Third generation biofuels: an overview

Chapter 10

Third generation biofuels: an overview Joa˜o Moreira Neto1, Andrea Komesu2, Luiza Helena da Silva Martins3, Vinicius O.O. Gonc¸alves4, Johnatt Allan Rocha de Oliveira5 and Mahendra Rai6 1

Department of Engineering, Federal University of Lavras (UFLA), Lavras, Brazil, 2Department of Marine Sciences, Federal University of Sa˜o Paulo (UNIFESP), Santos, Brazil, 3Department of Natural Sciences and Technology, State University of Para´ (UEPA), Bele´m, Brazil, 4Institute of Chemistry, Department of Physical Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 5Institute of Health Sciences, Nutrition College, Federal University of Para´ (UFPA), Bele´m, Brazil, 6Nanobiotechnology Lab, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, India

10.1 Introduction Rapid industrialization and high population growth are two major factors contributing to the global energy crisis.1 Today, approximately 88% of worldwide energy consumption is a consequence of the combustion of fossil fuels.2 Unfortunately, the massive use of fossil fuels has led to problems such as the depletion of reserves, price fluctuations, negative environmental impacts, and climatic change.1 Therefore alternative energy sources that are readily available, facilely accessible, and environment-friendly are very much needed.3 Biofuels have emerged as a promising alternative to fossil fuel resources. The term “biofuels” refers to energy enriched chemicals generated through biological processes or derived from the biomass of living organisms such as microalgae, plants, and bacteria.4 The energy in biofuel is stabilized through the process of biological carbon fixation, in which carbon dioxide (CO2) is converted into sugar that is only found in living organisms such as plants. In contrast to fossil fuels, biofuel is produced by, or derived from, living organisms in a relatively short period of time rather than being derived by the decomposition of organic matter over several million years.5 Currently, there are three generations of biofuels that have flourished and that are based on different feedstock. “First generation” biofuels such as Sustainable Bioenergy. DOI: https://doi.org/10.1016/B978-0-12-817654-2.00010-1 © 2019 Elsevier Inc. All rights reserved.

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bioethanol and biodiesels are produced directly from starch, sugar, and oil present in food crops like corn, wheat, and soybeans.5 Important characteristics of “first generation” biofuels include their ability to be blended with petroleum-based fuels and their efficiency in internal combustion engines, as well as their compatibility with flexible fuel vehicles.2 The most controversial issue with first generation biofuels is that it is necessary to choose between either fuel or food alternatives.5 Additionally, food crop feedstocks require large agricultural areas to produce sufficient quantities of biomass. In terms of environmental issues, such increased agricultural yield and subsequent harvesting may lead to enhanced land clearing, loss in biodiversity due to habitat destruction, water depletion, and air pollution.2 “Second generation” biofuels are generally produced from nonfood crops mainly lignocellulosic feedstocks such as grass, wood, and other organic wastes.5 The advantages of these feedstocks are their ease of availability, they do not compete with the food industry, and eventually have a lesser impact on the environment.1 Nevertheless, second generation biofuels also face steep challenges in the form of technical difficulties during pretreatment processes and inefficient conversion of lignocellulosic materials due to their complex structures.2 In the search for viable and cost-effective alternatives to fossil fuels, studies have reported superior capabilities of algae-derived biomass for the production of an improved version of biofuels: “third generation” biofuels.2 Algae (Alien to Light Green Antenna Entity) are highly diverse micro- and macro-plants that are found almost everywhere on planet Earth. They play an important role in many ecosystems; providing food, feed, and vital nutrients as well as supplying up to 60% of the required oxygen to all living beings vital for their survival.6 Lately, algal cultures have been used to produce vitamins, feed, and other products on an industrial scale. However, the rapid growth in world energy demand has led to the development and analysis of efficient sources capable of producing fuels and chemicals. Thus microalgae are reported to be a promising alternative for the production of fuels and chemicals due to their high photosynthetic conversion efficiency. These organisms are unicellular, contain carbon, hydrogen, oxygen, and nitrogen, and are categorized as aquatic biomass. The growth of microalgae depends heavily on the availability and intensity of light, the availability of nutrients like phosphorus and nitrogen, CO2 and O2 levels, temperature, and the type of culture system used.7 Algae represent a promising, everlasting source of fuel and other valuable products.6 Algae have the ability to produce crude oil which may, then, be easily processed into diesel and gasoline. Particular algae species which may be categorized as either microalgae or macroalgae may be genetically modified so that the carbon metabolic pathway facilitates the production of important end-products like ethanol.2 In addition, algae have the ability to produce

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more energy per acre of land compared to other conventional feedstock crops such as sugarcane and corn.8 For these reasons, microalgae are touted to have the maximum potential to act as a substitute for petroleum-derived transport diesel without adversely affecting food supply and other crop products.2 The main goal of this chapter is to explore the potential of algae for the production of different biofuels. Moreover, the physicochemical composition of microalgae and types of treatments applied for the deconstruction of microalgae are discussed.

10.2 Technologies for biomass conversion into biofuel The most widely used processes for transforming microalgal biomass into different energy sources can be classified as biochemical conversion, thermochemical conversion, chemical reactions, and direct combustion, as shown in Fig. 10.1.9 Biochemical conversion is a process of converting biomass into others fuels through anaerobic digestion and alcoholic fermentation. The example of processes belonging to biochemical conversion include fermentation for ethanol production10 and anaerobic digestion for methane11 and hydrogen production.12 Thermochemical conversion is a process of thermal decomposition of biomass into fuels products. Such thermochemical processes include gasification,13 pyrolysis,14 and liquefaction.14 An example of chemical conversion process involves the conversion of the extracted lipid from microalgae cells to biodiesel via a transesterification reaction.15 In the direct

FIGURE 10.1 Biofuels production via the microalgal biomass conversion processes. Adapted from Wan B, Li Y, Wu N, Lan CQ. CO2 bio-mitigation using microalgae. Appl Microbiol Biotechnol 2008;79:707 18 with copyright permission from Springer Nature.

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combustion, the algal biomass is burned in the presence of air, converting the chemical energy of the algal biomass into heat or electricity.14

10.3 Potential of microalgae as raw material for biofuels production Biofuels could be an alternative source of energy to meet the energy needs generated by the ever increasing world population.4 This scenario has led to the development and analysis of efficient sources to produce fuels and chemicals.16 The emergence of third generation bioethanol provides more benefits compared to first and second generation bioethanol. Third generation bioethanol is focused on the use of marine organisms such as algae.1 Several studies performed in the past have demonstrated that among different sources, microalgae have significant potential for the production of bioenergy and chemicals.16,17 This is due to their high lipid and carbohydrate contents, high proton conversion, easy cultivation in a wide variety of water environments, relatively low land usage and high carbon dioxide (CO2) absorption.18 With new approaches involving the production of microbial biofuels, the cultivation of microalgae has been recommended as a possible strategy to obtain higher amounts of biomass for the production of fuel. This biomass could be obtained by culturing biofilms of microalgae or cyanobacteria.19,20 The production of biofuels can be achieved by several species of algae, but species rich in carbohydrates, proteins, and lipids, are preferably used in the production of biofuels.21 Microalgae are considered as potential alternative feedstocks for biofuel production as they are a sustainable energy source and do not compete with other edible feedstocks. The cultivation of microalgae also creates a carbon sink for greenhouse gasses, thus, making it environment-friendly.3 Sugar is the basic molecular substrate for the production of bioethanol and biomethanol.22 The use of photosynthetic organisms as a source of biofuel is cheap and feasible, and is based on the main component found in this type of material; carbohydrate.21 Compared to other traditional biofuel crops, it is a better source for oil extraction and may be utilized to synthesize a multitude of diverse biofuels such as biomethane (via anaerobic digestion) and biodiesel (using microalgal oil), as well as biohydrogen (via photobiological synthesis).8 Their photosynthetic efficiency is higher than that of other plants, and some species are considered to be among the fastest growing plants in the world.23 It has been reported that the photosynthetic efficiencies of algae range from 3% to 8%, compared with the 0.5% of many terrestrial crops.24 Algae are simple plants ranging from microscopic (microalgae) to large seaweeds (macroalgae), from small green dots to larger cyanobacteria and the tinsel diatoms. There are nine major groups of algae which include

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cyanobacteria (Cyanophyceae), green algae (Chlorophyceae), diatoms (Bacillariophyceae), yellow-green algae (Xanthophyceae), golden algae (Chrysophyceae), red algae (Rhodophyceae), brown algae (Phaeophyceae), dinoflagellates (Dinophyceae), and “picoplankton” (Prasinophyceae and Eustigmatophyceae).25 Microalgae like Spirulina, Chlorella, Dunaliella, and Haematococcus are currently cultivated commercially to produce photosynthetically grown biomass from a few tons to several hundred tons annually. They grow at an exceptionally fast rate; 100 times faster than terrestrial plants, and they can double their biomass in less than 1 day.26 Harvesting microalgal biomass is a major challenge because of their small size and their low concentration in culture media. In the overall production process, microalgae are initially grown to reach a maximum biomass concentration of 0.02% 0.5%. From the culture medium, the biomass is concentrated to 15% 20%, either in a single step or in a series of concentration steps, before they can be processed further via drying, extraction, or other downstream processing steps.8,27,28

10.4 Physicochemical composition of microalgae The existence of nearly 300,000 species of algae, with varied oil contents and growth rates, was reported by Scott et al.29 Microalgal biomass is mainly constituted of lipids, carbohydrates, and proteins. A microscope is required in order to observe microalgae because of their size, being either unicellular or multicellular, but always smaller than 0.4 mm in diameter. The category includes groups such as diatoms and cyanobacteria that have high growth rates.5 The three most abundant classes of microalgae are Bacillariophyceae, Chlorophyceae, and Chrysophyceae.5 Different microalgal species have significantly different compositions, where the average lipid oil content typically varies between 8% and 31%.2 The carbohydrate content was also found to be relatively high which can reach up to 50% of the dry weight for some species such as Scenedesmus, Chlorella, and Chlamydomonas.1 Table 10.1 summarizes the dry weight composition of protein, carbohydrate, and lipid of some species of microalgae. Factors such as light, temperature, nutrient content, pH, O2, and CO2 level, salinity, and toxic chemicals influence the lipid and carbohydrate contents of microalgae.1 The lipid content of microalgae varies in accordance with the culture conditions. It is possible to increase the lipid concentration by almost 80% above natural levels, by optimizing the growth determining factors.5 Some of the common components in the cell wall of microalgae are cellulose, protein, lignin, pectin, hemicelluloses, and other carbohydrates which can be converted into monomers through acid or enzymatic hydrolysis to produce bioethanol.1

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TABLE 10.1 Composition of different species of microalgae on a dry matter basis. Microalgal species

Protein

Carbohydrate

Lipid

Anabaena cylindrica

43 56

25 30

4 7

Chlamydomonas reinhardtii

48

17

21

Chlorella pyrenoidosa

57

26

2

Chlorella vulgaris

51 58

12 17

14 22

Dunaliella bioculata

49

4

8

Porphyridium cruentum

28 39

40 57

9 14

Prymnesium parvum

28 45

25 33

22 39

Scenedesmus dimorphus

8 18

21 52

16 40

Scenedesmus obliquus

50 56

10 17

12 14

Spirogyra sp.

6 20

33 64

11 21

Spirulina maxima

60 71

13 16

6 7

Spirulina platensis

46 63

8 14

4 9

Synechococcus sp.

63

15

11

Tetraselmis maculata

52

15

3

Source: Adapted from Um BH, Kim YS. Review: a chance for Korea to advance algal-biodiesel technology. J Ind Eng Chem 2009;15:1 7 with copyright permission from Elsevier.

10.5 General use of bioethanol Bioethanol is mostly used in motor vehicles as fuel and as a gasoline additive. More than any other country, Brazil relies on ethanol as a motor fuel for several motor vehicles. Gasoline sold in Brazil contains at least 25% anhydrous ethanol. Hydrated ethanol (95% ethanol) can be used as fuel in more than 90% of new cars sold in the country. The United States uses mixtures of ethanol/gasoline, known as Gasohol (with a maximum of 10% ethanol), and 85% ethanol. Ethanol can also be used as rocket fuel in lightweight rocket-powered aircrafts.30 Australian law limits the use of pure ethanol from sugarcane residues to 10% in automobiles. It has been recommended that older cars should have their valves updated or replaced.30 Direct hydrated ethanol as an automotive fuel has been widely used in Brazil for pure ethanol vehicles and more recently for flexible vehicles. Ethanol fuel is distilled near to the azeotropic mixture of 95.63 wt% ethanol and 4.73 wt% water. By the end of 2012, there were approximately 20 million flexible vehicles running on Brazilian highways. Hydrated ethanol imposes a limitation on the normal operation of these vehicles because the

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lower evaporative pressure of ethanol (compared to gasoline) causes problems when the engine starts cold at temperatures below 288K. For this reason, both pure and flex-fuel vehicles are built with an additional small gasoline reservoir inside the engine compartment to aid in starting the engine when cold, initially by injecting gasoline.30

10.6 Bioethanol production In general, bioethanol production from biomass involves pretreatment, enzymatic hydrolysis, fermentation, and distillation.10 The pretreatment of lignocellulosic feedstocks is a necessary step to unwind the cellulose from the hemicellulose and lignin in which cellulose is embedded, to make the cellulose more susceptible in order to be easily accessed by enzymes during enzymatic hydrolysis, and to enhance the rate and yield of reducing sugars.31,32 Based on the type of treatment process involved, lignocellulosic biomass pretreatment methods are broadly classified into different categories: physical (e.g., milling, microwave, ultrasound, etc.,), chemical (e.g., dilute acid, organosolv, mild alkali, ionic liquid, etc.,), physicochemical (e.g., steam explosion, liquid hot water, ammonia based, oxidative pretreatment, etc.,), and biological (e.g., bacterial, brown, white, and soft-rot fungi, etc.,) methods.32 Such pretreatments, depending on the process conditions, can generate a range of inhibitory byproducts (e.g., hydroxymethylfurfural, furfural, phenolic compounds, or organic acids) which can impact on the viability of yeast in subsequent fermentation processes.33,34 Due to the absence of lignin and low hemicellulose content, algae generally require relatively mild conditions for processing as compared to lignocellulosic biomass.5 The composition of carbohydrates in algal biomass may differ significantly from species to species.1 It is of great importance to select algae with high carbohydrate productivity as well as suitable sugar composition for bioethanol production. Although macroalgae are a potential source of sugar, the majority of carbohydrates in macroalgae biomass (e.g., brown macroalgae) are present in the form of polysaccharides such as laminarin, mannitol, carrageenan, and alginate.34 The degradation of these polysaccharides is slow and requires specific enzymes.35 The development of conversion methods to release monosaccharides from these polysaccharides and the choice of industrial microorganisms for bioethanol production from macroalgae are bottlenecks to the feasibility of the utilization of seaweed as a feedstock.34 Wargacki et al.36 were capable of co-fermenting alginate, mannitol, and glucan from a brown algal species (Saccharina japonica) using Escherichia coli, yielding 0.41 g of ethanol/g of carbohydrate sugars. Sunwoo et al.37 performed a fermentation using mixed yeast, i.e., Saccharomyces cerevisiae KCTC 1126 and Pichia angophorae KCTC 17574, to galactose and mannitol from waste seaweed obtained from Gwangalli beach, Busan, Korea. The

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maximum ethanol concentration and ethanol yield obtained were 13.54 g/L and 0.45%, respectively. Carbohydrates (cellulose) in microalgae biomass are mainly present in the cell wall and starch in the plastids which can be readily converted into fermentable sugars that more suitable for ethanol production.38 The harvesting of microalgae is more expensive than macroalgae. Macroalgae can be harvested using nets which requires less energy, while microalgae are harvested by conventional processes which include filtration, flocculation, centrifugation, sedimentation, froth floatation, and ultrasonic separation.39 Several studies have been conducted on the utilization of different microand macroalgae for the production of bioethanol, such as Chlorella vulgaris,40 Gracilaria verrucosa,41 Gracilaria salicornia,42 Gelidium amansii,43 Sargassum spp.,10 Laminaria digitate,34 Chondrus crispus,34 Palmaria palmata,34 and Kappaphycus alvarezii.44 Saccharification is usually a rate-limiting step in biofuel production using lignocellulosic materials or microalgal biomass that contain a cellulose source.45 Saccharification or hydrolysis consist of the depolymerization of the polysaccharide contents (such as starch, cellulose, alginates, fucans, laminarin, and carrageenans) in algal cell walls into free monomer molecules which can be fermented into bioethanol.1 The absence or near absence of lignin and hemicellulose contents in algae, in comparison to lignocellulosic biomass, makes the enzymatic hydrolysis of algal cellulose easier.46 The main methods that are applied to produce sugars from microalgae can be categorized into two groups: enzymatic saccharification and acid saccharification.45 In acid hydrolysis, a wide range of acids have been used, of which sulfuric acid (H2SO4) is most preferred. It has been found that the polysaccharides of the three classes of macroalgae (brown, red, and green) can be effectively hydrolyzed to monosaccharides by treatment with diluted H2SO4 at high temperatures. The acid is responsible for breaking the bonds of the long chains of the polysaccharides. In the initial stage, the destruction of hydrogen bonds occurs in order to break up the chains of polysaccharides, thus transforming them into molecules in the amorphous state. Polysaccharides are extremely susceptible to acid hydrolysis at this point. The acid will then serve as a catalyst where the cleavage of the polysaccharide will occur by the hydrolysis of the glycosidic bonds. At the end of this process, any addition or dilution with water at a moderate temperature will provide complete hydrolysis and the hydrolysate will be rich in monosaccharides.1 In enzymatic hydrolysis, cellulases are employed as enzymes to degrade polysaccharides and can be categorized into three major types, namely endoglucanases, exoglucanases, and β-glycosidase. Among these, endoglucanases have the ability to hydrolyze the complex sugars of the raw material, attacking the inner parts of the amorphous region of the cellulose. Exoglucanases degrade cellulose by the cleavage of cellobiose units (union of two glucose molecules) from the nonreducing end of a cellulose fiber which allows for

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enzymatic attack. With the combined efforts of β-glycosidase, the residues of cellobiose finally split into two units of glucose.1 Although chemical hydrolysis usually gives a higher sugar production rate, this method requires harsh reaction conditions and the hydrolysates may contain inhibitory byproducts (such as hydroxymethylfurfural and furfural) to the subsequent bioethanol fermentation. On the other hand, enzymatic hydrolysis produces high amounts of fermentable sugars under mild conditions without producing sugar-degradation products or toxic byproducts.47 In order to improve the production of high concentrations of fermentable sugars, acid and enzymatic saccharification can be combined. As red seaweeds contain galactan and glucans and galactan cannot be hydrolyzed by the same enzyme used for the hydrolysis of glucans, Yanagisawa et al.48 combined the acid hydrolysis of galactan to produce galactose with the subsequent enzymatic hydrolysis of glucan to obtain glucose. There are different groups of microorganisms like yeast, bacteria, and fungi that can be used for the fermentation of saccharified algal biomass under anaerobic conditions for the production of bioethanol.39 S. cerevisiae is the most commonly employed strain in the fermentation of bioethanol due to its high selectivity, low accumulation of byproducts, high ethanol yield, and high rate of fermentation.1 In order to increase the production rate in the fermentation process, new process configurations were developed such as separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). In SHF, the saccharification and fermentation of the biomass are performed in two distinct reactors. The saccharification is conducted first to degrade the biomass into monomer sugars using an enzyme. This process is followed by the fermentation reaction which utilizes the sugars formed in the hydrolysis stage. However, one major problem associated with the SHF process is the end-product inhibition by sugars that are formed during the hydrolysis stage.1 In SSF, the saccharification and fermentation processes are conducted simultaneously in a single reactor. During the reaction, the biomass, enzyme, and yeast are put together in an orderly manner so that the sugars released are rapidly converted into bioethanol. SSF can limit end-product inhibition by removing the residual sugar.1 Thus SSF processes can achieve better ethanol yields than methods that apply separate hydrolysis and fermentation processes.31 El-Dalatony et al.49 studied both SHF and SSF processes for bioethanol production from microalgal (Chlamydomonas mexicana) biomass. SSF was selected as an efficient process to enhance bioethanol yields through repeated batches using immobilized S. cerevisiae YPH499 cells. The combined sonication and enzymatic hydrolysis of C. mexicana generated 10.5 and 8.48 g/L of ethanol in SSF and SHF, respectively. Similarly, Tan and Lee50 found that the SSF of seaweed solid wastes with S. cerevisiae was more effective as compared to SHF for bioethanol production. They were able to obtain a bioethanol yield of 55.9% for the SHF process and a yield of 90.9% for the SSF process.

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10.7 Production of fuel from microalgae through pyrolysis Thermochemical processes depend on the degradation of biomass following chemical reactions occurring at moderate to high temperatures. There are three common thermochemical processes for converting biomass into biofuels and chemicals: pyrolysis, liquefaction, and gasification. The main thermochemical route for biomass conversion is pyrolysis which is conducted in the absence of oxygen and at temperatures between 300 C and 700 C. This process converts biomass into pyrolytic oil (or bio-oil), char (also called biochar), and light gases (H2, CO, CO2, and C1 C4 hydrocarbons). Biochar is similar to conventional coal but contains some oxygen. Pyrolytic bio-oil is a complex mixture containing several oxygenated compounds. Generally, pyrolysis bio-oils contain mainly water, aldehydes, carboxylic acids, carbohydrates, phenols, furfural, alcohols, and ketones. The water content in biooil is relatively high (up to 40 wt.%) which often creates an aqueous-rich phase and an organic-rich phase.51 The operational conditions for pyrolysis are varied and the process is mostly classified into fast, flash, and slow and catalytic pyrolysis. The operational conditions can be set in order to favor the production of biochar, bio-oil, or gas fractions. For example, fast pyrolysis and flash pyrolysis (low residence times) produce higher yields of bio-oil (up to 80%). In contrast, slow pyrolysis produces larger amounts of char due to the increased degradation time of the biomass components. Another possibility is to use microwave assisted pyrolysis which shows advantages over traditional processes such as rapid and uniform heating which reduces residence times and accelerates chemical reactions. Regarding the use of fuels and chemicals, bio-oil is preferable due to the variety of compounds in its composition and its flexibility in upgradation, thus being a platform for both renewable chemicals and fuels. In addition, the use of a catalyst is generally related to decreases in the reaction temperature, the removal of the oxygen content, and obtaining more interesting fractions related to certain fuels. The dynamics of biomass production as well as its habitat are important factors for a sustainable chain of renewable fuel. Thus the production of biofuel from algae is of great interest given the high growth rate of some species, their interesting ability to sequester CO2, their robustness in growing in places that do not compete with arable lands, and their noncompetition with human alimentation.51 Promising aspects of the pyrolysis of many algal species such as Chlorella protothecoides, C. vulgaris, Dunaliella, Emilianiahuxleyi, Nannochloropsis residue, Plocamium, Sargassum, Spirulina, Synechococcus, Scenedesmus, Spirulina platensis, Tertiolecta, and Tetraselmis have been reported. Studies showed encouraging results that depict potential future industrial applications.52 Since lignocellulosic biomasses and algae are different in composition; algal bio-oils are quite different compared to lignocellulosic bio-oils. The

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pyrolysis of algae produces a bio-oil with a considerably higher number of linear hydrocarbons, fatty oxygenates, and nitrogenated compounds resulting from the cleavage of lipid and protein bonds. In addition, since algae have no lignin in their composition, the number of phenolic compounds is low. It is also reported that the pyrolysis of algae produces less biochar and a more stable bio-oil with improved properties which could be more feasibly upgraded.53 It is noteworthy that the use of micro- and macroalgae as raw materials for biofuel production is constantly increasing. However, large-scale aspects concerning environmental impacts need to be further studied. Moreover, the direct application of bio-oil (either from algae or lignocellulose) is of major concern due to its high oxygen content which leads to several undesired properties such as poor heating values, corrosiveness, and high viscosity. In this context, bio-oil production through catalytic pyrolysis and bio-oil hydrotreatment upgrading are major research topics in the production of liquid fuels or chemicals in the biorefinery context. Concerning the complexity of bio-oil, upgrading algae bio-oil shows advantages compared to lignocellulosic bio-oil.54

10.8 Protein biofuels Microalgae offer great potential for the large-scale and cost-effective production of recombinant proteins. Huo et al.55 studied the feasibility of the use of proteins as raw material for the bioreflex process through the application of a metabolic engineering strategy centralized in nitrogen. They integrated the merits of microorganisms, including rapid growth and ease of cultivation, with those from higher plants in posttranslational modification and photosynthesis.6,56 Large-scale protein production using microalgae through natural selection under frequent or continuous harvesting conditions may favor robust and fast-growing microorganisms which generally contain a high protein content and are fully adapted to the local environment. Biofuels are currently produced from the carbohydrates and lipids found in this raw material. Proteins, in contrast, have not been used to produce fuels due to the difficulties of deaminating protein hydrolysates. Thus recombinant strategies have proven to be viable for the large-scale production of enzymes that can be used to produce biofuels. Huo et al.,55 as mentioned by Gajraj et al.,6 applied metabolic engineering to generate E. coli that can deaminate protein hydrolysates, allowing cells to convert proteins into C4 and C5 alcohols which present a theoretical yield of 56%. They introduced three exogenous cycles of transamination and deamination which provided an irreversible metabolic force to drive the deamination reactions to conclusion.6 Huo et al.55 demonstrated that S. cerevisiae, E. coli, Bacillus subtilis, and microalgae can be used as protein sources; producing up to 4035 mg/L of

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alcohols from biomass containing B22 g/L amino acids. These results showed the feasibility of using proteins in biorefineries, in which high protein microalgae could be used as feedstocks, with the possibility of maximizing algal growth and total CO2 fixation. To test the viability of algal and bacterial proteins as raw materials, Huo et al.55 cultivated green algae (C. vulgaris), red algae (Porphyridium purpureum), blue-green algae (S. platensis), and cyanobacterium (Synechococcus elongatus), as well as E. coli and B. subtilis, in 1 L of fluorine or a 30 L tank. The biomass was harvested, digested with protease, and used as a feedstock for the production of biofuel with the engineered E. coli strain YH83. In all experiments, the protein concentration was adjusted to 21.6 g/L which was equivalent to the amount of protein in 4% of yeast extract.6

10.9 Direct combustion For this process, the algal biomass is burned in the presence of air in a furnace, boiler, or steam turbine, converting the chemical energy of the algal biomass into heat or electricity. The direct combustion of microalgae is only feasible with a moisture content of less than 50% of its dry weight. However, the process suffers from a limitation because it requires pretreatment of the algae (like drying) which can lead to a decrease in energy efficiency. Instead, the production of algae and coal-biomass is an effective way to generate electricity. During algal biomass aging, algae are cultivated and fed with a CO2 recycling stream from a biorefinery plant, and the cultured algae are fed to the plant together with charcoal.14

10.10 Ultrasonication to aid biofuel yields According to Chye et al.,2 the ultrasound process requires the incorporation of acoustic energy with frequencies in the range of 10 kHz to 20 MHz which is applied to form microbubbles in constant expansion and shrinkage. The microbubbles destabilize as the acoustic energy exceeds a certain limit. When it resonates with the natural frequency of the microbubbles, it collapses, causing high-speed microdata of more than 100 m/s which carry more than 100 MPa of pressure toward the surface of the biomass. This causes cavitation and the process improves biochemical reactions and may help maximize lipid yields during the lipid extraction of microalgae or cause the rupture of the cell wall to aid in the hydrolysis step for bioethanol fermentation or anaerobic digestion. Suali and Sarbatly57 demonstrated that ultrasound is able to reduce the lipid extraction time by one-tenth of the conventional methods and reach a 50% to more than 500% yield.

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10.11 Conclusions Microalgae and macroalgae are promising raw materials for the production of biofuels. Some technologies discussed in this chapter show the suitability and applicability of biofuel products which may be alternatives to fossil energy. The physicochemical composition of algal biomass based on their lipid, carbohydrate, and protein contents and types of treatments applied for the deconstruction of microalgae are crucial factors for obtaining products of interest in the chemical, food, and pharmaceutical industries. Although microalgae are reported to be a promising alternative for the production of fuels and chemicals due to their high photosynthetic conversion efficiency, further research and development are necessary to establish an economical industrial-scale production of algal biofuels.

References 1. Jambo SA, Abdulla R, Azhar SHM, Marbawi H, Gansau JA, Ravindra P. A review on third generation bioethanol feedstock. Renew Sustain Energy Rev 2016;65:756 69. 2. Chye JTT, Jun LY, Yon LS, Pan S, Danquah MK. Biofuel production from algal biomass. In: Konur O, editor. Bioenergy and biofuels. Boca Raton, FL: CRC Press, Taylor & Francis Group; 2018. p. 87 117. 3. Leong W, Lim J, Lam M, Uemura Y, Ho Y. Third generation biofuels: a nutritional perspective in enhancing microbial lipid production. Renew Sustain Energy Rev 2018;91:950 61. 4. Rodionova MV, Poudyal RS, Tiwari I, Voloshin RA, Zharmukhamedov SK, Nam HG, et al. Biofuel production: challenges and opportunities. Int J Hydrogen Energy 2017;42:8450 61. 5. Alaswad A, Dassisti M, Prescott T, Olabi AG. Technologies and developments of third generation biofuel production. Renew Sustain Energy Rev 2015;51:1446 60. 6. Gajraj RS, Singh GP, Kumar A. Third-generation biofuel: algal biofuels as a sustainable energy source. In: Kumar, et al., editors. Biofuels: greenhouse gasmitigation and global warming, A. Springer (India) Pvt. Ltd; 2018. p. 307 25. 7. Moncada J, Tamayo JA, Cardona CA. Integrating first, second, and third generation biorefineries: incorporating microalgae into the sugarcane biorefinery. Chem Eng Sci 2014;118:126 40. 8. Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25(3):294 306. 9. Wang B, Li Y, Wu N, Lan CQ. CO2 bio-mitigation using microalgae. Appl Microbiol Biotechnol 2008;79:707 18. 10. Borines MG, Leon RL, Cuello JL. Bioethanol production from the macroalgae Sargassum spp. Bioresour Technol 2013;138:22 9. 11. Sangeetha P, Babu S, Rangasamy R. Potential of green alga Chaetomorpha litorea (Harvey) for biogas production. Int J Curr Sci 2011;1:24 9. 12. Park JH, Yoon JJ, Park HD, Kim YJ, Lim DJ, Kim SH. Feasibility of biohydrogen production from Gelidium amansii. Int J Hydrogen Energy 2011;36:13997 4003. 13. Onwudili JA, Lea-Langton AR, Ross AB, Williams PT. Catalytic hydrothermal gasification of algae for hydrogen production: composition of reaction products and potential for nutrient recycling. Bioresour Technol 2013;127:72 80.

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