## Results and Discussion  
Metabolic Characterization of Yarrowia lipolytica LMBF Y-46: Effect of Different Glycerol Sources under Nitrogen Limitation  
In the first part of this report, it was desirable to evaluate the performances of \(Y\). _lipolytica_ LMBF Y-46 in shake-flask experiments in media in which pure or crude glycerol (\(Gly_{0}\) \(\approx\) 40 g/L) had been employed. Initial nitrogen concentration used in the medium was (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) at 1.5 g/L and yeast extract at 1.0 g/L (initial molar ratio \(\approx\) 48 moles/moles). The kinetics of both experiments are shown in Figure 1a-d. For both cases, DCW production almost reached a plateau (\(X\) = 10-11 g/L) at \(t\) = 120 h after inoculation. Onset of the secretion of polyols had been given earlier (\(t\)\(\approx\) 50-55 h after inoculation). At the very same fermentation step (\(t\approx\) 50-55 h after inoculation) ammonium nitrogen had barely disappeared from the growth medium (initial NH\({}_{4}\)+ concentration \(\approx\) 400 mg/L, NH\({}_{4}\)+ concentration at \(t\geq\) 50-55 h after inoculation \(\approx\) 25 \(\pm\) 10 mg/L). Thus, after that given fermentation point, cultures were performed under nitrogen-limited conditions, providing evidence that the onset of the accumulation of polyols into the medium occured after nitrogen had been depleted from the medium. \(X_{\text{max}}\) concentration was slightly higher in the case of pure glycerol compared with the raw feedstock (11.2 vs. 10.5 g/L; see Figure 1a). The maximum quantity of polyols produced in this set of experiments was \(c\). 13.5 g/L irrespective of the use of crude or pure glycerol. Mannitol was the principal polyol synthesized (\(c\). 80-84% _w/w_ of the _Pol_ synthesized) whereas, other polyols that were synthesized, in notably lower quantities, were erythroid and to lesser extent arabitol. For both experiments, polyols were re-consumed at the late culture steps, when glycerol concentration into the medium presented essentially low concentrations (Figure 1b,c). The assimilation of glycerol for both trials was almost linear (see kinetics of remaining into the medium glycerol in Figure 1b) and the rate of glycerol consumption (r\({}_{\text{Gly}}\)), calculated after linear regression of the experimental data of remaining in the medium glycerol vs. time for both trials, seemed unaffected by the utilization of the pure or the crude substrate. For both fermentations, r\({}_{\text{Gly}}\) was =0.24 g/L/h (Figure 1b). This result, together with the observations concerning the production of polyols and DCW that seemed not to differ between the two fermentations carried out, indicate that the impurities of the substrate did not seem to have any important impact upon the physiological  
Figure 1: Kinetics of dry yeast cell mass production (X, g/L) (a), glycerol assimilation (_Gly_, g/L) (b), total polyols synthesis (_Pol_, g/L) and (c) lipid in dry cell weight (%, _w/w_), (d) by _Yarrowia lipolytica_ LMBF Y-46, during growth on pure and biodiesel-derived glycerol in shake-flask experiments under nitrogen-limited conditions. Culture conditions: growth in 250 mL conical flasks at 180 \(\pm\) 5 rpm, initial pH = 6.0 \(\pm\) 0.1, pH ranging between 4.5 and 6.1, incubation temperature \(T=28\pm 1\)\({}^{\circ}\)C. Each experimental point is the mean value of two independent determinations (SE \(\leq\) 18%). behavior of the strain. In agreement with these results, for a number of wild-type microbial strains (i.e., _Klebsiella oxytoca, Mortierella ramantimana_, some strains of the species _Y. lipolytica_) cultivated on glycerol deriving from various origins, the impurities of the feedstock did not seem to have any impact upon the physiological behavior of the mentioned microorganisms [22; 23; 26; 27]. In contrast, in other cases (i.e., _Clostidium butyricum_, _C. pasteurianum, Meyerozyma caribica, Rhodotorula minuta_, other strains belonging to the species _Y. lipolytica_, etc.