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

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), physicochemical (steam explosion/autohydrolysis, ammonia fiber explosion, CO<sup>2</sup> explosion and SO2 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

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>

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].

**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:


dependent isocitrate dehydrogenase (cICDH) present in some eukaryotic organisms (citrate/

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

isocitrate/2-ketoglutarate cycle) [16, 32, 33].

ATP:citrate lyase; AMPD: AMP deaminase; cICDH: NADP<sup>+</sup>

140 Advances in Biofuels and Bioenergy


and extractive compounds present in the lignocellulosic biomass [45, 49–51]. The most common aromatic compounds in the lignocellulose acid hydrolysate are vanillin, syringaldehyde, 4-hydroxybenzoic acid, ferulic acid, etc. [45, 51]. Formation of degradation by-product strongly depends on the plant source and pretreatment process (temperature, pressure, reaction time and presence of catalyst) [46–48, 51].
