It is known that when microorganisms are cultivated on fat-type substrates (e. g. long-chain free-fatty acids, TAGs, fatty-esters, etc.) production of (intra­cellular, cell-bounded or extra-cellular) lipases is performed as a physiological response to the presence of fatty materials into the growth medium (Fickers et al., 2005). This secretion is obligatory in the case that TAGs or fatty-esters are used as substrates (Fickers et al., 2005; Papanikolaou and Aggelis, 2010). In contrast, a large variety of microorganisms are capable of utilizing soaps as well as free — fatty acids as sole carbon and energy source, regardless of the lipolytic capacities of the microorganisms used in order to break down fatty materials (Ratledge and Boulton, 1985; Papanikolaou and Aggelis, 2010). Specifically, for the case of the yeast Y. lipolytica, its culture on TAG-type substrates is accompanied by secretion of an extra-cellular lipase called Lip2p, encoded by the LIP2 gene (Pignede et al., 2000). This gene encoded for the biosynthesis of a precursor premature protein with Lys-Arg cleavage site. The secreted lipase was reported to be a 301-amino — acid glycosylated polypeptide which belongs to the TAGs hydrolase family (EC 3.1.1.3) (Pignede et al., 2000; Fickers et al., 2005). The Lip2p precursor protein was processed by the KEX2-like endoprotease encoded by the gene XPR6, whereas deletion of the above gene resulted in the secretion of an active but fewer stable pro-enzyme (Pignede et al., 2000). Simultaneously, other intra-cellular lipases (Lip7p, Lip8p) may also be produced and secreted into the culture medium, that present different fatty acid specificities, with maximum activity being displayed against D918:1 (oleic acid), 6:0 (capronic) and 10:0 (caprinic) fatty acids (Fickers et al., 2005).

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 for the case of Y. 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 (lauric acid-12:0 and myristic acid-14:0) or unsaturated (A918:1 and linoleic acid-A9,1218:2) fatty acids is significantly higher than that of principally stearic (18:0) and to lesser extent palmitic (16:0) 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 bio-transformations (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, 2003b, 2010).

The intra-cellular dissimilation of the various catabolized fatty acids is performed by reactions catalyzed by the various intra-cellular 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 Y. lipolytica (Fickers et al, 2005). In fact, it has been revealed that the aforementioned biochemical process is a multi-step 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; Mlickova et al. 2004a, 2004b; Fickers et al. 2005). Aox3p is specific for short chain acyl-CoAs, Aox2p preferentially oxidizes long-chain acyl-CoAs while Aox1p, Aox4p and Aox5p do not appear to be 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 (Mlickova et al. 2004a, 2004b). These strains were subjected to disruptions of the genes implicated in the encoding of various intra­cellular Aox. The genetically engineered strains, hence, either under-expressed or did not at all express several of the enzymes implicated in the catabolism (^-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 (Mlickova et al. 2004a, 2004b). Therefore, the above-mentioned studies as well as various others reported in the literature (Aggelis and Sourdis, 1997; Papanikolaou et al, 2003; Szczesna-Antczak et al., 2006; Mantzouridou and Tsimidou, 2007) indicate that external addition of fat (ex novo lipid accumulation) can significantly enhance the bio-process of SCO production in various oleaginous microorganisms, but external utilization of fat mainly serves for the ‘improvement’ and ‘upgrade’ of a fatty material utilized as substrate [e. g. 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 illipe butter, shea butter, sal fat (Papanikolaou and Aggelis, 2010)] and not for the use of the SCO produced in the manufacture of bio-diesel.