**1. Introduction**

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

microorganism since they grow much faster than the microalgae. Unlike microalgae, they can use more diverse sugars and other carbon sources for their growth and lipid synthesis monosaccharides (glucose and xylose), amino sugars (N-acetylglucosamine), disaccharides (lactose, galactose, mannose, cellobiose and sucrose), alcohols (glycerol, ethanol and methanol), polysaccharides (starch and pectin) and organic acids (volatile fatty acids and acetic

In order to increase economic feasibility of the process production, different low-cost substrates have been used for the production such as crude glycerol, lignocellulose biomass (hydrolysate sweet sorghum bagasse, rice straw hydrolysate and corn stover hydrolysate), molasses waste, waste streams from food industry (whey permeate, olive pomace oil and olive oil mill wastewaters) and waste spent yeast from the brewing industry [5, 25–31].

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

regulated. 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)

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].

)-depended isocitrate dehydrogenase (ICDH) is down-

Production of Microbial Lipids from Lignocellulosic Biomass

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acid) [5, 8, 20–24].

and the activity of NAD+

**2. Biochemistry of lipid accumulation**

(NADP+

and exported to mitochondria *via* CMT [16, 32].

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 microorganism since they grow much faster than the microalgae. Unlike microalgae, they can use more diverse sugars and other carbon sources for their growth and lipid synthesis monosaccharides (glucose and xylose), amino sugars (N-acetylglucosamine), disaccharides (lactose, galactose, mannose, cellobiose and sucrose), alcohols (glycerol, ethanol and methanol), polysaccharides (starch and pectin) and organic acids (volatile fatty acids and acetic acid) [5, 8, 20–24].

In order to increase economic feasibility of the process production, different low-cost substrates have been used for the production such as crude glycerol, lignocellulose biomass (hydrolysate sweet sorghum bagasse, rice straw hydrolysate and corn stover hydrolysate), molasses waste, waste streams from food industry (whey permeate, olive pomace oil and olive oil mill wastewaters) and waste spent yeast from the brewing industry [5, 25–31].
