## Results and Discussion
It was also decided to utilize the pure substrate as a carbon source (pure glycerol therefore was employed instead of crude). This decision was taken in order to ascertain that all potential changes observed in the quantitative cellular metabolism in these experiments were due only to the increment in \(Gly_{0}\) concentration (and also to the increase in the initial C/N ratio) employed into the medium and not to the potential accumulation of crude glycerol impurities into the medium, specifically at the higher \(Gly_{0}\) concentrations imposed. The obtained results of this set of experiments are shown in Table 1. As was anticipated, higher total DCW quantities were produced when compared with the previous experiment, since increased initial nitrogen concentrations were employed in the former case. Likewise, glycerol consumption was equally linear for this series of trials; linear regression of the experimental data of remaining glycerol concentration vs. incubation time for the points in which non-negligible glycerol quantity remained unconsumed into the medium, revealed a glycerol consumption rate value (r\({}_{\rm Gly}\), g/L/h) (see also Section 3.1). This value noticeably decreased as the \(Gly_{0}\) concentration increased in the medium. However, the r\({}_{\rm Gly}\) value was always higher than in the previous set of experiments in which a lower initial nitrogen concentration was employed; in all trials and irrespective of the \(Gly_{0}\) concentration in the medium, linear regression of the remaining glycerol (\(Gly\)) \(f\) (t) revealed an \(r^{2}\) value \(\geq\) 0.95 (linear regressions not presented) providing evidence of the linearity of consumption of glycerol by _Y. lipolytica_, with r\({}_{\rm Gly}\) ranging between 0.54 g/L/h (\(Gly_{0}\)\(\approx\) 70 g/L) and 0.37 g/L/h (\(Gly_{0}\)\(\approx\) 150 g/L). Despite the high concentration of glycerol in some runs (i.e., in the experiment with the highest \(Gly_{0}\) concentration into the medium this quantity was \(\approx\)155 g/L), the microorganism presented an appreciable DCW production, providing evidence again of the suitability of this substrate for strains of this species [8; 11; 16; 29; 44; 45; 46; 47; 48]. Though, the DCW\({}_{\rm max}\) quantity somehow decreased with the \(Gly_{0}\) concentration increase in the medium, indicating the potential inhibition exerted due to the high \(Gly_{0}\) quantity added into the medium (in accordance with reports for other _Y. lipolytica_ strains; see i.e., [PERSON] et al. [8; 18; 21]). In all cases, and despite the fact that the pH in the medium remained always in values \(\geq\)4.5 \(\pm\) 0.1, carbon catabolism was mostly directed towards the synthesis of polyols, while indeed low quantities of organic acids (mostly citric acid) were found into the medium; \(CA\) concentrations were \(\leq\)3.0 g/L for all culture phases and all \(Gly_{0}\) concentrations rested. In most cases in the literature in which _Y. lipolytica_ strains have grown under nitrogen limitation on glycerol-based media and medium pH remaining \(>\)4.5, this event normally favored the biosynthesis and secretion into the medium of citric acid. In contrast, relatively low quantities of polyols were simultaneously synthesized [8; 23; 40; 46; 48]. However, in agreement with the behavior of the strain LMBF Y-46 that mostly produced polyols instead of citrate, despite the maintenance of pH in values ranging between 4.5-6.1, in recent years, some reports have indicated similar physiological behaviors of other wild-type _Y. lipolytica_ strains cultivated on slightly acidic conditions (medium pH between 4.5-6.5) with glycerol employed as the substrate, in which mostly polyols have been synthesized [15; 16; 18; 36; 37]. As indicated, the glycerol uptake rate noticeably decreased with \(Gly_{0}\) increment into the medium. Despite significant extension of the culture time (i.e., up to 280 h that corresponds to \(\approx\)11.5 days) for the