## 4 Discussion  
### Lipid Production in Phosphorus-Limited Media  
Phosphorus is one of the basic elements for the cultivation of microorganisms, including oleaginous ones. Due to its incorporation into phospholipids, nucleic acids or coenzymes, the analyzed component has been considered crucial for the structure of yeast cells as well as their functioning. Phosphorus may also be stored as polymetaphosphate in biomass. Interestingly, the non-conventional yeast _Y. lipolytica_ contains nearly half as much phosphorus compared to the common yeast species _Sachamomyces cerevisiae_[39]. According to [PERSON] et al. [31], phosphorus limitation has the potential to be used as a regulator of lipid production by microorganisms, also in the presence of nitrogen-rich media. In other words, this approach provides new opportunities for microbial lipid biosynthesis in a more economical manner, due to the use of complex waste materials as a carbon source. Before the rapeseed oil after fish frying was used as substrate, in the present experiment, it was mechanically purified from the residue of the fried product suspended in it. In view of the above, the waste was not a source of nitrogen, but only a lipid carbon source. [PERSON] et al. [29] were the first to attempt to investigate the potential of cheese whey as a substrate for microbial oil production by _Y. lipolytica_ B9. The analyzed by-product consisting of nearly 93% water and about 7% dry matter is particularly rich in phosphorus. One of the factors examined was the effect of supplementation of KH\({}_{2}\)PO\({}_{4}\) as an additional phosphorus source on biomass growth and lipid accumulation levels. Similar to the current study, the authors found that phosphorus limitation increased the lipid content in yeast cells. The additional phosphorus source significantly decreased the efficiency of lipid substance accumulation in yeast cells compared to the medium without supplementation, when the efficiency was 44% on a dry weight basis. The addition of 1 and 2 g/dm\({}^{3}\) KH\({}_{2}\)PO\({}_{4}\) contributed to a cellular lipid content of 37 and 26%, respectively. Figure 3: Fatty acid composition [% \(w/w\)] of cellular lipids in _Y. lipolytica_ KKP 379 grown in **M1**, **M2**, **M3** and **M4** medium with 5% rapeseed waste post-frying oil as a carbon source. The effect of limiting access to mineral salts including potassium phosphate on SCO production by _Y. lipolytica_ yeast was also studied by [PERSON] et al. [35]. In the study, waste produced by ethanol distilleries named Distillery Spent Wash (DSW) was used. After all, the lipid accumulation efficiency was only 9.15% using a medium with 7.0 g/dm\({}^{3}\) KH\({}_{2}\)PO\({}_{4}\) and DSW. Moreover, phospholipids accounted for 34% of all lipids. However, this accumulation efficiency result was higher compared to the culture when raw DSW was used without additional phosphate supplementation (7.18%). The addition of 7 g/dm\({}^{3}\) KH\({}_{2}\)PO\({}_{4}\) and 1.5 g/dm\({}^{3}\) MgSO\({}_{4}\) to the culture medium resulted in the highest biomass yield of 7.34 g\({}_{\text{d.m.}}\)/dm\({}^{3}\). It is worth noting that the best growth of _Y. lipolytica_ yeast was in DSW with the addition of KH\({}_{2}\)PO\({}_{4}\) in the range of 7.5-8.0 g/dm\({}^{3}\). The results of the current study appear to be consistent. Using M4 medium with 7.0 g/dm\({}^{3}\) KH\({}_{2}\)PO\({}_{4}\), 1.5 g/dm\({}^{3}\) MgSO\({}_{4}\) and 4 g/dm\({}^{3}\) (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\), the biomass yield was 7.45 g\({}_{\text{d.m. }}\)/dm\({}^{3}\), but it was not the highest score. The yeast showed the best growth in medium with limiting additions of phosphorus source, 3.0 g/dm\({}^{3}\) KH\({}_{2}\)PO\({}_{4}\) and 1.1 g/dm\({}^{3}\) Na\({}_{2}\)HPO\({}_{4}\), with a relatively higher nitrogen source addition. A valuable experiment was conducted by [PERSON] et al. [31], whose results, similar to the current work, showed that better microbial lipid production efficiency correlated with low phosphorus source doses and higher C/P molar ratio.