Omics Approaches in Biofuel Technologies

C H A P T E R

18 Omics Approaches in Biofuel Technologies: Toward Cost Effective, Eco-Friendly, and Renewable Energy Vikas Y. Patade, Lekha C. Meher, Atul Grover, Sanjay M. Gupta and Mohammed Nasim Defence Institute of Bio-Energy Research, Haldwani, India

18.1 INTRODUCTION The global energy demand is steadily increasing with the economic growth combined with the population explosion. Among the energy consuming sectors, transportation consumes about 30% of the primary energy. The major sources of the energy are fossilderived fuels including petroleum, coal, and natural gases. According to the BP Statistical Review of World Energy conducted in 2010, the crude oil and natural gas, the major energy resources, may be run out respectively in another 45 and 60 years, with the current global energy consumption policy. Thus, the continued use of fossil-based fuels is not sustainable owing to its limited availability and emission of the greenhouse gases and other air contaminants including carbon dioxide (CO2), carbon monoxide (CO), sulfur dioxide (SO2), nitrogen oxides (NOx), particulate matter and volatile organic compounds, upon combustion. Furthermore, the volatile prices of the fossil-derived fuels are affecting the economic growth in the developing and underdeveloped countries. Therefore, for environmental and economic sustainability, renewable and carbon neutral efficient biofuels are needed to displace or supplement in long run, and complement the fossil-derived fuels in near future.

Omics Technologies and Bio-engineering: Towards Improving Quality of Life DOI: https://doi.org/10.1016/B978-0-12-815870-8.00018-8

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18.2 BRIEF OVERVIEW OF THE FIRST-GENERATION BIOFUEL TECHNOLOGIES First-generation biofuels are the fuels derived from sugar, starches, or vegetable oils. Biodiesel and bioethanol are widely known as first-generation or conventional biofuels. The use of vegetable oil as a fuel dates back to more than a century when Rudolph Diesel invented the compression-ignition (CI) engine. In order to lower the viscosity and improve volatility, the triglyceride oils require some chemical modifications such as transesterification or emulsification. Transesterification of the oil with a short-chain alcohol is preferred method to derive biofuel from the oil. The fatty acid methyl esters (FAME) obtained by transesterification of oil with methanol are popularly known as biodiesel. There is decrease in viscosity and improvement in the volatility as well as other fuel properties after transesterification. The physical properties of FAME resemble to petroleum-derived diesel fuel. Presently, biodiesel is derived from food-grade edible oils in many developed and developing countries, that is, soybean oil in the United States, canola in Canada, rapeseed in Europe, palm in Malaysia and Indonesia, and so on. India being one of the largest edible oil importers, it cannot divert these edible sources for fuel purpose. The nonedible oilseeds, that is, Karanja (Pongamia pinnata) and Jatropha (Jatropha curcas), are suitable feedstock for biodiesel in India. Recently, Center for Jatropha Promotion (CJP) has also identified other plants like Simarouba, Camelina, etc., to be equally potent sources of deriving biodiesel in India. The characteristic of biodiesel (B100) should be in accordance to the norms specified by EN 14214, ASTM D 6751, or IS 15607 as shown in Table 18.1. The biodiesel has similar fuel characteristics and can be blended with conventional diesel to fuel the CI engines. The bioalcohols are produced from sugar and starches through the process of fermentation by enzyme or microorganisms. Ethanol is the most common bioalcohol where biobutanol is known to a lesser extent. Bioethanol can be used in sparkignition engines in blends with gasoline. The biofuels are free from sulfur, aromatic compounds, and burns cleanly in the engine as a result, there is no SOx, unburnt hydrocarbon, or polyaromatics in the exhaust emissions. The catalyst used in the transesterification of oil plays the crucial role in process of synthesis of biodiesel. The hydroxides or alkoxides of sodium and potassium are used as catalyst for industrial scale biodiesel production. The use of above alkali catalysts requires the feedstocks to have specific quality having minimum level of free fatty acids and moistures. The acid catalyst process is suitable for high free fatty containing vegetable oils. The acid-catalyzed method has not gained much interest due to the fact that the reaction proceeds slowly, liquid acids corrode the reaction vessel and disposal of liquid acids have environmental concerns. The transesterification of oil catalyzed by heterogeneous catalyst has been reported in the literature (Kulkarni et al., 2006). The use of biocatalyst for vegetable oil transesterification is an alternate method to prepare biodiesel. Lipases from Mucor miehei, Candida antarctica, Thermomyces lanuginosus, Candida rugosa, Pseudomonas cepacia, etc., are known biocatalyst for biodiesel synthesis from oils (Table 18.2). The lipases are immobilized in solid support for their repeated use for biodiesel synthesis. The lipases-catalyzed transesterification proceeds with stepwise addition of methanol since the excess short-chain alcohol in the reaction medium inactivates the lipase. The lipase from C. antarctica is nonspecific and catalyzes the vegetable oil transesterification without acyl migration. The yield of esters

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TABLE 18.1

Specification of Biodiesel (B100) in European Countries, the United States, and India Specifications

S. no. Properties

Unit

EN 14214

ASTM D 6751

IS 15607

1

FAME content, min.

% (m/m)

96.5

n.s.

96.5

2

Density at 15 C

kg/m3

860 900

n.s.

860 900

3

Viscosity at 40 C

cSt

3.5 5.0

1.9 6.0

2.5 6.0

4

Flash point, min.



101

93

120

5

Sulfur content, max.

mg/kg

10

15

50

6

Carbon residue, max.#

% (m/m)

n.s.

0.05

0.05

7

Cetane number, min.

51

47

51

8

Sulfated ash content, max.

% (m/m)

0.02

0.02

0.02

9

Water content, max.

mg/kg

500

500

500

10

Total contaminants

mg/kg

24

n.s.

24

11

Copper strip corrosion (3 h at 50 C)

rating

Class 1

Class 3

Class 1

12

Oxidation stability, 110 C, min.

h

8

3

6

13

Acid value, max.

mg KOH/g 0.50

0.50

0.50

14

Iodine value, max.

gI2/100g

120

n.s.

To report

15

Linolenic acid methyl esters, max.

% (m/m)

12

n.s.

n.s.

16

Polyunsaturated ($4 double bonds) methyl esters, max.

% (m/m)

1

n.s.

n.s.

17

Methanol content, max.

% (m/m)

0.2

0.2

0.20

18

Monoglyceride content, max.

% (m/m)

0.7

0.4

n.s.

19

Diglyceride content, max.

% (m/m)

0.2

n.s.

n.s.

20

Triglyceride content, max.

% (m/m)

0.2

n.s.

n.s.

21

Free glycerol, max.

% (m/m)

0.02

0.020

0.02

22

Total glycerol, max.

% (m/m)

0.25

0.240

0.25

23

Group I metals (Na 1 K), max.

mg/kg

5.0

5

To report

24

Group II metals (Ca 1 Mg), max.

mg/kg

5.0

5

To report

25

Phosphorus content, max.

mg/kg

4.0

10

10

26

Cold soak filterability, max.

seconds

n.s.

200

n.s.

Cloud point



C

n.s.

To report

n.s.

Distillation temperature (AET, 90% recovery), max.



C

n.s.

360

n.s.

27 28

C

n.s., not specified. # On 10% distillation residue.

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TABLE 18.2 Biodiesel Production With Various Lipases Lipase source (immobilized)

Oil

Alcohol

Yield %

Reference

C. antarctica

Rapeseed

Methanol

91

Watanabe et al. (2007)

Rhizopus oryzae

Soybean

Methanol

80 90

Kaieda et al. (2001)

Chromobacterium viscosum

Jatropha

Ethanol

92

Shah et al. (2004)

M. miehei

Sunflower

Ethanol

83

Selmi and Thomas (1998)

P. cepacia

Palm kernel

Ethanol

72

Abigor et al. (2000)

T. lanuginosus

Sunflower

Methanol

90 97

Dizge et al. (2009)

Bacillus sp. S23

Microalgae

96

Surendhiran et al. (2015)

during enzymatic transesterification in solvent-free medium is listed in Table 18.2. In enzyme-catalyzed reaction, there is no negative influence of free fatty acids in the oil as free fatty acids are also converted into fatty acid alkyl esters. The immobilized lipases are easily recovered after completion of transesterification and reused several times. Bioethanol is the fuel, which has globally gained much interest and widely used in Brazil and the United States since last couple of decades utilizing the by-products of sugarcane. The ethanol industries in Brazil use sugarcane exclusively, the Brazilian sugarcane system of agro-energy represents the most efficient system. Currently 80% of vehicles are running with ethanol and also some jet engines. Ethanol has high octane rating than gasoline, which allows increase in engines’ compression ratio for increased thermal efficiency. The feedstocks commonly used for deriving ethanol are sugars and starches. The process of bioethanol production involves pretreatment, enzymatic digestion of starches to sugars followed by fermentation and further purification by distillation and drying. The pretreatment step includes washing, size reduction, extracting the juice, and separating the bagasse. The fermentation process involves the conversion of fermentable sugars, that is, hexoses to ethanol by the action metabolism of microorganisms. When sucrose is the substrate, fermentation is carried out by the yeast Saccharomyces cerevisiae while the bacteria Zymomonas mobilis is employed for fermentation of glucose to ethanol. The theoretical yield of ethanol is 0.511 g ethanol/g hexose and the real yield is around 0.485 g ethanol/g hexose. The fermentation is performed at temperature below 32 C, pH between 4 and 5, sugar concentration to be less than 16 Bx. The ethanol content in the fermentation medium is 7% 7.5% (w/w) and needs further distillation processes in several steps to obtain bioethanol (Soccol et al., 2011).

18.3 SECOND-GENERATION BIOFUEL TECHNOLOGIES The first-generation biofuels compete with inputs for food, so the alternative may be the advanced or second-generation biofuels, which uses cellulosic products such as wood, straw, long grass, or wood waste for biofuel production (Sims et al., 2010). Furthermore,

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these can meet the demand for fuel in a fair and eco-friendly manner. The advantage of second-generation biofuels is the ability to use the whole plant and not just its parts (e.g., grains), as is the raw material for the first generation. All plants contain lignin, hemicellulose, and cellulose. Lignocellulosic ethanol is made by freeing the sugar molecules from cellulose using enzymes, steam heating, or other pretreatments. These sugars can then be fermented to produce ethanol in the same way as first-generation bioethanol production (Limayema and Ricke, 2012). The by-product of this process is lignin, which can be burned as a carbon neutral fuel to produce heat and power for the processing plant and other purposes. Second-generation biofuel feedstocks that mainly include cereal and sugar crops, specifically grown energy crops, agricultural and municipal wastes and waste oils, etc. Green waste such as forest residues or garden or park waste may be used to produce biofuel via different routes. Examples include biogas captured from biodegradable green waste, and gasification or hydrolysis to syngas for further processing to biofuels via catalytic processes. Second-generation biofuels from lignocellulosic biomass can be broadly obtained through biochemical and thermochemical processes (Fig. 18.1) (Menon and Rao, 2012). Biochemical conversion uses biocatalysts, such as enzymes, in addition to heat and other chemicals, to convert the carbohydrate portion of the biomass into an intermediate sugar stream. Biochemical processes typically employ pretreatment to accelerate the hydrolysis process, which separates out the lignin, hemicellulose, and cellulose (Philbrook et al., 2013). Once these ingredients are separated, the cellulose fractions can be fermented into alcohols. Liquid biofuels from biomass can be obtained through thermochemical processing or by chemical treatment. Thermochemical treatment comprises thermal decomposition and chemical transformation of substrates by the action of the temperature in the presence of various concentrations of oxygen. The advantage of thermal treatment in relation to the

Technology

Biochemical/Physical Conversion -Chemical processing -Physical processing -Biochemical processing Lignocellulosic Biomass

End product

Second generation ethanol

Value added products Thermochemical Conversion -Pyrolysis -Gasification -Torrefaction

BtL Fuels

FIGURE 18.1 Second-generation biofuel processing ways and products from cellulosic biomass.

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biochemical is able to convert all organic ingredients, not just the polysaccharides, as is the case with chemical treatment (Thomas et al., 2009). Carbon-based materials can be heated at high temperatures in the absence (pyrolysis) or presence of oxygen, air, and/or steam (gasification). These thermochemical processes yield combustible gas and solid char. The gas can be fermented or chemically synthesized into a range of fuels, including ethanol, synthetic diesel, or jet fuel. Conversion of biomass into bio-oil, biochar, syngas, and others requires entirely thermochemical processes, such as torrefaction, carbonization, thermal liquefaction, pyrolysis, and gasification. The relative advantage of thermochemical conversion over biochemical is due to higher productivity and compatibility with existing infrastructure facilities (Zhang et al., 2010). However, the majority of these processes are still under development phase and trying to secure a market share due to various challenges, right from suitable infrastructure, raw material, technical limitations, government policies, and social acceptance (Elliott, 2008).

18.4 THIRD-GENERATION BIOFUEL TECHNOLOGIES The biofuels derived from microalgae, the unicellular algae, are referred as thirdgeneration biofuels. Microalgae are a promising feedstock for biofuels owing to their rapid growth rate and higher lipid productivity than the best oil producing terrestrial plants. Furthermore, the higher photosynthetic efficiency and wider adaptability to different environmental conditions are the other reasons for interest in microalgae for biofuels (Chisti, 2007). Microalgae do not need fertile land and can be grown in sewage water, thus eliminating or minimizing the competition with food crops for resources, consequently avoiding the food versus fuel conflict. In addition to source of different types of biofuels, microalgae are useful as nitrogen-fixing biofertilizers and in phyto-remediation (Munoz and Guieysse, 2006).

18.4.1 Microalgae Cultivation Microalgae can be cultivated in open/raceway ponds or closed photo-bioreactors (Fig. 18.2). In case of pond cultivation, operating cost is low but contamination risk and

FIGURE 18.2

Cultivation of microalgae under partially controlled condition for biofuel production.

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weather dependence are high, in addition to the low reproducibility, low biomass concentration and hence the higher harvesting cost. Contamination risk is low with closed photobioreactor cultivation and biomass concentration is higher along with lower harvesting cost. However, operating cost as well as start-up capital cost is very high. Therefore, cultivation system may be chosen depending on the requirements. Water is an important input for algae cultivation and depending on the microalgae species, freshwater or seawater can be used. Wastewater can be treated using microalgae for removal of nitrogen and phosphate from the effluents, which would otherwise result in eutrophication. Besides this, applications of microalgae for removal of several heavy metal contaminants have also been proven. Inadequate supply of carbon in the form of carbon dioxide is often the limiting factor in the microalgae productivity. To produce one unit of biomass with average chemical composition, algae use approximately 1.8 units of CO2. Naturally dissolved CO2 in water is not enough; therefore, bubbling of air into water to improve the dissolution is practiced as the pure CO2 is expensive. Alternatively, the waste source of CO2 like flue gases that typically contains 4% 15% CO2 can also be added to algae ponds without any harmful effects (Doucha et al., 2005). Besides CO2, the dissolved NOx can also be used by algae as nitrogen source. However, the requirement of flue gas needs to be optimized depending on the algae species, light intensity, and temperature. Furthermore, the altered pH due to dissolution of CO2 and SO2 needs to be controlled or buffered. Being photoautotrophic organism, microalgae rely on solar radiation for photosynthesis and thereby its growth. Therefore, depth of all the algae-culture systems is designed in a way to allow efficient harvesting of light by the algae. Closed photo-bioreactors are equipped with artificial light in the photosynthetic active radiations (PAR) (400 700 nm). Only 45% of solar radiation spectrum is PAR and maximum efficiency to use it during photosynthesis is 27% in algae. Therefore, the maximum theoretical conversion of light energy to chemical energy is approximately 11% (Gao et al., 2007). Furthermore, up to 25% of the photosynthates produced during the day time are utilized at night, as photosynthesis does not occur in dark/night, depending upon temperature and other conditions (Chisti, 2007). Besides, the carbon source and light-, micro-, and macronutrients are required for algae to grow. Among these, nitrogen and phosphorous are the most important nutrients. The nutrients can be supplemented in the form of pure chemicals, but can add significantly to the cost of cultivation. Synthetic media such as CHU, BG 11, and BBM suitable for microalgae cultivation are based on the pure chemicals for nutrient sources. Commercially available agricultural fertilizers can be a relatively low-cost alternative. Furthermore, wastewater effluents rich in these nutrients from different sources can also be used for economic cultivation of microalgae. Microalgae strains exhibit wider tolerance to temperature and other environmental conditions; however, average temperature of 25 C is favorable for optimal growth in general.

18.4.2 Microalgae Biomass Harvesting The smaller size of few micrometers makes harvesting and further concentration technically challenging and thus expensive contributing 20% 30% of the total cost of biomass production (Molina Grima et al., 2003). Gravity settling of the biomass is the simplest

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FIGURE 18.3 Harvesting of microalgae biomass by the process of flocculation.

technique for harvesting but the process is slow therefore requires substantial time in addition to the space. Filtration separates the algae cells from liquid medium by passing a suspension through permeable medium onto a screen of given aperture size that retains the cells. However, due to small mesh size (,100 µm), a pressure of compressed air or vacuum is required to apply across the medium in order to force fluid to flow through the filter. Filtration and microstraining may be cost-effective methods for harvesting microalgae but require large surface areas, high cost, formation of a filter-cake, which substantially increases head loss, prone to clogging, and require frequent maintenance. Centrifugation is commonly used to concentrate small-sized unicellular high-value algae, but generally is considered expensive for biofuel production as it contributes nearly 40% of the production cost. Alternatively, combination of gravity settling followed by centrifugation of the dense biomass as a secondary harvest method would reduce the cost considerably. Most of the algae are characterized as negative-charged surfaces. Because of the identical surface negative charges, microalgae cells repel each other, remain suspended, and do not get settled easily. The surface charge can be blocked by the treatments with flocculants, allowing the cells to adhere each other generating aggregates/flocs, thereby facilitating the sedimentation (Fig. 18.3). In case of electro-flocculation, electric charge is applied to aggregate microalgae cells. The technique has been proven to effectively remove up to 95% of algae in freshwater (Poleman et al., 1997). Main advantage of the technique is no need for flocculants; however, the technique suffers with a disadvantage that the cathodes are prone to fouling. Recently, bioflocculation of nonflocculating microalgae has been evaluated as a simple, effective, economic, and environmentally friendly promising alternative effective method for harvesting of microalgae (Ndikubwimana et al., 2016).

18.4.3 Lipid Extraction and Biodiesel Production The harvested biomass is first pretreated to alter degree of cell disruption, residual moisture content, and particulate size, which are known to affect the microalgal lipid extraction. The ideal technology for lipid extraction microalgae should be specific for

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lipids to minimize co-extraction of contaminants such as protein and carbohydrates. Furthermore, the technique should be more selective for acylglycerols than other lipid fractions to minimize downstream purification. Organic solvent and subcritical organic solvent are the commonly followed lipid extraction methods for microalgae biomass. Organic solvent extraction is based on the basic chemistry concept of “like dissolve like,” which means nonpolar (neutral) organic compounds are dissolved in the nonpolar solvents such as petroleum ether, and vice-versa. In recent years, efforts are being made by scientific community to enhance the kinetics of the lipid extraction by organic solvent method through various modifications including microwave assisted and supercritical organic solvent extraction. In case of supercritical solvent extraction method, the accelerated extraction kinetics and cellular disintegration are achieved at critical temperature and pressure values of the solvent. The emerging green technology appears suitable for lipid extraction from microalgae and has potential to replace the existing organic solvent extraction due to various benefits over the existing one. One of the major challenges for an industrial scale economic production of microalgaederived biodiesel is the nonavailability of the energy efficient cum economic microalgae harvesting, dewatering, and lipid extraction method. Alternate novel method, which does not require harvesting/dewatering the algal cells for biofuel production, is based on the concept of milking or in situ extraction (Yadugiri, 2009). In the process, biocompatible organic solvent (e.g., n-heptane) is re-circulated through the aqueous phase for mixing and lipid extraction purpose. The microalgae cells on repeated exposure to biocompatible solvent retained metabolic activity to continuously produce the compounds of interest without sacrificing of the cells. Feasibility of milking and re-milking the cells of microalgae species with biofuel potential for lipids or other readily usable forms of biofuels has been demonstrated (Zhang et al., 2011). OriginOil, Inc., developed a method of milking known as Live Extraction, in which algae cells are electrically stimulated for continuous oil extraction. The projected benefits of the method are: applicability to wide range of feedstock saves significant energy and time as dewatering of algae biomass is not required, chemical-free process therefore solvent recovery is not required, and the high-throughput method is highly scalable. Simultaneous extraction and transesterification of lipids is popularly known as in situ or direct transesterification. In this process, acid catalyst (sulfuric acid/acetyl chloride is commonly used) and methanol are added to the microalgal biomass. Lipids extracted with methanol are transesterified by the acid catalyst to produce FAME. The downstream processing steps followed are similar to that of the traditional transesterification. Microalgae cell debris is removed out by filtration, and methanol is recovered by distillation of the reaction mixture. After settling, the biodiesel or un-transesterified lipids form top phase, whereas glycerol settles down as a bottom phase. The top phase is decanted off and washed repeatedly with water to eliminate any acid catalyst. Use of solvents with higher polarity has resulted in higher FAME conversion yield (Im et al., 2014). H2SO4 has been proved very effective as a reaction catalyst for converting fatty acids and triacylglycerols (TAGs) from wet microalgae biomass. Performance of the process is regulated by key operating parameters such as ratio of methanol to dried biomass and reaction temperature.

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18.5 PRACTICAL CHALLENGES AHEAD IN BIOFUEL TECHNOLOGIES The first-generation biofuels are derived from food reserves such as vegetable oils, sugars, and starches. This creates food versus fuel scenario globally. In the context of developing countries such as India, which import edible oil to supplement the food demand, it is less likely to derive biofuels from food crops. Furthermore, the feedstocks for first-generation biofuel compete with food crops for land and water resources. The resources for first-generation biofuels are also limited for competing with fossil fuel. Therefore, the alternative may be the second-generation biofuels, which uses cellulosic products such as forest- or agro-based plant residues for biofuel production. Furthermore, the whole plant and not just its parts can be utilized for producing these biofuels, therefore feedstock availability is not a problem. However, the process involved requires huge capital investment and relatively simple conversion processes are not available consequently, and there is no proven commercial technology available today. Microalgae are a promising feedstock for the third-generation biofuels owing to their rapid growth rate and higher lipid productivity than the best oil producing terrestrial plants. Furthermore, the higher photosynthetic efficiency and wider adaptability to different environmental conditions are the other reasons for interest in microalgae for biofuels (Chisti, 2007). Microalgae, which can be grown even in wastewaters, successfully eliminate competition with food crops. However, the major challenges are cost-effective microalgae cultivation and harvesting technique for biofuel production. Furthermore, rapid and economical technique for biomass drying, extraction of lipid, and further conversion into biodiesel or other biofuels are not yet available. However, development of model-integrated production and biorefinery system will bring viable algae-based biofuels into the market.

18.6 OMICS ADVANCEMENT AND APPROACHES FOR COST-EFFECTIVE PRODUCTION OF RENEWABLE ENERGY Over the past few years, a massive amount of omics data has been generated to gain insight into biofuel production. Omics advancements contribute to the development of the fourth-generation biofuels from genetically engineered species (Grover et al., 2013, 2014a,b). The omics technological advancement has tremendous future scope to extract deeper biological knowledge and thereby cost-effective production of renewable energy. It mainly includes key area of research such as new strain development, improved cultivation, low-energy harvesting, and high-yield extraction-conversion technology. In order to advance the economic feasibility of the microalgae or other feedstocks, much attention is being given on genetic and metabolic engineering to increase the yield of biofuel relevant lipids without compromising the growth. The lipid accumulation in microalgae is usually triggered by exposure to some forms of stress including nutrient deficiency, salinity, temperature, etc. Therefore, research efforts to identify the lipid triggers and further engineering the algal strains with potential to produce more lipids

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throughout the whole growth cycle have received significant attention. Earlier efforts to increase lipid content and modify the fatty acid profile to optimize microalgae as a biodiesel feedstock by introducing heterologous plant fatty acid synthesis enzymes have resulted in relatively lower success. This emphasizes the necessity of comprehensive understanding of the algal fatty acid biosynthetic machinery. At present, scant available information on algal metabolic pathways and nonavailability of stable transformation protocol are the major challenges in engineering microalgae for high lipid content. Algal fatty acid biosynthesis pathway has been deduced based on homology with well-characterized plant system and the fatty acid synthase enzyme encoding genes have been annotated in many of the genome sequenced algae. Lei et al. (2012) cloned five genes viz. acylase carrier protein (ACP), ketoacyl-ACP synthetases (KAS), thioesterase (TE), fatty acid desaturase (FAD), and malonyl ACP transacylase (MAT) involved in fatty acid biosynthesis from Haematococcus pluvialis. Among the genes, transcript expression levels of ACP, KAS, and TE were in linear relationship with fatty acid synthesis in response to the different lipid biosynthesis-triggering stressors, thus were proposed as key rate limiting genes. As mentioned earlier, in general, microalgae accumulate lipids as an energy storage molecule on exposure to the environmental stresses; however, net lipid yield is reduced due to severe decline in biomass growth on exposure to the stresses. Fan et al. (2014) reported differential lipid accumulation and transcript expression of genes involved in, in response to nitrogen, phosphorous, or iron depletion in an oleaginous microalgae Chlorella pyrenoidosa. The transcript abundance of genes encoding ME (malic enzyme), Accase (acetyl CoA carboxylase), and DGAT (di-acyl glycerol acyl transferase) showed significant correlation with lipid accumulation. Thus, these genes are likely to exert great influence on lipid biosynthesis. ME being considered as a major supplier of a critical factor for intracellular fatty acid content, that is, NAPDH, overexpression of the encoding gene could have enhanced lipid content. Accase catalyzes the first rate-limiting step in the fatty acid biosynthesis pathway through formation of malonyl CoA from acetyl CoA, whereas DGAT enzyme plays an important role in acylation process of diacylglycerol into TAG in lipid biosynthesis. Therefore, the genes encoding these enzymes could be important targets for metabolic engineering of microalgae strains for enhanced biofuel production. Trentacoste et al. (2013) demonstrated the application of targeted metabolic engineering toward the enhancement in lipid accumulation in eukaryotic microalgae Thalassiosira pseudonana without compromising growth. In this study, expression of multifunctional lipase/phospholipase/acetyl transferase was silenced through targeted antisense knockdown approach. The knockdown microalgae strains thus developed exhibited greater than threefold higher lipid content as compared with the wild type without compromising growth during exponential growth phase. The lipid content was fourfold higher than the wild type after 40 hours of silicon starvation. In order to extract the lipids from microalgae for biofuel production, the biomass is handled in energy intensive steps of harvesting, drying, and then organic solvents extraction, severely affecting overall economics of the derived biofuels (Molina Grima et al., 2003). Therefore, to skip these steps, Liu et al. (2011) genetically engineered the cyanobacteria to produce and continuously secrete the free fatty acids, which can be further collected from the culture medium. Acyl-ACP thioesterase I encoded by tes A gene in

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Escherichia coli is normally a periplasmic protein due to presence of a signal sequence peptide. In absence of the signal peptide, the synthesized fatty acids are secreted in the culture medium (Cho and Cronan, 1995). The same concept is being industrialized for biofuel production using engineered E. coli by the biofuel company LS9 (Steen et al., 2010). Recently, Liu et al. (2011) applied the concept to cyanobacteria because being photosynthetic organism cyanobacteria has a big advantage over the E. coli. Alkanes (C4 C23) possess higher energy density and compatibility with existing liquid fuel engines and are the major constituents of gasoline, diesel, and jet fuels. Wang et al. (2013) genetically engineered cyanobacteria, which exhibit higher photosynthetic efficiency and growth rate than the eukaryotic microalgae, to achieve efficient photosynthetic production of alka(e)nes. Alkane biosynthetic genes [acyl-acyl carrier protein reductase (AAR), aldehyde deformylating oxygenase (ADO)] were over-expressed under the regulation of strong promoter Rubisco in cyanobacteria Synechocystis sp. PCC 6803. The overexpression resulted in more than eightfold enhanced alka(e)ne production in the engineered strains on dry weight basis than the wild-type strains. Furthermore, feasibility of the enhanced alka(e)ne production in the engineered strains was demonstrated by redirecting the carbon flux to acyl-ACP. Thus, the results demonstrate the power of metabolic engineering strategies to overproduce alka(e) nes in cyanobacteria. However, enhanced understanding on physiological roles and regulatory mechanism of native alka(e)nes in cyanobacterial cells need to be developed. As medium-chain alkanes are less toxic to the cells than the other nonnative products, huge scope lies in engineering cyanobacteria for enhanced alkane production. Genetic engineering approach has been widely used for improvement in biofuel traits in terrestrial plants as well. Members of NAC family genes contribute to enhanced stress tolerance and in secondary growth of the plants, thereby building biomass. Thus, overexpression of NAC transcription factor gene provides a possibility to tailor biofuel plants. Several studies on overexpression of NAC genes have shown improved biotic and abiotic stress tolerance as well as enhanced biomass production in the transformed plants (Singh et al., 2016). Phenomenon of photorespiration causes considerable losses in photosynthetic productivity of most C3 plants. Recently, Dalal et al. (2015) reported significant impact of photorespiratory bypass approach on increasing seed productivity for biofuel crop Camelina. On overexpression of photorespiratory bypass genes, photorespiration was reduced coupled with increased photosynthesis in the bypass expressing Camelina lines. In these lines, seed yield was increased by up to 70% without any loss in seed quality. Furthermore, the transgenic plants also produced more biomass with earlier flowering, seed setting, and maturity than the wild types. Thus, the approach may be useful in other C3 biofuel plants for early and higher biomass yield for biofuel production. Commercial application of genetically engineered species is, however, subject to strict biosafety regulations. In case of an aquatic species like microalgae, biosafety requirements such as sterilization of entire cultivation system to prevent release in open environment will have a detrimental impact on overall economics and energy balance of the system. Thus, the large-scale application of engineered microalgae in the current biosafety regimes does not appear viable.

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18.7 CONCLUSION AND FUTURE PERSPECTIVES Natural petroleum resources synthesized over millions of years are likely to be exhausted shortly. Although, a number of alternative fuels have been discovered, none of them is as usable as biofuels are, primarily because we do not need to change the way we currently use our fuels or energy resources. Both the cultivation of the raw material for biofuels and harnessing the yield are challenging as these so-called biofuel crops are relatively naı¨ve to our agricultural systems and understandings. Thus, huge amount of investments on research and development of these resources are required. For the ease of classification of various biofuel technologies, these have been classified into the first, second, third, and fourth generations, which in anyway does not indicate the superiority of one over the other. The limited potential of first-generation biofuels to make a significant contribution to displace fossil fuels and reduce GHG emissions highlighted by several studies unleashed a sense of urgency for the transition toward second-generation biofuels (Limayema and Ricke, 2012). While dedicated energy crops would still be competing for land with food crops, it is envisioned that either by using lesser quality soils (Jatropha) or by providing more utilizable biomass per unit of land (e.g., Camelina, switchgrass, or short tree rotations), the pressure for prime quality soils will be reduced. As compared to the case of first-generation biofuels, where feedstock can account for over two-thirds of the total costs, the share of feedstock in the total costs is relatively lower (30% 50%) in the case of second-generation biofuels. To date, there is no large-scale commercial production of second-generation biofuels. If external costs of production of fossil fuels were considered, the cost difference will generally be lower for many second-generation biofuels. At present, the second-generation biofuel technologies look most promising, as they do not compete for resources with the food crops, no special requirements for cultivation of the raw material exist, and technology for conversion of biomass to biofuels is nearing commercialization. Furthermore, the major drawbacks of these technologies that are energy input for thermochemical conversion can be overcome by maturation of third- and fourth-generation biofuels. Given the current state of technology, and the uncertainty remaining about the future breakthroughs that would potentially make some advanced-generation biofuels cost competitive, policymakers need to carefully consider what goals are to be pursued in providing support to different biofuels. Biofuels that simultaneously advance multiple policy goals could warrant greater support when designing incentive mechanisms. An integrated approach combining economically sustainable rural development, climate change mitigation, and alternative energy provision provides a good policy framework for advanced-generation biofuels. It is also necessary to consider regional and international developments in policies and trade in order to maximize the potential benefits achievable through the policies implemented (Pacheco, 2007).

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Philbrook, A., Alissandratos, A., Easton, C.J., 2013. Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass. In: Petre, M. (Ed.), Environmental Biotechnology New Approaches and Prospective Applications. InTech. Available from: http://dx.doi.org/10.5772/55309. Available from: https://www.intechopen.com/books/environmental-biotechnology-new-approaches-and-prospective-applications/ biochemical-processes-for-generating-fuels-and-commodity-chemicals-from-lignocellulosic-biomass. Poleman, E., De Pauw, N., Jeurissen, B., 1997. Potential of electrolytic flocculation for recovery of micro-algae. Resour. Conserv. Recycling 19, 1 10. Selmi, B., Thomas, D., 1998. Immobilized lipasecatalyzed ethanolysis of sunflower oil in a solvent free medium. J. Am. Oil Chem. Soc. 75, 691 695. Shah, S., Sharma, S., Gupta, M.N., 2004. Biodiesel preparation by lipase-catalyzed transesterification of jatropha oil. Energy Fuels 18, 154 159. Sims, R.E.H., Mabee, W., Saddler, J.N., Taylor, M., 2010. An overview of second generation bio-fuel technologies. Bioresour. Technol. 101, 1570 1580. Singh, S., Grover, A., Nasim, M., 2016. Biofuel potential of plants transformed genetically with NAC family genes. Front. Plant Sci. 7, 22. Soccol, C.R., Faraco, V., Karp, S., Vandenberghe, L.P.S., Soccol, V.T., Woiciechowshi, A., et al., 2011. Lignocellulosic Bioethanolbioethanol: Current status and future perspectives. In: Pandey, A. (Ed.), Biofuels: Alternative Feed Stocks and Conversion Processes. Academic Press, Salt Lake City, UT, ISBN: 978-0-12-385099-7. Steen, E.J., Kang, Y., Bokinsky, G., Hu, Z., Schirmer, A., McClure, A., et al., 2010. Microbial production of fattyacid-derived fuels and chemicals from plant biomass. Nature 463, 559 562. Surendhiran, D., Sirajunnisa, A.R., Vijay, M., 2015. An alternative method for production of microalgal biodiesel using novel Bacillus lipase. 3 Biotech 5, 715 725. Thomas, D.F., Andy, A., Dutta, A., Phillips, S., 2009. An economic and environmental comparison of a biochemical and a thermochemical lignocellulosic ethanol conversion processes. Cellulose 16, 547 565. Trentacoste, E.M., Shrestha, R.P., Smith, S.R., Gle, C., Hartmann, A.C., Hildebrandt, M., et al., 2013. Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proc. Natl. Acad. Sci. 110, 19748 19753. Wang, W., Liu, X., Lu, X., 2013. Engineering cyanobacteria to improve photosynthetic production of alka(e)nes. Biotechnol. Biofuels 6, 69. Watanabe, Y., Pinsirodom, P., Nagao, T., Yamauchi, A., Kobayashi, T., Nishida, Y., et al., 2007. Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase. J. Mol. Catal. B Enzym. 44, 99 105. Yadugiri, V.T., 2009. Milking diatoms—a new route to sustainable energy. Curr. Sci. 97, 748 750. Zhang, F., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2011. Screening of biocompatible organic solvents for enhancement of lipid milking from Nannochloropsis sp. Process Biochem. 46, 1934 1941. Zhang, L., Xu, C., Champagne, P., 2010. Overview of recent advances in thermo-chemical conversion of biomass. Energ. Convers. Manage 51, 969 982.

Further Reading Maeda, Y., Tateishi, T., Niwa, Y., Muto, M., Yoshino, T., Kisailus, D., et al., 2016. Peptide mediated microalgae harvesting method for efficient biofuel production. Biotechnol. Biofuels 9, 10. Oey, M., Ross, I.L., Stephens, E., Steinbeck, J., Wolf, J., Radzun, K.A., et al., 2013. RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii. PLoS One 8, e61375. Park, W., Feng, Y., Ahn, S.J., 2014. Alteration of leaf shape, improved metal tolerance, and productivity of seed by overexpression of CsHMA3 in Camelina sativa. Biotechnol. Biofuels 7, 96. Radakovits, R., Eduafo, P.M., Posewitz, M.C., 2011. Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum. Metabolic Eng. 13, 89 95. Yao, L., Qi, F., Tan, X., Lu, X., 2014. Improved production of fatty alcohols in cyanobacteria by metabolic engineering. Biotechnol. Biofuels 7, 94.

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