## 2 Materials and Methods
### Analytical Methods  
Flasks were periodically removed from orbital shaker, the culture broth was measured volumetrically (this was needed due to moisture loss attributed to the evaporation), the volume was corrected to 50 \(\pm\) 1 mL and cells were harvested, as precipitate, through centrifugation at 15,000\(\times\) g, at \(T\) = 2 \({}^{\circ}\)C for 15 min (Hettich Universal Centrifuge, Model 320-R, Merck KGaA, Darmstadt, Germany) and washed twice with distilled water. The supernatant was collected for further analyses. Special treatment was required for the case of _P. laurentii_ NRRL Y-2536 cultivation, since fermentation broth was particularly viscous, due to the extra-cellular polysaccharides' secretion. The exopolysaccharide (EPS), in form of capsule and/or released polysaccharides in fermentation broth, as well as the cells were both concomitantly precipitated by anhydrous acetone (purity \(\geq\) 99.5%), but only when the carbon source had been totally consumed. In this case, total dry precipitate consisted of both dried cells and polysaccharides. Acetone was recycled by evaporation (Flash Evaporator/Rotavapor R-114, BUCHI Labortechnik AG, St. Gallen, Switzerland). Residual lactose (S, g/L), total dry weight (TDW) of biomass (X, g/L), and cellular lipids (L, g/L) were determined (the corresponding methods and assays are analyzed below). pH, yield or substrate to biomass coefficient (Y\({}_{\text{X/S}}\), g of total biomass produced per g of substrate consumed), substrate to lipids coefficient (Y\({}_{\text{L/S}}\), g of cellular lipids produced per g of substrate consumed), dry biomass productivity (P\({}_{\text{X}}\), total dry biomass concentration produced per hour expressed in g/L/h), lipid productivity (P\({}_{\text{L}}\), total lipid concentration produced per hour, expressed in g/L/h), and lipid content in dry biomass (K\({}_{\text{L/X}}\), g of cellular lipids per g of total biomass) were calculated, according to [PERSON] et al. [44]. As far as the experiments in second CW were concerned, the following extra analyses were occasionally performed; Free Amino Nitrogen (FAN, mg/L), Total Kjeldahl Nitrogen (TKN, g/L), Residual Protein (RP, g/L), and Inorganic Phosphorus (IP, g/L) assays (as described below). In cases of intra-cellular and/or extra-cellular polymeric substances production, total polysaccharides (TPS, g/L) were determined (as described below). The total dry biomass (X, g/L) was gravimetrically determined after drying the precipitate at \(T\) = 80 \({}^{\circ}\)C until constant weight (usually for 26-30 h). Total polysaccharides (TPS, g/L), (intra-cellular (IPS) and extra-cellular polymeric substances (EPS)), were determined for _P. laurentii_ NRRL Y-2536, according to the slightly modified method of [PERSON] et al. [45] and [PERSON] et al. [46]. In brief, 50 mg of aggregate (dry weight of yeast cells mass and EPS) were mixed with 2 mL of hydrochloric acid (HCl, 2.5 M) and were allowed to boil (\(T\) = 100 \({}^{\circ}\)C) for 30 min. Cellular debris were removed by centrifugation (as described above) and the supernatant was neutralized with 2 mL of sodium hydroxide (NaOH, 2.5 M). The final volume was adjusted to 5 mL, and the total polysaccharides were quantitatively determined and expressed as glucose equivalents, through the assay \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Total Solids** **(\%)** & **Lectose** **(g/L)** & **FAN** & **Protein (Bradford)** & **IP** & **TKN** **(g/L)** & **pH** \\ \hline \(7.2\pm 1.0\) & \(60.9\pm 1.8\) & \(63.0\pm 2.5\) & \(1.10\pm 0.15\) & \(0.68\pm 0.10\) & \(1.14\pm 0.13\) & \(6.25\pm 0.21\) \\ \hline \end{tabular}
\end{table} 
Table 1: Characteristics of centrifugated and filtrated second cheese whey. of 3,5-dinitrosalicylic acid [46]. Moreover, the determination of individual saccharides of TPS was performed in selected samples through High-Performance Liquid Chromatography.