## 4 Discussion
### Lipid Production in Nitrogen-Limited Media  
Nitrogen is considered to be a special component, the limited amount of which in the medium has the greatest effect on the induction of lipogenesis in the cells of oil-producing species. In other words, when the culture medium is deficient in nitrogenous compounds or when its pool is depleted, the synthesis of nucleic acids and proteins is inhibited and the rate of biomass growth decreases [40]. According to [PERSON] et al. [32], in addition to magnesium limitation, the restriction of cellular access to nitrogen is the factor for efficient cellular lipid biosynthesis. Continuous culture of the wild yeast strain _Y. lipolytica_ ACA-DC50109 resulted in an accumulation efficiency of 47.5% with a biomass yield of 12.2 g/L. Furthermore, lipid accumulation at high levels was provided by low nitrogen doses, but at levels that allowed _Y. lipolytica_ yeast cells to grow. This correlation was related to the maintenance of the pentose-phosphate pathway properly function, which provides the coenzyme NADPH for the mechanism of lipogenesis. For this reason, understanding the physiology of oleaginous microorganisms is of great importance in efficient cellular lipids production. Inorganic (NH4)2 SO4 was the only source of nitrogen in the present study. The current experiment assumed the use of a medium containing only mineral components, excluding the carbon source. There are reports that the presence of organic nitrogen sources in the medium may stimulate microbial synthesis of lipid components [41; 42]. Neither the preliminary study stage nor the bioreactor experiments proved a clear relationship between efficient cellular lipid accumulation via ex novo pathway and the level of nitrogen supplementation. In M3 medium (10 g/dm3 (NH4)2 SO4), the lipid accumulation efficiency was 32.67%, while in M2 (8 g/dm3 (NH4)2 SO4) it was only 20.90%. It is worth noting that in the medium with a lower dose of ammonium sulfate the biomass yield reached more than twice the value (Table 3). When the low addition of a nitrogen source in the M4 culture medium was compensated by a higher level of a phosphorus source, the accumulation efficiency was close to that for the phosphorus-limited M3 medium. This result further strengthened the hypothesis that the ratio of the two analyzed elements nitrogen and phosphorus can be considered an important factor for both yeast cell growth and lipid accumulation capacity. The oil-producing species of the yeast _Trichosporon oleaginous_, in the presence of xylose and limited access to nitrogen compounds, accumulated over 50% of lipids in the dry mass of cells. The type of limitation used (-N, -C and -CN) had a significant effect on the fatty acid profile of the oil extracted from yeast cells. The content of linoleic acid (C18:2 in the -cis configuration) showed the greatest variation depending on the conditions applied. When the researchers limited the access of the cells to nitrogen and carbon, the C18:2 content was 7.31% and 7.45% (\(w/w\)), respectively. When carbon source limitation was applied alone, the content of linoleic acid (as the main component of the cell membrane) increased threefold, accounting for 21.58% of all fatty acids [43]. In the current study, the linoleic acid content of the extracted oils was a maximum of 22.58% of total fatty acids. Notably, nutritionally valuable linoleic acid was present in each of the oil samples, with the highest percentage from M2 cultivation. In the experiment, the highest percentage of SCO production by _Y. lipolytica_ yeast was obtained for culture medium with 3.0 g/dm3 (NH4)2 SO4 and 3.0 g/dm3 \(\mathrm{K}\mathrm{H}\mathrm{z}\mathrm{PO}_{4}\) and molar ratios of C/P = 114.9:1 and C/N = 71.8:1. A molar C/N ratio of 20:1 is considered to be the minimum for promoting lipid accumulation in oleaginous yeast cells [44], in the experiment, was not lower than 21.5. As found, simultaneous limited access to phosphorus and nitrogen promoted lipid accumulation, but inhibited biomass growth.