni

Swine wastewater

700

T-N = 48% NH4+-N = 98%

An et al. 2003

Chlamydomonas reinhardtii

Municipal

2000

N = 55.8 mg/L/day P = 17.4 mg/L/day

Kong et al. 2010

Dunaliella tertiolecta

Industrial carpet mill

T-N & P = 99%

Chinnasamy et al. 2010

Scenedesmus obliquus

Brewery wastewater

T-N-97% P-74%

Leonilde Marchão et al. 2018

Chlorella sorokiniana CY-1

Palm oil mill effluent (POME)

2.07% (TN), 47.09% (COD) and 30.77% (TP)

Wai YanCheah et al. 2018

40

28 0.2 g ash free dry weight/day 2.12 g/L

Regarding effectiveness, well-designed and appropriateness for the pre-treatment of microalgal biomass to release the oil/lipid, the energy demand, cost-effectiveness and the system design are the major factors at the industrial level. The hybrid or combination of more than one method or system for the pre-treatment of algal biomass is extra efficient in cell wall disruption via microwaves sonication is tracked by high-speed spinning bead mills treatment to free the oil (Barros et al. 2019). The formulation of the growth medium used in aerobic growth analysis is the result of optimizing earlier synthetic media used for the particular selected potential strain (e.g., Natronomonas pharaonis). The carbon sources were tested using different carbon sources, including all of the amino acids, acetate, glycerol, citrate cycle intermediates and mixtures of various concentrations with different combinations (Gonzalez et al. 2010, Xin Zhang et al. 2014).

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Fig. 9.5. Scale-up process of microalgae cultivation.

Fig. 9.6a. Algae biomass to fuel conversion possible pathways.

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Fig. 9.6b. Pathways for Algae biomass to fuel.

Fig. 9.6c. Metabolic pathways for microalgal biomass to biofuel bio-hydrogen.

7.1 Bioreactor, Biomass Harvesting and Centrifugation The bioreactors used were either open systems (e.g., ponds) or closed system of photobioreactors (PBRs), which are the two major kinds of microalgae culture systems. The shallow channel pond is one, in which the suspension is blended with a paddle wheel and is the most widely used among the current microalgae culturing devices, because it is comparatively simple and inexpensive to build and operate (Chaumont 1993, Doucha and Lívanský 2006). At present more than 90% of the world, microalgae biomass production are found in large raceway ponds. Although, the small biomass productivity at the field level was a fatal drawback, considering the competition of land for traditional crops when commercial developments of microalgae biofuel

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are encouraged (Chen et al. 2013). They are effectively bound by contamination (by another group of algae and bacteria). The extent of which depends on climatic conditions, which is especially not easy to retain an open algal culture system in the tropics throughout the rainy season (Xin Zhang et al. 2014). After cultivation, the biomass harvesting, the microalgae biomasses have to be separated from its growth medium and recovered for downstream processing. On the other hand, the algae grow in dilute suspension and the negative surface charge results in dispersed constant algal suspensions (Sanyano et al. 2013). Most of the oleaginous microalgae species are small size single-cell microorganism such as Scenedesmus sp. and Chlorella sp. (Phukan et al. 2011, Wang et al. 2013). Microalgae harvesting remains a major hurdle for industrial-scale processing, and it was estimated to account for 20–30% of the total biomass production cost (Molina Grima et al. 2003). Therefore, microalgae harvesting is an active area for research, promising to develop a suitable and cost-effective harvesting system (Xin Zhang et al. 2014). The harvesting methods include, (1) bulk harvesting (known as primary harvesting) to separate microalgae from suspension, such as sedimentation, flocculation and flotation; (2) thickening (known as secondary dewatering)—to concentrate the microalgae slurry after bulk harvesting, such as centrifugation and filtration (Grima et al. 2003, Lam and Lee 2012, Sharma et al. 2013). An optimizing harvesting technique is supposed to be independent of the cultured species, consume less energy and few chemicals, and do not spoil the valuable products in the extraction process (Chen et al. 2011a, Salim et al. 2011, Milledge and Heaven 2013, Ahmad et al. 2014a). The centrifugation process, where by solid–liquid separation is driven by a much superior force (gravity) to promote accelerated settling of microalgae cells and can be used for almost all microalgae types consistently and without trouble (Pires et al. 2012). Though, centrifugal recovery is only feasible if the targeted metabolite is a high-value product, as the process is extremely energy concentrated. In addition, using this technique at a large scale is difficult because of high power consumption, which increases production costs (Xin Zhang et al. 2014).

7.2 Filtration, Flocculation, Floatation Filtration is a physical separation process by filter (membrane), which is characterized by its efficiency, reliability and safety for the solid–liquid separation. The membrane filtration removes the debris and the microalgae cells from the culture medium totally releasing recycling water. Different membrane materials {example, polyvinyli dene fluoride (PVDF), polyvinylchloride (PVC), polysulfonemembrane (PS)} (DeBaerdemaeker et al. 2013, Sun et al. 2013a) and membrane pore size [microfiltration, ultrafiltration (UF)] were tested for algae harvesting and PVDF was the superior polymer, while UF showed better fouling resistance (De Baerdemaeker et al. 2013, Xin Zhang et al. 2014). Flocculation is normally performed before secondary dewatering processes to reduce the cost of harvesting microalgae. Flocculation can be attained by different methods and adding up of chemicals known as flocculants to counter the surface charge on the algae is widely applied. The inorganic coagulants are typically in the form of aluminum and ferric salts, which includes aluminum sulfate, ferricsulfate and ferricchloride (Grima et al. 2003). The inorganic flocculants need acidic or alkaline pH for optimal microalgae flocculation. In the process up to date, organo clay has proved as an ovel flocculant for fast and well-organized harvesting of microalgae. Flotation can capture particles with a diameter of less than 500 lm (lumen) by the collision between a bubble and a particle and the subsequent adhesion of the bubble and the particle (Chen et al. 2011a). Based on bubble sizes used in the flotation process, the applications can be split into Dissolved Air Flotation (DAF), dispersed flotation and electrolytic flotation. The flotation process where the microalgae float to the surface of the medium is prone to harvest in microalgae mass culture, and has been used for specific strains (Xin Zhang et al. 2014).

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7.3 Drying, Sedimentation The drying process is helpful to separate the solid biomass from the medium in liquid suspension. The percentage of water enclosed in algal paste after secondary dewatering is supposed to not exceed 50% before oil extraction (Kumar et al. 2010). And extensive drying of microalgae biomass is necessary for biofuels production as the existence of water interferes with the extraction or conversion of algal lipids to biodiesel. There are some general methods for drying microalgae after secondary dewatering which includes spray drying, drum drying, freeze-drying and solar drying (Richmond 2008).

7.4 Lipid Extraction and Transesterification The Lipid extraction process for biodiesel production, lipids and fatty acids has to be extracted from the microalgae biomass. The lipids’ extraction efficiency is directly associated with the overall process effectiveness in biodiesel production. Hence, the extraction process is very significant before the transesterification of the lipid takes place. It is essential to develop resourceful and inexpensive extraction processes to reach industrial biodiesel production at suitable costs. The extraction of microalgae lipids is generally performed using a chemical solvent such as hexane Soxhlet extraction (Soxhlet 1879) and mixed methanol–chloroform (2:1v/v; Bligh–Dyermethod) (Bligh and Dyer 1959). Hexane Soxhlet extraction is normally used to capture high-quality lipids such as triglycerides and fatty acids, which are simply esterified in to biodiesel (Demirbas 2008, Kanda et al. 2013). The Bligh–Dyer method is valuable for extracting oily substances because a broad range of oily components can be isolated from microalgae. These two processes are efficient in extracting microalgae lipids, but the extraction competence depends on microalgae strains. Chemical solvent has high selectivity and solubility toward lipids and hence, even inter lipids can be extracted through diffusion across microalgae cell wall (Xin Zhang et al. 2014). The Transesterification process, it is the most accessible technology reported for biodiesel production, and currently is the transesterification reaction in which triglycerides (lipids compounds) react with short-chain alcohol (e.g., methanol or ethanol) in the presence of the catalyst and the final reaction products are known as biodiesel Fatty Acid Methyl Esters (FAMEs) and glycerol (by-product) (Lam et al. 2010). The most commonly used catalysts for microalgae lipids transesterification are discussed next (Xin Zhang et al. 2014). The homogeneous alkaline and acid catalysts and the heterogeneous catalysts are the main types of catalysts used in biodiesel production (Xin Zhang et al. 2014). The homogeneous alkaline catalysts (e.g., KOH and NaOH) have been the most commonly used route for biodiesel production as it catalyzes the reaction at low temperatures and atmospheric pressure. In addition, high conversion yield can be achieved in a short time (minutes), thus being the most economical way to catalyze the transesterification reaction (Meher et al. 2006, Sharif Hossain et al. 2008). On the other hand, the formation of soaps in the presence of the FFAs (> 1%) and water (> 0.06%) will lead to lower biodiesel yield and increase the difficulty to separate biodiesel from the co-product (Hidalgo et al. 2013). Hence, homogeneous acid catalysts have been proposed to overcome the limitations of high FFAs content as the catalysts are not sensitive toward FFAs level in oil. The commonly used acid catalysts in the transesterification process are H2SO4 and HCl (Xin Zhang et al. 2014). Based on present available technologies, the combination of both acid and base catalysts (two-step reaction) has been proposed to produce biodiesel from lipids with a high FFAs content (Canakci 2007, Francisco et al. 2010). Microalgae lipids are first subjected to acid pre-treatment so that its FFAs level is condensed to less than 1% weight, followed by the second transesterification step performed by using an alkaline catalyst (Xin Zhang et al. 2014). The heterogeneous catalysis (e.g., CaO, MgO, a mixed form of CaO–Al2O3, etc.) has also been developed for biodiesel production. Heterogeneous solid catalysts have numerous advantages as it is non-corrosive and it can be recycled. The catalysts propose facile product separation through filtration and consequently minimize product contamination and the number of the water washing cycle. Though, the use of heterogeneous catalysts can result in low biodiesel

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conversion yields in comparison with homogeneous catalysts, which may be due to the fact that homogeneous catalysts can dissolve in the bulk liquid, while heterogeneous catalysts do not dissolve in the bulk liquid producing mass transfer limitations (Liu et al. 2007, Lam et al. 2010, Xin Zhang et al. 2014).

8. Conclusion and Future Perspectives Microalgal technology advancement leads to an increased efficiency to reduce pollutants and produce energy and materials consumption. This proves that technologies at large scale and in different settings are needed. Microalgae are renewable, sustainable and economical sources for the production of biofuels, bioactive products and food supplements. It has extensive applications in the bioenergy, nutraceutical and pharmaceutical industries. It has a synergistic effect of both biomass and protein feed stock production and biofuel besides with pollutant removal (also helpful in carbon dioxide and carbon reduction) from wastewater through cost-effective and eco-friendly technology.

Acknowledgement The authors wish to extend their gratitude to editor and the publishers of the book.

Conflict of Interest The authors declare that they have no conflict of interest.

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10 Microbial Corrosion and It’s Current Mitigation Strategies Reena Sachan,1,* Ajay Kumar Singh,1 Madan Sonkar 2 and Shiv Bharadwaj 3

1. Introduction Corrosion is defined as oxidation of metals caused by electrochemical reactions occurring in its vicinity (Davis 2000). The term corrosion originated from a Latin word ‘corrodere’ which means gnaw of pieces. It is a natural process which accelerates the deterioration of metallic/non-metallic materials. The corrosion cycle involves the destruction of metal due to the chemical reactions of material with environmental factors like chemicals, water, wind, etc. Consequently, all metals exist in natural form as their respective different compound forms, commonly known as ‘ores.’ The whole process moves towards the lower energy state (Fig. 10.1) (Park and Fray 2009). The first report related to corrosion and its economic importance was published in 1948. After that, during the 1970s many studies reported the economic losses due to corrosion. In 2002, the U.S. Federal Highway Administration (FHWA) reported a survey of economic losses caused by corrosion in various industries (Koch et al. 2002). This study revealed that in the USA, the total annual cost of corrosion was approximately USD 276 billion, which is equivalent to 3.1% of GDP (Gross Domestic Product). According to the National Association of Corrosion Engineering (NACE) impact study, in the USA, the global cost of corrosion was approximately USD 2.5 trillion, equivalent to 3.4% of GDP (2013) (Koch et al. 2016). A published report by NACE, in India, estimated USD 26.1 billion as the direct cost caused by corrosion and the indirect cost of corrosion was USD 39.8 billion, which was approximately 3.6% of GDP (Bhaskaran et al. 2014). The direct cost of corrosion includes the cost of replacing corroded structures machinery or their components such as condenser tubes, pipelines, etc.; the additional cost of corrosion resistant alloys instead of mild steel, etc. While the indirect cost of corrosion includes contamination of products, loss of efficiency, unit shutdown, loss of products, etc., the assessment of the indirect cost of corrosion is more complicated than the direct cost of corrosion.

Department of Applied Science and Engineering, Indian Institute of Technology, Roorkee. Department of Paper and pulp, Indian Institute of Technology, Roorkee. 3 Department of Biotechnology, College of life and applied sciences, Yeungnam University, Republic of Korea. * Corresponding author: [email protected], [email protected] 1 2

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Fig. 10.1. Corrosion cycle of metal in nature.

1.1 Microbial Influenced Corrosion (MIC) Microorganisms can influence the corrosion of metals and non-metals directly or indirectly, depending on the microbial strains, materials and solution of electrolytes (Fig. 10.2). Microbial influenced corrosion is defined as deterioration of metals or non-metals as a result of the metabolic activities of different microbes such as algae, fungi, archaebacteria, bacteria, etc. (Little and Lee 2014). Sometimes, MIC is also termed as biocorrosion, biological corrosion or microbial corrosion. MIC is not a separate or new type of corrosion, rather a different agent that causes metal corrosion. In this regard, various bacterial species have been logged as the main perpetrators of MIC. Hence, MIC is a major concern for a wide range of industries because of associated economic losses.

Fig. 10.2. Effects of microbes, metals and media in MIC.

Microbial Corrosion and It’s Current Mitigation Strategies 221

1.1.1 Economic Impacts of MIC Bacteria are ubiquitous and responsible for corrosion in various industries such as oil and gas industries (Taleb-Berrouane et al. 2018, Xu and Gu 2015), process plants (Festy et al. 2001), paper and pulp industries (Baird et al. 2016), fire sprinkler system (Clarke and Aguilera 2007), water distribution system (Zhu et al. 2014), pipelines (Li et al. 2000, Muthukumar et al. 2003, Usher et al. 2014), etc. Annual MIC related industrial losses in Australia were estimated upto AUD 6b (Javaherdashti 1999). In Canada, heat exchanger tubing of nuclear power generating plants has been estimated to cost the corporation US$300,000 per unit/day in replacement energy costs (Brennenstuhl and Doherty 1990). In oil and gas industries, it was estimated that 34% of corrosion experienced due to MIC (Jack et al. 1992). According to Booth’s report (Booth 1964), in the UK, 50% of corrosion failures in pipeline industries were related to microbes while Flamming suggested that 20% of all corrosion damages were due to microbes (Flemming 1996). In 1977, an early reported incident of refrigerated propane tank explosion was related to MIC. This incident was suspected to be weld failure by Sulfate Reducing Bacteria (SRB) and the estimated loss was US$179 million (Chang and Lin 2006). Another incident of natural gas pipeline leakage and explosion due to MIC was reported in Carlsbad, New Mexico, United States (Abdullah et al. 2014). In 2006, MIC contributed by SRB was suspected to cause leakages of Alaska oil pipelines (Jacobson 2007). Recently, in a catastrophic MIC failure attributed by methanogens bacteria, about 100,000 tons methane was leaked from storage field in Aliso Canyon, CA USA and caused a huge impact on the climate (Conley et al. 2016). 1.1.2 Bacteria Involved in MIC Various microorganisms such as algae, fungi, bacteria, etc., are involved in the deterioration of engineering materials (Geweely 2011, Javaherdashti et al. 2009, Usher et al. 2014). Although algae and fungi are not well studied in MIC, some reports related to corrosion due to these microbes were published. Liu et al., reported that the corrosion of carbon steel was four times higher in the presence of Chlorella vulgaris (Liu et al. 2015). Moreover, a high rate of corrosion was recorded during the daytime compared to night. Fungi were also held responsible for the deterioration of different metals such as carbon steel, copper, aluminum and stainless steel (Cojocaru et al. 2016, Lugauskas et al. 2016). Fungi can induce corrosion of metals by producing organic acids on the fungi-metal interface, consume oxygen and make a suitable environment for anaerobic bacteria by adsorption of metal on their cell wall and create a galvanic cell on metals surface (Das et al. 2009, Little et al. 2001, Usher et al. 2014). Hence, bacteria has been secluded as the major contributors in metal corrosion; the other reported bacteria for MIC are discussed below. (a) Sulfate-Reducing Bacteria (SRB) SRB have been extensively investigated as major MIC causative agent for decades because of presence of sulfate in various systems like brackish water, oil and gas industries, seawater, etc. (Jia et al. 2017, Li et al. 2016). SRB are non-pathogenic and anaerobic bacteria that utilize sulfate as a terminal electron acceptor and reduce it to sulfide ions (Keller and Wall 2011, Ollivier et al. 2007). Many SRB and archaea can reduce other inorganic sulfur compounds such as elemental sulfur and utilize them as terminal electron acceptors; these are a diverse group that consists of autotrophic and heterotrophic microorganisms. SRB can also act as a catalyst in the reduction reaction of sulfate compounds to H2S, and hence, lead to the corrosion by production of some enzymes (Beaton 2007, Little et al. 1998). Li et al., reported corrosion rate more than 20% in the presence of SRB in the soil by comparison to the control (Li et al. 2001). Different mechanisms of corrosion due to SRB had been proposed earlier such as cathodic depolarization by hydrogenase enzyme (Kuhr and Van der Vlugt 1934), King’s mechanism (iron sulfides) (King and Miller 1971), anodic depolarization (Obuekwe

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et al. 1981a), three stage mechanism (de Romero 2005), etc. However, the relative contribution of different corrosion mechanisms due to SRB remains controversial (Yuan et al. 2013), and probably depends on the type of SRB species (Enning and Garrelfs 2014). SRB reduces sulfate to form H2S, which is slightly soluble in water and rapidly oxidizes metallic iron to iron sulfide (FeS). Dissolved sulfide plays an important role in influencing corrosion (Dall’Agnol et al. 2014). For instance, resultant H2S and H2 are responsible for embrittlement of steel (Biezma 2001). Electrochemical reaction involved in the production of sulfide ions by SRBs are given below. 4Fe → 4Fe2+ + 8e- (anodic reaction)

(1)

8H + 8e ↔ 8H (ads) (cathodic reaction)

(2)

8H2O ↔ 8H+ + 8OH-– (water dissociation)

(3)

SO4 + 8H ↔ S + 4H2O (bacterial involvement)

(4)

Fe2+ + S2– ↔ FeS (corrosion product)

(5)

+

–

+

2–

2–

4Fe + SO4 + 4H2O ↔ 3Fe(OH)2 + FeS + 2OH (Net reaction) 2–

–

(6)

(b) Sulfur Oxidizing Bacteria (SOB) These bacteria are aerobic or anaerobic, acidophilic or neutrophilic in nature (Friedrich et al. 2001). SOB oxidizes elemental sulfur, sulfides, H2S, and produced sulfuric acid, which is an aggressive corrosive agent. General reactions for SOB metabolism are given below. S + 4 H2O → H2SO4 + 6H+ + 6e–

(7)

S + 6 H2O → SO3 + 3 H2O + 6H + 6e 2–

2–

+

(8)

–

S2O32– + 5 H2O → 2SO42– + 10 H+ + 8e–

(9)

S2O6 + 2H2O + O2 → 2HSO5 + 2H + 2e 2–

–

+

–

(10)

Due to sulfuric acid, the acidity of solution and hydrogen penetration increases which results in enhanced corrosion (Little et al. 2000). Sulfur Oxidizing Bacteria (SOB) can also encourage the growth of SRB by producing essential products for their growth (Norlund et al. 2009). Dong et al., reported that severe corrosion in high grade stainless steel caused by Acidithiobacillus caldus (Dong et al., 2018). (c) Acid-Producing Bacteria (APB) Acid-producing bacteria are heterotrophic and facultative bacteria. APB utilize organic substances and produce organic acids such as acetic acid, formic acid, lactic acids, etc. fermenters

(CH2O)n → (organic acids, alcohols, CO2, H2, and H2O)

(11)

Due to these organic acids, an acidic environment is produced on the metal biofilm interface, which was suggested to be responsible for the deterioration of metals (Cote Coy 2013). (d) Nitrate Reducing Bacteria (NRB) These bacteria are facultative microbes, also known as denitrifying bacteria, which utilizes nitrate ions as terminal electron acceptors in anaerobic conditions; for example, some species of genus Pseudomonas and Acronobacter (Beech et al. 2000). Under anaerobic environment, nitrate reduction is the main metabolism of NRB, which can be described using the following reaction. NO3– + 2H+ + 2e– → NO2– + H2O

(12)

Microbial Corrosion and It’s Current Mitigation Strategies 223

Nitrate addition stimulates the growth of NRB, especially in the oil and gas industries. Production of nitrite and nitrous oxides by NRB suppresses the growth of SRB and biogenic sulfide production by SRB (Gieg et al. 2011, Greene et al. 2003). Anaerobic microbes such as Paracoccus denitrificans and E. coli reduces nitrate to ammonium ions and elemental iron act as an electron donor under anaerobic environment (Kielemoes et al. 2000). Fe → Fe2+ + 2e–

(13)

NO3 + 8e + 10H → NH4 + 3H2O

(14)

4Fe + NO3– + 10H+ → 4Fe2+ + NH4+ + 3H2O

(15)

–

–

+

+

(e) Methanogens Methanogens (e.g., genus Methanococcus and Methanobacterium) produce methane by utilizing carbon dioxide or hydrogen and organic compounds (Beese-Vasbender et al. 2015, Kato 2016). Generally, methanogens utilize hydrogen coupled with CO2 reduction. When the hydrogen supply is limited, then these bacteria switch to iron as an electron donor. 4Fe → 4Fe2+ + 8e–

(16)

HCO3– + 9H+ + 8e- → CH4 + 3H2O

(17)

4Fe + HCO3 + 9H → 4Fe + CH4 + 3H2O

(18)

–

+

2+

Some methanogens can oxidize Fe0 as a source of energy for their growth through cathodic depolarization (Uchiyama et al. 2010). Previously, various studies were reported related to pitting corrosion of steel pipes due to methanogenic archaea (Larsen et al. 2010, Park et al. 2011). (f) Iron Reducing Bacteria (IRB) IRB are facultative bacteria and also known as iron respiring bacteria; for example, Shewanella sp. They use oxygen aerobically and switch to anaerobic conditions. In anaerobic conditions, these microbes reduce insoluble ferric ions to soluble ferrous ions. Fe3+ +e– → Fe2+

(19)

Fe3+ ions act as a terminal electron acceptor in their metabolism for the generation of ATP (Lee and Newman 2003). Albeit, the role of IRB in corrosion is still not clear but some researchers suggested that IRB can inhibit corrosion (Dubiel et al. 2002, Lee and Newman 2003) while others have reported IRB induced corrosion (Dawood and Brözel 1998, Obuekwe et al. 1981b). (g) Metal Oxidizing Bacteria (MOB) These bacteria are also known as metal depositing bacteria includes manganese-oxidizing bacteria (e.g., Leptothrix discophora) and iron-oxidizing bacteria (e.g., Gallionella, proteobacteria, etc.) (Linhardt 2006, Linhardt 2010, Liu et al. 2017). Leptothrix genus can oxidize manganese to MnO2 (Boogerd and De Vrind 1987) with MnOOH as an intermediate (Shi et al. 2002). Biomineralized manganese oxide deposited over the steel surface act as cathodic reactants and recycled to Mn(II) (Dickinson and Lewandowski 1996a, Olesen et al. 2000). Manganese oxides are responsible for the electrochemical behavior of stainless steel in the presence of manganese-oxidizing bacteria. MnO2 + H+ + e– → MnOOH

(20)

MnOOH + 3H+ + e– → Mn2+ + 2H2O

(21)

Manganese oxides are present in Extracellular Polymeric Substances (EPS) secreted by them. In Leptothrix discophora, EPS found to be in structured sheaths or freely in medium (Ghiorse

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1984, Mulder and Van Veen 1963). In Leptothrix discophora, biomineralized MnO2 which were deposited in a biofilm formed over the Stainless Steel (SS) cathode increased eight times the corrosion rates of galvanic coupled mild steel than the control without manganese (Boogerd and De Vrind 1987). After a few minutes, MnO2 discharged and the corrosion rate of mild steel decreased compared to biofouled cathode without manganese. Dickinson and Lewandowski, 1996b reported that manganese oxide deposited by Leptothrix discophora was responsible for the ennoblement of stainless steel. Maruthamutha et al., and Rajasekar et al., reported corrosion behavior of manganeseoxidizing bacteriain diesel and naphtha pipelines respectively, and gave the hypothesis on the role of these bacteria on the API 5LX steel (Maruthamuthu et al. 2005, Rajasekar et al. 2005). Manganeseoxidizing bacteria can be divided into three groups; Group (I) consists of the manganese-oxidizing bacteria dissolved Mn2+ ions using oxygen as a terminal electron acceptor, Group (II) oxidizes prebound Mn2+ ions using oxygen as a terminal electron acceptor, and Group (III) oxidizes Mn2+ ions using hydrogen peroxide as their oxidant. All the three groups of manganese-oxidizing bacteria affect MIC (King et al. 2008). The second category of metal-oxidizing bacteria is Iron-Oxidizing Bacteria (IOB). Among the corrosive microbes, IOB plays a very significant role in corrosion (Rao et al. 2000, Xu et al. 2007). IOB produces an iron based film over the metal surface, acts as super crevice and stabilizes the pitting corrosion (Suleiman and Newman 1994). Another study reported that in natural spring water, IOB could cause minor localized corrosion on the stainless steel 304 L (Chamritski et al. 2004). The cool age circuit of Fast Breeder Test Reactor (FBTR) in Kalpakkam had many problems like pipeline blockage due to tubercle formation in pipelines, pipe leakage, the high corrosion rate of carbon steel due to IOB (Rao et al. 1993). The most troublesome group of bacteria involved in tubercle formation are Leptothrix sp., Crenothrix sp., and Sphaerotilus sp. IOB consists of morphologically and phylogenetically heterogeneous prokaryotes. Although IOB species were found in different phyla, most of the IOB belong to proteobacteria. IOB can be divided into four main physiological groups; (i) phototropic iron oxidizers, (ii) acidophilic aerobic iron oxidizers, (iii) neutrophilic aerobic iron oxidizers and (iv) neutrophilic anaerobic iron oxidizers. Some species of IOB are capable of reducing ferric ions and oxidizing ferrous ions depend on the environmental conditions. IOB have become a point of attraction for researchers not only for the involvement of these bacteria in the iron cycle and industrial applications but also the discoveries of novel genera or species which are capable of iron oxidation (Emerson et al. 2010). Acidophilic, Aerobic Iron Oxidizers Acidophilic IOB can survive in an acidic environment. These bacteria mainly obtain energy from the oxidation of ferrous ions to ferric ions in aerobic conditions for their growth. A. ferrooxidans is the most widely studied species in this category. Corrosion behavior of carbon steel 1010 in the presence of Acidithiobacillus ferrooxidans was studied by Wang et al. They concluded that severe corrosion occurred in the carbon steel in the presence of Acidithiobacillus ferrooxidans. This bacteria uses the oxidation reaction of ferrous ions to ferric ions for its growth to fix the carbon dioxide (CO2) in low pH range 2 to 4.5 (Leathen et al. 1956) according to the reaction given below. 24Fe2+ + 6CO2 + 24H+ → 24Fe3+ + C6H12O6 + 6H2O

(22)

Neutrophilic, Aerobic Iron Oxidizers Neutrophilic aerobic IOB mainly belongs to the class proteobacteria (Emerson et al. 2010). Proteobacteria can be divided into two categories—(a) freshwater species (betaproteobacteria) and (b) marine species (zetaproteobacteria) (Emerson et al. 2007). Gallionella ferrunginea is well known neutrophilic IOB. It can grow autotrophically or mixotrophically using ferrous ions as an electron donor (Hallbeck and Pedersen 1991). For example, Sideroxydans sp. and Ferritrophicum sp. are

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also aerobic, neutrophilic IOB. Joyce M. McBeth et al., reported that corrosion of mild steel was higher in the presence of zetaproteobacteria such as Mariprofundus ferrooxydans by comparison to sterile control (McBeth et al. 2011). Higher corrosion was reported in the case of stainless steel 304 L in the presence of Spherotilus sp. than the control media (Starosvetsky et al. 2008). Spherotilus sp. was responsible for the disruption of the passive layer of resistant stainless steel according to the mechanism of crevice corrosion. Recently, Pseudomonas/Pseudoalteromonas were isolated from the volcanic seamount and these can oxidize iron (Sudek et al. 2009). Neutrophilic, Anaerobic (Nitrate Dependent) Iron Oxidizers Bacteria that coupled iron oxidation and nitrate reduction can be divided into two groups: (i) autotrophs and (ii) heterotrophs. Straub et al., isolated and identified three gram negative bacterial strains from genus Acidovorax, Aquabacteriu and Thermomonas, which could oxidize iron with nitrate reduction in anaerobic environments (Straub et al. 2004). In these bacteria, the rate of iron oxidation was observed to be slow in the absence of organic acid. The end product of nitrate reduction was nitrogen gas with nitrous oxides in small amounts. Iron was deposited as ferrihydrite mineral according to the equation given below. 10FeCO3 + 2NO3– + 10H2O → Fe10O14(OH)2 + 10HCO3– + N2 + 8H+

(23)

Phototropic Iron Oxidizers Most of the phototropic iron-oxidizing bacteria belong to class Alphaproteobacteria except Thiodictyon. This group of bacteria used ferrous ions as a source for the reduction of carbon dioxide according to equation given below. 4Fe2+ + CO2 + 11H2O + hν → CH2O + 4Fe(OH)3 + 8H+

(24)

Rhodobacter sp. strain SW2 was the first iron oxidizer, which oxidizes ferrous ions in the presence of organic carbon sources. It was also capable of utilizing hydrogen and organic compounds (Ehrenreich and Widdel 1994). 1.1.3 Role of Biofilm in Microbial Influenced Corrosion Biofilm is referred to as the structured community of microbial cells enclosed in a slimy matrix known as Extracellular Polymeric Substances (EPS) and adhered to a wide number of surfaces (Costerton et al. 1995). The nature of metallic surfaces can influence the rate of bacterial adherence to the surface (Fletcher and Loeb 1979). Surface microbial metabolic activity within the biofilm deposited over the metallic/nonmetallic surfaces can affect the kinetics of electrochemical reactions (cathodic/or anodic reactions) (Jones and Amy 2002). It can also modify the chemistry of the passive layers on the surfaces, leading to either acceleration or inhibition of the corrosion process (Little and Ray 2002). MIC is a synergistic effect of metal surfaces, corrosion products, bacterial cells and their metabolites. In this study, thermophilic and thermotolerant bacteria were isolated from hot springs. The corrosion rates were observed higher in the presence of various combinations of bacteria than pure culture (Valencia-Cantero et al. 2003). Biofilms are generally found in every environment, both natural and artificial, where microbes and moisture are present. The composition of biofilm may vary with environmental conditions. Biofilms are usually composed of water, extracellular polymeric substances, including proteins, polysaccharides, lipids, nucleic acids, etc., and bacterial cells. The biofilm composition is shown in Table 10.1. From Table 10.1, water is the major component of biofilm (roughly 95% and 3% of EPS). So it is generally acknowledged that EPS is the most important factor of biofilm in the point of MIC (Allison 2003, Costerton et al. 1995). Biofilm development occurs on the metal surface through five stages (Fig. 10.3).

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% Total Up to 95%

Microbial cells

2–5%

Polysaccharides

1–2%

Proteins Phospholipids Nucleic acid (DNA and RNA)

< 1–2% 1–2% < 1–2%

Description

Origin

Characteristics determined by dissolved solutes Neutral and polyanionic; homopolysaccharides or heteropolysaccharides

Extracellular

enzymes and other regulatory proteins

Extracellular and cell lysis Extracellular and cell lysis Cell lysis

Fig. 10.3. Various stages for the formation of biofilm on the metal surface adapted from (Toyofuku et al. 2016) with modifications.

Various enzymes involved in metal corrosion are substanial in the biofilm. For example, in SRB, the hydrogenase enzyme were reported to play a crucial role in MIC (Da Silva et al. 2002). The corrosion rate was dependent on the hydrogenase enzyme of the biofilm rather than the bacterial population in the case of SRB (Bryant et al. 1991). Other enzymes such as catalase, peroxidase and superoxide dismutase were also suggested to be involved in oxygen reduction reactions. Hence, SRB were advised to facilitate corrosion by accelerating reduction reactions. In addition, oxygen reduction by these enzymes also depends on the surface film chemistry. Busalmen et al., reported that the reduction kinetics of hydrogen peroxide produced by oxygen reduction could be influenced by the catalase enzyme (Busalmen et al. 2002). The EPS composition can also influence the corrosion process. EPS is the structural component of the biofilm and provides a framework for it. EPS is found in two forms; (i) capsular—EPS intimately associated with the cell surface and (ii) slime—is loosely associated with the bacterial cells. Bacterial EPS is highly heterogeneous and highly bioactive due to the presence of various anionic functional groups (e.g., carboxyl, phosphate, glycerate, pyruvate, sulfate, etc.). These anionic functional groups are found in carbohydrates and proteins, which are the chief components of the biofilm. Due to these anionic groups EPS binds with metals. This EPS-metal binding play important role in MIC (Kinzler et al. 2003) and this binding depends on both bacterial strains as well as the type of metal ions (Lens et al. 2003). The binding affinity of multidentate anionic groups

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with multivalent cations such as Fe2+, Fe3+, Ca2+, Mg2+, etc., can be very strong. Metal ions present in different oxidation states in the biofilm are responsible for standard reduction potential shifts. EPS bound metal ions were recorded to act as an electron shuttle and opened a new redox reaction pathways in the biofilm/metal system, and hence, affected the corrosion of metals (Beech and Sunner 2004). 1.1.4 Factors Affecting MIC 1.1.4.1 Environmental Factors Various environmental factors such as rainfall, soil moisture, temperature, the topography of soil, water levels, pH, ions concentration, oxygen concentration and organic carbon availability for microbes can affect the corrosion of metals (Cole and Marney 2012, Katano et al. 2003, Mansfeld and Little 1991). Fluid dynamics can also affect the MIC. The surface layer of biofilms and corrosion products are stable at a low velocity rate while at a high flow rate only the corrosion products layer is dominant. Fluid flow can also affect the growth of microbes in the biofilm by limiting oxygen availability and lowering the nutrients exchange in the biofilm (Stewart 2012, Thomen et al. 2017). MIC can also get affected by temperature because the bacteria requires optimum temperature for their growth. For example, the optimum temperature for SRB is ~ 298.15 K, but it can also survive at 333.15 K (Ismail et al. 2014). Corrosion causing microbes mainly belong to the category of mesophiles that can survive between 293.15–318.15 K. Microbial growth also depends on the pressure; for example, SRB can tolerate 50.6 Mpa pressure (Kumar and Libchaber 2013, Li et al. 2015). A study reported that negative pressure can decrease the microbial growth rate and inhibits biofilm formation (Li et al. 2015). Optimum pH is a very important factor for microbial growth pH depends on the partial pressure of carbon CO2 or H2S, organic acids produced by microbes, etc., pH can inhibit the microbial growth outside the optimum range (Ibrahim et al. 2018). Availability of gases can affect the microbial growth; for example, methanogens and SOB utilize CO2 to derive carbon for their growth (Ibrahim et al. 2018). Besides CO2, microbes vary in their requirements for oxygen. Aerobic bacteria thrive in presence of oxygen and catalase it as terminal electron acceptors such as APB, SOB and IOB while anaerobic bacteria cannot grow in the presence of oxygen such as SRB. These aerobic and anaerobic bacteria play crucial role in MIC (Fig. 10.4).

Fig. 10.4. An outline of various factors affecting MIC (Ibrahim et al. 2018).

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1.1.4.2 Corrosion Products MIC can also get affected by corrosion products. Metal oxides deposited on the metal surface helps in microbial adhesion and tubercle formation. Metal oxides facilitate cell adhesion due to surface roughness, charge and hydrophobicity (Li and Logan 2004). In the case of anaerobic environments, the main corrosion products were FeS, FeS2 and Fe3S4 in the presence of SRB (Videla et al. 1999) while in aerobic environments, the major corrosion products were Ironhydroxides (α-FeOOH, β-FeOOH, γ-FeOOH, Fe3O4) (Cornell and Schwertmann 2003). 1.2.3 Chemical Environment Microbes require the nutrients for their growth such as carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, potassium, magnesium, calcium, manganese, traces of zinc, cobalt, copper and molybdenum. However, nitrate, phosphates and sulfates may act as inhibitors for corrosion (Little 2003). By uptake of various nutrients (SO42–, NO3–, PO43–, NO2–), microbes can influence metal degradation (Little 2003). In the presence of various anions, the critical pitting potential may shift either more active values or noble values; for instance, critical pitting ability shifts towards more active values (negative) when the concentration of chloride ions increases while critical pitting potential shifts towards noble values (positive) in presence of other anions (SO42–, NO3–, PO43–, OH–, etc.) (Little 2003). Critical pitting potential is the potential below which pitting or localized corrosion is unstable. To determine the effect of nutrients on corrosion, researchers of the Russian Federation exposed carbon steel in SRB and oil oxidizing bacteria. They used glucose mineral medium with peptone as a nutrient media and observed that more corrosion occurs in the presence of bacteria than in the control media. However, in the presence of enriched media with the same biofilm, less corrosion was observed (Rodin et al. 2005). 1.2.4.4 Microbiological Factors Metal surface and minerals are a point of attraction point for bacteria in their colonization (Costerton et al. 1995). Researchers categorize the MIC mechanisms in different ways such as indirect and direct mechanisms. Direct mechanisms involve specific biochemical mechanisms such as catalyzing oxidation and affects the corrosion, while indirect mechanism involves the general metabolic activities like metals binding (Gadd 2010). Gu defined three categories of biocorrosion: (i) electrons removal from steel by microbes via nanowires, (ii) fermentative microbes produce organic acids as byproducts and oxidize steel but do not have any catalytic function, and (iii) heterotrophic microbes degrade organic materials such as polyurethane. Many microbes are motile and are attracted towards energy sources; for instance, corroding pipe releases ferrous ions which attract the IOBs. After the establishment of microbes on the metal surface, they modify the local environment of the metal vicinity and affect the corrosion (Lens et al. 2003). Microbes obtain energy from environmental sources by various carriers such as intracellular components ferredoxins, flavoproteins and cytochromes that can be either oxidized or reduced and form the redox couple. The reduced carriers act as electrons/hydrogen donors while oxidized carriers act as electrons acceptors. However not every donor-acceptor combination has to be thermodynamically possible. Both mechanisms are important for MIC. In direct electron transfer, surface bound proteins facilitate the transfer of electrons to the cell cytoplasm while in indirect electron transfer, it is dependent on electron shuttles such as formate, flavin, riboflavin, nicotinamide adenine dinucleotide (NAD), etc. Several researchers have reported that mediators produced by microbe can accelerate electron transfer (Rabaey et al. 2005). Electron transfer starts with electron donors which can be organic electron donors (e.g., acetate, lactate, etc.) or inorganic electron donors (e.g., molecular hydrogen, Fe0, S0, etc.). The term Chemical Microbially Influenced Corrosion (CMIC) was coined by Ennings et al., to describe the effect of biogenic H2S on corrosion and concluded that sulfate and other electron donors can cause the CMIC (Enning et al. 2012). They also reported that under certain circumstances SRB can also

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act as an electrogenic. When metallic iron is used as only a source of electrons and carbon dioxide as a source of carbon, then SRB grown in contact with iron surface electrons were transferred for sulfate reduction (Enning et al. 2012). In Electrical Microbial Influenced Corrosion (EMIC), direct electron uptake involves the outer membrane proteins, e.g., c-type cytochromes which interact with extracellular electron donors and acceptors. Not all microbes have to conserve energy from the reduction process. Hamilton (Hamilton 2003) identified and suggested that in microbial respiration, terminal electron acceptors played a role in MIC. Microbes that can use oxygen only for the electron acceptor are known as aerobic microbes while microbes that cannot use oxygen are known as anaerobic microbes. Facultative microbes use oxygen or other electron acceptors (e.g., NO3–, NO2–, SO42–, Fe3+, etc.). 1.2.5 Mechanism of MIC 1.2.5.1 Differential Aeration Cells Differential aeration cells are created via uptake of oxygen by microbes or alteration of oxygen by producing masses of biological slime, mats metabolites accumulated on the surface and colonies; all appear in the biofilm formed on the metal surface. When the electrochemical cells are formed, the anaerobic regions under the deposits act as an anode and the surroundings of deposits act as a cathode. Accumulation of corrosion products along with the biofilm in the case of IOB was reported to provoke the corrosion reactions by cathodically reactive ferric oxides and oxygen consumption by respiration in the deposits (Fig. 10.5) (Tiller 1983). The tubercles produced by MIC diminish oxygen diffusion to the surface of the metal and leads to corrosion by differential aeration cells (Kajiyama and Koyama 1997, Rao et al. 2000). The biofilm acts as a barrier for restricted oxygen diffusion and creates differential aeration cells (Little et al. 1991). Additionally, under the biofilm, microbes utilize oxygen and create an anaerobic environment that is suitable for the growth of anaerobic bacteria like SRB. In aerated chlorine containing water, IOB establishes differential oxygen concentration in the case of stainless steel 300 series and initiate localized corrosion (Ray et al. 2010). The rate of corrosion does not dependent on the cell number in the deposits. It depends on the metallurgy and physical/chemical properties of the electrolytes such as dissolved oxygen, chloride ions concentration, etc.

Fig. 10.5. Simple schematic diagram of biofilm-formed differential oxygen concentration cell (Tiller 1983).

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1.2.5.2 Galvanic Cells Microbes can adsorb metals by EPS, capsules and cell walls (Gadd 2010). Adsorption of metals to the microbial cells is important for microbial-metal interactions and responsible for the formation of galvanic cells on the metal surface (Barkay and Schaefer 2001). Additionally, EPS and bacterial cell walls are chemically active and have the ability to precipitate or adsorb different dissolved metal ions, assisted by a large surface area to volume ratio and high surface charge densities (Langley and Beveridge 1999). The extent of metal binding and precipitation was also affected by the biofilm growth and its surface reactivity. Metal rich phases of biofilm close to the metal surface can induce the formation of galvanic cells between the metal surface and biofilm. Cells have the metal binding ability that can alter the conductivity of low frequency currents (Konhauser 2009). Ray et al. demonstrated in carbon piling of Duluth-Superior Harbor (DSH), copper was deposited under the deposits of IOB and a galvanic couple was established between the carbon steel and copper layer (Ray et al. 2010). Minerals (sulfides, iron and manganese oxides) precipitated by microbes can support the cathodic reactions. Hence, these minerals present in the vicinity of the anodic site on the metal surface can establish electrochemical cells and be responsible for shifting of corrosion potential either in positive or negative directions (Little et al. 1998). 1.2.5.3 Innoblememnt It is well established that in passive alloys, the biofilm is responsible for the ennoblement of corrosion potential (Dexter and Zhang 1990, Ito et al. 2002). The biofilm can change the electrochemical behavior of metals and these electrochemical changes collectively termed as ennoblement in which corrosion potential increases towards noble values (Dickinson et al. 1997). The elevated corrosion potential encourages corrosion initiation in the case of SS304L immersed in seawater (Dexter 1995). The ennoblement potential has been correlated with the biological activity of the biofilm, cell density, electron transport activity and content of lipopolysaccharides (Dexter and Zhang 1990, Mollica et al. 1989). Another study demonstrated the partitioning of manganese oxides between the planktonic cells and sessile cells to determine the extent to which manganese-depositing bacteria encourage ennoblement and mineral composition (Dickinson and Lewandowski 1996a, Shi et al. 2002). 1.2.5.4 Acidification Microbial cells present in the biofilm can alter the local pH in the vicinity of metals by secreting various metabolites and promote the corrosion process (Biezma 2001). One important factor for the corrosion can be the acidity caused by microorganisms (Campaignolle and Crolet 1997, Rajasekar et al. 2010). Additionally, heterotrophic microbes produce a wide range of organic acids like citric acid, oxalic acid, succinic acid, coumaric acid, etc., and are capable of enhancing metal dissolution (Francis 1998). Barker et al., demonstrated a decrease in pH near the bacterial cells inside the cracks present in corroded metal than the bulk solution (Barker et al. 1998). Some microbes like methanogens and SRB utilized the hydrogen as an electron donor, and hence, their growth and corrosion due to these microbes may be increased with an increment of hydrogen ion concentration (Boopathy and Daniels 1991). 1.2.5.5 Nanowires Some bacteria consist of electrically conductive appendages, e.g., filaments or pilli, termed as nanowires, may transfer the electrons from bacterial cells to Fe3+ bearing iron oxides and reducing them (Gorby et al. 2006, Malvankar et al. 2011). These nanowires can act as bridges between bacterial cells and distribute the electrons between diverse microorganisms in the environment (Gorby et al. 2006). There are very few studies related to nanowires and interactions amongst microorganisms to understand the biofilm functioning, biocorrosion and bioleaching. It is not essential that all pilli are conductive in nature (Vargas et al. 2013).

Microbial Corrosion and It’s Current Mitigation Strategies 231

1.2.5.6 Direct Consumption of Electrons from Steel Some researches demonstrated that consumption of hydrogen by microbes does not increase the corrosion rate significantly (Mori et al. 2010, Venzlaff et al. 2013). Instead of highly corrosive microbes such as SRB and methanogens can catalyze electrons directly from steel by oxidation reactions (Dinh et al. 2004, Uchiyama et al. 2010). This mechanism of direct uptake of electrons from metals has been referred to as Electrical Microbiologically Influenced Corrosion (EMIC) (Enning et al. 2012). 1.2.5.7 Microbial Metabolites and Enzymes Bacteria produce a wide range of compounds such as hydrogen sulfides, phosphene, ammonia, highly oxidative hydrogen peroxides that can affect the electrochemical behavior of metals (Li et al. 2012). They also secrete enzymes such as hydrogenases, lyases, oxidoreductases, catalases, etc. (Busalmen et al. 2002, Lens et al. 2003, Little et al. 2000). Hydrogenase enzyme of microbes consist of iron active sites and can catalyze the oxidation of hydrogen/reduction of H+ ions (Da Silva et al. 2002, Gu 2012). Some enzymes are also found on the cell wall of microbes or found free in EPS. These enzymes can remain active after bacterial cell death and contribute to the corrosion process (Beech et al. 2005). 1.2.5.8 Microbial Alteration of the Passive Film on the Metal Surface After exposure of base metal to the corrosive medium, some microbes start eating the protective passive layer on the metal surface. On the other hand, eating passive films by microbes may create different concentration cells in the vicinity of metal. Microbes eat the protective layer of metal surface unevenly and produce a patchy biofilm in these areas. Generally, bacteria that eat or dissolve the protective layers are metal reducing type, e.g., Shewanella sp. (Myers and Nealson 1988). 1.2.5.9 Extracellular Electron Transfer (EET) Microbes can interact with the metal surface via Direct Electron Transfer (DET) or Mediated Electron Transfer (MET). In DET, electronic states of surface material and active centers of microbial enzymes overlap, resulting in increasing the electron transfer probability across the interface. While in MET, natural or artificial electron transferring agents can participate in redox reactions of biological components (Chaubey and Malhotra 2002). Electrostatic interactions, suitable redox potential, pH of the solution and ionic strength are the major factors that play an important role in MET (Dominguez-Benetton et al. 2012). Electrochemically active microorganisms, with electrochemically active biofilm are capable of electric current production or exhibiting electron exchange activity through the working electrode and play an important role in efficient electron transfer (Parot et al. 2008). 1.2.6 MIC Diagnosis For the diagnosis of MIC, the identification of corrosive bacteria is very essential. The identification of corrosive bacteria can be done by traditional or modern methods. The traditional method consists of cultural techniques, biochemical tests, etc., while the modern methods include the molecular technologies such as sequencing technique. Ribosomal RNA sequencing has been used to identify the corrosive bacteria in various scientific fields (Lens et al. 2003). These sequences can be deposited in online database like GenBank database and these sequence data are publically available which allow to compare the sequence data of other microbes using the search tool Basic Local Alignment Search Tool (BLAST). After comparision of the phylogenetic tree can be constructed to find evolutionary relationships of corrosive microbes. Fluorescent strains, Confocal Laser Scanning Microscopy (CLSM), electron microscopy and spectroscopy are important tools for MIC diagnosis. Fluorescent strains (DAPI and Sytox) bind with

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the components of microbes or DNA and help in visualization of distribution of microbes in corroded samples. CLSM is used to determine the microbes’ distribution and their three dimensional images in corroded samples (Little et al. 2006). It can also be used to measure accurate dimensions of the objects by laser light. Electron microscopy can be used for the biofilm development, composition and distribution along with corrosion products formed on the metal surface. Other techniques such as light microscopy, immunofluorescence techniques, Atomic Force Microscopy (AFM), etc., can also used to diagnose MIC. MIC can not be diagnosed by the morphology of pits formed on the metal surface and metallographic features. Bacterial cells adhered to the metal surface (sessile bacterial cells) play important role in MIC. These cells proliferate in the biofilm formed on the metal surface and affect the chemical reactions, pH value, concentration of dissolved oxygen, etc., on the vicinity of metal by their metabolic products. Consequently, the corrosion products formed on the metal surface was suggested to be different in the presence of bacteria and abiotic conditions. For example, corrosion products formed in the absence of bacteria are generally iron oxides and oxyhydroxides. In constrast, corrosion products formed on the metal surface in the presence of bacteria may different such as ferrihydrite (in the presence of iron oxidizing bacteria, e.g., Lepidocrocite sp. and Gallionella sp.) (Toner et al. 2012), iron sulfite (In IOB Pseudomonas sp. DASEWM1 strain) (Sachan and Singh 2019), etc. A very simple method for the diagnosis of MIC is to scrap some corroded material from the surface, heat it and check the smell. If the smell of burnt material is like burnt hair or meat, indicates the presence of protein. After that more sophisticated and commercially available protein tests are recommended. Kits for adenosine triphosphates (ATP) are also available commercially and can help to diagnose MIC because all living organisms consists of ATP. Recently, various probes (BIOX, ALVIM, etc.) were used to detect the biofilm. These probes are commercially available. 1.2.7 Control/Prevention/or Inhibition of MIC 1.2.7.1 Physical Treatment MIC can be controlled using two methods; (i) pigging, (ii) UV irradiation, and (iii) ultrasonic treatment. i. Pigging This is a physical cleaning method and used against corrosion. In this method some devices were used which are similar to plugs or sponge balls. These devices are moved inside the clogged pipes to remove the clogs, tubercles and corrosion products formed inside the pipes (Cote et al. 2014, Javaherdashti 2017b). The pigging process has the some limitations such as it can only be applied to piggable lines having a constant diameter. ii. UV irradiation UV light can kill the microbes by formation of thymine dimer in the DNA of microbes. Only those microbes can be killed by UV light which were directly exposed to this light. If microbes present inside the biofilm then it is very difficult to destroy them using UV light. While it is practical implementation, but is difficult in industries. For example the internal surface of pipelines are not suitable for UV light exposure. iii. Ultrasonic treatment In water, ultrasound produces bubbles and when these bubbles collapse they can destroy the microbes. Ultrasound can be used to kill or inhibit the microbes, but its implementation is difficult for the industries.

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1.2.7.2 Chemical Treatment (a) Biocides Biocides treatment are used in industries such as oil and gas industry, water distribution system, etc. Two types of biocides (oxidizing and non-oxidizing) are used for the mitigation of MIC in industries. Oxidizing biocides Chlorine is used as oxidizing biocides in wastewater treatment plants and water utilities. In various industries, the cooling systems are very important. Chlorine can release the very active free redicles which are lethal for microbes but they can also corrode equipment (Kahrilas et al. 2015). Chlorine treatment is very frequently used to control the biofilm. It can destroy the microbes present inside the biofilms and can also react with the EPS and destroy it (Oliveira et al. 2016). Chlorination is a good option to mitigate the microbes due to its low cost and good efficacy (Fagerlund et al. 2016). Non-oxidizing biocides Glutaraldehyde and Tetrakis (hydroxymethyl) phosphonium Sulfate (THPS) are commonly used in oil and gas industries due to their broad spectrum activity, cost effectiveness, etc. (Javaherdashti 2017a, Xu et al. 2017). Glutaraldehyde has some limitations due to its corrosive nature for carbon steel, poor performance and non-target toxicity (Eid et al. 2018). Quaternary ammonium or amine compounds and alkyldimethylbenzylammonium chloride are used as cationic biocides. These biocides are used frequently to destroy the microbes by damaging their cell membrane action (Ioannou et al. 2007). In biofilm, it is very difficult to eradicate sessile bacterial cells than planktonic cells because sessile cells are more tolerant. The biofilm is a consortium of different communities of bacteria which have different defense mechanisms against harsh conditions (Xu et al. 2017). So, higher concentration of biocides is required for the eradication of sessile cells. Keeping these points in the mind, bicide enhancers were introduced. Biocide enhancers are used to enhance the biocide activities to destroy the bacteria. Generally, surfactants were used in biocide blend to enhance their activity. (b) D-amino acids D-amino acids are found in nature as a part of proteins. They exists in living organisms such as microbes, plants, humans, etc. D-amino acids play role in the regulation of biological functions (Aliashkevich et al. 2018). Kolodkin-Gal et al., stated that the biofilm formation of Bacillus subtilis can be inhibited by the mixture of D-amino acids (nanomolecular concentration) (Kolodkin-Gal et al. 2010). Pseudomonas sp. and Desulfovibrio sp. can also be inhibited by D-amino acids (Emma 2011). It was noted that D-amino acid alone have the limited ability of bacterial dispersion but when it is combined with biocides its efficiency of bacterial dispesion accelerates (Jia et al. 2017). The mechanism of dispersal of biofilm is not fully understood yet. According to the hypothesis, the D-ala of bacterial cell wall is substituted by the other D-amino acid and this substitution of amino acid leads the dispersal of the biofilm (Kolodkin-Gal et al. 2010). (c) Chelators For biofilm inhibition, chelating agents can be used as biocide enhancers. For the treatment of biofilms formed on the medical catheters, ethylenediaminetetraacetic acid (EDTA) can be used to accelerates the activity of biofilm (Raad et al. 2007). The acidic form of this chelating agent can be corrosive at a very high concentration. To reduce this corrosive nature, either sodium salt can

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be used with EDTA or the pH of solution can be maintained to nearly neutral, i.e., pH 7. Another chelating agent ethylenediaminedisuccinate (EDDS) can be used as a chelating agent instead of EDTA due to its biodegradable nature. EDDS is non-toxic and biodegradable. Due to these qualities EDDS can be use in industries to reduce biofilm formation. 1.2.7.3 Biological Treatment (a) Norspermidine Norspermidine is a kind of polyamine and present in some algae, bacteria and plants and play a role in mitigation of MIC by either disassembeling the fully mature biofilms or inhibition of biofilm formation (Si and Quan 2017). It was reported that the higher concentration of (100 µM) norspermidine may inhibit the mature biofilm formed by S. epidermidis. Another study reported that the lower concentration (5 µM) of norspermidine can also mitigate the biofilm formation in S. mutants. Norspemidine can inhibit the cell viability in the biofilm and can also change the biofilm structure (Ou and Ling 2016). Qu Lin et al., reported that high dose of norspermidine (0.1 and 1 mmol/L) can inhibit the formation of biofilm (24 hours mature) in Pseudomonas aeruginosa. (Qu et al. 2016). Some researchers also reported that norspermidine can accelerate the biofilm formation in some bacterial strains such as Vibrio cholera (Parker et al. 2012). Norspermidine with the combition of D-tyr can also disassemble the 6 months old biofilm formed in wastewater system by biofilm dispersion (Si et al. 2014). According to these research studies, norspermidine can be used for the treatment of biofilms in industries. (b) Bacteriophage treatment Bacteriophage is a virus which infect bacterial cells. It uses the depolymerase enzyme and lyse the bacterial cells, and can also attack bacterial cells present in the biofilm (Parasion et al. 2014). Phages can act alone or in combination to inhibit the formation of biofilm. Three bacteriophages, i.e., LiMN4p, LiMN4L and LiMN17, in combination can reduce the sessile cells of Listeria monocytogenes adhered on the stainless steel surface (Arachchi et al. 2013). Bacteriophage can also work with the combination of other agents (honey) to inhibit the biofilm formation, this combination has show good antibacterial activity for E. coli (Oliveira et al. 2017). Phage-antibiotic synergy can also be used for the treatment of biofilm. Rahman et al., reported that the combination of a bacteriophage (SAP-26) and rifanpicin antibiotic can reduce the sessile cells of S. aureus (Rahman et al. 2011). Each bacteriophage has its specific host range of bacteria. Due to this reason it is a big challenge to apply bacteriophages for the treatment of biofilm in an industrial level. (c) Quorum sensing approach The Quorum Sensing Inhibition (QSI) approach can be used to control the biofilm formation effectively. Quorum Sensing (QS) is a mechanism of cell to cell communication. The QSI approach is helpful to disrupt QS by (i) inhibition of gene responsible to form QS signal molecule, (ii) distrupt the QS signal molecule and (iii) modulation of signal-recepter binding site, etc. (Lade et al. 2014). Pseudomonas aeruginosa is the most studied bacteria for quorum sensing. Many natural inhibitors (ajoenagrom garlic, iberin from horseradish, etc.) also have the ability of QSI for P. aeruginosa (Jakobsen et al. 2012). Many natural compounds found in plants such as alkaloids, phenolics, quinines, tannins, etc., have been investigated for QSI (Paczkowski et al. 2017). Natural inhibitors are produced in very small quantities, so synthetic chemicals are used for the QSI. Synthetic chemical are cost effective and more practical (Kalia et al. 2014). 1.2.7.4 Coatings Coating on the surface of corrosion sensitive substratum isolates their surface from the electrolyte solutions. Sometimes, the coatings can be mechanically disrupted or degraded biologically. Due to the destruction in coatings, small portions of substratum comes in contact with electrolyte

Microbial Corrosion and It’s Current Mitigation Strategies 235

solution and act as small anodic sites. These anodic sites can attract the corrosive microbes and may accelerate localized corrosion. Coatings can be modified using several strategies such as biocide leaching coatings, fouling and adhesion resistant coacting, contact killing and conductive coatings, graphine coatings, vivianite coatings, etc. Biocidal leaching compounds such as biocidal agents or biogenic compounds were incorporated with different polymers. Biocides can be released from the polymer and inhibit the bacterial cells. A major problem with the biocidal leaching coatings is the environmental damage due to leached biocides. Another problem is that the biocides have a limited life span in the coatings. Adhesion resistant coatings are used to control the MIC. This approach is based on surface properties and inhibition of bacterial adhesion on the surface of materials These types of coatings include hydrophilic, hydrophobic and amphiphillic compounds. Biomimetic coatings contain those substances which can mimic the compounds present in antifouling sufaces. Contact killing coatings include the positively charged compounds which are immobilized in a polymer matrix (Klibanov 2007). Positively charged groups such as quaternary ammonium salts, chitosins, peptides, etc., can interact with negatively charges cells of bacteria and disrupt their cell wall (Guo et al. 2018, Klibanov 2007). Conductive coatings contain conductive polymers such as polyaniline, polythiophene, etc. These coatings have antifouling properties and can produce the mixed oxide layer and protect steels. Graphene and vivianite coatings are used to control the MIC. Graphene is an allotrope of carbon and form the honeycomb lattice. It is the lightest material, thinnest and strongest compound, and also a good heat conductor. Graphene coating is very delicate and can be easily disrupted. Once this coating damages, the probability of pitting and galvanic corrosion increases and create major problems. Vivianite is phosphate rich compound and inhibit MIC (Breur et al. 2002, Volkland et al. 2000). Volkland et al., and Cote et al., reported the presence of vivianite formation in the presence of iron reducing bacteria on mild steel (Cote et al. 2015, Volkland et al. 2000). Cote et al., concluded that vivianite was more protective than iron oxide produced in absence of bacteria (Cote et al. 2015).

2. Conclusion and Future Prospects Currently, the concern of microbial corrosion has led research to elucidate the mechanism of MIC, i.e., initiation of MIC, factors affecting MIC and type of microbes involved in this process. In the natural environment, microorganisms do not occur singly or as an axenic culture but are found as a consortium. Accordingly, one needs to segregate and identify various bacteria in the natural environment, e.g., in soil/underground, water, etc., followed by a test for their corrosivity. A generation of this data will help to understand and predict the MIC in the natural environment, which is essential for many industrial activities, e.g., oil exploration, maritime activity, etc. Nevertheless, the effect of flow rate of media on MIC should be investigated, which is a well known substantial factor to affect the biofilm formation and hence, the extent of microbial influenced corrosion. Although most work related to the bacterial effect on metal indicates the role of biofilm in accelerating corrosion attack due to bacteria; however, recent work shows, in some instances, the development of biofilms which inhibits the MIC. Hence, studies on establishing control of MIC by the development of inhibitor biofilm will pave the way for an environment-friendly approach to MIC control with the use of bio inhibitors.

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11 Extremophile Biofilm Behavior, Characterization and Economical Applications Shashi Bhushan,1,# Jayakrishnan U.,2,# Shaon Raychaudhuri 3 and Halis Simesk 1,*

1. Introduction Life on Earth had been continuously evolving for millennia, with living organisms colonizing every suitable ecosystem on the biosphere. Prokaryotes are the apex life form in this evolutionary track that has pushed the boundaries in which life can exist. Microscopic life forms have been thriving under extreme conditions on the planets like high pressure, salinity, radiation, desiccation, nutrient limitation lower, higher temperature and pH. Extremophiles were isolated from various geochemical locations such as volcanic acidic lakes and hot springs, deep-sea sediments and thermal vents, sea ice and permafrost, deserts, and arid regions, nuclear-contaminated sites, etc. Recent studies with some polyextremophiles have shed light on life beyond the planetary boundaries and to the panspermia hypothesis (Merino et al. 2019). The limits of extreme conditions classify microorganisms into extremophile and extremotolerant. Extremophiles grow optimally under extreme conditions, while extremotolerant are facultative extremophiles that can survive with minimal growth under extremity. Extremophiles have evolved genotypic and phenotypic adaptions that neutralize the deleterious effects of the extreme conditions mediated by metabolites like extremozymes, extremolytes, exopolysaccharides (EPS), pigments, etc. Exopolysaccharides are high molecular weight polymer of the sugar moiety, homo or hetero monomeric units, consisting of protein, lipids and nucleic acid that contribute to the overall structural and functional integrity. EPS is synthesized and secreted by extremophiles in response to the fluctuation in extreme conditions providing additional resistance for their survival by encapsulating them. For the majority of extremophiles, EPS production is concurrent with biomass growth, while production starts during the early stationary phase for others. These EPS producing extremophiles had been identified from both the prokaryotic domains—bacteria and archaea. Extremophile EPS draws large biotechnological applications owing to its broad spectrum of properties (Nicolaus et al. 2010, Yin et al. 2019).

Agricultural and Biosystems Engineering, North Dakota State University, Fargo, ND 58108-6050 (USA). Centre for Environment, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039 (India). 3 Department of Microbiology, Tripura University, Suryamaninagar, Tripura, 799022 (India). * Corresponding author: [email protected] # These authors contributed equally to the manuscript. 1 2

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Most of the prokaryotes produce EPS under the exposure of proper stimulation. However, extremophiles are non-pathogenic natural producers of EPS with the ability according to their ecological niche (Nicolaus et al. 2010). This EPS synthesis facilitates the formation of an encased and organized cluster of the microbial communities in the form of a biofilm, further strengthening the resistance to adverse environmental conditions. Fossilized biofilm embedded with finely preserved microbial fossils discovered from some of the deep-sea volcanic mud, thermal vents, hot springs, etc., indicates the biofilm’s resilience. Microbial cells move towards the surface using chemotaxis. These microbes then get adhered to or adsorbed to the surface mediated by cell surface glycoconjugates. The attachment to the surface triggers EPS production, encapsulating cells forming, thereby colonizing the entire surface. Extremophiles can lead to faster biofilm formation due to innate EPS production overcoming these rate-limiting steps (Yin et al. 2019). Several quorum sensing molecules and secondary messengers modulate biofilm formation and maintenance in extremophiles (Díaz et al. 2018). The cell-to-cell communication mediated by these molecules integrates the cellular function. Thereby, the biofilm microbial communities act in unison as a single functional group to achieve a common goal. The microenvironment created inside the biofilm ensures the availability of substrate, nutrients, reducing equivalents, etc., for microbial growth. Continuous recirculation of microbial cells takes place in the biofilm. The microbial cells attach, grow and detach until an equilibrium reaches between the planktonic and biofilm cells (Yin et al. 2019). The affixing of the microbial cell in the biofilm prevents biomass washout and allows better growth due to increased bioavailability of resources. This leads to increased biomass holdup and homogenous distribution enabling the system to withstand higher hydrodynamic process conditions. The higher cell density imparts process stability, shock load resistance and thereby enhanced outcome (Pasupuleti et al. 2014). The biofilm potential of extremophiles has not been adequately explored. However, EPS production by various extremophiles is appropriately enumerated. The ability to self-immobilization using the naturally produced EPS, the microorganism and its EPS characteristics makes it a potential area for investigation. This chapter is a comprehensive outlook on the biofilm formation by extremophiles and its characteristics assessed by available analytical tools, thereby deliberating the potential applications of extremophilic biofilm in various segments.

2. Extremophiles in Biofilm Formation and their Advantage Extremophiles synthesize EPS in planktonic state and biofilm in the sessile state to survive the extreme ecological condition that they exist in. The protective covering helps to balance fluctuations in the extremities to maintain optimal growth. This property has been used for biotechnological production and enhancement of EPS. Extremophiles are classified according to the extreme condition in which metabolically and biochemical functions are optimum such as thermophile, acidophile, alkaliphile, halophile, psychrophile, pedophile, radiophile. Both Bacteria and Archaea domains contribute to prokaryotic extremophiles. Polyextremophiles can optimally function under two or more extreme conditions like thermoacidophile, haloalkaliphile, psychropiezophile, etc. Polyextremophiles are common between halophile, psychrophile, barophile microbes due to their common source of existence, i.e., marine origin. Many investigations on EPS production from extremophile projects the immense industrial prospects of extremophilic biofilms (Table 11.1).

2.1 Thermophiles Thermophiles had been mostly isolated from hot/geothermal springs spread across the world. Many are facultative thermophiles that can tolerate temperatures up to 50ºC, while obligate thermophiles like Bacillus sp., Geobacillus sp., Brevibacillus sp., and Aeribacillus sp. can grow at a temperature range of 55–65ºC. Extreme thermophiles like in genus Thermus can optimally grow

Table 11.1. Recent investigations into the extracellular polysaccharide production by extremophiles, its characteristics and bioprocess condition. Microorganism

Source of Isolation

EPS Conc. (gL–1)

Carbon and Nitrogen Source

Monosaccharide Profile1

Geobacillus tepidamans V264

60

0.111

Maltose/(NH4)2 HPO4

Glucose, galactose, fucose, fructose (1:0.07:0.04:0.02)

1000

280ºC

(Kambourova et al. 2009)

Aeribacillus pallidus 418

55ºC pH 7.0

0.095

Maltose/NH4Cl

EPS1-Mannose, glucose, galactosamine, glucosamine, galactose, ribose (1:0.16:0.1:0.09:0.07:0.06:0.04) EPS2-Mannose, galactose, glucose, galactosamine, glucosamine, ribnsose, arabinose

700

Thermostable at 226ºC

(Radchenkova et al. 2013)

Behaved as Newtonian fluid, high viscosity with increasing Ca2+

(Yasar Yildiz et al. 2014)

Hot springs

Brevibacillus thermoruber 423

Molecular Property Weight (kDa)

Reference

100

55ºC, pH 6.5

0.863

Maltose/peptone

Glucose, galactose, mannose, galactosamine, mannosamine (1:0.3:0.25:0.16:0.04)

n.a

Brock medium/ tyrptone

glucose, galactose, mannose and N-acetylglucosamine

n.a

n.a

(Koerdt et al. 2010)

–

N-acetylgalactosamine, galactofuranose, galactopyranose (1:1:2) (1:0.5:0.5)

500

Imunomodulatory effect

(Lin et al. 2011)

Sucrose

Fructose, fucose, glucose, galactosamine, mannose (1.0:0.75:0.28:tr:tr)

1000

Thermostable at 240ºC

(Spanò et al. 2013)

Marine broth and Lactose

Xylose, glucose, arabinose (1:1.57:3.72) (1:0.27:0.42) Glucose, arabinose, xylose, mannose

80 (avg.)

n.a

(Sardari et al. 2017)

Maltose/yeast extract, tryptone, NH4Cl

Mannose, glucose, galactose, xylose, rhamnose (1:0.97:0.2:0.18:0.17

–

–

(Atalah et al. 2019)

Sulfolobus Acidocaldarius DSM639

n.a

76ºC, pH 3.0

Thermus aquaticus YT-1

n.a

60ºC

Bacillus licheniformis T14

Shallow hydrothermal vent

55ºC, pH 8.0

Rhodothermus marinus DSM4252T Rhodothermus marinus MAT493

Shallow marine hot spring

65ºC

Anoxybacillus and Staphylococcus

Corroded aeroplane engine

–

0.366

8.82 13.72

50ºC

–

–

Table 11.1 contd. ...

Extremophile: Biofilm Behavior, Characterization and Economical Applications 245

Extreme Condition

246

...Table 11.1 contd. Source of Isolation

Extreme Condition

EPS Conc. (gL–1)

Carbon and Nitrogen Source

Monosaccharide Profile1

Salipiger mucosus strain A3T

Hypersaline soil from solar saltern

32ºC, 7.5% salinity

Halomonas almeriensis M8T

Soil from coastal region

Vibrio neocaledonias NC470

Molecular Property Weight (kDa)

1.35

MY medium with glucose

Mannose, glucose, galactose, fucose (1:0.58:0.97:0.39)

250

Low viscosity, psuedoplastic behavior and emulsifying

(Llamas et al. 2010)

32ºC, pH 7.0, 7.5% salinity

1.7

MY complex medium with glucose

Mannose, glucose (1:0.43) Mannose, glucose, rhamnose (1:0.38:0.007)

15

Low viscosity, psuedoplastic behavior and emulsifying and chelating capacity

(Llamas et al. 2012)

Biofilm on a marine nonvertebrate animal

35ºC, pH 7.0, salinity –50 gL–1

2.0

Glucose+Zobell marine broth

Glucose, N-acetyl glucosamine, N-acetyl-galactosamine, galacturonic acid, glucuronic acid (1:0.94:0.69:0.19)

672

n.a

(Chalkiadakis et al. 2013)

Pantoea sp. BM39

Sea water

28ºC, 40% NaCl

20

Glucose

Homopolymer of glucose

830

n.a

(Silvi et al. 2013)

Alteromonas macleodii PA2

Water sample form sea

30ºC

23.4

Zobell marine broth, lactose

n.a

n.a

n.a

(Mehta et al. 2014)

Labrenzia aggregate PRIM-30

Deep sea

32ºC, pH 7.2, 7.5% salinity

0.84

MY complex medium

Glucose, arabinose, galacturonic acid, mannose (1:0.08:0.07:0.04)

269

Emulsifying and antioxidant properties

(Priyanka et al. 2014)

Halomonas stenophila HK30

Soil from saline wetland

32ºC, 5.0% salinity

3.89

MY complex medium with glucose

Glucose, glucuronic acid, mannose, fucose, galactose, and rhamnose (1:0.31:0.23:0.19:0.05:0.04)

82

Flocculating, emulsifying and biofilm forming ability

(Amjres et al. 2015)

Nitratireductor kimnyeongensis PRIM-31

Sub surface water

32ºC, 7.5% salinity

0.65

MY complex medium

Arabinose, glucose, xylose, glucuronic acid, galactose (1:0.53:0.32:0.2:0.15)

Antioxidant and ferric reducing capability

(Priyanka et al. 2016)

Pseudoalteromonas sp. MD12-642

Ocean sediments

28ºC, sea water

4.4

Glucose, yeast extract, peptone

Glucuronic acid, glucosamine, galacturonic acid, rhamnose (1:0.32:0.54:0.61)

n.a

(Roca et al. 2016)

6300

1400 90

≥ 1000

Reference

Extremophiles: Wastewater and Algal Biorefinery

Microorganism

Marine sediment

75% sea water

n.a

Glucose

Glucose, galactose, glucuronic acid (1:0.63:0.56)

Chromohalobacter canadensis 28

Salterns

30ºC, pH 8.5, 15% NaCl

0.172

Lactose, peptone

Glucosamine, glucose, rhamnose, xylose, unidenfied sugar (1:0.88:0.69:0.05:0.11)

Pantoea sp. YU16-S3

Offshore area

32ºC

4.28

Zobell marine broth

Mix culture

Acid mine drainage solution

⁓ pH 1.0

0.15

Antioxidant, metal chelating, antitumor capability

(El-Newary et al. 2017)

≥ 1000

High swelling behavior, emulsifying, stabilizing, and foaming capability

(Radchenkova et al. 2018)

Glucose, galactose, N-acetyl galactosamine, and glucosamine (1:0.53:0.21:0.01)

175

Thermal stability at 200ºC, shear-thickening Newtonian behaviour

(Sahana and Rekha 2020)

Acid mine drainage

DS1: Heptose, hexose, glucose, galactose, rhamnose, mannose, arabinose (1:0.90:0.71:0.43:0.46:0.42:0.04) DS2: Mannose, glucose, galactose, rhamnose, arabinose, hexose, heptose (1:0.79:0.26:0.81:0.19:0.89:0.55)

n.a

n.a

(Jiao et al. 2010)

Emulsifying activity, cryoprotective effect, heavy metal tolerance

(Caruso et al. 2018a)

0.34

37.6

4ºC, pH 7.0, 3% NaCl

202.34

Glucose, peptone, yeast extract

Glucose, mannose, galacturonic acid, arabinose, galactose, glucosamine, glucuronic acid (1:0.5:0.3:0.25:0.1:0.1:0.1)

n.a

4ºC, pH 7.0, 3% NaCl

453.95

Sucrose, peptone, yeast extract

Mannose, arabinose, galacturonic acid, glucuronic acid galactose, glucose, glucosamine (1:0.9:0.4:0.3:0.2:0.2:0.01)

n.a

Colwellia sp. strain GW185

15ºC, pH 6.0, 3% NaCl

0.3857

Sucrose, peptone, yeast extract

Glucose, mannose, galactose, galactosamine, glucuronic acid, galacturonic acid (1:1:0.7:0.7:0.3:0.04)

n.a

Shewanella sp. CAL606

4ºC, pH 7.0 3% NaCl

0.30613

Sucrose, peptone, yeast extract

Glucose, galactose, mannose, galactosamine, glucuronic acid, galacturonic acid (1:1:0.9:0.6:0.3:0.1)

n.a

Winogradskyella sp. CAL384

Winogradskyella sp. CAL396

Antarctic marine sponge

Table 11.1 contd. ...

Extremophile: Biofilm Behavior, Characterization and Economical Applications 247

Bacillus amyloliquefaciens 3MS 2017

248

...Table 11.1 contd. Source of Isolation

Extreme Condition

EPS Conc. (gL–1)

Carbon and Nitrogen Source

Monosaccharide Profile1

Pseudoalteromonas sp. MER144

Antartic sea water

4ºC, pH 7.0, 3% NaCl

0.319

Sucrose

Glucose, mannose, N-glucosamine, arabinose, glucuronic acid, galacturonic acid, galactose (1:0.36:0.26:0.06:0.06:0.05:0.03)

Pseudomonas sp. ID1

Marine sediment near Antarctica

11ºC

Glucose, peptone and yeast extract

Glucose, galactose, fucose (1:0.5:0.14)

n.a

Molecular Property Weight (kDa) 250

≥ 2000

Reference

Chelating activity

(Caruso et al. 2018b)

Emulsifying and cryoprotectant capability, psuedoplastic behavior

(Carrión et al. 2015)

n.a: not available, 1: The composition is normalized with respect to the monosaccharide with the highest composition, 2: mg/mg cell dry weight.

Extremophiles: Wastewater and Algal Biorefinery

Microorganism

Extremophile: Biofilm Behavior, Characterization and Economical Applications 249

between 65–80ºC, while hyperthermophiles like the Thermotoga genus are viable at temperature ≥ 80ºC (Kambourova et al. 2016). The EPS production for some thermophiles initiates in the early stationary phase. Geobacillus tepidamans strain V264 isolated from Velingrad Hot spring produced EPS in an early stationary phase with high molecular weight and stable at 280ºC (Kambourova et al. 2009). Meanwhile, spore-forming thermophiles like Aeribacillus pallidus 418, Geobacillus toebii 419, Brevibacillus thermoruber 425, Anoxybacillus kestanbolensis 415 (Radchenkova et al. 2013), Bacillus licheniformis T14 (Spanò et al. 2013) and Brevibacillus thermoruber 438 (Yasar Yildiz et al. 2014) synthesize maximum EPS during exponential phase. In Rhodothermus marinus DSM4252T and MAT493, the EPS production started during exponential phase using maltose as the carbon source and extended to the stationary phase using lactose with the stationary phase resulting in maximum EPS production from both the strains (Sardari et al. 2017). A recent study found the biofilm formed on a corroded airplane engine to be colonized primarily by Anoxybacillus sp. and Staphylococcus sp., when isolated at 50ºC (Atalah et al. 2019). Sulfobales species like S. acidocaldarius, S. solfataricus and S. tokodaii are acidohyperthermophile/crenarchaeal microbes investigated for their biofilm-forming ability. The microbes attained optimal growth at 76ºC and pH of 3.0–4.0, while maximum biofilm synthesis was at 60ºC and pH 6.0. Biofilm formation might have insulated Sulfolobus sp. from alkaline pH and provided the microenvironment for iron oxidation on the substrate. Biofilm formation was abolished for S. solfataricus at higher iron concentrations and pH as survivability was reduced. However, there was a 4 fold and 10 fold increase in biofilm formation for S. tokodaii and S. acidocaldarius, respectively, at pH 6 and 0.065 gL–1 iron with maximum biomass content sessile compared to total cell mass (Koerdt et al. 2010). Genotypic and phenotypic insights into thermophiles’ EPS production like Geobacillus WSUCF1 and Thermococcus eurythermalis A501 (hyperthermophilic, conditional piezophilic) can improve the prospects of biofilm aided biotechnological application of thermophiles (Wang et al. 2019, Zhao and Xiao 2015). EPS synthesis by these microbes has a high thermostability of 200–280ºC (acting as a thermoprotectant) and high therapeutic value (Lin et al. 2011, Kambourova et al. 2016) (Table 11.1). The biofilm formation at elevated temperatures can deliver a contamination-free and high biomass density process. Therefore, thermophiles’ growth associated EPS production can immobilize the microbes on the required substratum to derive their biotechnological possibilities.

2.2 Acidophile Particular categories of microorganisms, like acidophiles, can survive and grow optimally at pH ≤ 3.0 (Baker-Austin and Dopson 2007). These chemolithotrophs are found naturally in hyper acidic lakes, volcanic hot springs or anthropogenic sources like Acid Mine Drainage (AMD) (Merino et al. 2019). They grow using reduced inorganic sulfur compounds, metal sulfides, ferrous iron (Bellenberg et al. 2012). The biofilm formation by these acidophiles is an extensively investigated area. Acidothiobacillus sp. and Leptospirullum sp. are some of the iron/sulfur and iron oxidizing autotrophic acidophiles, respectively (Kay et al. 2014). These obligate acidophiles can resist low pH (1.0) of the acid mine drainage. The genomic analysis of a biofilm formed in AMD revealed Leptospirullum group II to be dominant and Leptospirullum group III to be a minority microbial community. The monosaccharide composition of the mid-developmental stage (DS1) and mature biofilm (DS1) was different. Metals present in the AMD also find presence in the biofilm with Fe as the major one (Jiao et al. 2010). Bellenberg et al. (2012) observed the induction of capsular polysaccharide production by ferrous iron-grown Acidothiobacillus ferrooxidans in the presence of pyrite, metal sulfide degradation product, glucose and phosphate limitation. However, biofilm formation by Sulfobacillus thermosulfidooxidan was initiated by regularly exchanging exhausted media for over a month as pH, ferric ion, phosphate starvation had no significant effect (Li et al. 2016). A recent study with Acidianus sp. DSM 29099 showed the attachment and biofilm growth to

250 Extremophiles: Wastewater and Algal Biorefinery be dependent on the type of substrate and that a large portion of the surface fails to be covered by the biofilm (Zhang et al. 2019). Extensive investigations have described the biofilm formation by acidophiles on metal ores. Chemotaxis leads acidophiles towards the substrate resulting in surface adhesion through cell surface glycoconjugate rich in fucose, mannose and glucose. EPS production gets initiated after the attachment forming a biofilm. This encapsulation provides structural and organization coherence among the community for the required functioning (Zhang et al. 2019). The quorumsensing system mediates cellular level communication, and thereby the structural and functional integrity of the biofilm. A. ferrooxidans possess the AfeI/R (qsI) quorum sensing system with afeI-orf3-afeR operon. The afeI gene produces N-acyl homoserine lactone synthetase (acyl-HSL, C8–C16) regulated by AfeR protein. Overexpression of qsI gene leads to increased EPS production and biofilm formation, while the deletion of the operon had an insignificant effect on the above traits. Meanwhile, the external addition of long acyl-chain HSL signaling molecules like C14-HSL increased A. ferrooxidans adhesion from a mixed community to pyrite inducing biofilm production (González et al. 2013, Gao et al. 2020). Besides quorum sensing, Acidithiobacillus thiooxidans biofilm had elevated intracellular second messenger, cyclic diguanylate (c-di-GMP), content and pel gene transcription level. The presence of a higher amount of c-di-GMP represses cell mobility and increases biofilm formation. c-di-GMP binds to the cytoplasmic side of the membrane protein, PelD increasing the glycosyltransferase activity (Díaz et al. 2018). Anaerobic sludge (mix culture) is an ideal source of a broad spectrum of microbial community which can be converged depending on the acclimatized condition. Tailored acid-tolerant consortia from anaerobic mix culture that can tolerate pH in the range of 3.0–12.0 have varied applications. The inherent EPS production for floc formation by anaerobic sludge can assist in biofilm formation with an appropriated substrate. Spore-forming acidogenic consortia were enriched from anaerobic mix culture under pH 3.0 for 24 hours for Volatile Fatty Acid (VFA) production from food waste (Sarkar et al. 2016). A recent study found Ethanoligenens harbinense dominating the anaerobic sludge inoculum in a fixed-bed reactor performing acidogenic digestion of sucrose at a pH ≤ 3.0. The biomass distributed between the biofilm on the bed and the suspended culture contributed to the reactor performance (Mota et al. 2018).

2.3 Alkaliphile To date, the maximum pH tolerance observed was for microbial communities belonging to Serpentomonas sp. under a pH of 12.5. The obligate alkaliphile isolated from the terrestrial serpentizing system had an optimal growth at pH 11.0. A moderately haloalkaliphilic Halomonas campisalis, isolated from a soda lake, had an optimal activity at a wide pH range (6.0–12.0). The source of alkaliphiles also extends to various sites across the world like hyper alkaline sediments from lime working sites, steel slag contaminated site, deep groundwater from granite formations, etc. (Suzuki et al. 2014, Merino et al. 2019). An anaerobic, hydrogenotrophic-methanogenic biofilm community dominated by Clostridia developed over cellulose cotton incubated at a pH of 12.0 in the anaerobic zone of lime kiln waste site (Charles et al. 2015). Similarly, alkaline to hyperalkaline condition is preferred for acidogenic digestion of solid wastes as alkaline pH enhances the hydrolysis and solubilization (Lee et al. 2014). Therefore, anaerobic mix communities diverted to biofilm can improve the process outcome at highly alkaline conditions. A mix culture in a cementitious disposal facility for radioactive waste synthesized EPS followed by floc formation to protect and sustain the microbes under high alkalinity up to 13.0. The flocs dominated by Alishewanella and Dietzia sp. were rich in mannose. The anionic surface of EPS (monosaccharides, protein, nucleic acid) prevents the migration of OH– ions to the hydrophobic core. This barrier exposes the microbes to an internal pH lesser by 1–1.5 units than the external (Charles et al. 2017). Irradiated graphite panels from a nuclear power plant developed a sub-surface biofilm dominated by Alcaligenes sp. and Dietzia sp. in an alkaline microcosm

Extremophile: Biofilm Behavior, Characterization and Economical Applications 251

maintained at pH 9.0 and 11.0, respectively. The biofilm was also able to retain 14C isotope from the graphite panels (Rout et al. 2018). These investigations point towards the advantage of EPS/biofilm formation on the survival of microbial communities under high pH and its implication in managing radioactive waste. Further studies are required to open up new avenues for biofilm formation by alkaliphiles.

2.4 Halophilic The hypersaline condition requires specialized organisms that can survive and grow. Biofilm production acts as an additional osmoprotectant to aid the survival of halophiles. However, the available literature regarding halophilic biofilms is scarce. Meanwhile, many researchers have documented EPS production by halophiles isolated from different marine sources thoroughly. This natural EPS production at their optimal growth condition makes halophilic biofilm a suitable option. Marine bacteria involved in EPS production primarily belong to Halomonadaceae, Alteromonadaceae, Rhodobacteracea and Vibrionaceae family under Gammaproteobacteria and Alphaproteobacteria (Llamas et al. 2012). Mostly explored and investigated halophiles for EPS production belong to the genus Halomonas like H. eurihalina, H. maura, H. ventosase, H. anticarinensis, H. almeriensis, H. smyrnensis, H. stenophila, H. xianhensis, etc. Generally, EPS production from Halomonas sp. is observed during the exponential growth phase (Béjar et al. 1998, Biswas and Paul 2017). The EPS production by halophilic microbes like Salipiger mucosus A3T (Llamas et al. 2010), Pantoea sp. YU16-S3 (Sahana and Rekha 2020), Nitratireductor kimnyeongensis PRIM-31(Priyanka et al. 2016), is also related to biomass growth, i.e., early exponential phase. These microbes can be considered as a better biofilm former, since the biofilm formation is associated with high biomass growth. Nonetheless, the EPS and substrate properties for attachment will also play a vital role in biofilm characteristics. Many recent studies had used a salinity of 7.5% (w⁄v) to elicit optimal EPS production (Table 11.1). Halomonas almeriensis M8T and Halomonas stenophila HK30 had optimal EPS production with a higher and lower molecular weight fraction at 7.5% salinity. The molecular weight influences the rheological property of the EPS (Amjres et al. 2015, Llamas et al. 2012). Similarly, Pantoea sp. BM39 strain produced maximum EPS at the highest salinity of 40% NaCl (Silvi et al. 2013), while Alteromonas macleodii PA2 had the highest EPS production (23.4 gL–1) by an extremophile in the recent period (Mehta et al. 2014). Some categories of halophiles start EPS production during the early stationary phase. Biofilm formation could be slower for these microbes with an imbalance of cell viability among the microbial community. This might have some controlling effects on the possible biotechnological application of these halophiles, like Alteromonas hispanica F23T, Idiomarina fontislapidosi F32T and Idiomarina ramblicola R22T (Mata et al. 2008), Labrenzia aggregate PRIM-30 (Priyanka et al. 2014), Vibrio neocaledonias NC470 (Chalkiadakis et al. 2013), Pseudoalteromonas sp. MD12-642 (Roca et al. 2016), etc. Some halophile like Pseudoalteromonas tunicate produces an autocidal protein (AlpP) during biofilm formation (carbon depletion). Biofilm from such halophile could be affected as the protein disperses phenotypically divergent and metabolically dynamic planktonic subpopulation (Mai-Prochnow et al. 2006). Similarly, longer exposure to chlorine resulted in the detachment of the biofilm formed by Pseudoalteromonas ruthenica on a glass slide using TrisG medium (Saravanan et al. 2006). The EPS produced by halophiles have low-high viscosity, emulsifying and metal chelating capability, as well as therapeutic implications (Table 11.1). Some special characteristics of certain halophile projects great importance in the therapeutic application (Kambourova 2018). Further research to exploit these varied features of halophile and halophilic EPS for biofilm application is required.

2.5 Piezophiles Piezophiles or barophiles are microorganisms that optimally grow above atmospheric pressure but under 60 MPa, while hyperpiezophiles can survive and thrive above 60 MPa (Kusube et al. 2017).

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Obligate piezophiles optimally grow at a pressure range of 10–50 MPa, while extreme piezophilic requires 50–100 MPa to survive. At the same time, most of the identified piezophiles are also psychrophilic. The common source of piezophile is at the deep seafloor, depth of 2000–5000 m (Kato et al. 1998). Shewanella benthica is one of the first piezophile to be isolated and identified from the Mariana trench, Challenger deep at a depth of 11 Km (Kato et al. 1998). The optimal growth for S. benthica Strain KT99 isolated from the Tonga-Kermadec trench, South Pacific Ocean (9856 m), was at 90 MPa and 10ºC (Lauro et al. 2013). Similarly, the psychropiezophilic characteristics of Colwellia sp., Moritella sp., Psychromonas sp., Dermacoccus abyssi MT1.1T had also been investigated (Kusube et al. 2017). The genomic analysis of thermopiezophilic Pyrococcus yayanosii CH1 at different hydrostatic pressure revealed upregulation of some clusters of CRISPRcas system, while others got down-regulated at 20 and 80 MPa pressure. A few studies had shown the role of cas genes in biofilm and spore formation as a response to stress (Michoud and Jebbar 2016). This genetic regulation indicates an EPS response to hydrostatic pressure. However, EPS or biofilm production by piezophiles is a potential area yet to be explored.

2.6 Psychrophilic Psychrophiles are adapted to survive and optimally grow at temperatures below 15ºC. Psychrotrophs or psychrotolerant grow optimally between 15–20ºC with the capacity to survive at low temperatures (Moyer and Morita 2007). These microbes are generally of marine origin sourced from deep sea sediments, the Arctic and Antarctic seawater and ice. Many Pseudoalteromonas sp. and Colwellia sp. have been isolated from these sources producing EPS concurrent with biomass growth upon adding carbohydrates as a carbon source. A decrease in temperature from its optimal condition triggers psychrophiles to scale up EPS production, thereby acting as a cryoprotectant to survive and grow in extremely cold conditions (Carrión et al. 2015, Caruso et al. 2018a, Qin et al. 2007). Meanwhile, EPS production in Colwellia psychrerythraea 34H increased in response to a decrease in temperature and an increase in pressure and salinity from the optimum condition of growth (Marx et al. 2009). EPS also provides stability to cold-adapted vital protease from autolysis (Qin et al. 2007). The EPS produced by Pseudoalteromonas strain SM20310 at 15ºC imparted a cross-cryoprotection to the co-cultured Escherichia coli indicated by its improved freeze-thaw tolerance (Liu et al. 2013). The psychrophilic EPS possesses properties like emulsifying ability, metal chelating effect, etc., (Caruso et al. 2018a). The cryoprotective-biofilm formation aided the microcolonies of Acidithiobacillus ferrivorans ACH to attach and grow over pyrite under extreme pH and temperature of 1.7 and 4ºC, respectively, enabling pyrite mineralization at 10ºC, though slower at low temperatures (Barahona et al. 2014). Therefore, the psychrophilic biofilm has greater untapped potential for therapeutic and industrial application.

2.7 Radiophile Few microbial communities grow optimally under radionuclide or radioactive environments like soil with radionuclide deposits, volcanic hot springs, nuclear power plant sites, hot arid deserts, air particles, northern regions, etc. The EPS, secondary metabolites, like carotenoid pigments produced in response to radiation, impart tolerance and shield the microbial colonies from the radiation’s lethal effects. Meanwhile, radiotolerance in certain species belonging to Bacillus, Serratia, Arthrobacter, Pseudomonas, Rhodococcus, etc., employ mechanisms like bioaccumulation, biosolubalization, chelation, complexation, etc. (de Carvalho 2017, Enyedi et al. 2019, Shukla et al. 2017). These radiophiles can survive desiccation, high temperature, acidity and salt conditions, which could damage DNA or the protein. Hence, called polyextremophiles (Enyedi et al. 2019). Some of the prominent polyextremophiles reported are members of genus Deinococcus and Rubrobacter that can survive radiation dose higher than 25 × 103 Sv, while that of some Chroococcidiopsis sp. was 15 × 103 Sv (Shukla et al. 2017).

Extremophile: Biofilm Behavior, Characterization and Economical Applications 253

The dried cells of Deinococcus aetherius ST0316 (thickness ≥ 500 μm) exposed to extreme temperature and radiation of space had survived (Yamagishi et al. 2018). Enyedi et al. (2019) identified extreme radiation-resistant genera of Paracoccus, Marmoricola, Dermacoccus and Kytococcus (after exposure to 15 kGy doses of gamma-rays) in the biofilms collected from spring cave walls and water surface having a high level of radium activity. Multiple glycoconjugates mediate the adhesion and biofilm formation by Deinococcus geothermalisī E50051 on glass and acid-proof stainless steel, thereby aiding in the resistance to multiple extreme conditions (Peltola et al. 2008). This capacity of the biofilm is evident from the enhanced survival of dried biofilm of three different strains of Chroococcidiopsis sp. exposed to low earth conditions by EXPOSE-R2 at the international space station for 2.5 years (Billi et al. 2019). Therefore, biofilm production improves the extraordinary resistance of radiophile against a plethora of harsh conditions, making them one of the toughest life forms on the Earth. Some environmental conditions related to the extreme conditions generate biofilm response, like a resistance mechanism against antibiotic and antibacterial components (Yin et al. 2019), for efficient use of scarcely available nutrients (Metcalf and Eddy 2003), to survive under low water activity (xerophiles) (Merino et al. 2019). Therefore, changing climatic and geotectonic conditions could generate different types of highly resilient microbes, which might have desirable characters that could find potential industrial application.

3. Microscopic Techniques in Biofilm Assessment As early as 1936, ZoBell and Anderson (1936) observed that the bacterial count in stored seawater increased significantly in the presence of attachment surface and was dependent on surface area per unit volume, oxygen concentration and the volume of liquid (de Carvalho 2018) since a significant fraction of natural bacterial population in seawater were sessile (ZoBell 1943). Further research continued on these lines, and it was in 1978 that the biofilm-theory was proposed (Costerton et al. 1978). The detailed understanding of the formation of biofilm (Landini 2009, Rabin et al. 2015), its maintenance (de Carvalho 2017, 2012, Gu et al. 2013) and its prevention (de Carvalho and da Fonseca 2007, De La Fuente-Núñez et al. 2012) are of immense importance and are extensively studied by different groups. Under the clinical condition, it is a hazard (Donlan 2001, Gilbert et al. 2002) and needs to be understood more for the point of preventing its formation and progression (Donlan and Costerton 2002, Hall-Stoodley et al. 2004, Høiby et al. 2010, Parsek and Singh 2003). From the environmental perspective, biofilm formation has both desirable and nondesirable aspects. It is undesirable when formed in drinking water supply pipelines, which causes corrosion, resulting in a reduced life span of pipes; compromised quality of the water with enhanced odor, decreased dissolved oxygen, change in taste, and color (due to bacterial action) (Characklis and Marshal 1990). Microbial biofilms in drinking water pipelines might become sources for transmitting antibiotic resistance (Balcázar et al. 2015, Schwartz et al. 2003). The submerged surface of ships and boats develop microbial biofilms, which led to the deterioration of the construction material (Kavitha and Raghavan 2018) due to biocorrosion (Little et al. 2008) and biofouling (Cao et al. 2011) enhancing the operation (Schultz et al. 2011) and maintenance cost (Lobelle and Cunliffe 2011) for many establishments like aquaculture (Fitridge et al. 2012, Floerl et al. 2016), maritime transport (Fernandes et al. 2016), water desalination (Maddah and Chogle 2017) as well as oil and gas industry (Skovhus et al. 2017). Microbial biofilms on the inert metal surfaces help in metal polishing (Das et al. 2012) while biofilm reactors in wastewater treatment enhance the shelf life and performance of the system (Biswas et al. 2019, Halder et al. 2020, Qaderi et al. 2011, Ray Chaudhuri et al. 2016b, 2016a, Saha et al. 2018). Interference Reflection Microscopy (IRM) has also been used to investigate biofilm (Marshall et al. 1989). Bacterial biofilm formed on crystals of zinc selenide and germanium has been detected

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Extremophiles: Wastewater and Algal Biorefinery

by Fourier Transform Infrared Spectrometry (FTIR) (Mattila‐Sandholm and Wirtanen 1992). Experiments with Nuclear Magnetic Resonance (NMR) (McLean et al. 2008) and Scanning Confocal Laser Microscopy (SCLM) (Shunmugaperumal 2010) have revealed important information on biofilms (Mattila‐Sandholm and Wirtanen 1992). Profilometer (Cross et al. 2009) and ellipsometer with Bruster Angular Microscopy (BAM) (Busalmen et al. 1998, Hoenig and Moebius 1991) are some sophisticated technologies, which measure the roughness, height and refractive index of the biofilm respectively. During biofilm formation, these technologies can also reveal the correlation between the cells (Bhushan et al. 2019). The extent of information about different biofilm aspects that can be obtained using some of the techniques mentioned above is detailed below. Simple biochemical methods of staining (Martín et al. 2008) (Fig. 11.1a and b) gives a quantitative measure of the biofilm thickness. This is done through quantifying the stain adsorbed on the biofilm, which is proportional to the thickness of the biofilm layer (Bhushan et al. 2019, Martín et al. 2008). A similar biofilm could also be visualized under the light microscope to visualize the cells embedded in the matrix, quantified using the biochemical assay. A further detailed structural analysis of these cells’ morphological identification could be done through SEM analysis of the biofilm in the environmental mode followed by Field

Fig. 11.1. A 24 well microtiter plate epiphytic biofilm formation with and without addition of leaf phytochemical (a) (Bhushan et al. 2019). (b) biofilm progression with time by Bacillus sp. (DebRoy et al. 2013) measured at 620 nm. Fluorescent microscopic image (Fluorescent In situ Hybridization FISH) of epiphytic biofilms in absence (c) and presence (d) of phytochemicals showing selective biofilm stimulation by leaf phytochemical on epiphytic microbes (Bhushan et al. 2019).

Extremophile: Biofilm Behavior, Characterization and Economical Applications 255

Emission SEM. The gradual development of a biofilm on uniform surface can also be visualized through SEM and compared with the biochemical data (Fig. 11.1b). The disruption of the biofilm in response to antibiofilm agents can also be visualized using a similar approach (Ray Chaudhuri et al. 2021, Sarkar et al. 2021), namely SEM analysis. Atomic Force Microscopy reveals greater details of the surface structure (Chakraborty et al. 2018) including bacterial biofilms (Wright et al. 2010). To get an idea about the density of the cells in the biofilm entrapped among the EPS matrix, Fluorescent In-situ Hybridization technique (FISH) could be employed. FISH could also facilitate in understanding biofilm progression with time (Fig. 11.1c and d). Fluorescent microscopy is also used for biofilm visualization (Harrison et al. 2014). Precise quantification of the biofilm’s depth with the extent of cell distribution in different layers could be assessed through scanning confocal laser microscopic analysis. SEM analysis of the biofilm at different magnifications helps us understand the impact of chemicals/phytochemicals on biofilm formation by bacteria (Bhushan et al. 2019, Ghosh et al. 2016). Leaf extracted phytochemicals were seen to stimulate biofilm formation by epiphytic bacteria. At the same time, inhibit the growth of non-epiphytic bacteria of the same bacterial genus, pointing towards the plants’ epiphyte selection mechanism (Ghosh et al. 2016). The initiation of biofilm formation is known to occur in response to certain stimuli, which could also be a kind of stress (Costerton et al. 1978). Adhesion of bacteria to solid surface starts after an hour of incubation, while macromolecules stick to the surface within seconds of immersion in liquid (de Carvalho 2018). Various compounds, biogenic carbon-rich components stick to the surface and are used by the bacteria for its growth and metabolism (Costerton et al. 1995, Dang and Lovell 2016, Furey et al. 2017). Figure 11.2a represents this state of biofilm formation. Figure 11.2b shows a well-established biofilm with EPS and macromolecules’ deposits, as expected during biofilm development under submerged conditions. Under stress, the bacterial cell responds by manifesting certain cytoskeletal changes like the elongation of cells to effectively decrease the surface area exposed to the pollutant, shortening of the cells when in a position to accumulate the pollutant further (like metal), arrested cell division, development of a woolly coat to prevent entry of further pollutants as well as secretion of thick layer of EPS in response to stress (Mishra et al. 2012, Ray Chaudhuri et al. 2010). EPS production is aptly demonstrated through Transmission Electron Microscopy (Fig. 11.2c, d) and so is heavy metal accumulation inside bacterial cells (Fig. 11.2c) the rest of the manifestations can be revealed through SEM analysis. These techniques are well documented and extensively used for biofilm assessment. To understand the formation of biofilm, surface structure and roughness are crucial parameters. Use of profilometry seems promising in investigation of biofilm formation as it provides topographical data. The variation in the height of the biofilm formed during its development can be assessed using this technique. A representative data of the biofilm formed by a Bacillus sp. (WBUNB004) from the environmental origin with simultaneous removal of nitrate and phosphate from wastewater is shown in (Fig. 11.3a, b). The image represents the roughness due to biofilm formation on the glass slide pieces of uniform dimension with time. The most interesting property of this technique is predicting the correlation between the cells during biofilm formation. The correlation is calculated through Hurst exponent computation (Bhushan et al. 2019). Figure 11.3c, d represents the Hurst exponent calculated using the profilometry and ellipsometry data. A value above 0.5 represents a positive correlation, which is a persistent behavior representing long-range dependence. It means that the biofilm forming behavior by the isolate has a definite trend, and it is possible to predict the system’s performance in the long run. However, for real-time biofilm measurements under moist conditions, refractive index-based assessment of roughness in terms of height measurement can be carried out using ellipsometry (Fig. 11.4) with Brewster Angular Microscopy (Fig. 11.5). Time series analysis (Hurst exponent calculation) of the generated data can be used for predicting the correlation between the cells during

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Fig. 11.2. Electron Miroscopy images of biofilm. Scanning Electron Microscope image during the initiation of biofilm formation (a) and with formed biofilm (b) by Bacillus sp. (platinum coated) (DebRoy et al. 2013). Transmission Electron Micrograph of biofilm (c) oil degradating Pseudomonas aeruginosa (GenBank Acc. No FJ788518), grown in the presence of silver nitrate solution bacterial, within matrix (Mishra et al. 2012) (d) (uranyl acetate stained) phosphate accumulating bacteria under high concentration of phosphate (DebRoy et al. 2013).

the formation of biofilm. Like the profilometry data, ellipsometry also reflects strong biofilm formation by the strain with persistent behaviors.

4. Economic Application of Extremophile Biofilm 4.1 Wastewater Treatment Biological processing during wastewater treatment is a principal component in environmental engineering. It has evolved gradually from activated sludge to biofilm-based processes (Pan

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Fig. 11.3. Images representing the roughness of the biofilm formed at a time 1 hour (a) and 10 hours (b) by Bacillus sp. WBUNB004 using profilometry and the Hurst Exponent calculation (1 hour to 10 hours) generated from (c) profilometry and (d) ellipsometry (Bhushan et al., 2019).

et al. 2014). Extensive research (Davey and O’toole 2000, Lettinga et al. 1980, Lewandowski and Boltz 2011) has gone into evolving this transition due to the limitations of the activated sludge process, namely biomass sensitivity to variation in nature and concentration of pollutant (Clark Ehlers and Turner 2012) a large volume of sludge generation requiring multiple downstream processing steps before the discharge making the process laborious, cumbersome, energy and cost intense (Guzzon et al. 2019). The biofilm-based system has a higher resistance to toxins in wastewater, protects the cells in the biofilm (Quintelas et al. 2011) and enhances performance due to the combined effect of adsorption (on EPS), biotransformation (by the living cells) by enhanced biomass in the biofilm. Genetic modification due to enhanced gene transfer within the biofilm’s diverse members may also be responsible for the biofilm reactor (Gieg et al. 2014, Horemans et al. 2013, Singh et al. 2006). Various types of biofilm reactors, namely Membrane Biofilm Reactor (MBR), Fluidized-Bed Reactors (FBR), Trickling Filter (TF), Moving-Bed Biofilm Reactor (MBBR) and Microbial Fuel Cells (MFCs) (Asri et al. 2018, Rodgers et al. 2003), with different carriers for immobilization (Asri et al. 2017, Jeong and Chung 2006, Kariminiaae-Hamedaani et al. 2003) were developed keeping in mind the properties/performance of the biological material used for bioremediation as well as the nature of wastewater (Leyva-Ramos et al. 2008, Shammas 2005). Recalcitrant pollutants

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1h

2h

3h

4h

5h

6h

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Fig. 11.4. Delta map representing the data extracted from the BAM images through ellipsometry representing the biofilm progression of Bacillus sp. WBUNB004 during its growth with time (1–10 hours) as the change in height (micron). The data shows the change in the biofilm thickness with time. For each hour, five different spots of uniform dimension were measures with a large number of readings within each spot. The time series was calculated using more than 1000 data.

get immobilized on the biofilm, diffuse to the cells increasing the pollutant’s bioavailability to the cells leading to more efficient bioremediation (Quintelas et al. 2010, Singh et al. 2006, Teitzel and Parsek 2003). A BOD:COD ratio of 0.5 or above makes wastewater suitable for biological treatment (Metcalf and Eddy 2003). Biological treatment is eco-friendly and often economical (Nilanjana et al. 2012), but the major problem is the slow performance, which often does not reach the discharge level as per the Environmental Protection Agency Norms. An appropriate selection of microbes with requisite combination with the physicochemical process can address the problem (Lin et al. 2012). It was assumed that wastewater with biodegradable COD below 1000 mgL–1 is suitable for aerobic treatment and those with COD of 4000 mgL–1 or more (Cakir and Stenstrom 2005, Chan et al. 2009) treatable anaerobically. However, of late, it is understood that the selection of anaerobic or aerobic system depends on the microorganisms selected for the treatment (Biswas et al. 2019, Halder et al. 2020, Saha et al. 2018). The first documented evidence of biofilm technology for wastewater treatment (trickling filters in England) dates back to 1893 (Lohmeyer 1957). Different biofilmbased systems have been deployed for organic, inorganic and refractory (heavy metals, textile dyes, petroleum, pesticides) compounds (Chen et al. 2008, Hai et al. 2015, Mitra and Mukhopadhyay 2016, Quintelas et al. 2013). Extremophilic microorganisms create a biofilm structure to survive in harsh environmental conditions, including in acidic or alkaline conditions and high or low temperatures. Hence, they are used to remove toxic pollutants from industrial wastewaters. However, available literature on extremophilic biofilm for wastewater treatment are limited to elucidate the relationships

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Fig. 11.5. Image representing the biofilm network progression with time (1–10 hours) formed by phosphate and nitrate removing Bacillus sp. WBUNB004 using Brewster Angular Microscope.

between extremophilic microorganisms and their bioremediation processes. The distribution of such microbial communities also gets restricted during the conventional biofilm-aided waste treatment. Under ambient or moderate conditions, extremophiles could get outperformed due to their requisite extreme condition for optimal growth. Hence the utility of extremophiles comes where a

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process needs to be handled under unique or harsh conditions. Such conditions could accelerate the treatment process, improves the system’s resilience against environmental perturbation, reduces contamination, etc. The biofilm system with extremophiles has shown advantages for the microbial electrochemical system, acidogenic fermentation, etc. Meanwhile, EPS characteristics from extremophiles projects their capacity to be implementable for remediation of heavy metal contamination, hydrocarbon degradation, treating electroplating and battery industry waste, acid mine drainage, etc. There is are few studies available to explain biodegradation of hydrocarbons, textile dyes and metals using extremophile microbes. Hydrocarbons originate from domestic and agricultural use of herbicides and pesticides. Extremophilic microorganisms play an important role in biodegradation in extreme environmental conditions and produce the necessary enzymes for hydrocarbon degradation. For instance, extremophile fungi can be used on the biodegradation of hydrocarbon in an extremely cold environment. Similarly, extremophile black yeasts can be adapted in harsh habitats and can tolerate various pH and temperature to achieve biodegradation of industrial wastes (Duarte et al. 2018, Giovanella et al. 2020, Park and Park 2018). The enzymes produced from extremophilic microorganisms are applied on textile dye to decolorize and detoxify textile dyes. The concentration of dyes in textile industry is high and contain hydroxy or amino functions. They are manufactured from either natural sources or synthetic compounds. Due to the characteristics of their hydrogen bounds, dyes have low solubility and generally contain heavy metals and high salt. Extremophilic microorganisms can tolerate high salt concentration in the textile dye because of their adaptation capability to survive in saline environment. For instance, laccases and azoreductases in textile dye can be degraded by extremophilic microorganisms (Giovanella et al. 2020, Rossi et al. 2017). Heavy metal contamination in the environment is very common because of industrial advancement. Heavy metals are toxic to the environment and accumulate in the biota. Several extremophile microorganisms including Sulfolobus solfataricus, Laptospirillum ferriphilum and Metallosphaera sedula have the ability to degrade heavy metals. Extremophile fungi Aspergillus flavus and Sterigmatomyces halophilus were found to be successful for metal biosorption, precipitation and reduction. The performance of bioremediation by extremophile microorganisms of metals can be assessed by measuring the pollutant concentration before and after the remediation (Chatterjee et al. 2010, Takeuchi et al. 2001). Extremophiles have shown application in heavy metal removal under extreme conditions primarily through adsorption, biosorption, a reductive process like bioprecipitation, etc. Geobacillus sp. ID17 synthesized and accumulated gold nanoparticles when exposed to Au3+. Acidophiles like At. ferrooxidans, A. thiooxidands, L. ferrooxidans, etc., have shown efficient removal capabilities against heavy metals like U4+, Cr4+, As3+, Hg2+, etc. Halophile, like Halomonas nitroreducens 11ST, reduces selenite to elemental selenium, while arsenic and chromium were removed efficiently by Exiguobacterium sp. and Microbacterium sp. Nesterenkonia lacusekhoensis EMLA3 at high alkaline condition degrades azo dyes in the presence of heavy metals (resistance) Ni2+, Cr4+ and Hg2+. Similarly, several psychrophiles like Pseudoalteromonas sp. have also shown resistance to heavy metals. Many extremophilic EPS have metal-chelating properties (Table 11.1) that could allow the biofilm-aided system to improve metal removal (Orellana et al. 2018). Earlier, At. ferrooxidans was used to remove Cr, Ni and Li from wastewater produced during the recycling of a spent lithium primary battery. As a result, Fe2+ got oxidized to Fe3+ with a concomitant decrease in pH (Yoo et al. 2010). Remediation of oil spillage and related contamination is an area for the application of extremophilic biofilm. Acidithiobacillus and Sulfobacillus strains had shown the possibility of hydrocarbon degradation at low pH. Extremophiles make hydrocarbon degradation at high temperature (≥ 50ºC) as a feasible option, indicated by hexadecane degradation by Geobacillus

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thermoleovorans T80 at 60ºC. Similarly, microbes capable of degrading crude oils below freezing points are also reported. Oil spills and other petroleum-related leakages ceate havoc in marine and aquatic ecosystems. Here, hydrocarbon-degrading halophiles open up a new avenue in the effort to mitigate such disasters. Halomonas organivorans can degrade a broad spectrum of aromatic hydrocarbons. Halophiles like Exiguobacterium aurantiacum also show pesticide degradation. Halophiles with lignocellulosic material, chlorophenols, formaldehyde degrading capability have been reported. The N2-fixing capability of Mastigocladus sp. CHP1 at 60ºC indicates the possible biofilm aided remediation of nitrate contaminated industrial effluent (Orellana et al. 2018). Microbial Electrochemical System (MES) requires biofilm formation over the electrode surface, enhancing the electron transfer rate/electrocatalysis via conductive proteins from microbes to electrode and vice versa. An increase in thickness is proportional to electron transfer resistance due to increased mass transfer resistance that decreases the electrocatalysis (Rathinam et al. 2019). Extremophiles are ideal candidates to drive MES due to their EPS producing characteristics. Besides, the complexity of the process depends upon the type of effluent/waste treated. This caters to extreme conditions of operation and microorganisms that can survive these conditions. Complex wastes like a distillery, brewery, meat industry wastes, pathogenic waste or hot waste streams need a higher temperature of operation, requiring the service of thermophiles. Meanwhile, the pulp and paper industry, tanneries, textile industry, oil and gas, aquaculture waste requires functioning at high salinity or alkalinity, where halophiles perform better (Shrestha et al. 2018). Waste management through value-add product conversion had been considered a desirable option. Complex wastewaters and waste residues are being treated or minimized through biogas production, organic acid, ethanol, etc., production. In this respect, thermophiles and thermophilic conditions ensure improved kinetic rates, mass transfer rates, reduced viscosity contamination-free, better mixing, etc. A recent work by Mota et al. 2018 demonstrated a biohydrogen producing biofilm system on a fixed bed reactor working under pH ≤ 3.0. The valorization products from waste can also be tailored by adjusting the acidophilic condition and, thereby, the activity of acidophiles. Radioactive and related wastes also require microbes adapted to growing optimally under these conditions. Effluents from nuclear plants, radioactive mining sites, etc., could be potentially treated by radiophiles immobilized as biofilms. Biofilms of bacteria, fungi, yeast and algae were developed for wastewater treatment (Abzazou et al. 2016, Badia-Fabregat et al. 2017, Cong et al. 2014, Hoh et al. 2016). This opens to the possibility of a combined extremophilic biofilm system as well. The ideal candidates for biofilm development were slow growers that performed their function but with slow, sustained biofilm development so that the sloughing off would be delayed (Lee et al. 2006). A mixed population of biofilm was found to be useful in bioremediation. Biofilm photobioreactors (Kesaano and Sims 2014) with microalgae and bacteria are used successfully in municipal wastewater treatment, which functions by the algae producing the oxygen through photosynthesis and the bacterial community using this oxygen and producing carbon dioxide through respiration while releasing mineral nutrients through the degradation of organic matter. The algal community during photosynthesis utilizes these degradation products for its biomass growth. The nutrients (nitrate and phosphate) in the municipal wastewater are converted into biomass and treated water through this approach. This results in the reduction of chemical oxygen demand of the treated water. The produced algal biomass is a substrate for biodiesel production, bio-alcohol production, biofertilizer and an aquaculture feed (Ferreira et al. 2018, Rawat et al. 2011). A significant advantage of these algal biofilm photobioreactors is the reduction in biomass harvesting cost post-treatment, which accounts for up to 40% of biodiesel production cost (Miranda et al. 2017). Factors stimulating biofilm formation (Ham and Kim 2018) were assessed, and the information available has been

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implemented for bioreactor development (Asri et al. 2017, Hoh et al. 2016). Such extremophilic biofilm systems can be used for faster and more efficient treatment of some effluents produced in large quantities with a variable load of COD: the municipal wastewater (low COD), tannery, mining effluent, dairy effluent and petrochemical effluent. A few cases will be discussed in the following paragraph to emphasize the efficiency of the biofilm reactors in wastewater treatment. Some cases that explored microbes that could tolerate some harsh conditions for achieving the removal of certain pollutants are discussed in the following paragraph. The removal efficiency of some pollutants is improved using specific extremotolerant microbes. Soluble sulfate reduction from 1600 mgL–1 (influent concentration) could be achieved in an anaerobic biofilm reactor within 3.5 hours (below 500 mgL–1) using a well-characterized bacterial consortium composed of Desulfovibrio sp., Clostridium sp., and bacterial enrichment clones (Nasipuri et al. 2010). The consortium was selectively enriched from wastewater fed aquaculture slurry from East Kolkata Wetland, Kolkata, India, in sulfate reducing bacteria-specific medium with lactic acid as the carbon source. The process was scaled up to 1.32 m3d–1 processing capacity using synthetic wastewater, mining and tannery effluent (Ray Chaudhuri et al. 2016a, 2015). The system ran continuously for 18 months in the laboratory setup before being discontinued. In order to make the system self-sustainable, a two-reactor setup was designed in which the first reactor produced lactic acid from where water using a novel lactic acid bacteria in biofilm, and the lactic acid, in turn, became the carbon source for the second reactor in series with SRB immobilized on the matrix (Ray Chaudhuri and Thakur 2013). Modified minimal medium development have been carried out to make the technology’s adoption at the industrial scale economically viable (Chanda et al. 2020). The case is similar for refractory petrochemical wastewater with high COD/BOD consisting of components like benzene, toluene, ethylbenzene, xylene (BTEX), volatile organic compounds (VOCs), dissolved minerals, phenols, polycyclic aromatic hydrocarbons along with emulsified oil. This makes the petrochemical industry effluent fall in the red category of pollution. The existing physicochemical process (Ebrahimi et al. 2010, Fajardo et al. 2017, Farajnezhad and Gharbani 2012, Lin et al. 2001, Painmanakul et al. 2010, Piekutin and Skoczko 2016) alone cannot cause the remediation. Appropriate eco-friendly technologies (Medina-Bellver et al. 2005, Oliveira et al. 2013, Qaderi et al. 2018, 2011, Rusten et al. 1999, Wessman et al. 2004) have been developed with Proteobacteria, Firmicutes, Sphingobacteria as the principal players (Floodgate 1995, Geiselbrecht et al. 1996, Head et al. 2006, Yang et al. 2012). Petrochemical wastewater treatment has been extensively using MBBR (Campo et al. 2016, Dong et al. 2011, Hatika Abu Bakar et al. 2018). Laboratory scale bioreactor with activated sludge and MBBR in series gave 97% BOD removal from petrochemical wastewater under the ambient condition within 23 hours with aeration at 3.33 Lmin–1. Though it was rapid, there was sludge generation (Qaderi et al. 2018). To develop a rapid sludge free bioremediation system (Ray Chaudhuri et al. 2016b, Saha et al. 2018) the microbial community of petrochemical ETP sludge was isolated combined into a consortium. The consortium, at the industrial level of 12 m3day–1, attained 96% BOD removal within 18 to 20 hours at the ambient condition with two MBBR in series with 1/3rd surface area (unpublished, communicated data) of that reported by Qaderi et al. (2018). The treated water was suitable for gardening purposes. The MBBR has been running since 2016 in the industry without any breakdown with one-time charging (Fig. 11.6a, b).

4.2 Value Added Products EPS metabolism gets upregulated as a response to the extreme condition and its fluctuation, which acts as a protective clothing encapsulating microorganisms. Carbohydrate, protein, and nucleic acid present in composite polyglycan transmit different attributes to EPS depending on the type and

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Fig. 11.6. Three moving bed biofilm reactors (MBBR) in series at the ETP (a). Photograph of the top view of one MBBR (b).

relative composition. EPS from extremophiles possesses many properties like emulsifying ability, metal chelating capability, viscosity, cryoprotectant, osmoprotectant, thermal stability, antioxidant immunomodulatory, antitumor, anti-inflammatory, antiviral, etc. (Table 11.1). These characteristics have enabled various application of extremophilic biofilm and the potential influence in different industrial and biomedical sectors. Several value-added compounds, like acids, enzymes, alcohols, secondary metabolites, gases, etc., have been produced using extremophilic biofilm in different reactor configurations (Germec et al. 2020). The EPS produced by extremophiles contains some rare and pharmaceutically valuable monosaccharide components. Fucose is one such uncommon carbohydrate present in EPS. Fucose has high potential in the pharmaceutical and cosmetic industry, even though it a hard to produce carbohydrate (Carrión et al. 2015). Glucuronic acid is a common component of extremophilic EPS having potential use in surgery, tissue engineering, therapeutic drugs, etc. (Roca et al. 2016). The biofilm-based process enables continuous production with a higher yield of such valuable carbohydrates due to increased cell density and efficient bioseparation (Germec et al. 2020). One of the most strategic uses of extremophilic biofilm is in biohydrometallurgy. Acidophilic/thermoacidophilic, iron-oxidizing/iron-sulfur oxidizing bacteria and archaea catalyze the process. These specialized microbes enhance the extraction of minerals at favorable conditions of extremely low pH and high temperature. They adhere to the surface of metal ore, like pyrite followed by EPS production resulting in biofilm formation. The biofilm microenvironment generates the oxidizing reagents, Fe3+ or H+ (acidic) for mineral solubilization through the oxidation of Fe2+ or reduced inorganic sulfur compounds. The minerals get extracted from their ores through several corrosion pits formation. This bioleaching or biooxidation is advantageous for low-grade ore where the mineral content is lower for which conventional extraction is expensive. Copper, uranium, gold, zinc, nickel and cobalt are the usual targets for bioleaching (Kaksonen et al. 2018, Zhang et al. 2019). Similarly, Leptospirillum ferriphilum dominated biomass that grew on jarosite particles on continuous aeration enhanced the bioleaching of iron through FeSO4 production (Germec et al. 2020). Production of some value-added products enhances through extreme conditions depending on the use of extremophile or its enrichment from mixed sources. Biohydrogen is one of the highly valuable next-generation fuels. It is a by-product evolved during the production of industrially relevant VFAs like acetic acid, propionic acid, butyric acid, etc., through acidogenic fermentation using a mix as well as a pure culture. A pH lower as 3.0 (acidophile) and high as 12.0 (alkalophile) facilitates acidogenic bioconversion. The self-immobilization by anaerobic sludge (pretreated

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or untreated) or inducing biofilm formation in pure culture on a preferred surface had been implemented for acidogenic fermentation (Lee et al. 2014, Mota et al. 2018). Thermophiles promoted the enhancement of VFA and hydrogen production used under moderate to hyperthermophilic conditions. Similarly, extremophiles support the working of Microbial Electrochemical Systems (MES) under harsh physiochemical conditions, thereby suppressing methanogenic activity, improving current generation and value addition in the form of metabolites, hydrogen, etc. In MES, electroactive microbes attach and develop biofilm over anodic electrode enabling direct electron transfer via conductive protein between microorganism and electrode, leading to an increase in the electrocatalysis. Halophiles and thermophiles have found huge potential in this direction (Rathinam et al. 2019, Shrestha et al. 2018). Biogas production is one of the primary value-added products with methane as a major component. The thermal and alkaline condition increases substrate solubilization. However, biomethanation requires strict buffering of the system to enable carbon transfer to methane as methanogens are active in a narrow pH range of 6.8–7.5 (Lee et al. 2014). In this context, a haloalkaliphilic anaerobic consortium collected from soda lake sediment shows high promise. This polyextremophilic consortium was able to improve biogas production from the digestion of Spirulina at pH 10.0 and 2 M Na+. The alkaline medium also acts as CO2 and H2S scrubber, thereby enriching biogas with more of CH4 (Nolla-Ardevol et al. 2015). Simultaneously, higher temperatures (50–70ºC) have shown to improve biogas yield by accelerating metabolic and bioprocess rates (Westerholm et al. 2018). Thermotoga maritime, a hyperthermophilic EPS producing bacteria, had increased cell density when co-cultured with Methanococcus jannaschii. The hydrogen transfer (increased acid production) in the system improved methane production (Kambourova et al. 2016). The self-biofilm-forming capability of extremophilic or mix culture microbes can be of advantage for scaling up biogas production. Biofilm produced by extremophiles also acts as an anticorrosive agent. Various non-sugar moieties like uronic acid, sulfate groups, protein, nucleic acid create a negative charge over the biofilm, enabling them to bind to positively charged metal ions. This chelating ability of extremophilic EPS is well understood. Some studies had shown the chelating ability of marine extremophiles to be in the order Fe > Cu > Zn > Cd > Hg (Caruso et al. 2018b). The EPS produced by Vibrio neocaledonicus KJ841877 exhibited a corrosion inhibitory activity of 95.1% for the carbon steel exposed to seawater for 5 hours. The strong binding affinity led to the formation of a compact absorbed film of Fe-EPS complex on the surface, acting as a barrier to oxygen and other reactive species from penetrating, hindering anodic/cathodic reaction and immobilizing the generated Fe3+ (Moradi et al. 2018). Similarly, biofilm produced by alkalophiles had shown to prevent the leakage of radioactive 14C from irradiated graphite moderators from nuclear plants (Rout et al. 2018). Extremolytes are compounds released by extremophiles to adapt to extreme and stress conditions acting as a protection towards cells. Extremolytes prevent damage to protein and nucleic acid (structural and chemical integrity) from UV radiation and Reactive Oxygen Species (ROS), desiccation, osmotic imbalance, heating, freezing, etc. Ectoine, mannosylglycerate, α-diglycerol phosphate, mycosporin like amino acid, carotenoids, etc., are some of the highly investigated extremolytes. Due to the protective characteristics, extremolytes have extensive biotechnological and industrial application. It is being used in skincare and other cosmetic products, the food and beverage industry, for protein and nucleic acid stability, as immunomodulators, antioxidants and pharmacopoeial precursors for pharmaceuticals, etc. (Lentzen and Schwarz 2006, Raddadi et al. 2015). Biofilm formation can be used for optimal bioprocessing for pilot as well as industrial-scale production to meet the demand of extremolyte. The growth-associated EPS production capability of extremophiles can assist in the natural encapsulation and immobilization of the cells on a suitable surface. Biofilm formation could retain a higher amount of biomass per unit volume with active cells enabling more straightforward bioseparation/downstream processing and better hydrodynamic

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working conditions. In this way, biofilm formation on a polyurethane foam enhanced 1,3-propanediol production from crude glycerol by immobilizing Pantoea agglomerans DSM 30077 (Germec et al. 2020). The unique characteristics of some of these metabolites provide the extremophilic biofilm as a biofertilizer role. The rhizoinoculated halophilic Kocuria flava AB402 and Bacillus vietnamensis AB403 formed a biofilm on the roots resulting in adsorption/intracellular sequestration of arsenic from soil and production of phytohormones like indole acetic acid/siderophore under salt stress. This effect promoted the growth of rice seedlings (Mallick et al. 2018). The EPS produced by extremophiles, especially halophiles, is reported to possess immunomodulatory, anticancer, anti-inflammatory, tissue regeneration, etc. The biofilm produced by extremophiles has potent use in biomedical applications. The sulfate and carboxylate groups in EPS provide a net negative charge enabling interaction with the cationic amino acids of cell proliferative proteins such as fibroblast growth factors, cytokines, etc. This property makes the biofilm an excellent candidate for scaffold for cellular regeneration, wound healing. Antioxidant activity of the biofilm could aid in cellular migration (Priyanka et al. 2016). The EPS has also shown antiproliferative activity towards the glioblastoma cell line. Over-sulfated EPS also induced selective apoptosis of malignant T-cell (Ruiz-Ruiz et al. 2011, Priyanka et al. 2016). This suggests the anticancer potency for the biofilm scaffold. The antiviral activity of EPS produced due to the induction of specific cytokines also points towards the broad scope of extremophile biofilm in a medical scenario (Arena et al. 2006). Halopsychrophilic biofilm can also be used as a cryoprotectant. It can minimize the damage caused by repeated freezing and thawing. This becomes of particular use where cold storage is vital for preservation as the EPS imparts protection to extracellular proteins as well as co-cultured microbes against the cold environment (Qin et al. 2007, Liu et al. 2013). The biofilm has a cryoprotectant that can find application in food and beverage industry, pharmaceutical and medical application, molecular and microbial works, and other uses requiring cold temperature for operation. The radiophilic biofilm enhances the extraordinary survival of radiophiles in different harsh conditions. The biofilm growth Deinococcus geothermalis DSM 11300 had better survival but reduced culturing ability compared to the planktonic grown cell when exposed to a mixture of simulated space and martian like harsh conditions (Frösler et al. 2017). A recent study showed the enhanced survival of a dried biofilm of three different Chroococcidiopsis sp. strains in low Earth orbit due to excess dried EPS encapsulating the cyanobacteria (Billi et al. 2019). Thus, radiophilic biofilms are ideal candidates for extra-terrestrial applications like space missions. The attachment of radiophiles and biofilm growth on radionuclide ores can assist in biomining nuclear power plant ingredients like uranium, thorium, etc. Deinococcus radiodurans R1 metabolically engineered to harbor a nonspecific acid phosphatase encoding (phoN) gene maintained uranium bio-precipitation ability even after being exposed to 6 kGy of 60Co gamma rays (Appukuttan et al. 2006).

5. Conclusion The harsh environmental conditions spread across the planet are the convergence of highly resilient and functionally versatile extremophiles that can survive and grow normally in varied conditions. Extremophiles can produce EPS naturally as additional support to survive in harsh conditions. These EPS impart functions like cryoprotection, osmotolerant, anti-desiccant, thermostability, metal chelation, etc. Extremophilic EPS have high value for industrial and medical applications. The EPS production diverted to biofilm formation with a suitable matrix would improve their survivability and functioning. Extremophiles have substantial industrial importance in areas like extremozymes, extremolyte, wastewater treatment, etc. Natural encapsulation and fixing of extremophiles in biofilm expand its application and improve outcomes. Biofilms of acidophiles and thermoacidophiles exponentially are highly explored for bioleaching and treatment of wastewater like acid mine

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drainage. Extensive studies on biofilm and its specific impact on the industrial process would further increase the current scope of extremophiles.

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12 Extremophiles for Textile Industry Shalini Singh,* Sujata Das and Charu Khanna

1. Extremophiles in Nature As per estimates, only about 1% of microorganisms, the most ubiquitous and the most diverse organisms on Earth, have been discovered as yet present in about every part of the blue planet. As per estimates only about 1% of the total microorganisms present on Earth have been discovered so far, and there still are unexplored habitats where, these omnipresent organisms may be present (Arora and Panosyan 2019, Rampelotto 2013). Of these, extremophiles, the largely unexplored group of microbes, distinctively associated with their ability to survive in extremes of environmental conditions, have been fascinating the scientific fraternity since long. Where other life-forms fail, extremophiles have been found to luxuriously thrive in highly acidic as well as alkaline conditions, high salt concentrations, high as well very low temperatures, high pressure conditions, etc. Their presence and ‘ease’ of growth in inhospitable environments, ranging from, but not limited to, toxic waste materials, heavy metals, etc., have all opened up potential avenues for using their metabolic abilities of such organisms for various industrial applications as well. The ability to adapt to multiple physicochemical conditions (polyextremophilic nature) among many extremophilic microorganisms, further adds to the importance of extremophiles as model organisms for industrial activities (Cowan et al. 2015, Cardenas et al. 2010, Navarro et al. 2013, Dopson and Holmes 2014, Sarethy et al. 2011, Arora and Panosyan 2019). In general, extremophiles belong to a complex group of organisms, owing to the high degree of phylogenetic diversity observed amongst the group’s members. Interestingly, certain groups include both extremophiles as well non-extremophiles (Rampelotto 2013). The members may be dispersed in the evolutionary tree, even if they are adapted to the same type of extreme environment, and certain members of the same family might be adapted to different extremes of environmental conditions. All these variables impressively add to the complexity of extremophiles, which is credited to enormously diverse genetic and metabolic abilities, complex and highly variable physiological abilities and existence of specialized metabolic activities (like those involving methane, sulfur, etc.) (Rampelotto 2013).

School of Bioengineering and Biosciences, Lovely Professional University, Punjab-144411. * Corresponding author: [email protected], [email protected]

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Often classified on the basis of their adaptive abilities or in other words, the conditions in which they grow, thermophiles/hyper thermophiles, acido/-alkali-philes, halophiles, barophiles, psychrophiles, have exhibited amazing abilities to survive (extremotolerant) and thrive (extremophiles) in high/extremely high temperatures, acidic/alkaline/xerophillic conditions, conditions (hot acids capable of dissolving even steel, alkaline fluids capable of use as floor strippers), including acid mine drainage sites, acidic lakes, alkaline lakes and deserts, high pressure conditions (as in autoclaves), high salt concentrations, low temperature, respectively. Apart from the above, extremophiles in deep-sea hydrothermal vents, solfataric fields, soda lakes, saline waters, hot and cold deserts, metals, nuclear waste, etc., (Rampelotto 2013, Nee 2007, Arora and Panosyan 2019, Oren 2013, DeMaayer et al. 2014, Kawamoto et al. 2011, Zhang et al. 2015, Johnson 2014, Orell et al. 2013) have been reported too. Few major genera of the extremophiles include, Halobacterium, Methanolobus, Methanococcoides, Halalkalicoccus, Halobiforma, Halorubrum, Natrialba, Natronococcus, Natronorubrum, Shewanella, Psychromonas, Photobacterium, Colwellia, Thioprofundum, Moritella, Thermococcus, Sulfolobus, Pyrococcus, Acidithiobacillus, Leptospirillum, Alicyclobacillus, Acidiphilium, Acidimicrobium, Ferrimicrobium, Sulfobacillus, Ferroplasma, Acidiplasma, Metallosphaera, Acidianus, Deinococcus, Bacillus, Rubrobacter, Kineococcus and the members of the family Geodermatophilaceae and cyanobacteria including, Nostoc and Chroococcidiopsis (De Maayer et al. 2014, Bowers and Wiegel 2011, Zhang et al. 2015, Johnson 2014, Dopson and Holmes 2014, Brim et al. 2003, Gtari et al. 2012, Bagwell et al. 2008, Gabani and Singh 2013). A number of adaptive features/mechanisms in extremophiles like, the presence of sturdy/stable and even unique proteins (mainly, stable enzymes) in organisms like, thermophiles and psychrophiles, well adapted and stable cell membranes and cell walls in cold/hot loving organisms, contribution of inorganic ions and compatible solutes to adapt to specific environments in halophilic organisms, existence of specific mechanisms to maintain internal pH in pH stable organisms, and a distinct combination of any of the above, help these organisms to adapt to their specific niches and environments. Further a unique genome and ability to multiply under extreme conditions, also characterize these organisms (Arora and Panosyan 2019). Though extremophiles spread through all three domains of life yet, most of the members of this group comprise of Archaebacteria. A wide range of excellent adaptive abilities to flourish in varying extremes of environmental conditions has been witnessed in members of the Archaea. Eubacteria, though not as a prominent contributor to the group, show adaptations to certain extreme environments as well. Cyanobacteria for example, offer comfortable growth in the presence of extremes of temperatures, salt concentration, xerophilic environment, high metal concentration, etc. Still, its adaptation to very low pH is not commonly reported (Rampelotto 2013). Among eukaryotes, tardigrade, both in hibernating as well as active form, is one of the most important extremophiles showing adaptability to various types of extreme environments including, extremes of temperatures, pHs, salt concentration, heavy metals, high pressures, radiations, etc. Overall, fungi exhibit great variability in adaptations to extreme environments like, acidity, metal concentrations, salt concentrations, but their response to high temperatures is not impressive as they cannot survive at very high temperatures (Rampelotto 2013). Thermophiles have attracted the most attention among the known extremophiles. These can be generally classified as those that grow best between 50–60ºC (moderate thermophilic organisms), between 60–80ºC (extreme thermophilic organisms), between 80–11ºC (hyperthermophilic organisms) (Rothschild and Manicinelli 2001). Extreme thermophilic organisms mainly include genera Bacillus, Clostridium, Thermoanaerobacter, Thermus, Fervidobacterium, Rhodothermus, Thermotoga and Aquifexi (Adams et al. 1995, Niehaus et al. 1999). Hyperthermophiles mainly include phyla, Crenarchaeota, Euryarchaeota, Korarchaeota and Nanoarchaeota (Adams et al. 1995, Niehaus et al. 1999). Thermophiles produce chaperonins which are thermostable proteins and proteins of thermophiles, denatured at high temperature, are refolded by the chaperonins, thus storing their native form and function. The cell membrane of thermophiles consists of saturated

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fatty acids, which increase protein core hydrophobicity and keep the cell rigid enough to survive at high temperatures (Sterner and Liebl 2001, Piardini et al. 2002). Moreover, hyperthermophiles have membranes containing lipids linked with ether to their cell walls, attributing to thermal stability of these membranes. In addition, proteins of thermophiles have increased surface charge and less exposed thermo-labile amino acids, exhibiting increased ionic interaction and hydrogen bonds, increased hydrophobicity, decreased flexibility and smaller surface loops (Sterner and Liebl 2001, Piardini et al. 2002). Psychrophiles are cold-loving or cold-adapted organisms found to inhabit environments of the Earth where temperatures never exceed 5°C. Many psychrophiles live in biotopes having more than one stress factor, such as low temperature and high pressure in deep seas (piezo-psychrophiles) or high salt concentration and low temperature in sea ice (halo-psychrophiles) (Cavicchioli et al. 2002, Margesin et al. 2003). These organisms span from Gram negative (like, Pseudomonas) to Gram positive (like, Arthrobacter), Archaea (like, Halorubrum), yeast (like, Cryptococcus) and filamentous fungi (like, Penicillium). Their ability to grown at low temperatures is attributed to different metabolic adaptations (Cavicchioli et al. 2002). Their proteins for example, show increased structural flexibility in the presence of low temperatures and thus, remain active (Margesin et al. 2003, Feller and Gerday 2003). The cell membranes of these organisms have more unsaturated fats as compared to saturated ones, which help in maintaining membrane functions under low temperatures. The ability to synthesize specialized enzymes and proteins which remain active even in very low temperatures (Feller and Gerday 2003), further helps these organisms to adapt to their environments. Cold-loving enzymes such as, lipases, amylases, cellulases, etc., from such organisms, have been exploited for industrial applications in detergent, textile, leather, food and juice, pharmaceutical industries successfully (He et al. 2004, Cavicchioli et al. 2002, Margesin et al. 2003, Onyshchenko et al. 2002). These enzymes have also shown promise in environment pollution control applications (Gomes and Steiner 2004). Metallophiles have the ability to grow in the presence of high metal concentrations, colonizing industrial sediments, soils and wastes with high metal content. Several members of the genus Ralstonia, are members of the group and they have adapted well to harsh environments of such kinds (Mergeay et al. 2003). Often associated with the presence of one or two large mega-plasmids containing metal resistant genes, members of these metal-resistant Ralstonia are able to grow in the presence of metals like zinc, cadmium, cobalt, etc. (Gomes and Steiner 2004). Many microorganisms like, Ralstonia, could be used for mitigating heavy metal pollution as such contamination that causes hazards to public health, aquatic life and wildlife (Valls and de Lorenzo 2002). The microbes can be employed to biosorb such polluting metals, or change/transform these harmful metals through enzymatic ativities or other biological reactions, into less harmful species/non-toxic species (Mergeay et al. 2003, Valls and de Lorenzo 2002). Alkaliphiles, falling under the mesophilic organism’s category, include alkaliphiles (need a pH of 8 or higher optimum being around 10 for their growth) and haloalkaliphiles (require pH 8 or more as well as high salt concentration to grow) (Wiegel and Kevbrin 2004). Exhibiting polyextremophilic characters, alkaliphiles growing optimially at pH 8 or above as well as high temperatures (50–85ºC) (alkalithermophiles) and even those requiring high salt concentrations for growth have been found (Gomes and Steiner 2004). The habitats of these organisms vary from alkaline hot springs, alkaline hydrothermal vents, to mildly acidic to even neutral habitats (mesobiotic) and even acidic environments, to extremely alkaline saline habitats (Wiegel and Kevbrin 2004, Horikoshi 1999). On the other end of the spectrum are the acidophiles, which are capable of growth at pH values as low as 0.7 (Gomes and Steiner 2004). Acidothermophiles on the other hand, not only grow in the presence of low pH but also high temperatures. Acidophiles and alkaliphiles use proton pumps to maintain their internal pH and hence, intercellular enzymes need not be adapted to low/ high pHs. While extracellular enzymes require to be stable under the above mentioned extreme environments (the mechanisms are not clearly understood) (Gomes and Steiner 2004). Apart from this, a number of adaptive mechanisms are adopted by these organisms to survive in extremes of

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environments (Wiegel and Kevbrin 2004, Horikoshi 1999, Kar and Dasgupta 1996). Enzymes like, amylases, pullulanases, lipases, pectinases, peroxidases, etc., with huge commercial potential have been obtained from these organisms (Wiegel and Kevbrin 2004, Horikoshi 1999) and have been applied in detergent, leather, paper, textile industries in both upstream as well as effluent treatment processes (Wiegel and Kevbrin 2004, Horikoshi 1999, Maier et al. 2004). They are found luxuriantly growing in salt rich environments of the Earth including, Great Salt Lake, the Dead Sea, etc., halophilic microbes accumulate sodium or potassium chloride up to equilibrium conditions with the environment. Well adapted to up to 4 M KCL and over 5 M NaCl concentrations, the proteins of these organisms have amino acids that are stable and active at high ionic strength. With few exceptions (Mijts and Patel 2002), these proteins have a high concentration of acidic amino acids, which play a crucial role in reducing surface hydrophobicity and decrease the tendency to aggregate at high salt concentration (Mevarech et al. 2000). These proteins show instability in low salt concentrations but remain active in high salt concentration (Mevarech et al. 2000). These organisms use different ways to remain stable in high osmotic pressure conditions. Few adaptations include accumulation of K+ & compatible solutes (Danson and Hough 1997, Mevarech et al. 2000). Halozymes have shown promising potential in commercial applications like, in aqueous/organic and non-aqueous media (Marhuenda-Egea and Bonete 2002) (use of these enzymes in organic solvents is still limited (Sellek and Chaudhuri 1999). These enzymes in real time applications, as in the manufacture of chemicals and even in bioremediation, have shown immense promise (Marhuenda-Egea and Bonete 2002). Radiophiles are known to exhibit resistance to high levels of ionizing and UV radiations (Gomes and Steiner 2004). Deinococcus radiodurans, one of the most important radiophiles, shows high resistance towards chemicals, oxidative damage, high levels of radiation and even dehydration (Sandigursky et al. 2004). It is found to contain a wide array of genes that help it to repair DNA damage effectively. Other radiophiles like, Deinococcus radiophilus, Thermococcus radiotolerans, etc., are also important representatives of the group. These species especially, Deinococcus radiodurans has been used to detoxify nuclear waste materials as well as halogenated organic compounds, heavy metals, etc., from various types of mixed waste (Gomes and Steiner 2004). Peizophiles or barophiles, prefer high-pressure conditions for growth, and are found in world’s oceans (Abe and Horikoshi 2001). The enzymes from these organisms are found to be stable at high pressures and have been isolated from thermophiles as well as psychrophiles and have optimum growth above one atmosphere of pressure (Abe and Horikoshi 2001, Yano and Poulos 2003). Interestingly, microbes in deep sea are not exposed to pressures exceeding 120 MPa (Gros and Jainicke 1994), which means that their proteins need not exhibit specific pressure related adaptations (Gros and Jainicke 1994), Still, few reports indicate that some high-pressure related adaptations in proteins have been observed in these organisms (Pledger et al. 1994). The major genera include Shewanella, Colwellia, Moritella, Methanococcus, Pyrococcus and Thermus (Abe and Horikoshi 2001, Yano and Poulos 2003). Commercially, enzymes from such organisms can well be used in industrial applications where high-pressure operation conditions along with high temperature conditions prevail (Abe and Horikoshi 2001, Yano and Poulos 2003). Examples include, food processing like, food preservation units. Practical applications of these organisms and their products, though offer certain issues as the organisms are not easily cultivable (Gomes and Steiner 2004). Certain members from fungi, lichens and algae known as xerophiles, have the ability to grow in very dry conditions. Such organisms are often associated with spoilage of dried food items like, grains, nuts, etc., but their biocatalytic potential is still not well known, thereby limiting its applications for commercial exploitation (Madigan and Marrs 1997). The uniqueness of extremophiles makes them one of the best candidates for industrial applications as well as for investigating the working of extreme ecological environmental systems

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which in turn, throw important insights to bioflora of such ecological habitats as well the movement of different substances through biotic and abiotic factors (Arora and Panosyan 2019). A combination of methods including, both the conventional techniques of isolation/growth, as well as advanced and more sophisticated methods like, those based on molecular biology, have been successfully used to study and characterize extremophiles present in various environmental conditions (Rampelotto 2013, Egorova and Antranikian 2005, Ferrer et al. 2007). Being one of the ideal candidates to understand conditions existing through origin and evolution of life, extremophiles have provided interesting and significant data on ancient life forms, prevailing conditions in those times and even possible life conditions on other planets (astrobiology) as well (Arora and Panosyan 2019, Rampelotto 2013).

2. Extremophiles for Industrial Applications With enzyme applications forming an integral part of industrial sector worldwide, numerous sources of the same have been explored (Khajuria and Singh 2020, Singh 2018, Singh et al. 2011a, Singh et al. 2014a, Singh et al. 2011b, Singh et al. 2009, Singh et al. 2011). Among the microbial sources, extremophiles like, archaeabacteria, in comparison to bacteria and even fungi in the mesophillic range, form a small group of enzyme producers for industrial use. Interestingly, the applications of enzymes/metabolites from non-extremophiles often end up in becoming ineffective or suboptimal in performance when subjected to real time industrial conditions, which themselves are many times, harsh and difficult. This clearly indicates that extremophiles need to be extensively explored for potential uses in industries, as that would exponentially improve the use of such metabolites/enzymes in industries (Cardenas et al. 2010, Lopez-Lopez et al. 2014, Yildiz et al. 2015). The unique characteristic features of such organisms have made them all the more important, much beyond the ecological significance they hold, to their applications in industries of various kinds, which are much more promising than the use of mesophilic organisms and their products produced under such conditions. Their ease of adaptation to extremes of environment existing in various industries has easily made them more attractive in comparison to the mesophilic organisms. Use of such microbes in restoring polluted environments, producing industrial products under difficult cultural conditions, etc., is being explored worldwide. Their strong enzyme systems, specially, adapted to extremes of temperatures, salt concentration, pH, high pressure, etc., make them the favorites among organisms to be employed for industrial uses for processes, like those involving biodegradation and bioremediation, production of bioenergy, etc. Thus, extremozymes and their applications have been proven to be the most important contributors of biotechnology in the industrial sector. Further, as already highlighted, many of these microbes show adaptability and stability towards more than one type of extreme conditions. This enhances their applicability manifolds. The advent of biotechnological tools and their advancements thereafter have further helped in identifying and using these microbes in different ways (Arora and Panosyan 2019, Rampelotto 2013, Ferrer et al. 2007, López- López et al. 2014).

3. Extremozymes for Industrial Applications With the already booming industrial enzymes’ market, with over 3000 enzymes isolated from microbes, many of which, have already entered the market, the participation of extremozymes is bound to sky rocket industrial enzymes’ demand manifolds. The growing threat from diverse and increasingly dangerous pollutants, rapidly deteriorating the environmental quality and threatening our ecosystem, is further adding to the demand of extremozymes, worldwide. Thermophilic enzymes have attracted the most attention (Adams et al. 1995) as they are better suited for harsh industrial processes. As industrial processes conducted at high temperatures offer increased solubility of many polymeric substrates, resulting in decreased viscosity, faster reaction,

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decreased chances of microbial contamination, etc., thermozymes have become one of the most sought after enzymes in industries. Thermozymes like, Taq polymerase from organisms such as, Thermus aquaticus, Thermococcus litoralis and Pyrococcus furiosus are already known for their application in PCR based diagnostics. ‘Pretaq’, a protease, is another example of thermophilic enzyme which is used to cleanup DNA prior to PCR amplification and even thermolysin, again a protease, is applied in synthesis of peptides (Bruins et al. 2001, Jayakumar et al. 2012, Albers et al. 2006, Leis et al. 2013, Purcell et al. 2017). A genetically engineered strain of E. coli expressing a thermozyme coded by a gene isolated from thermophilic bacteria, Coprothermobacter proteolyticus, was found to exhibit multiextremophilic properties, with high thermostability in the presence of detergents, solvents, variable pHs. The enzyme well transmitted ability to bioremediation of solid waste (Toplak et al. 2013). A strain of Aeropyrum pernix has been used as source of thermostable phosphorylase which is being applied for synthesis of nucleoside analogues and used in antiviral therapy (Zhu et al. 2013). Amylases and other DNA processing thermozymes have also been reported (Bruins et al. 2001, Jayakumar et al. 2012). Produced from thermo-and hyper-thermophiles, thermozymes often exhibit stability against denaturing agents, solvents and even high salt concentration. Their use in industrial processes face lesser risk of contamination, lower viscosity and higher solubility of substrates. Thermostable phytases are also reported to be evaluated for use in animal feed for the release of digestible phosphorous (Eichler 2001, Haki and Rakshit 2003). Psychrophilic organisms are not far behind in popularity and utility, with a number of cold active enzymes being explored for industrial purposes including, those applied in production of fine chemicals or used as detergents and food applications (Cavicchioli et al. 2011). With activities at even very low temperatures and lesser cost intensive nature, exhibiting greater energy economy, as compared to thermozymes, cold-loving enzymes are a promising option for many industrial uses. Though obtained from cold loving, Halorubrum lacusprofundi, a β-galactosidase was found to be stable from −5 to 60°C, indicating its wide range of thermostability. It is expressed well even in high salinity, and in the presence of different types of organic solvents. These characteristic features suggest that it can well be used for production of sugars under low water activity as well as at different ranges of temperatures (Karan et al. 2013). Numerous whole-cell extremophilic biocatalysts and even isolated enzymes (extremozymes), like, hydrolytic enzymes, are being used in food industries, petroleum and energy industries, detergent and textile industry, pulp and paper industry, leather industry, biomining industry and many more (Hough and Danson 1999). Halophilic enzymes like the above, are active and maintain stability under low water activity and also organic solvents—a significant character of these enzymes for use in harsh conditions of a similar kind prevailing in certain types of industries (Raddadi et al. 2013, Datta et al. 2010). Thus, a stable market for enzymes like, amylases, esterases, cellulases, peptidases, lipases and xylanases already exist (Raddadi et al. 2013, Bhalla et al. 2013, Du et al. 2013, Elleuche et al. 2014). Raddadi and co-workers (Raddadi et al. 2013) isolated cellulases from Paenibacillus tarimensis, a halophile from the Sahara Desert, which was found to exhibit activity across a pH of 3–10.5, was stable even at a temperature 80°C and could even withstand high salt concentrations as well. The CMCase activity was maintained in the presence of organic solvents, detergents, heavy metals and even under highly alkaline conditions. Halophilic enzymes have been successfully applied for production of fatty acids for the food industry and production of biofuel (Litchfield 2011, Schreck and Grunden 2014). Li and co-workers (Li et al. 2014) reported efficient use of an extremotolerant lipase from a halophile for production of biodiesel from Jatropha. Applications of extremozymes from alkaliphilic bacteria in detergent and laundry industries have also been reported (Sarethy et al. 2011). Pressure-loving microbes have been characterized (Abe and Horikoshi 2001, Mota et al. 2013) but their potential industrial applications are rarely investigated (Zhang et al. 2015). Pressure loving and pressure tolerant organisms have been used for production and de-contamination of food at

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different pressure conditions. Lamosa and co-workers identified specific organic solutes from Persephonella marina, with potential role in imparting piezophilic effect in certain extremophiles (Lamosa et al. 2013). With the help of genetic and chemical manipulations, the productivity, efficiency and stability of these enzymes can be improved for industrial processes (Arora and Panosyan 2019). Microbes from extreme environments have been successfully used in the extraction of metals from rock ores and mine waste. As many of them grow luxuriantly in the presence of heavy metals and/or love acidic environments, metalophillic and/or acidophillic microbes are now being used for metal-polluted sites as well (Navarro et al. 2013, Johnson 2014, Orell et al. 2013). Various species of Deinococcus have been successfully tested for their bioremediative ability while being used for radiotactive waste treatment (Brim et al. 2003, Appukuttan et al. 2006). Owing to their radio-tolerant enzymes like, lipases, genetically engineered strains of E. coli using specific lipid degrading enzyme’s genes, have been found to resist radiations. Some of the above strains even exhibit thermostability as well as stability in the presence of surfactants and solvents (Shao et al. 2013). The application of drought-resistance promoting microbiome in farming practices in desert areas has also been reported (Marasco et al. 2012). Similar applications of root-associated microflora were found to help plant productivity in desert cultivation by transmitting drought resistance in plants (Rolli et al. 2015). The use of alcohol dehydrogenases, another important extremozyme (Demirjian et al. 2001) for biocatalytic activity has been investigated (Resch et al. 2011, Demirjian et al. 2001) while Chen and co-workers (Chen et al. 2009) found the same enzyme to be useful in converting nitriles to high value acids and amides.

4. Extremolytes for Industrial Applications Though extremozymes have been quite popular amongst researchers for their potential as industrial enzymes, other products from many extremophilic organisms are also being investigated now for industrial purposes. This has further expanded the avenues for applications involving extremophiles. Some organic compounds produced under extremes of conditions have been exploited for industrial purposes. These organic compounds, termed as extremolytes, make up to 25% of dry cell weight and are produced in stressed environment. Few polyol derivatives like, ectoine, hydroxyectoine, betaine, carbohydrates such as, firoin, firoin-A, different amino acids, derivatives of inositol and glycerol, UV- radiation protective compounds, etc., have been found in extremophiles (Borges et al. 2002, Lentzen and Schwarz 2006, Singh and Gabani 2011, Empadinhas and da Costa 2011, Esteves et al. 2014, Alarico et al. 2013, Lamosa et al. 2013, Bougouffa et al. 2014, Singh et al. 2010, Gabani and Singh 2013, Rastogi and Incharoensakdi 2014). Extremolytes have primarily been used in cosmetics and have the potential for application to the pharmaceutical sector and food industry (de la Coba et al. 2009, Soule et al. 2009, Singh and Gabani 2011, Choi et al. 2014, Avanti et al. 2014, Ryu et al. 2008, Faria et al. 2013, Kanapathipillai et al. 2005, Pastor et al. 2010, Autengruber et al. 2014, Cencic and Chingwaru 2010, Klein et al. 2007).

5. Extremophiles for Textile Industry As highlighted above, extremophiles hold great of promise as sources of enzymes and metabolites for use in industrial processes. The textile industry, one of the most chemical intensive and highly environment polluting industries worldwide, has been one of the major industries attracting the use of enzymes/metabolites from microorganisms, and extremophiles are the most attractive options available for the same due to the obvious. Usually, enzymes like those obtained from mesophiles/ active in mesophilic range of temperature, can be used at low temperatures and at near-neutral pHvalues (Sarrough et al. 2012), but with the advent of exploring extremophiles and their enzymes/ metabolites, a wide range of working conditions can be used for working of the enzymes, with better

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adaptability and integrity with the usual textile processing. Interestingly, the use of enzymes in the textile industry is not of recent origin and has been in place for quite some years now, but the use of extremophilic enzymes is still in its nascent stages. By and large, extremolytes have primarily been used in pharmaceutical and cosmetic sectors but still, no already known extremolytes have been found to be used in textile processing. The textile industry thus, primarily targets few extremozymes for various applications or use whole cells directly (Raddadi et al. 2015). Psychrophilic enzymes that are active at low or moderate temperatures offer many opportunities to be used in industrial processes (Demirjian et al. 2001, Van den Burg 2003, Eichler 2001, Cavicchioli et al. 2002, Deming 2002, Margesin et al. 2003, Feller and Gerday 2003, Georlette et al. 2004). Organisms from deep-sea have been a source to cold-adapted amylase enzymes and they have potential applications in the detergent, textile and food industries (Kuddus et al. 2011, Zhang et al. 2016, Qin et al. 2014, Dou et al. 2018). In the same way, cold active cellulose degrading enzymes like, glucosidases might be useful in industries including, the textile industry. They have exhibited characteristic features like, enzyme activity more than half of the maximum even at 4ºC of temperature and just a loss of about 20% activity of the maximum even at pH 11 for more than 10 hours (Chen et al. 2010, Mao et al. 2010), or more than 60% of its maximum activity at 10ºC and a pH range of 6.6–9.0 (Mao et al. 2010). Cellulases and hemicellulases from psychrophiles have also been found which can well be studied for applications in detergent industry, ethanol industry, food and feed industry or textile industry (Tao et al. 2010). Fibrobacter succinogenes S85 has been used as a source of cellulase, with a Vmax (temperature) value of 1200/min (4ºC) and has shown promise for animal feed, textile processing and detergent industry applications (Iyo and Forsberg 1999). Cold-stable xylanases have also been reported from certain Cladosporium sp. (Del-Cid et al. 2014). Psychrophilic lipases also have the potential in various industries including, their application in bioremediation process the chemical, pharmaceutical, cosmetic, food, laundry detergent and environmental remediation industries. Esterases of a similar nature (cold loving) can also be explored for industrial use in industries like, chemical and for bioremediation processes (Cavicchioli et al. 2011). Esterase, stable at 10ºC, in high salt concentration, with improved activity in the presence of chemicals like, some alcohols, dimethyl sulfoxide (DMSO) and acetonitrile, have been studied (Wu et al. 2015). Such esterase can well be explored for applications in harsh conditions, as those existing in textile industries. Psychrophilic proteases and pectinases are being explored for applications in the textile industry (denim washing) and dehairing of hides and skins and food processing (meat tenderization, fruit juice processing, respectively (Cavicchioli et al. 2011, Margesin et al. 2003, He et al. 2004). Cold active alkaline proteases, especially those that are stable against more than one type of extremes of conditions, hold great value for various industries like detergents, textiles and leather (Sharma et al. 2016). Vibrio rumoiensis has been found to produce cold-active catalase, with Vmax (temperature) of 4100 U/mg (30ºC) and showing promise to be used in textile industries along with dairy, paper, food industries (Yumoto et al. 1999). The EU Fourth Framework research program has sponsored a project through which many enzymes like, alphaamylase, cellulase, etc., have been obtained from organisms isolated from the Antarctica that have promising potential in textile and detergent industries along with other industries (Cavicchioli et al. 2002). On the other hand, thermophilic enzymes, which have wide applicability in industrial use and have long been considered of high industrial importance, either as intact organisms or as a source of thermostable enzymes, which can catalyze specific reactions at high temperatures. In fact, in some cases, these organisms and/or their enzymes are already being applied at a commercial level. A number of enzymes like, amylases, pullulanase, glucoamylases, glucosidases, cellulases, xylanases, proteases, chitinases, etc., with potential applications in various industries have been found in thermophiles (Collins et al. 2005, Demirjian et al. 2001, Van den Burg 2003, Eichler 2001, Cavicchioli et al. 2002, Deming 2002, Margesin et al. 2003, Feller and Gerday 2003, Georlette et al. 2004, Wu et al. 2006). Lignocellulolytic applications like those using cellulases, active and

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stable at high temperatures, are being used in different industries including, textile industries, along with detergent industries, food industries, paper industries, pharmaceutical industries, etc. (Dalmaso et al. 2015, Cavaco-Paulo 1998, Tolan and Foody 1999 and Ikeda et al. 2006). A potentially highly valuable extremophilic cellulase has also been found from hydrothermal vents with activity against a variety of cellulosic/carbohydrate substrates and activity at even very high temperature (92ºC) (Gao et al. 2015, Li et al. 2013, Wu et al. 2015). Horikoshi (Horikoshi 1999) has also reported the use of alkaline thermostable cellulases for mild treatment in the textile and detergent industry. Cellulases from Clostridium cellulovorans were well characterized by Doi et al. (1998). Geobacillus pallidus and Caldibacillus cellulovorans have exhibited production of thermostable cellulases, active at 60ºC (Baharuddin et al. 2010) and 80ºC (Huang and Monk 2004), respectively. Glucanases of cellulolytic thermophile Clostridium stercorarium was investigated by Bronnenmeier and coworkers (Bronnenmeier et al. 1991). Several other enzymes amylopullulanases, arylsulfatases and xylanases have also been obtained from such extreme environments as deep-seas and can very well add to the search of powerful enzymes for industrial applications (Gao et al. 2015, Li et al. 2013, Wu et al. 2015). Powerfully thermostable xylanases with optimum activity in the range of 80°C to 105°C have also been described in different species of Thermotoga (Niehaus et al. 1999). Kim et al. (2014) found Gloeophyllum trabeum to produce xylanase at 70°C under acidic pH (4–7). Acid tolerant xylanases have been found Neocallimastix frontalis (Hebraud and Fevre 1990). Sharma et al. (2016) reported a polyextremophilic strain of Bacillus which was found to be active at both high temperature as well as high pH, thereby indicating its industrial importance (Sharma et al. 2016). Pyrococcus furiosus was found to show optimum amylase activity at about 100ºC and even some activity was reported up to a temperature of 130ºC (Koch et al. 1990). Another thermophilic amylase was reported by Sharma and Satyanaryana (2012) to exhibit appreciable enzyme activity at 80ºC and was also found to be stable in the presence of acids. Methanococcus jannaschii has shown extreme thermostability with an optimum activity at 12ºC (Kim et al. 2001). Thermostable subtilases are considered to be among the important proteases with potential applications in food, textile, detergent, pharmaceutical and leather industries (Toplak et al. 2013). Toplak et al. (2013) expressed the gene coding for subtilase obtained from a thermophile into a strain of E. coli. The enzyme so produced, exhibited significant thermostability at varying ranges of pH. Further, it was stable in the presence of organic solvents and detergents. Lipases are used in fat hydrolysis, esterification, interesterification, trans-esterification and organic biosynthesis, removal of pitch from pulp produced in the paper industry, the hydrolysis of milk fat in the dairy industry, the removal of non-cellulosic impurities from raw cotton before fabric dyeing, the removal of subcutaneous fat in the leather industry, etc. Valuable features like, thermostability, stability in the presence of harsh chemicals and stability in the presence of varying pH, impart better adaptability of lipases in the conventional chemical processes. A strain of Thermococcus kodakarensis has yielded a pullulanase that works best at boiling temperature (100ºC) (Han et al. 2013). Certain thermophilic fungi have also offered potential enzymes for use in various applications. Chaetomium thermophile, Humicola insolens, Humicola grisea var. thermoidea, Talaromyces emersonii, Thermoascus aurantiacus, Myriococcum albomyces, etc., for thermostable cellulases are some noted examples (Maheshwari et al. 2000, El-Gindy 1991, Chung 1971). Melanocarpus albomyces has been found to be a cellulase, hemicellulose as well as a laccase producer. Similarly, both endoglucanases as well exoglucanases with optimum activity between 50ºC–80ºC from thermophilic fungi, and amylases from fungi for textile and detergent industry have been reported (Singh 2016, Singh and Khajuria 2017, Singh et al. 2014b, Tyagi et al. 2011, Singh et al. 2014c, Qureshi et al. 1980, Prabhu and Maheshwari 1999, Kiiskinen et al. 2002). Halophilic enzymes, with their ability to act under low water activity and even sometimes in the presence of organic substances, also have great potential to be used in varied types of industries like, food, textile, pulp and paper and detergent industry (Raddadi et al. 2013, Datta et al. 2010). Their utilization

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can well be done in bioremediation too. Such enzymes are often associated with stability in the presence of organic solvents and sometimes even at high or low temperatures, along with activity in the presence of high salt concentration (Yin et al. 2015, Raddadi et al. 2013, Datta et al. 2010, Dou et al. 2018, Wu et al. 2015). Halophilic cellulases, xylanases and amylases have been reported (Raddadi et al. 2013, Bhalla et al. 2013, Du et al. 2013, Elleuche et al. 2014). Halophilic amylases have been obtained from Haloarcula hispanica, Halomonas meridian, Natronococcus amylolyticu, etc. (Coronado et al. 2000, Hutcheon et al. 2005, Yadav et al. 2015). Sahay et al. (2012) studied halo-alkalophilic bacteria from hyper saline Sambhar Lake, India and proposed that these organisms can well be used for different applications. A thermophilic, acid stable cellulase from a strain of Paenibacillus tarimensis was reported by Raddadi et al. (2013), which was stable in the presence of harsh chemicals like, organic solvent, detergents, etc., seems to be a good candidate for applications in detergent, textile and pulp and paper industries (Raddadi et al. 2013, Raddadi et al. 2015). Azlina and Norazila (2013) reported such protein degrading enzymes which could well be used in the detergent industry. Walker et al. (2006) reported alkalotolerant cellulases from a strain of Nocardiopsis which was found to be active at pH 8.0 and 40ºC, while Shanmughapriya et al. (2010) showed production of alkalo-tolerant cellulase from marine sponge. Alkalitolerant Bacillus strains have been isolated and characterized from sources like, leather waste (Anandharaj et al. 2016), and reported to hold potential for industries like, leather, textile and food industries (Pant et al. 2015). Metal loving whole organisms have been reported to be of value for treatment of environment pollution as well as for industrial applications like, biomining or bioleaching processes. As such organisms tend to lower down the concentration of heavy toxic metals in their environment, they can well be used for bioaccumulation, biomineralization and bioremediation processes (Khare and Fleishman 2013, Navarro et al. 2013). Martin et al. (2001) evaluated a number of alkaline and serine proteases, pectinases, cellulases, amylases, lipases and xylanases which showed extremophilic characters of more than one kind suggesting that such enzyme profiles of extremophiles can well be explored for variety of applications in different industries. A number of enzymes like, cellulases, pectinases, hemicellulases, lipases, serinicases, proteases, polyesterases, catalases, amylases, galactosidases, glucanases, pullulanases, etc., are being used/have potential to be used for different stages in textile processing (Gulrajani 1992, Kundu et al. 1991, Pikuta et al. 2007, Yoon et al. 2002). Several such enzyme producing organisms include members from Bacillus, Corynebacterium glutamicum, Pseudomonas, Aletromonas, Halobacterium, Shewanella, Psychrobacter, Pseudoalteromonas, Arthrobacter, Colwellia, Gelidibacter, Marinobacter, Psychroflexus, Moritella, Halomonas, Photobacterium, Colwellia, Thioprofundum, Methanolobus and Methanococcoide (Chen and Jiang 2017, Zhang et al. 2015, De Maayer et al. 2014, Dopson et al. 2014, Gabani and Singh 2013, Gtari et al. 2012) and some of their enzymes have been either tested for application in textile processes along with other industrial applications, and some hold significant potential for the same to be evaluated accordingly. As described by Kozubal et al. (2008), extremophiles like, Methanococcoides burtonii, reported to be cold loving as well as halophilic organisms, as also members of Sulfolobales (such as Acidianus, Metallosphaera) or Thermoplasmatales (like, Thermogymnomonas acidicola) are a few examples of such potential industrially relevant organisms which shall be investigated further for their commercial use. In some steps of the textile industry, acidic and psychrophilic as well as alkali and thermotolerant cellulases are needed (Zeng et al. 2006, Bhat et al. 2013). There are two well-established enzyme applications in the textile industry. Firstly, in the preparatory finishing area amylases are commonly used for the desizing process and secondly, in the finishing area cellulases are used for softening, bio-stoning and reducing of pilling propensity for cotton goods. At present, applications of pectinases, lipases, proteases, catalases, xylanases, etc., are used in textile processing. There are various applications which entail enzymes that include fading of denim and non-denim, bio-scouring, bio-polishing, wool finishing, peroxide removal,

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decolorization of dyestuff, etc. (Cavaco-Paulo and Gubitz 2003, Chelikani et al. 2004, Barrett et al. 2003, Sharma 1993, Nalankilli 1998, Shenai 1990, Etters and Annis 1998, Heine and Höcker 1995). Textile desizing is an important step where the size material from warp yarns after a textile fabric is woven, is removed. This is carried out by synthetic as well as natural sizing agents (Mojsov 2012, Mojsov 2014). Enzymatic desizing is a classical desizing of cotton fabrics and amylases have been used for the same. The enzyme breaks down the starch size over the textile surface into amylose and amylopectin. This leads to the removal of starch from the fabric surface as the enzymatic action make is water soluble. With high enzymatic specificity, enzymes/microbial cells offer no harmful effect on the fiber as such (Etters and Annis 1998). Another variation of microbial usage, fermentative desizing, uses microbial cells directly impregnated into the fabric for microbial enzymes to work. Though not very popular currently, this method is more economical than the other methods (Cavaco-Paulo and Gübitz 2003). Xylanase from Bacillus stearothermophilus SDX have been used in textile industries for desizing of cotton and micropoly fabrics (Saurabh et al. 2008). Stone washing is a process applied to denims and other fabrics to improve their appearance and handle and give a ‘faded’ effect. Conventionally done by a pumice stone (sodium hypochlorite or potassium permanganate) which are required in very large amounts, stone washing by enzymes/ biowashing is being used to get a stone-wash effect nowadays (Sariişik 2004, Arjun et al. 2013). An environment friendly alternative, with no chemical residue left behind on the fabric, fabric surface being free from surface hairiness and neps with good handle and flexibility, and the enzymes are required in very little amount, biowashing with cellulases and hemicellulases. Cellulases are also being used for softening of textiles. Cold-stable proteases have also been reported, and are being investigated for stone washing in the textile industry (Cavicchioli et al. 2011, Margesin et al. 2003). Some types of ligninases even provide a good alternative to the conventional treatment (Pederson and Schneider 1998, Campos et al. 2001, Pazarloglu et al. 2005). Biofinishing or biopolishing, a permanent treatment process using enzymes, removes protruding fibers and slubs from the fabric surface, reduces the pilling effect and softens the fabric, and even provides a sheen to the fabric. Also, bio-polishing being a sustainable alternative of chemical processes helps in reduction of the use of chemicals and reduces energy and water consumption. Less chemical waste further translates to less environmental pollution. This treatment involves cellulases and hemicellulases (Steward 2005, Cavaco Paulo 1998, Cavaco-Paulo et al. 1996, Lenting and Warmoeskerken 2001) that act on the surface of cellulosic materials. Cellulases basically break down 1–4 β-glucosidic bonds of cellobiose chain. Thermophilic cellulases have been used in the treatment of raw cotton fabric (Svetlana et al. 2008). Mori et al. (1995, 1996) highlighted certain limitations of cellulase treatment of textile fabric. They explained that the dyeing affinity of fibers in response to cellulase treatment and proposed that the dyeing mechanism of enzyme treated as well untreated cotton fabrics were not different from each other. Carbonization of wool, for removing cellulosic impurities (vegetable matter and wool grease) from wool is conventionally done by chemicals like, acids. Nowadays enzymes such as, cellulases, xylanases and pectinases, in conjunction with some chemicals, are being used for the same (ElSayed et al. 2010). Natural wax, non-fibrous impurities or added soil/dirt are removed from the fiber through the process of scouring. Conventionally done with harsh alkalis, bioscouring enzymes like, pectinases, and cellulases have become popular for obvious benefits, viz., ease of operation, environment friendliness, no potential health hazards, no harmful effects on fiber, etc. The pectinases remove cotton cuticle by acting on pectin while cellulases digest the primary wall cellulose immediately after the cuticle cotton. Polygalacturonase lyases, obtained from thermophilic microbes with temperature optima between 50–70ºC have been reported to be of value in bioscouring (Truong et al. 2001, Araujo et al. 2008). Animal fibers are also modified under scouring using proteases

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(Rocky 2012, Mamun et al. 2017). Not only the process itself, but even the effluent water generated, is less polluted than the conventional alkaline scouring process. The process with improvement in strategic approaches, a combined scour-desize process is also being used nowadays (Pawar et al. 2002). Bacillus stearothermophilus has been used as a source of thermostable xylanases which helps in lowering the wetting time of fabrics, exhibit bioscouring efficiency and reducing the weight loss of the fabrics, thereby retaining the fabric strength (Saurabh et al. 2008). Degumming of silk fibers is done to improve the shine, color handle and texture of silk by removing the sericin or silk gum, from silk. Acid and alkali as well as enzymatic degumming are done commonly. With high specificity in reaction, lesser risk of over degumming, lesser fiber weakness, economical, environment friendliness, etc., associated, trypsin, papain and proteases are commonly used for degumming of silk fibers. Pectinases are used in degumming of ramie fibers too (Wiegel and Kevbrin 2004, Gangwar et al. 2013, Horikoshi 1999, Maier et al. 2004, Paar et al. 2003, Thompson et al. 2003, Rinsey Antony and Karpagam Chinnammal 2012). Enzymes like, proteases are also used for wool processing for improvement of its characteristic features. Conventional wool processing requires higher energy than enzymes for the same purpose (Heine and Höcker 1995, Walawska et al. 2006, Ammayappan and Gupta 2011, Cardamonme 2002, Ammayappan 2013). Lipases are commonly used in the textile industry to remove size lubricants, for providing better absorbency of fiber for improved dyeing. Polyesterases are being used for polyester treatment for improved fabric properties. Lipases, by their action on fats and oils, improve hydrophilicity of polyester (Rowe 2001, Andualema and Gessesse 2012). Bleaching, where the natural pigments (Hedin et al. 1992, Ardon et al. 1996) are removed from a cotton fabric for a pure white appearance, is usually done with hydrogen peroxide and other bleach chemicals. Such treatment often leads to damage of the fiber, thereby weakening it, and even add to industrial pollution, with the release of such harmful chemicals in wastewater. Peroxidases, pectinases, glucose oxidases, amyloglucosidases, laccases are a few enzymes that are being explored for use in textile bleaching (Pereira et al. 2005, Tzanov et al. 2003). Laccases have been found to provide a bleaching effect on cotton (Tzanov et al. 2003, Pereira et al. 2005) due to oxidation of flavonoids within a short span of time. A combination of ultrasound-laccase treatment has also been successfully evaluated for its role in cotton bleaching (Basto et al. 2007). Cold-stable proteases are being investigated in waste management and can be used for improving the quality of textile wastewaters as well (Cavicchioli et al. 2011, Margesin et al. 2003). A thermostable genetically engineered thermo- and pH-stable subtilase, exhibiting stability towards organic solvents and detergents, also can be used for solid waste treatment Toplak et al. (2013). Catalase and peroxidase or oxidoreductase can help reduce residual hydrogen peroxide present in bleach liquor and released into textile wastewaters (Wiegel and Kevbrin 2004, Mojsov 2011, Horikoshi1999, Maier et al. 2004, Paar et al. 2003, Thompson et al. 2003). Ligninolytic enzymes like, manganese peroxidase acts on lignin and other phenolics and is being explored for textile wastewater treatment especially for dye pollution remediation. Similarly, laccases that oxidize, decarboxylate or demethylate substrates like, ortho and paradiphenols, aminophenols, polyphenols, polyamines, lignins and aryldiamines, can well be applied for bioremediation of textile effluents as well as lignin. Lignin peroxidases act upon halogenated phenolic compounds, polycyclic aromatic compounds and other aromatic compounds (Karigar and Rao 2011). Certain applications of encapsulated halophilic enzymes can be evaluated for remediation of dye pollution in textile effluents. They are also active against recalcitrant substances, salts and solvents released into the wastewaters (Marhuenda-Egea and Bonete 2002, Marhuenda-Egea et al. 2002). Psychrophilic enzymes can also be used as environmental biosensors (like, dehydrogenases) (Onyshchenko et al. 2002, Singh and Khajuria 2020) or in biotransformation (such as, methylases, aminotransferases and alanine racemase), and can well be investigated for incorporation in textile industries as

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well (Onyshchenko et al. 2002). Though piezophiles have not been extensively explored (Abe and Horikoshi 2001, Yano and Poulos 2003), some like, Ralstonia, could be used in heavy metal bioremediation (Mergeay et al. 2003, Valls and de Lorenzo 2002). An indirect application of enzymes like, cold-stable proteases, lipases, amylases and cellulases include, their use in detergent formulations for cold washing. This helps in reducing wear and tear of textile fibers, and improving life of the fabric, along with reducing energy consumption as well (He et al. 2004). Genencor, headquartered in New York, USA, is credited to commercialize one of the first industrial cellulase obtained from an alkaliphilic bacteria isolated from a soda lake for use in textile detergents (Podar and Reysenbach 2006).

6. Conclusion A large number of significant discoveries have been made in the world of extremophiles, with many of their enzymes and/or metabolic products being explored for numerous of exciting industrial applications. Agriculture, food industry, feed industry, pharmaceutical sector, detergent and textile industries, leather industry, paper industry, etc., are some such major industrial sectors. Still, extremophiles and their products have to offer a lot and so, extensive characterization of extremophiles and their commercial potential still needs to be further explored. Though metagenomics has helped in studying uncultivable extremophiles yet, for their further use in processes of economic value, exhaustive characterization of these organisms is required. Cultivation of extremophiles through the services of genetic engineering where, the desired gene of interest from extremophiles can be expressed in mesophilic hosts might help in overcoming the problems associated with cultivation of such organisms. Biotechnology itself and further the use of extremophiles in the textile industry will prove to be a boon to the given industrial sector and will drastically help in cutting down the use of harmful chemicals in textile processing as well. At the same time, environmental pollution could be well reduced. The importance of enzymes, as effective catalytic agents, that enhance the economic value of the processes in which they are applied in an environment friendly way, is already known. The use of various enzymes in different stages of textile processing as well effluent treatment in effective and innovative way need to be extensively evaluated. For this, better understanding of the textile substrates and mechanisms of catalytic reactions is required. Textile preparatory process like desizing, scouring and bleaching are few attractive areas to use enzymes and extremozymes can play a big role here. Further, the use of extremophilic products and extremozymes face cost challenges that need to be addressed seriously and if their cost can be reduced, enzymes can play a great role in the textile wet processing and even textile effluent treatment. Despite great advantages of extremozymes, the majority of the enzymes used in industries are from mesophiles. Interestingly, the application of such enzymes faces issues of limited stability at the extremes of temperature, pH and ionic strength, which make extremozymes all the more important. Biotechnology has thus, many avenues to evaluate in the given field with a rapid increase in understanding in the field of metagenomics, proteomics, metabolomics, etc., and with the ever increase demand of powerful yet cheap biocatalysts by industries. Advancements in molecular and computational biology, new sources of enzymes, combinatorial methodologies and biochemical engineering of single and multicomponent enzyme systems will further support the applications of extremophiles in industries. To date, extremolytes have been explored for application in pharmaceutical, cosmetic and food industries, but potential avenues for extremolytes in textile industry are still very limited. Novel extremolytes, that can provide a competitive edge to the conventional chemical-based methods, would greatly impact the industrial developments in textile wet processing.

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Index A

H

Anode 45, 47, 53, 55–57

High-value bioproducts 167, 170, 171

B

I

Bioactive metabolites 100 Biocides 233, 235 Biodiesel 1–3, 5–7, 9, 10, 17 Bioelectricity 6, 11, 13, 14, 16, 17 Bioethanol 1–3, 5, 6, 11, 16, 17 Biofilm 222, 224–235, 243, 244, 246, 249–266 Biofuel 80, 81, 83, 86, 88, 98, 103–107, 151–153, 155, 158–162, 187, 191, 193, 194, 197, 199, 202, 203, 206, 208, 209 Biomass 187, 191–209 Bioproducts 199, 202 Biorefinery 1, 2, 11 Bioremediation 26, 27, 30, 31, 33, 105–107, 151, 152, 156, 160 Biotechnological applications 167

Marine microbes 115 Metabolic engineering 104, 106, 107 Microalgae 5–13, 16, 17, 80, 81, 83, 86–92, 94–96, 98–107, 151–163 Microbial electrochemical system 44 Microbial fuel cell 11, 16, 34, 36 Microbial influenced corrosion 220, 225, 229, 235 Microorganisms 277, 279, 283

C

P

Carbon dioxide reduction 209 Coatings 234, 235 Cultivation 81, 95–98, 100, 105, 107

Phylogeny 81 Pigments 80, 85, 101–103

E

Remediation 4, 7, 8, 10, 12

Environment 23–31, 33–35, 37 Enzymes 115, 116, 118, 123, 125–127, 129, 132, 136, 138, 140, 141 Extremolytes 283, 284, 289 Extremophiles 1, 2, 4, 6, 8, 13–15, 17, 43, 44, 46–49, 51–54, 57, 58, 80, 81, 87, 88, 90, 93, 95, 96, 98, 104, 106, 115, 116, 119, 126–131, 133, 135–139, 141, 243–245, 251, 256, 259–261, 263–266, 277, 278, 280, 281, 283, 286, 289 Extremophilic microalgae 167–171, 173, 175–178 Extremophilic organisms 151, 152, 162

G Growth engineering systems 169

Industrial applications 167, 175, 277, 279–283, 285, 286, 289 Industrial biotechnology 151, 152, 162

M

R

S Seaweed hydrolysis 116, 132 Stress tolerance 81 Stressed habitats 168, 169, 173 Sustainability 3, 6

T Textile industry 277, 282–284, 286–289 Thermophiles 43, 44, 46, 48, 49, 52, 53, 57 Thermozymes 282

W Wastewater 1, 2, 4–17, 23, 24, 26–36 Wastewater treatment 151, 155–157, 159, 162, 163, 253, 256, 258, 261, 262, 265