## Results and Discussion
), glycerol impurities seemed to (drastically) modify the cellular metabolism of the implicated microorganisms [28; 29; 30; 31; 32; 33; 34]. Therefore, the impurities found in the crude glycerol feedstock did not seem to have a clear positive or negative effect upon the production of dry yeast biomass and metabolites by _Y. lipolytica_ yeast. However, in some cases it must be pointed out that the impurities found in the medium seemed to enhance the production of the metabolites by various yeast strains including _Y. lipolytica_[29; 32; 33; 34]. From the above-mentioned analysis, therefore, it seems that the biosynthesis of metabolites by _Y. lipolytica_ strains cultivated on glycerol, appears to be strain dependent in relation to the origin of the feedstock implicated in the bioprocess performed. According to theory, polyols are synthesized as secondary anabolic compounds with their biosynthesis being coupled with nitrogen limitations in the growth medium [3; 10; 13; 14; 35; 36; 37]. Though, the current investigation, as well as other limitations in the literature [18; 21] indicate that the total biomass biosynthesis may occur simultaneously with the biosynthesis of polyols into the medium. In fact, in the current investigation it was seen that typically after nitrogen depletion from the medium (at \(t\approx\) 50-55 h after inoculation), polyols were secreted, while an almost linear profile of polyols biosynthesis occurred between 50 and 150 h after inoculation (see Figure 1c). On the other hand, in spite of nitrogen depletion that, as stated occurred at \(t\approx\) 50-55 h after inoculation (at that fermentation step \(X\) concentration was \(\approx\)7.0 g/L), some supplementary biomass production occurred between 50-55 h and \(t=\) 120 h after inoculation; as indicated, DCW production plateaued at \(t=\) 120 h, and when biomass concentration reached its plateau \(X\) concentration was \(\approx\)10-11 g/L. This supplementary total biomass production that occurred at the late growth stages did not indicate that cells \"fattened\" as fermentation proceeded; in fact, insignificant quantities of total lipids were accumulated inside the yeast cells for both trials performed, despite the fact that cultures were performed under nitrogen limitation (Figure 1d). The kinetic results of the current investigation indicate that somehow elevated lipid quantities were synthesized at the relatively early growth stage of the culture, with lipid in DCW values decreasing as the fermentation proceeded (Figure 1d) This is in accordance with various literature reports in which _Y. lipolytica_ strains (wild-type or mutant ones) had been cultured batch-wise in glycerol-based media [18; 38; 39; 40]. The total dry biomass production increase that occurred after nitrogen had been depleted from the culture (_viz._ between 50-55 h and 150 h after inoculation), could suggest the accumulation of intra-cellular polysaccharides inside the cells occurring after nitrogen deprivation from the medium. This assumption is in accordance with the reports of several microorganisms like other _Y. lipolytica_ strains or other yeast species/genera like _Apiotrichum curatum_, _Rhodosporidium toruloides_, _Metschnikowia_ sp. [16; 37; 41; 42; 43]. Metabolic Characterization of Yarrowia lipolytica LMBF Y-46: Effect of Glycerol Concentration in Media with Constant Initial Nitrogen in Shake-Flask Trials  
In the next approach, and in order to more profoundly quantify the metabolism of _Y. lipolytica_ LMBF Y-46, another set of shake-flask cultures was carried out. In this set, a higher initial nitrogen concentration was set into the medium (ammonium sulfate and yeast extract at an initial concentration of 2.0 and 3.0 g/L, respectively) in order to further \"boost\" the production of the total biomass, and potentially to increase the consumption rate of glycerol. Variable \(Gly_{0}\) concentrations with higher initial substrate quantities than the previous experiment, were tested (\(Gly_{0}\) quantities adjusted between \(\approx\)70 and \(\approx\)150 g/L).