In the case of SCO utilization for biodiesel production, research interest is focused only upon the process of de novo lipid accumulation. In this case, there is continuously increasing interest upon the potentiality of transforming abundant renewable materials (such as waste glycerol, flour-rich waste streams, and cellulose and hemicellulose hydrolysates) into SCO that will be further transformed into biodiesel. The process of ex novo lipid accumulation aims at adding value to low-cost fatty materials so that speciality high-value lipids (eg, cocoa-butter or other exotic fats substitutes) will be produced (Papanikolaou et al., 2001, 2003; Papanikolaou and Aggelis, 2003a,b, 2010).

Lipids produced by oleaginous microorganisms are mainly composed of neutral fractions (principally triacylglycerols, TAGs, and to lesser extent steryl-esters, SEs) (Ratledge, 1994; Ratledge and Wynn, 2002). As a general remark it must be stressed that when growth is carried out on various hydrophobic substances, the microbial lipid produced contains lower quantities of accumulated TAGs compared with growth elaborated on sugar-based substrates (Koritala et al., 1987; Guo et al., 1999; Kinoshita and Ota, 2001; Papanikolaou et al., 2001, 2002a; Fakas et al., 2006, 2007, 2008a). In any case, accumulation of storage lipids is accompanied by morphological changes in the oleaginous microorganisms, since “obese” cells with large lipid globules can generally appear during the lipid-accumulating phase (Fig. 8.1). Storage lipids, unable to integrate into phospholipid bilayers, cluster to form the hydrophobic core of the so-called “lipid bodies” or “oil bodies” (Mličková et al., 2004a,b). Lipid bodies of the oleaginous Yarrowia lipolytica yeast are illustrated in Fig. 8.2. As previously stressed, the biochemical pathways of de novo and ex novo lipid accumulation process present fundamental differences. These differences will be presented, explained, clarified, and comprehensively discussed in the following sections.

The excessive decrease of intra-cellular AMP concentration alters the Krebs cycle function; the activity of both NAD⁺ and NADP⁺-isocitrate dehydrogenases, enzymes responsible for the transformation of iso-citric to α-ketoglutaric acid, loss their activity, since they are allosterically activated by intracellular AMP, and this event results in the accumulation of citric acid inside the mitochondrion (studies performed in the oleaginous microorganisms Candida sp. 107, Rhodosporidium toruloides, Yarrowia lipolytica, Mortierella isabellina, M. alpina, Mucor circinelloides, and Cunningamella echinulata) (Botham and Ratledge, 1979; Evans and Ratledge, 1985; Wynn et al., 2001; Finogenova et al., 2002; Papanikolaou et al., 2004b; Marki et al., 2010). When the concentration of citric acid becomes higher than a critical value, it is secreted into the cytosol. Finally, in the case of lipogenous (lipid-accumulating) microorganisms, cytosolic citric acid is cleaved by ATP-citrate lyase (ACL), the key-enzyme of lipid accumulation process in the oil-bearing microorganisms, in acetyl-CoA and oxaloacetate, with acetyl-CoA being converted, by an inversion of β-oxydation process, to cellular fatty acids. In contrast, nonoleaginous microorganisms (eg, various Y. lipolytica and Aspergillus niger strains) secrete the accumulated citric acid into the culture medium (Ratledge, 1994; Anastassiadis et al., 2002; Papanikolaou et al., 2002b) instead of accumulating significant quantities of reserve lipid. In general, production of citric acid by citrate-producing strains is a process carried out when extra- and hence intracellular nitrogen is depleted (overflow metabolism phenomenon—see Anastassiadis et al., 2002), while studies of the intracellular enzyme activities and coenzyme concentrations have somehow identified and clarified the biochemical events leading to citric acid biosynthesis (Finogenova et al., 2002; Morgunov et al., 2004; Makri et al., 2010) and indeed it has been demonstrated that citric acid secretion and SCO accumulation are processes indeed identical into their first steps.

Various strains of Yarrowia lipolytica, when growing on glycerol, under nitrogen-limited conditions, do not show features of typical oleaginous microorganisms. Other Y. lipolytica strains (eg, ATCC 20460) have been reported to show increased Y_L/DCW at the beginning of the culture (Y_L/DCW ≈ 32% w/w 48 h after inoculation) (Sestric et al., 2014), while some strains show a high accumulation of lipid (eg, >10 g/L with simultaneous Y_L/DCW > 30% w/w) during growth on glycerol. In the first growth phase, and during nitrogen-excess conditions (balanced growth phase), strains accumulate some storage lipid. Thereafter, and despite the carbon excess in the anabolism of the yeast Y. lipolytica as well as progressive exhaustion of nitrogen, the Y_L/DCW values are depleted, while simultaneously low molecular weight metabolites (citric acid, acetic acid or polyols) are secreted. The concentration of available nitrogen is important for SCO production in Y. lipolytica, since some quantities of nitrogen are crucial for lipid accumulation, whereas when the nitrogen concentration falls below a threshold value, secondary metabolites, and notably citric acid, are produced, with lipid biodegradation being observed (Papanikolaou et al., 2013).

In a third category of microorganisms, the accumulated (inside the cytosol) citric acid provokes inhibition of the enzyme 6-phospho-fructokinase, and the above fact results in the intracellular accumulation of polysaccharides based on the 6-phospho-glucose (Evans and Ratledge, 1985; Galiotou-Panayotou et al., 1998). Schematically, the intermediate cellular metabolism resulting in the synthesis of either citric acid or storage lipid is presented in Fig. 8.3 (Ratledge, 1994; Ratledge and Wynn, 2002; Papanikolaou and Aggelis, 2009).

After the biosynthesis of intracellular fatty-CoA esters, an esterification with glycerol takes place in order for the reserve lipids to be stocked in the form of TAGs (Ratledge, 1988, 1994). This synthesis in the oleaginous microorganisms is conducted by virtue of the so-called pathway of α-glycerol phosphate acylation (Ratledge, 1988; Davies and Holdsworth, 1992; Athenstaedt and Daum, 1999; Müllner and Daum, 2004; Fakas et al., 2009b). In this metabolic pathway, free-fatty acids are activated by coenzyme A and are subsequently used for the acylation of the glycerol backbone to synthesize TAGs. In the first step of TAGs assembly, glycerol-3-phosphate (G-3-P) is acylated by G-3-P acyltranferase (GAT) at the sn-1 position to yield 1-acyl-G-3-P (lysophospatidic acid-LPA), which is then further acylated by lysophosphatidic acid acyltransferase (also named 1-acyl-G-3-P acyltransferase-AGAT) in the sn-2 position to yield phosphatidic acid (PA). This is followed by dephosphorylation of PA by phosphatidic acid phosphohydrolase (PAP) to release diacylglycerol (DAG). In the final step DAG is acylated either by diacylglycerol acyltransferase or phospholipid diacylglycerol acyltransferase to produce TAGs (Ratledge, 1988; Davies and Holdsworth, 1992; Athenstaedt and Daum, 1999; Müllner and Daum, 2004; Fakas et al., 2009b).

As far as the structure of the microbial TAGs produced is concerned, although their final composition could theoretically be a random substitution of acyl-CoA groups into glycerol, in the case of the oleaginous microorganisms that have been examined, the glycerol sn-2 position is almost always occupied by unsaturated fatty acids (production of vegetable-type TAGs; see Ratledge, 1988, 1994; Guo and Ota, 2000). Therefore, various oleaginous microorganisms (principally yeasts belonging to the species Rhodosporidium toruloides, Apiotrichum curvatum, and Yarrowia lipolytica) have been long considered as promising candidates for the production of equivalents of exotic fats (fats that are principally saturated but containing unsaturated fatty acids esterified in the sn-2 glycerol position) (Moreton, 1985, 1988; Moreton and Clode, 1985; Ykema et al., 1989, 1990; Davies et al., 1990; Lipp and Anklam, 1998; Papanikolaou et al., 2001, 2003; Papanikolaou and Aggelis, 2003b, 2010).

The free-fatty acids (existed as initial substrate or produced after lipase hydrolysis of the TAGs/fatty-esters) will be incorporated, with the aid of active transport, inside the microbial cell. It is interesting to state that in the case of Yarrowia lipolytica yeast, the various individual substrate fatty acids would be removed from the medium (and hence incorporated inside the microbial cell) with different rates (Papanikolaou et al., 2001, 2002a; Papanikolaou and Aggelis, 2003b). Specifically, regardless of the initial concentrations of the extra-cellular fatty acids, the incorporation rate of the lower aliphatic chain fatty acids, lauric acid (C12:0) and myristic acid (C14:0), or the unsaturated fatty acids ^Δ9C18:1 and ^Δ9,12C18:2, is significantly higher than that of stearic (C18:0) and to lesser extent palmitic (C16:0) acid (Papanikolaou et al., 2001; Papanikolaou and Aggelis, 2003b). Moreover, the incorporated fatty acids will be either dissimilated for growth needs or become a substrate for endo-cellular biotransformations (synthesis of “new” fatty acid profiles which did not exist previously in the substrate) (Ratledge and Boulton, 1985; Koritala et al., 1987; Aggelis and Sourdis, 1997; Guo et al., 1999; Kinoshita and Ota, 2001; Papanikolaou et al., 2001, 2002a, 2007b; Papanikolaou and Aggelis, 2003a,b, 2010).

The intracellular dissimilation of the various catabolized fatty acids is performed by reactions catalyzed by the various intracellular acyl-CoA oxidases (Aox). A significant amount of experimental work has been performed in relation with the elucidation of the above-mentioned reactions by using strains of the nonconventional yeast Yarrowia lipolytica (Fickers et al., 2005). In fact, it has been revealed that the aforementioned biochemical process is a multistep reaction requiring different enzymatic activities of five acyl-CoA oxidase isozymes (Aox1p through Aox5p), encoded by the POX1 through POX5 genes (Luo et al., 2002; Mličková et al., 2004a,b; Fickers et al., 2005a). Aox3p is specific for short-chain acyl-CoAs, Aox2p preferentially oxidizes long-chain acyl-CoAs, while Aox1p, Aox4p, and Aox5p do not appear of being sensitive in the chain length of the aliphatic acyl-CoA chain (Mauersberger et al., 2001; Luo et al., 2002; Fickers et al., 2005). It should also be noticed that genetically modified strains of Y. lipolytica, namely JMY 798 (MTLY 36-2P) and JMY 794 (MTLY 40-2P), have been created from the wild-type W29 strain (Mličková et al., 2004a,b). These strains were subjected to disruptions of the genes implicated in the encoding of various intracellular Aox. The genetically engineered strains, hence, either under—or did not at all express—several of the enzymes implicated in the catabolism (β-oxidation) of aliphatic chains. When cultures were performed on oleic acid utilized as the sole substrate, although the genetically engineered strains showed almost equivalent microbial growth compared with the wild strain (W29) from which they derived, in contrast with W29 strain they presented significantly higher formation of lipid bodies and, hence, increased lipid accumulation (Mličková et al., 2004a,b). Therefore, the above-mentioned studies as well as various others reported in the literature (Aggelis and Sourdis, 1997; Papanikolaou et al., 2003; Szczęsna-Antczak et al., 2006; Mantzouridou and Tsimidou, 2007) indicate that external addition of fat (ex novo lipid accumulation) can significantly enhance the bioprocess of SCO production in various oleaginous microorganisms, but external utilization of fat mainly serves for the “improvement” and “up-grade” of a fatty material utilized as substrate (eg, valorization of low-cost or waste fats so as to produce specialty lipids like cocoa-butter substitutes or substitutes of other high-added value lipids like illipé butter, shea butter, sal fat; Papanikolaou and Aggelis, 2010), and not for the use of the SCO produced in the manufacture of biodiesel.