## 3 Results
### Trials on Lactose-Based Media

Table 2: Quantitative data originated from kinetics of seven microbial strains, cultivated on a semi-defined medium, with lactose as carbon source, at initial lactose (S0) concentration adjusted to c. 56 g/L and initial molar ratio C/N \(\approx\) 58 moles/moles. Culture conditions as in Section 2; standard error for most experimental points was \(\leq\)17%. microorganisms _C.uzbekistanensis_ NRRL Y-44 and _T. ovoides_ ACA-DC 5052 were incapable of assimilating lactose and growing under these culture conditions. Despite considerations in the literature indicating that an initial C/N molar ratio adjusted to c. 60 moles/moles (as in the screening study presented in Table 2) would be sufficiently high to induce noticeable production of lipids inside the yeast cells [54,55], this seemed not to be the case for the microorganisms used in the present investigation. The microorganism _C. curvotus_ ATCC 20509, due to higher production of biomass and lipids and the relatively higher lipid content in dry cell basis as compared to the other strains (see Table 2), was chosen to be batch-cultured on lactose-based substrates in 250-mL Erlenmeyer flasks, in various initial C/N molar ratio media, ranging from nitrogen-excess to significant nitrogen-limited conditions. Therefore, the C/N ratio in nitrogen excess medium was set to c. 14 moles/moles (employment of 46.3 g/L lactose, 5 g/L Y.E., 5 g/L A.S.). Nitrogen-limited experiments were conducted in four different initial lactose concentrations (S\({}_{0}\approx\) 58 g/L, 118 g/L, 107, and 153 g/L), while the initial C/N ratios for the respective S\({}_{0}\) concentrations were \(\approx\)124, \(\approx\)124, \(\approx\)337, and \(\approx\)482 moles/moles (see also Section 2). Therefore, in the second set of experiments, the impact of both the initial C/N molar ratio and the S\({}_{0}\) concentration of lactose were studied. The obtained results are summarized in Table 3. The cultivation of _C. curvatus_ ATCC 20509 in nitrogen excess medium resulted in high biomass production (X = 20.0 g/L) with only 8% lipid content in TDW; that is an expected result since, as indicated in the previous paragraphs, the de novo lipid production process is only enhanced under nitrogen-limited conditions [34,54,55]. Cultivation in C/N \(\approx\) 124 moles/moles led to X = 22.4 g/L and L = 7.3 g/L (K\({}_{\rm L/X}\) = 0.33 g/g) in the case of S\({}_{0}\approx\) 58 g/L, while for the significantly high S\({}_{0}\) = 118 g/L, TDW production was =44.0 g/L, with a 36% lipid content (the respective L value was =15.8 g/L). Higher nitrogen limitation, viz. the initial imposed C/N ratio \(\approx\) 337 moles/moles, led to production of 10.2 g/L of TDW containing 4.20 g/L lipid production (K\({}_{\rm L/X}\) = 0.41 g/g) and a volumetric lipid productivity =0.038 g/L/h, whilst the microorganism consumed only 36 g/L lactose, in a 111 h-long batch culture. Further incubation did not improve lactose assimilation from the growth medium (data not presented). At the highest initial C/N ratio imposed (\(\approx\)482 moles/moles), a TDW quantity =14.9 g/L containing 58.4% _w/w_ lipid content (8.7 g/L total cellular lipids) and 0.054 g/L/h lipid productivity, although lactose consumption, as in the previous trial, was somehow limited (c. 33 g/L within 160 h after inoculation, without tendency of further sugar consumption after more prolongated incubation). For the latter case, also of interest for further discussion is the elevated conversion yield of total lipid produced per unit of sugar consumed (Y\({}_{\rm L/S}\)) that was =0.26 g/g (see Table 3), which is one of the highest values ever reported in the international literature concerning the de novo lipid accumulation process by wild-type microbial strains [33,54,55,56,57, 33,54].