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Marine biomass toward biofuel production
Marine biomass toward biofuel production
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Jeevanandam Vaishnavi, Arumugam Arulprakash, Adikesavan Selvi and Aruliah Rajasekar Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Vellore, India
20.1
Introduction
Providing an adequate supply of clean energy for the society is one of the most challenging tasks the world is facing now, as it is directly linked with global stability and economy issues. Fuel usage in the form of transportation, manufacturing, and domestic heating applications contributes around 70% of the total global energy requirements. On the other hand, electricity accounts for only 30% of the global energy consumption. The world’s primary energy resource is dependent on fossil fuels, extraction of oil, and natural gas which leads to the emission of carbon dioxide, which in turn contribute to the greenhouse effect (Change, 2014). According to Vially et al. (2013), the risks of dwindling reserves are expected to last for probably 53 years (oil reserve) and 54 years (natural gas reserve). Climatic changes urge the need for an effective ecological change (Taylor et al., 2015). Therefore a bio-based approach, such as marine waste biorefinery, is considered as one of the innovative and alternative technologies that convert biomass into sustainable and economically valuable products (Demirbas, 2009). Petroleum refineries formed the basis to the emergence of biorefineries that have greatly replaced conventional refineries. Biorefineries have been considered as an alternative for the fossil fuels due to increased fuel usage, high fuel cost, use of nonrenewable resource, and secondary pollutant generation (Aristidou and Penttil¨a, 2000). Marine biorefinery is one such method that makes use of microbial community isolated from the marine environment for high-yield biofuel production. Major source of marine biorefinery includes microalgae (diatoms, green, golden, and blue/green algae), macroalgae (brown, red, and green seaweeds), yeast, and bacteria. Marine crops have long been recognized as a potential biofuel source due to its greenhouse gas abatement potential characteristics and CO2 adsorption capacity than terrestrial plants (Jong et al., 2010). During the process of biofuel (bioethanol, biodiesel, biomethanol) production, other value-added by-products, such as pharmaceuticals, food, feedstocks, enzymes, and pigments, are also produced (Coates et al., 2013). Therefore biorefinery technology plays a dual role
Refining Biomass Residues for Sustainable Energy and Bioproducts. DOI: https://doi.org/10.1016/B978-0-12-818996-2.00020-X © 2020 Elsevier Inc. All rights reserved.
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in reusing the waste and producing valuable products. Above all, this technology is believed to support economic growth and environmental sustainability. However, the operational cost of marine biorefineries still remains too expensive to consider them as a viable option for biofuel production and other related applications. Hence, biorefinery system should be designed with minimal investments, less energy usage, recycling options, easy separation, wide range of raw material availability, and transportation.
20.2
Biofuel generations
Based on the biomass used, biofuels can be categorized into first, second, and third generations that determine the flexibility and potential of the biofuel industry. Different generations of biofuel with their characteristics are shown in Table 20.1. Due to the ethical problems faced by first- and second-generation biofuels, the economic concern has shifted from terrestrial to marine biomass for the biorefinery process (Gouveia, 2011).
20.3
Sources of biofuel
20.3.1 Algae Algae are usually aquatic oxygen-emitting photosynthetic organisms with the simple structure of no roots, stems, or leaves. Since they don’t fit under a single monophyletic group, they cannot be easily defined. They exist as a group of ubiquitous, but individual species, thus occupying specific habitats. Algae exist in various forms, such as a few of the algae attached to plant substrates; few possess motility behavior like animals; few are suspended in water; few grow loosely in trees, soil, and animals; and some form symbiotic association with other organisms (e.g., lichens, corals). Microalgae lack complex multicellular structures with great variation in their internal cell structure. The blue-green algae or cyanobacteria have a prokaryotic cell structure that closely resembles bacteria. The demands for the biofuels lead to the search for feedstocks of micro and macroalgae for their production. Algae consist of 40% of lipids, which helps in rapid conservation of biofuel, thus making the process environment friendly and cost-effective (John et al., 2011). In addition, the cultivation of algae is also a relatively simple and easy technique. So this method has become globally attractive (Miao and Wu, 2004). Algal biomass can be classified as sugars, proteins, and triglycerides, which can be converted into various valuable products, such as pharmaceutical products, food, feedstocks, enzymes, and pigments along with biofuel (bioethanol, biodiesel, biomethanol). Owing to these advantages, petroleum refinery can be partially replaced by biorefinery. Therefore this section deals with different algal resources involved in biofuel production.
Table 20.1 Biofuel generations. S. no.
First generation
Second generation
Third generation
Produced from sugar, starch, vegetable oil, or animal fats Basic feed stocks are wheat, corn, and rape seeds and grains Bioethanol, biodiesel, starchderived biogas, vegetable oils, biomethanol, and boaters Anaerobic digestion
Produced from a variety of nonfood crops, such as lignocellulosic materials from agricultural, forestry, and industry
Produced from yeast, fungi, and algal biomass
FT diesel from biomass and bioethanol
Hydrogen and methane gas, bioethanol, butanol, and acetone
Thermochemical, flash pyrolysis, enzymatic
Acid hydrolysis, liquefaction, pyrolysis, gasification, extraction and transesterification, fermentation, and anaerobic digestion processes Eco-friendly and cost-effective
1.
Biomass source
2.
Biofuel type
3.
Conversion route
4.
Advantages
Reduced global warming emissions and fossil energy consumption
5.
Disadvantages
Compete with food and feed industries for use of biomass and agricultural land Give rise to ethical implication Increased fossil fuel price
FT, Fischer Tropsch.
Improved land-use efficiency and environmental performances Availability of widespread and cheap raw material Allow coproduction biofuels, chemical compounds Generation of electricity and heat Better energy, environmental, and economic performances Biomass residues used are still at precommercial stage
Usage of large volumes of water
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20.3.2 Microalgae and macroalgae The production of biofuel using microalgae includes selection of a particular microalgae cultivation of that specific species, harvesting followed by pretreatment methods that include acid transesterification, product separation, and anaerobic digestion (Assacute et al., 2018). Macroalgae are commonly known as seaweeds, it is generally found in subtidal and intertidal regions of the sea. It includes selection, cultivation, and harvesting of a particular macroalgae, in continuation with extraction of biodiesel along with the production of enzymes with alcoholic fermentation and anaerobic digestion. When compared to microalgae, macroalgae seems to be more complex. Same feedstock has been converted into different value-added products (Assacute et al., 2018). The production of biodiesel using microalgae and macroalgae is depicted in Figs. 20.1 and 20.2, respectively.
20.3.2.1 Chlorella The green microalgae, Chlorella has a high protein content and it can be used for human consumption, whereas it can accumulate large amounts of lipids when grown under stress condition. Due to this ability, it can be used for the production of biofuels. The main disadvantage of the cultivation of Chlorella includes high cost (Guccione et al., 2014). It is cultivated under photoautotrophic conditions predominantly in open ponds (Ramaraj et al., 2016). The largest autotrophic production of algae was started from the year 2000 in a system of 500 km in glass
Figure 20.1 Production of biofuel using microalgae.
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Figure 20.2 Production of biofuel using macroalgae.
tubing in Klotze, Germany, which produced approximately 100 t of chlorella annually (Pulz and Gross, 2004). Till date, chlorella production guarantees high quality which can be used safely and successfully (https://www.algomed.de/en/homepage). Two main Chlorella species, Chlorella vulgaris and Chlorella pyrenoidosa, are commercially cultivated. Chlorella species contains high amount of starch, which can be used for the production of bioethanol (Bra´nyikova´ et al., 2011) when supplemented with 50% of sulfur content. The Chlorella biomass is rich in carbohydrates, minerals, and proteins that can be used to produce many value-added by-products and several bioactive compounds after the extraction of biofuels (Brennan and Owende, 2010) and thus Chlorella helps to produce environment-friendly and sustainable biofuels. The main advantages in cultivating microalgae (Chlorella) are as follows: it does not need a fertile soil, limits the pollution, and efficient in utilizing the nutrients from wastewater. Algae require carbon dioxide for its growth (Guccione et al., 2014).
20.3.2.2 Botryococcus braunii The pear-shaped, bloom-forming green microalgae Botryococcus braunii grow in a cluster form that can be used for the production of biodiesel. Due to its blooming nature, there is an increased quantity of biodiesel production of high quality. The selection of strains is solely based on the high amount of lipid production. Unless
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grown in an optimum condition, their growth rate is too slow (Lassing, et al., 2008). It is one of the most promising biofuel producing microalgae due to its high lipid content. B. braunii is a little unusual in the secretion of lipids in the extracellular medium, whereas other microalgae have cytoplasmic lipids (Hirose et al., 2013).
20.3.2.3 Pleurochrysis carterae The unicellular microalgae, Pleurochrysis carterae, have a unique ability of calcification process occurring at the subcellular level to produce calcified scales. This type of microalgae can be commercially used for the production of biodiesel due to their lipid content. It is a fast growing organism with low risk of contamination (Rahbari, 2009).
20.3.2.4 Dunaliella salina The biflagellate green microalgae, Dunaliella salina, belong to the family Dunalliellacea. It is predominantly found in high salt regions commonly in marine waters. Due to its high amount of carotenoids and its antioxidant activity, it is regarded as a food source. It is also used for the biodiesel production due to its methylation of fatty acids, such as linolenic and palmetic acids (Oren, 2005).
20.3.3 Macroalgae 20.3.3.1 Gracilaria chilensis The red macroalgae Gracilaria chilensis have been reported to produce higher amounts of biomass when compared to other macroalgae (Wi et al., 2009). Due to the high content of polysaccharides, it is useful for the production of bioethanol by the method called hydrolysis. After the extraction of biomass, it can be used for producing other value-added by-products.
20.3.3.2 Sargassum angustifolium The brown algae Sargassum angustifolium are abundantly present in the Persian Gulf, which is used for the production of biodiesel and other value-added byproducts. After the extraction of biofuel, S. angustifolium is mainly used for the production of sodium alginate. The biomass obtained after the alginate production is used for bioethanol production by fermentation method. The macroalgal S. angustifolium biomass can be used as the substitute for yeast during ethanolic fermentation (Ardalan et al., 2018). The bioethanol production from S. angustifolium biomass should be pretreated with acid (Yazdani et al., 2015). This pretreatment step disrupts the recalcitrant structure completely and evolves nitrogen gas that can be used for the fermentation, thus cutting down the cost of nutrients required for the process.
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20.3.3.3 Sea lettuce: Ulva lactuca The green sea lettuce, Ulva lactuca, is used for bioethanol production and it is a renewable gas fuel. U. lactuca commonly refers to green tides due to the eutrophication (excess amount of nitrogen secretion) or algae blooms due to high lipid content (Allen et al., 2013). Sea lettuce generally contains minimal amount of cellulose from which biomethane is produced by anaerobic digestion (VergaraFerna´ndez et al., 2008). The shallow basins remain the most susceptible place for the growth of sea lettuce. Shallow topographs protect the algae from washout and it also keeps the pollutant, such as urea and nitrogen, from initiating the algal growth.
20.3.4 Other microbial biofuel sources Microorganisms are versatile living factories that can utilize numerous natural and synthetic compounds for their growth and convert them into different useful chemicals. Conversion of marine biomass into simple sugars is a necessary step in the production of biofuel. The major barrier for this process is the presence of high branched and recalcitrant compounds in marine biomass. However, these compounds can be subjected to pretreatment methods to release chemicals and enzymes that help in the conversion of biomass into biofuel. Pretreatment methods involve the application of microbial enzymes that help in breaking down of highly branched structures. Generally, marine biomass is composed of cellulose, hemicellulose, lignin, minerals, proteins, and oil (Wyman, 1999). Pretreatment of biomass using microbial enzymes is considered as a cost-effective and environment-friendly approach (Chaturvedi and Verma, 2013). However, pretreatment methods will vary according to the proportion of the chemicals present in different biomass. In earlier days, saccharification and fermentation process was considered attractive because of the combined addition of hydrolytic enzymes and microbes in the same environment (Punnapayak and Emert, 1986). So, this process has led to minimal inhibition, thus resulting in high yield of products at less cost (Wyman, 1999). On the other hand, solid-state fermentation (SSF) is employed at low pH and high temperature. But the microbes are subjected to adopt the ability to grow in this extreme condition. In order to obtain good results, microbes can be genetically engineered and employed in this process (Zhang et al., 1995). Later, SSF was replaced by separate hydrolysis and fermentation (SHF) process, in which the pretreatment of biomass was followed by enzymatic hydrolysis and then to a fermentation process. In this method, biomass undergoes each process independently under optimal conditions. This type of treatment of biomass leads to the additional discharge of waste, which can be used for producing other value-added products using biocatalysts (Chaturvedi and Verma, 2013). Unlike SHF, consolidated bioprocessing process combines both cellulose and hemicellulose fermentations in a single batch process (Lynd et al., 2002). Microbes that have the ability to produce cellulolytic enzymes can be employed in this process. Glucose and xylose cofermentation can be attained by employing genetically engineered microbial consortium (Zhang et al., 1995).
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20.3.4.1 Escherichia coli Escherichia coli has the natural ability to utilize a variety of sugars in both aerobic and anaerobic conditions. In this regard, E. coli is used for producing biodiesel and many other industrial products (Liu and Khosla, 2010). However, the development of new molecular technology tools to identify the genetic variation and genetic makeup of the organism helps in generating high amounts of biofuel (Ferrer et al., 2009). Currently, ethanol is dominating in biofuel as an alternative source. Additional advantage of using E. coli is its ability to metabolize both hexose and pentose sugars (by glycolysis), the components of lignocellulosic material, whereas many other microbes do not have the ability to use pentose sugar. Effective implementation of genetic engineering can overcome this issue. In a study by Zhang et al. (1995), the genes “pdc” and “adhB” from Zymomonas mobilis are inserted in E. coli. These genes have been expressed in operon from plasmid which is constitutively expressed in the production of ethanol using pET (expression plasmid for T7 RNA polymerase) promoter. So, the genetically engineered E. coli produces pyruvate decarboxylase and alcohol dehydrogenase II, which helps in producing 95% ethanol without redox imbalance. Continuous generation of ethanol can be achieved by constructing E. coli plasmid vector with “pods” and “adhB” gene inserts with selective chloramphenicol resistance gene and integrated through chromosome.
20.3.4.2 Zymomonas mobilis Gram-negative, facultative anaerobe, rod-shaped bacterium belongs to class Alphaproteobacteria that is a natural ethanologen which has many important environmental characteristics to replace the natural fossil fuel (Yang et al., 2016). It is regarded as the best alternative for fuel as it reduces the production cost of controlling aeration during fermentation method (Yang et al., 2016). The nitrogen fixation ability of this organism does not affect the ethanol production; rather it increases the productivity of ethanol. Z. mobilis has the ability to metabolize both C5 and C6 sugars. The first recombinant strain of Z. mobilis was reported in 1995. It was done by inserting “tal and tkt” genes from E. coli into Z. mobilis (Zhang et al., 1995). Furthermore, genetic engineering in Z. mobilis has led the organism to simultaneously utilize glucose, xylose, and arabinose for the production of ethanol by fermentation method (Deanda et al., 1996). The “pdl and adh” genes are the prominent genes that help in producing bioethanol and they have also been introduced into other microorganism, such as E. coli, as reported earlier (Piriya et al., 2012).
20.3.4.3 Bacillus subtilis It is a Gram-positive rod-shaped bacterium that has added advantages of withstanding low pH, high temperature, high salt concentrations, and other intolerant conditions (Dien et al., 2003). Bacillus subtilis has the ability to produce butanediol, lactate, acetate, and traces of ethanol in order to produce high amount of ethanol.
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The “pdc and adh” genes from Z. mobilis are inserted into B. subtilis which eliminated butanediol synthesis and inserting E. coli udhA transhydrogenase in “alsS” locus. This recombinant B. subtilis strain possesses the ability to produce ethanol as a sole product (Romero et al., 2007).
20.3.5 Yeast Yeast, a single-celled eukaryotic microorganism, is currently the leading microorganism that is highly employed for fuel production (Hahn-H¨agerdal et al., 2006). Yeast is generally grown slightly in acidic condition and used for the production of food. At present, the application of yeast in biofuel production by fermentation has the greatest demand due to their economic sustainability and environment-friendly nature. The sugars present in biomass can be converted into ethanol by breaking down of sugars by amylase enzyme and then allow it to ferment to produce ethanol. Most commonly, coculturing of yeast and recombinant yeast were reported to be very effective in utilizing both xylose and glucose to increase the bioethanol yield (Fu et al., 2009). But, the problem in coculturing is inhibition of one yeast by the other. Saccharomyces cerevisiae has the special compatibility to grow along with other yeast strains. It is well known for bioethanol production in spite of toxic chemicals present in biomass. S. cerevisiae is grown in optimal condition to give the maximum yield. Very few yeast strains, such as, Pachysolen tannophilus, Candida shehatae, and Pichia stipitis, were reported to have the ability to uptake both C5 and C6 sugars (Agbogbo and Coward-Kelly, 2008). When the biomass is subjected to pretreatment by biological or chemical method, it is reported to improve ethanol yield. Enzymes play an important role in converting biomass into biofuels and many other value-added by-products which is economically and environmental sustainable. In addition, it has various applications in food, feed, and textile industries. S. cerevisiae is grown along with other microbes to increase the production yield and to reduce the cost and time. Xylose reductase gene from Scheffersomyces stipites has been introduced into S. cerevisiae to increase the yield of ethanol production (Runquist et al., 2010).
20.4
Challenges and future perspective
Producing biofuels from marine resource is more challenging because of the biomass complexity and the problems during harvesting and transporting to the centralized refinery section. It also includes the pretreatment methods, microbial fermentation, and microbial contamination during the process. Reducing the cost of biofuel is also a major challenge faced by the biorefinery (Balan, 2014). If this cost issue is addressed, biofuels can be considered as "fuel of future" for transportation in the next few years. New companies, new competencies, and job opportunities are expected to emerge in upcoming years for the biorefineries. It also requires further innovation in abovementioned aspects (De Jong and Jungmeier, 2015).
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20.5
Refining Biomass Residues for Sustainable Energy and Bioproducts
Conclusion
Combustion of fossil fuel sources contributes high atmospheric carbon dioxide release, thus being one of the major causes of global warming. On the contrary, utilizing the nutrients obtained from waste sources could certainly help to develop sustainable environment. In this regard, biofuel obtained from marine sources offers various advantages of providing good content of energy production, consumes high carbon dioxide, and provides cheap fuel source. Therefore this chapter has compiled various marine sources involved in biofuel production, along with their properties, biofuel production procedures, and need for biosources for biofuel production. The marine biosources are found to be an excellent biooption for a sustainable environmental application of biofuel production. They are found to help in reducing cost of fuel and global warming thus contributing to environmental remediation along with various value-added potential by-products.
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Jeevanandam Vaishnavi, Arumugam Arulprakash, Adikesavan Selvi and Aruliah Rajasekar Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Vellore, India
20.1
Introduction
Providing an adequate supply of clean energy for the society is one of the most challenging tasks the world is facing now, as it is directly linked with global stability and economy issues. Fuel usage in the form of transportation, manufacturing, and domestic heating applications contributes around 70% of the total global energy requirements. On the other hand, electricity accounts for only 30% of the global energy consumption. The world’s primary energy resource is dependent on fossil fuels, extraction of oil, and natural gas which leads to the emission of carbon dioxide, which in turn contribute to the greenhouse effect (Change, 2014). According to Vially et al. (2013), the risks of dwindling reserves are expected to last for probably 53 years (oil reserve) and 54 years (natural gas reserve). Climatic changes urge the need for an effective ecological change (Taylor et al., 2015). Therefore a bio-based approach, such as marine waste biorefinery, is considered as one of the innovative and alternative technologies that convert biomass into sustainable and economically valuable products (Demirbas, 2009). Petroleum refineries formed the basis to the emergence of biorefineries that have greatly replaced conventional refineries. Biorefineries have been considered as an alternative for the fossil fuels due to increased fuel usage, high fuel cost, use of nonrenewable resource, and secondary pollutant generation (Aristidou and Penttil¨a, 2000). Marine biorefinery is one such method that makes use of microbial community isolated from the marine environment for high-yield biofuel production. Major source of marine biorefinery includes microalgae (diatoms, green, golden, and blue/green algae), macroalgae (brown, red, and green seaweeds), yeast, and bacteria. Marine crops have long been recognized as a potential biofuel source due to its greenhouse gas abatement potential characteristics and CO2 adsorption capacity than terrestrial plants (Jong et al., 2010). During the process of biofuel (bioethanol, biodiesel, biomethanol) production, other value-added by-products, such as pharmaceuticals, food, feedstocks, enzymes, and pigments, are also produced (Coates et al., 2013). Therefore biorefinery technology plays a dual role
Refining Biomass Residues for Sustainable Energy and Bioproducts. DOI: https://doi.org/10.1016/B978-0-12-818996-2.00020-X © 2020 Elsevier Inc. All rights reserved.
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in reusing the waste and producing valuable products. Above all, this technology is believed to support economic growth and environmental sustainability. However, the operational cost of marine biorefineries still remains too expensive to consider them as a viable option for biofuel production and other related applications. Hence, biorefinery system should be designed with minimal investments, less energy usage, recycling options, easy separation, wide range of raw material availability, and transportation.
20.2
Biofuel generations
Based on the biomass used, biofuels can be categorized into first, second, and third generations that determine the flexibility and potential of the biofuel industry. Different generations of biofuel with their characteristics are shown in Table 20.1. Due to the ethical problems faced by first- and second-generation biofuels, the economic concern has shifted from terrestrial to marine biomass for the biorefinery process (Gouveia, 2011).
20.3
Sources of biofuel
20.3.1 Algae Algae are usually aquatic oxygen-emitting photosynthetic organisms with the simple structure of no roots, stems, or leaves. Since they don’t fit under a single monophyletic group, they cannot be easily defined. They exist as a group of ubiquitous, but individual species, thus occupying specific habitats. Algae exist in various forms, such as a few of the algae attached to plant substrates; few possess motility behavior like animals; few are suspended in water; few grow loosely in trees, soil, and animals; and some form symbiotic association with other organisms (e.g., lichens, corals). Microalgae lack complex multicellular structures with great variation in their internal cell structure. The blue-green algae or cyanobacteria have a prokaryotic cell structure that closely resembles bacteria. The demands for the biofuels lead to the search for feedstocks of micro and macroalgae for their production. Algae consist of 40% of lipids, which helps in rapid conservation of biofuel, thus making the process environment friendly and cost-effective (John et al., 2011). In addition, the cultivation of algae is also a relatively simple and easy technique. So this method has become globally attractive (Miao and Wu, 2004). Algal biomass can be classified as sugars, proteins, and triglycerides, which can be converted into various valuable products, such as pharmaceutical products, food, feedstocks, enzymes, and pigments along with biofuel (bioethanol, biodiesel, biomethanol). Owing to these advantages, petroleum refinery can be partially replaced by biorefinery. Therefore this section deals with different algal resources involved in biofuel production.
Table 20.1 Biofuel generations. S. no.
First generation
Second generation
Third generation
Produced from sugar, starch, vegetable oil, or animal fats Basic feed stocks are wheat, corn, and rape seeds and grains Bioethanol, biodiesel, starchderived biogas, vegetable oils, biomethanol, and boaters Anaerobic digestion
Produced from a variety of nonfood crops, such as lignocellulosic materials from agricultural, forestry, and industry
Produced from yeast, fungi, and algal biomass
FT diesel from biomass and bioethanol
Hydrogen and methane gas, bioethanol, butanol, and acetone
Thermochemical, flash pyrolysis, enzymatic
Acid hydrolysis, liquefaction, pyrolysis, gasification, extraction and transesterification, fermentation, and anaerobic digestion processes Eco-friendly and cost-effective
1.
Biomass source
2.
Biofuel type
3.
Conversion route
4.
Advantages
Reduced global warming emissions and fossil energy consumption
5.
Disadvantages
Compete with food and feed industries for use of biomass and agricultural land Give rise to ethical implication Increased fossil fuel price
FT, Fischer Tropsch.
Improved land-use efficiency and environmental performances Availability of widespread and cheap raw material Allow coproduction biofuels, chemical compounds Generation of electricity and heat Better energy, environmental, and economic performances Biomass residues used are still at precommercial stage
Usage of large volumes of water
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20.3.2 Microalgae and macroalgae The production of biofuel using microalgae includes selection of a particular microalgae cultivation of that specific species, harvesting followed by pretreatment methods that include acid transesterification, product separation, and anaerobic digestion (Assacute et al., 2018). Macroalgae are commonly known as seaweeds, it is generally found in subtidal and intertidal regions of the sea. It includes selection, cultivation, and harvesting of a particular macroalgae, in continuation with extraction of biodiesel along with the production of enzymes with alcoholic fermentation and anaerobic digestion. When compared to microalgae, macroalgae seems to be more complex. Same feedstock has been converted into different value-added products (Assacute et al., 2018). The production of biodiesel using microalgae and macroalgae is depicted in Figs. 20.1 and 20.2, respectively.
20.3.2.1 Chlorella The green microalgae, Chlorella has a high protein content and it can be used for human consumption, whereas it can accumulate large amounts of lipids when grown under stress condition. Due to this ability, it can be used for the production of biofuels. The main disadvantage of the cultivation of Chlorella includes high cost (Guccione et al., 2014). It is cultivated under photoautotrophic conditions predominantly in open ponds (Ramaraj et al., 2016). The largest autotrophic production of algae was started from the year 2000 in a system of 500 km in glass
Figure 20.1 Production of biofuel using microalgae.
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Figure 20.2 Production of biofuel using macroalgae.
tubing in Klotze, Germany, which produced approximately 100 t of chlorella annually (Pulz and Gross, 2004). Till date, chlorella production guarantees high quality which can be used safely and successfully (https://www.algomed.de/en/homepage). Two main Chlorella species, Chlorella vulgaris and Chlorella pyrenoidosa, are commercially cultivated. Chlorella species contains high amount of starch, which can be used for the production of bioethanol (Bra´nyikova´ et al., 2011) when supplemented with 50% of sulfur content. The Chlorella biomass is rich in carbohydrates, minerals, and proteins that can be used to produce many value-added by-products and several bioactive compounds after the extraction of biofuels (Brennan and Owende, 2010) and thus Chlorella helps to produce environment-friendly and sustainable biofuels. The main advantages in cultivating microalgae (Chlorella) are as follows: it does not need a fertile soil, limits the pollution, and efficient in utilizing the nutrients from wastewater. Algae require carbon dioxide for its growth (Guccione et al., 2014).
20.3.2.2 Botryococcus braunii The pear-shaped, bloom-forming green microalgae Botryococcus braunii grow in a cluster form that can be used for the production of biodiesel. Due to its blooming nature, there is an increased quantity of biodiesel production of high quality. The selection of strains is solely based on the high amount of lipid production. Unless
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grown in an optimum condition, their growth rate is too slow (Lassing, et al., 2008). It is one of the most promising biofuel producing microalgae due to its high lipid content. B. braunii is a little unusual in the secretion of lipids in the extracellular medium, whereas other microalgae have cytoplasmic lipids (Hirose et al., 2013).
20.3.2.3 Pleurochrysis carterae The unicellular microalgae, Pleurochrysis carterae, have a unique ability of calcification process occurring at the subcellular level to produce calcified scales. This type of microalgae can be commercially used for the production of biodiesel due to their lipid content. It is a fast growing organism with low risk of contamination (Rahbari, 2009).
20.3.2.4 Dunaliella salina The biflagellate green microalgae, Dunaliella salina, belong to the family Dunalliellacea. It is predominantly found in high salt regions commonly in marine waters. Due to its high amount of carotenoids and its antioxidant activity, it is regarded as a food source. It is also used for the biodiesel production due to its methylation of fatty acids, such as linolenic and palmetic acids (Oren, 2005).
20.3.3 Macroalgae 20.3.3.1 Gracilaria chilensis The red macroalgae Gracilaria chilensis have been reported to produce higher amounts of biomass when compared to other macroalgae (Wi et al., 2009). Due to the high content of polysaccharides, it is useful for the production of bioethanol by the method called hydrolysis. After the extraction of biomass, it can be used for producing other value-added by-products.
20.3.3.2 Sargassum angustifolium The brown algae Sargassum angustifolium are abundantly present in the Persian Gulf, which is used for the production of biodiesel and other value-added byproducts. After the extraction of biofuel, S. angustifolium is mainly used for the production of sodium alginate. The biomass obtained after the alginate production is used for bioethanol production by fermentation method. The macroalgal S. angustifolium biomass can be used as the substitute for yeast during ethanolic fermentation (Ardalan et al., 2018). The bioethanol production from S. angustifolium biomass should be pretreated with acid (Yazdani et al., 2015). This pretreatment step disrupts the recalcitrant structure completely and evolves nitrogen gas that can be used for the fermentation, thus cutting down the cost of nutrients required for the process.
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20.3.3.3 Sea lettuce: Ulva lactuca The green sea lettuce, Ulva lactuca, is used for bioethanol production and it is a renewable gas fuel. U. lactuca commonly refers to green tides due to the eutrophication (excess amount of nitrogen secretion) or algae blooms due to high lipid content (Allen et al., 2013). Sea lettuce generally contains minimal amount of cellulose from which biomethane is produced by anaerobic digestion (VergaraFerna´ndez et al., 2008). The shallow basins remain the most susceptible place for the growth of sea lettuce. Shallow topographs protect the algae from washout and it also keeps the pollutant, such as urea and nitrogen, from initiating the algal growth.
20.3.4 Other microbial biofuel sources Microorganisms are versatile living factories that can utilize numerous natural and synthetic compounds for their growth and convert them into different useful chemicals. Conversion of marine biomass into simple sugars is a necessary step in the production of biofuel. The major barrier for this process is the presence of high branched and recalcitrant compounds in marine biomass. However, these compounds can be subjected to pretreatment methods to release chemicals and enzymes that help in the conversion of biomass into biofuel. Pretreatment methods involve the application of microbial enzymes that help in breaking down of highly branched structures. Generally, marine biomass is composed of cellulose, hemicellulose, lignin, minerals, proteins, and oil (Wyman, 1999). Pretreatment of biomass using microbial enzymes is considered as a cost-effective and environment-friendly approach (Chaturvedi and Verma, 2013). However, pretreatment methods will vary according to the proportion of the chemicals present in different biomass. In earlier days, saccharification and fermentation process was considered attractive because of the combined addition of hydrolytic enzymes and microbes in the same environment (Punnapayak and Emert, 1986). So, this process has led to minimal inhibition, thus resulting in high yield of products at less cost (Wyman, 1999). On the other hand, solid-state fermentation (SSF) is employed at low pH and high temperature. But the microbes are subjected to adopt the ability to grow in this extreme condition. In order to obtain good results, microbes can be genetically engineered and employed in this process (Zhang et al., 1995). Later, SSF was replaced by separate hydrolysis and fermentation (SHF) process, in which the pretreatment of biomass was followed by enzymatic hydrolysis and then to a fermentation process. In this method, biomass undergoes each process independently under optimal conditions. This type of treatment of biomass leads to the additional discharge of waste, which can be used for producing other value-added products using biocatalysts (Chaturvedi and Verma, 2013). Unlike SHF, consolidated bioprocessing process combines both cellulose and hemicellulose fermentations in a single batch process (Lynd et al., 2002). Microbes that have the ability to produce cellulolytic enzymes can be employed in this process. Glucose and xylose cofermentation can be attained by employing genetically engineered microbial consortium (Zhang et al., 1995).
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20.3.4.1 Escherichia coli Escherichia coli has the natural ability to utilize a variety of sugars in both aerobic and anaerobic conditions. In this regard, E. coli is used for producing biodiesel and many other industrial products (Liu and Khosla, 2010). However, the development of new molecular technology tools to identify the genetic variation and genetic makeup of the organism helps in generating high amounts of biofuel (Ferrer et al., 2009). Currently, ethanol is dominating in biofuel as an alternative source. Additional advantage of using E. coli is its ability to metabolize both hexose and pentose sugars (by glycolysis), the components of lignocellulosic material, whereas many other microbes do not have the ability to use pentose sugar. Effective implementation of genetic engineering can overcome this issue. In a study by Zhang et al. (1995), the genes “pdc” and “adhB” from Zymomonas mobilis are inserted in E. coli. These genes have been expressed in operon from plasmid which is constitutively expressed in the production of ethanol using pET (expression plasmid for T7 RNA polymerase) promoter. So, the genetically engineered E. coli produces pyruvate decarboxylase and alcohol dehydrogenase II, which helps in producing 95% ethanol without redox imbalance. Continuous generation of ethanol can be achieved by constructing E. coli plasmid vector with “pods” and “adhB” gene inserts with selective chloramphenicol resistance gene and integrated through chromosome.
20.3.4.2 Zymomonas mobilis Gram-negative, facultative anaerobe, rod-shaped bacterium belongs to class Alphaproteobacteria that is a natural ethanologen which has many important environmental characteristics to replace the natural fossil fuel (Yang et al., 2016). It is regarded as the best alternative for fuel as it reduces the production cost of controlling aeration during fermentation method (Yang et al., 2016). The nitrogen fixation ability of this organism does not affect the ethanol production; rather it increases the productivity of ethanol. Z. mobilis has the ability to metabolize both C5 and C6 sugars. The first recombinant strain of Z. mobilis was reported in 1995. It was done by inserting “tal and tkt” genes from E. coli into Z. mobilis (Zhang et al., 1995). Furthermore, genetic engineering in Z. mobilis has led the organism to simultaneously utilize glucose, xylose, and arabinose for the production of ethanol by fermentation method (Deanda et al., 1996). The “pdl and adh” genes are the prominent genes that help in producing bioethanol and they have also been introduced into other microorganism, such as E. coli, as reported earlier (Piriya et al., 2012).
20.3.4.3 Bacillus subtilis It is a Gram-positive rod-shaped bacterium that has added advantages of withstanding low pH, high temperature, high salt concentrations, and other intolerant conditions (Dien et al., 2003). Bacillus subtilis has the ability to produce butanediol, lactate, acetate, and traces of ethanol in order to produce high amount of ethanol.
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The “pdc and adh” genes from Z. mobilis are inserted into B. subtilis which eliminated butanediol synthesis and inserting E. coli udhA transhydrogenase in “alsS” locus. This recombinant B. subtilis strain possesses the ability to produce ethanol as a sole product (Romero et al., 2007).
20.3.5 Yeast Yeast, a single-celled eukaryotic microorganism, is currently the leading microorganism that is highly employed for fuel production (Hahn-H¨agerdal et al., 2006). Yeast is generally grown slightly in acidic condition and used for the production of food. At present, the application of yeast in biofuel production by fermentation has the greatest demand due to their economic sustainability and environment-friendly nature. The sugars present in biomass can be converted into ethanol by breaking down of sugars by amylase enzyme and then allow it to ferment to produce ethanol. Most commonly, coculturing of yeast and recombinant yeast were reported to be very effective in utilizing both xylose and glucose to increase the bioethanol yield (Fu et al., 2009). But, the problem in coculturing is inhibition of one yeast by the other. Saccharomyces cerevisiae has the special compatibility to grow along with other yeast strains. It is well known for bioethanol production in spite of toxic chemicals present in biomass. S. cerevisiae is grown in optimal condition to give the maximum yield. Very few yeast strains, such as, Pachysolen tannophilus, Candida shehatae, and Pichia stipitis, were reported to have the ability to uptake both C5 and C6 sugars (Agbogbo and Coward-Kelly, 2008). When the biomass is subjected to pretreatment by biological or chemical method, it is reported to improve ethanol yield. Enzymes play an important role in converting biomass into biofuels and many other value-added by-products which is economically and environmental sustainable. In addition, it has various applications in food, feed, and textile industries. S. cerevisiae is grown along with other microbes to increase the production yield and to reduce the cost and time. Xylose reductase gene from Scheffersomyces stipites has been introduced into S. cerevisiae to increase the yield of ethanol production (Runquist et al., 2010).
20.4
Challenges and future perspective
Producing biofuels from marine resource is more challenging because of the biomass complexity and the problems during harvesting and transporting to the centralized refinery section. It also includes the pretreatment methods, microbial fermentation, and microbial contamination during the process. Reducing the cost of biofuel is also a major challenge faced by the biorefinery (Balan, 2014). If this cost issue is addressed, biofuels can be considered as "fuel of future" for transportation in the next few years. New companies, new competencies, and job opportunities are expected to emerge in upcoming years for the biorefineries. It also requires further innovation in abovementioned aspects (De Jong and Jungmeier, 2015).
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20.5
Refining Biomass Residues for Sustainable Energy and Bioproducts
Conclusion
Combustion of fossil fuel sources contributes high atmospheric carbon dioxide release, thus being one of the major causes of global warming. On the contrary, utilizing the nutrients obtained from waste sources could certainly help to develop sustainable environment. In this regard, biofuel obtained from marine sources offers various advantages of providing good content of energy production, consumes high carbon dioxide, and provides cheap fuel source. Therefore this chapter has compiled various marine sources involved in biofuel production, along with their properties, biofuel production procedures, and need for biosources for biofuel production. The marine biosources are found to be an excellent biooption for a sustainable environmental application of biofuel production. They are found to help in reducing cost of fuel and global warming thus contributing to environmental remediation along with various value-added potential by-products.
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