## 2 Materials and Methods  
### Yeast strain and Culture Conditions  
In the current work, the yeast strain _Y. lipolytica_ KKP 379 from the Collection of Industrial Microorganisms Cultures belonging to Professor [PERSON] Institute of Agricultural and Food Biotechnology--State Research Institute in Warsaw (Poland) was used. The yeast culture was stored at \(-20\)\({}^{\circ}\)C using cryovials containing ceramic beads and cryoprotective agent (Protect Select, Technical Service Consultants Ltd., Heywood, UK).  
Inoculation culture was conducted in YPG medium with the following composition: 10 g/dm\({}^{3}\) yeast extract (Y), 20 g/dm\({}^{3}\) peptone (P) and 20 g/dm\({}^{3}\) glucose (G). The flasks were incubated at 28 \({}^{\circ}\)C for 24 h with a rotation amplitude of 140 rpm.  
The yeast cells were incubated in 500 cm\({}^{3}\) flasks at 28 \({}^{\circ}\)C with 140 rpm rotary shaker speed. Each flask contained 200 cm\({}^{3}\) of sterile medium. Batch cultures were also conducted in a BIOFLO 3000 laboratory bioreactor from New Brunswick Scientific (USA) with a working volume of 4 dm\({}^{3}\), fermentation temperature 28 \({}^{\circ}\)C and 0.025% (_v/v_) inoculum. Oxygen control was applied using compressed air to maintain the relative degree of oxygenation in the culture medium at a level not lower than 30% of the initial oxygen concentration (variable agitation speed 300-600 rpm). The dissolved oxygen level was measured using an oxygen electrode. Changes in pH values were also monitored using a selective electrode. Parameters of cellular lipid biosynthesis in a batch culture of _Y. lipolytica_ strain were calculated according to [PERSON] et al. [2].  
The yeast grew in a mineral medium containing 1.5 g/dm\({}^{3}\) MgSO\({}_{4}\), 0.16 g/dm\({}^{3}\) FeSO\({}_{4}\)\(\cdot\)H\({}_{2}\)O, 0.15 g/dm\({}^{3}\) CaCl\({}_{2}\), 0.08 g/dm\({}^{3}\) MnCl\({}_{2}\)\(\cdot\)4H\({}_{2}\)O, 0.02 g/dm\({}^{3}\) ZnSO\({}_{4}\) and KH\({}_{2}\)PO\({}_{4}\), Na\({}_{2}\)HPO\({}_{4}\), and (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) at different levels. All inorganic chemicals were purchased from Avantor Performance Materials Poland S.A (Gliwice, Poland). In all batch cultures, the carbon source was waste rapeseed post-frying oil at 50 g/dm\({}^{3}\). Waste oil, in which cod (_Gadus morhua_) fillets were fried at 170 \({}^{\circ}\)C in full immersion, came from a fish processing company in Podlaskie Voivodeship (Poland). The preliminary batch shaking flask cultures in duplicate have been conducted with statistical planning of experiments using a Latin square plan (4 \(\times\) 4) (Table 1).  
Bioreactor batch cultures have been conducted in media containing variable concentrations of KH\({}_{2}\)PO\({}_{4}\), Na\({}_{2}\)HPO\({}_{4}\) and (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) (Table 2)  
\begin{table}
\begin{tabular}{c c c c c} \hline \multirow{2}{*}{\(\mathbf{KH}_{2}\)PO\({}_{4}\)(g/dm\({}^{3}\))} & \multicolumn{4}{c}{**Duration of the Cultivations (Days)**} \\ \cline{2-5}  & 3 & 4 & 5 & 6 \\ \hline
3 & 4.0 & 6.0 & 8.0 & 10.0 \\ \hline
7 & 6.0 & 8.0 & 10 & 4.0 \\ \hline
9 & 8.0 & 10.0 & 4.0 & 6.0 \\ \hline
11 & 10.0 & 4.0 & 6.0 & 8.0 \\ \hline \end{tabular}
\end{table}
Table 1: Latin Square plan 4 \(\times\) 4-flask experiment design.  
\begin{table}
\begin{tabular}{c c c c c} \hline \multirow{2}{*}{**(g/dm\({}^{3}\))**} & \multirow{2}{*}{**M1**} & \multirow{2}{*}{**M2**} & \multirow{2}{*}{**M3**} & \multirow{2}{*}{**M4**} \\ \hline KH\({}_{2}\)PO\({}_{4}\) & 3.0 & 3.0 & 3.5 & 7.0 \\ \hline Na\({}_{2}\)HPO\({}_{4}\) & 1.1 & 1.1 & 2.5 & 2.5 \\ \hline (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) & 3.0 & 8.0 & 10.0 & 4.0 \\ \hline \end{tabular}
\end{table}
Table 2: Composition of culture media used in yeast cultivation in a laboratory bioreactor.