## 2 Materials and Methods
### Media  
The initial screening of yeast strains on a semi-defined medium included cultures on a nitrogen-limited medium (inducing the de novo lipid production process; see: [34; 35]), where the initial concentration of lactose (carbon substrate; S) was adjusted to c. 55 g/L,close to the concentration of lactose in second CW [27]. The molar ratio C/N was set at c. 56 moles/moles as a suitable ratio for lipid accumulation, according to [PERSON] et al. [27]. Yeast extract (Y.E., \(\approx\)10% \(w/w\) nitrogen content) was used as an organic nitrogen source (2.32 g/L), whereas the concentration of inorganic source, ammonium sulfate, was 1.09 g/L (A.S., \(\approx\)21% \(w/w\) nitrogen content). The medium salt concentration was (in g/L): MnSO\({}_{4}\)\(\times\) H\({}_{2}\)O 0.06, MgSO\({}_{4}\)\(\times\) 7H\({}_{2}\)O 1.5, ZnSO\({}_{4}\)\(\times\) 7H\({}_{2}\)O 0.02, CaCl\({}_{2}\)\(\times\) 2H\({}_{2}\)O 0.15, FeCl\({}_{3}\)\(\times\) 6H\({}_{2}\)O 0.15, KH\({}_{2}\)PO\({}_{4}\) 7.0, and Na\({}_{2}\)HPO\({}_{4}\) 2.5 [44]. The medium was transferred into 250-mL Erlenmeyer flasks (50 \(\pm\) 1 mL working volume), sterilized in autoclave (T = 121 \({}^{\circ}\)C, for \(\geq\)20 min), and then inoculated with 1.0 mL of 24-h exponential pre-culture (1-3 \(\times\) 10\({}^{7}\) cells; initial concentration of biomass--X\({}_{0}\) = 0.12 \(\pm\) 0.02 g/L in flasks). All pre-cultures were carried out in 250-mL flasks filled with 50 \(\pm\) 1 mL working volume of the following medium: 5 g/L D-Lactose, 5 g/L D-Glucose, 10 g/L Y.E., and 10 g/L peptone. The cultures were incubated in an orbital shaker (Lab-Line, Illinois, USA) at incubation temperature \(T\) = 30 \(\pm\) 1 \({}^{\circ}\)C and 180 \(\pm\) 5 rpm agitation rate for efficient oxygen transfer. It was necessary to maintain a pH value at 5.5 \(\pm\) 0.3 (pH/mV meter H1 8014, Hanna Instruments Hellas, Athens, Greece); therefore, an appropriate volume of NaOH (5M) or HCl (5M) was added periodically into the flasks, under aseptic conditions, when it was needed. The microorganism with the higher potential of production of dry biomass and lipids (viz. C. _curvatus_ ATCC 20509) was, furthermore, batch-cultured on lactose-based substrates with nitrogen excess and nitrogen limitation in flasks. The nitrogen excess medium contained initial lactose (S\({}_{0}\)) \(c\). 46 g/L as carbon source and Y.E. (5 g/L) and A.S. (5 g/L) as nitrogen sources, with C/N ratio equal to 14 \(\pm\) 1 moles/moles. The nitrogen-limited experiments were conducted in four S\({}_{0}\) concentrations, namely \(\approx\)58 g/L, \(\approx\)118 g/L, \(\approx\)107 g/L, and \(\approx\)153 g/L, and the nitrogen sources were added in the appropriate quantities (g/L), Y.E. 1.16 and A.S. 0.545; Y.E. 2.32 and A.S. 1.09; Y.E. 0.5 and A.S. 0.5; Y.E. 0.5 and A.S. 0.5, respectively, to achieve the respective C/N ratios \(\approx\)124, \(\approx\)124, \(\approx\)337, and \(\approx\)482 moles/moles. The medium salt concentration and incubation conditions were the same, as described previously. Second cheese whey (CW) was also employed as a carbon source. It was provided by the Laboratory of Dairy Research (Agricultural University of Athens). The cheese-making process included sheep milk, rennet (90% Chymosin, Vlachopoula SA, Athens, Greece), and lyophilized starter culture of lactic acid microorganisms (Laboratorio PRODOR, Mezzano Scotti, Italy), to produce Feta cheese. The main by-product generated by Feta cheese manufacture, namely CW, was thermally processed at \(T\) = 90 \({}^{\circ}\)C for half an hour, in order for the whey proteins to be flocculated and collected as whey cheese \"Mizithra\". The final by-product of Mizithra whey cheese manufacture is the partially deproteinized second CW.