**2. Biochemistry of lipid accumulation**

**1. Introduction**

138 Advances in Biofuels and Bioenergy

Biodiesel is renewable, biodegradable and non-toxic transport fuel composed of fatty acid methyl esters. It is produced by transesterification of triacylglycerols with alcohol (mostly methanol and ethanol) in the presence of alkaline catalyst (e.g. sodium hydroxide). Common feedstocks for the production of biodiesel are different vegetable oils including rapeseed oil, palm oil, cooking oil, soybean oil and sunflower oil [1]. Production of biodiesel increased steadily in the last few years. In year 2016, the United States and Brazil were the world's biggest biodiesel producers with a production volume of around 5.5 and 3 billion liters, respectively [2]. All existing diesel engines, vehicles and infrastructure can run on pure biodiesel (B100) or blends with petroleum diesel fuel without any change [1]. Use of biodiesel has positive environmental impact, improves energy supply security, stimulates economic development and generates employment especially in the rural areas [3]. It reduces harmful emission characteristic for diesel exhaust such as particulate matter, carbon monoxide and total unburned hydrocarbons. Additionally, emission of toxic compounds including vapor-phase hydrocarbons from C1 to C12, aldehydes and ketones up to C8 and polyaromatic hydrocar-

Microbial lipids are viewed as an alternative feedstock for the biodiesel production because fatty acid compositions of accumulated lipids are similar to vegetable oils currently used as feedstock for the production of first generation biodiesel. Microbial lipids are also known as single cell oils (SCO), and are produced by heterogeneous group of oleaginous microorganisms that include less than hundred species of different microbial species including yeasts, fungi, bacteria and algae [3, 4]. Oleaginous microorganisms have the ability to accumulate significant amounts of intracellular lipids (more than 20% of their dry weight), mostly triacylglycerols, under certain cultivation conditions. Yeast strains such as *Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula graminis, Rhodotorula glacialis* and *Trichosporon oleaginosus* can accumulate intracellular lipids from 50 to 80% (w/w) under certain cultivated conditions [5–8]. The fatty acid composition of lipids depends on the microbial strain and the cultivation conditions used. The most common fatty acids are palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acids [9]. Microbial lipids of some oleaginous microorganisms are valuable source of polyunsaturated fatty acids that are used as additive for different food products and in nutriceuticals. Following omega-3 and omega-6 fatty acids are commercially produced using different wild-type and genetically modified oleaginous microorganisms such as γ-linolenic acid (GLA, C18:3, n−6) by *Mucor circinelloides*; dihomo-gamma-linoleic (DGLA) (20:3, n−6) by *Mortierella alpina* 1S-4; eicosapentaenoic acid *(*EPA) (20:5, n−3) by *Mortierella alpina* ST1358 and *Yarrowia lipolytica*; docosahexaenoic acid (DHA, 22:6, n−3) by *Crypthecodinium cohnii*, *Schizochytrium* and *Ulkenia* sp. and arachidonic acid (ARA, 20:4, n−6) by *Mortierella alpina* [10–17]. Microbial lipids from oleaginous yeast strains can be used as substitute for cocoa-butter and shea butter [18]. In comparison to vegetable oils, biodiesel production from microbial lipids have a number of advantages such as heterotrophic oleaginous microorganism grow much faster than the terrestrial crops; no need for arable land for cultivation; growth as well as cultivation does not depend on whether conditions and elimination of conflict between food and food supply chain [19]. Yeasts and fungi are favored oleaginous

bons and nitrated polyaromatic hydrocarbons are also decreased [4].

The fatty acid biosynthetic pathway in most of oleaginous microorganisms is similar to nonoleaginous microorganisms. Two features of oleaginous microorganisms make them an efficient producer of lipid such as ability to efficiently produce precursor acetyl-CoA and cofactor NADPH needed for fatty acid synthesis. Process of lipogenesis could be divided in two steps, synthesis of precursor acetyl-CoA followed by biosynthesis of triacylglycerols (**Figure 1**). Nitrogen starvation in the presence of excess of carbon sources triggers *de nov*o synthesis of lipids in oleaginous microorganisms. Depletion of other media component like phosphorus or sulfur can efficiently induce lipogenesis [16]. Exhaustion of the nitrogen source induces a series of the consecutive biochemical reaction in the cell. The activity of AMP deaminase (AMPD) is upregulated. It cleaves the AMP to inosine monophosphate (IMP) and ammonia ions that cell can use as a nitrogen source. Consequently, concentration of AMP is reduced and the activity of NAD+ (NADP+ )-depended isocitrate dehydrogenase (ICDH) is downregulated. This enzyme in oleaginous microorganisms is allosterically regulated by its activator AMP. Isocitrate accumulates in mitochondria and isomerized to citrate by aconitase (A). Accumulated citrate is transported into the cytoplasm in exchange for malate (citrate/malate translocase, CMT). In the cytoplasm, ATP citrate lyase (found only in oleaginous microorganisms) converts citrate to acetyl-CoA and oxaloacetate [32]. The acetyl-CoA is used for fatty acid synthesis while oxaloacetate is converted to malate by malate dehydrogenase (MDH) and exported to mitochondria *via* CMT [16, 32].

The synthesis of lipids depends on efficient supply of NADPH, which is used for acetyl group reduction in the growing acyl chain. For the synthesis of 1 mol of a C18 fatty acid, 16 mol of NADPH is required. There is no unique metabolic route for generating NADPH in the oleaginous microorganism. Ratledge [33] described several routes for the synthesis of NADPH in the cytosol during lipogenesis. Transhydrogenase cycle which includes three enzymes pyruvate carboxylase (PC), MDH and malic enzyme (ME) has been proposed as a major route for the NADH production in the oleaginous microorganism. However, presence of ME in cytosol was not confirmed in some yeast species [16, 33]. In yeast *Y. lipolityca,* ME is located in the mitochondria and therefore cannot participate in the lipid synthesis [34].

triacylglycerols form lipid droplets on the lumenal and/or cytoplasmic side of the endoplas-

Production of Microbial Lipids from Lignocellulosic Biomass

http://dx.doi.org/10.5772/intechopen.74013

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Lignocellulose is complex biopolymer composed of the polysaccharides (cellulose and hemicellulose), amorphous polymer lignin and a remaining smaller part including pectin, protein, extractives and ash. The structural carbohydrates, which accounts for approximately two thirds of the total dry weight of the lignocellulosic biomass, can be used as carbon source for microbial production of biofuels after hydrolysis to fermentable sugars. Composition of lignocellulosic biomass varies depending on the plant source. For example, the agriculture residues like rice, rye and wheat straw contains less cellulose (approximately 30%) than hardwood including poplar, pinewood and spruce (>40%) [37–42]. However, digestibility of carbohydrates in the native lignocellulosic biomass by cellulases is low due to its structural features. Structural features of lignocellulosic biomass are determined by its chemical composition (content of lignin, hemicellulose and acetyl groups bound to hemicellulose) and physical characteristics (accessible surface area, i.e., porosity, crystallinity and degree of cellulose polymerization, the physical distribution of lignin in the biomass matrix, pore volume and biomass particle size) [43]. Lignocellulosic biomass is subjected to pretreatment process which breaks down the native structure and exposes cellulose fibers to hydrolytic enzymes improving the yield of fermentable sugars. For the past three decades, various methods for the pretreatment of the lignocellulosic biomass have been developed. The pretreatment process is considered as one of the most expensive steps in the production of lignocellulosic biofuels. The estimated cost of pretreatment process in bioethanol production is approximately 30 US cent per gallon of ethanol [44]. The pretreatment processes are classified in the following groups: physical (milling, grinding, pyrolysis, extrusion and gamma-ray irradiation), chemical (alkali hydrolysis, dilute acid hydrolysis, organosolv process and oxidative delignification), physi-

**3. Lignocellulose biomass as carbon source for microorganism** 

cochemical (steam explosion/autohydrolysis, ammonia fiber explosion, CO<sup>2</sup>

 explosion), biological (biochemical degradation using white-, brow- and soft-rot fungi and lignin-degrading enzymes) and combination of these methods. During pretreatment process, a number of degradation products are formed: furan aldehydes (furfural and 5-hydroxymethyl furfural), aromatic compounds (vanillin, syringaldehyde and 4-hydroxybenzoic acid), aliphatic acids (acetic, formic and levulinic) and inorganic compounds [44–46]. During the pretreatment process at high temperature and pressure, hemicellulose is hydrolysed mainly to xylose and lesser extent to glucose. Furan and 5-hydroxymethyl furfural (HMF) are formed by dehydration of released xylose and glucose, respectively [45, 47, 48]. Acetic acid is formed by hydrolysis of acetyl groups in hemicellulose. Formic and levulinic acid are derived from furan aldehydes during prolonged exposure to high temperature in an acidic environment. Formic acid is formed by furfural and HMF degradation, while levulinic acid is generated from HMF. Concentration of HMF in lignocellulosic hydrolysate is much lower than the furfural due to limited hydrolysis of hexose from lignocellulosic biomass. The third group of degradation product includes diverse phenolic compounds which are derived from lignin

explosion and

mic reticulum membrane [16].

**growth**

SO2

**Figure 1.** Overview of major metabolic pathways involved in lipid synthesis. The precursors for fatty acid synthesis, acetyl-CoA, malonyl-CoA and NADPH, are highlighted (red rectangles). Green and red arrows indicate upregulation and downregulation of the key enzyme for lipid accumulation. Abbreviations used for enzymes and metabolic intermediates: 6PGDH: 6-phosphogluconate dehydrogenase; AT: acyltransferase, cytosolic; ACC: acetyl-CoA carboxylase; ACL: ATP:citrate lyase; AMPD: AMP deaminase; cICDH: NADP<sup>+</sup> -dependent isocitrate dehydrogenase; CMT: citrate-malate translocase; D: desaturase; E: elongase; FAS: fatty acid synthetase; G6PDH: glucose-6-phosphate dehydrogenase; ICDH: isocitrate dehydrogenase; MDH: malate dehydrogenase; ME: malic enzyme; PC: pyruvate carboxylase; FA: fatty acid; IMP: inosine monophosphate; PUFA: polyunsaturated fatty acid; TAG: triacylglycerol. Adapted from [16, 33].

Furthermore, expression of this enzyme is not changed upon limitation of the cell growth by nitrogen source [35]. Recent studies confirmed that primary source of NADPH for lipid synthesis in *Y. lipolityca* is the Pentose phosphate pathway [34, 36]. The NADPH is generated by enzymes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Additional NADPH could be also provided by the cytosolic NADP<sup>+</sup> dependent isocitrate dehydrogenase (cICDH) present in some eukaryotic organisms (citrate/ isocitrate/2-ketoglutarate cycle) [16, 32, 33].

The *de novo* fatty acid biosynthesis takes place in cytosol on a multifunctional enzyme complex called fatty acid synthetase (FAS). FAS is fed by three precursors needed for the fatty acid synthesis such as acetyl-CoA, malonyl-CoA and NADPH. Malonyl-CoA is synthetized by carboxylation of acetyl-CoA with the enzyme acetyl-CoA carboxylase (ACC). The end products are saturated fatty acids C16 (palmitic cid) or C18 (stearic acid) depending on the microorganism. Fatty acid are further elongated and desaturated by specific elongases (E) and desaturases (D) in the endoplasmic reticulum leading to fatty acid of different chain length and degree of unsaturation. The final step is triacylglycerol formation from glycerol-3 phosphate and fatty acids catalyzed by specific acyltransferases (AT). Neutral lipids including triacylglycerols form lipid droplets on the lumenal and/or cytoplasmic side of the endoplasmic reticulum membrane [16].
