## 4 Discussion
### Cellular Oxygen Demand in Microbial Lipid Production  
Based on the authors' knowledge, there is no clear statement on the effect of the oxygenation degree on the efficiency of lipid accumulation. The link between dissolved oxygen in the medium and accumulation capacity by _Y. lipolytica_ yeast is inconclusive. However, as a result of thought-provoking observations, the issue of oxygen demand by _Y. lipolytica_ yeast appeared to be worth exploring. The concentration of oxygen available in the culture medium is a crucial variable in both the context of growth and efficient microbial lipid production by strictly aerobic yeast _Y. lipolytica_. It is predicted that an increase in substrate oxygenation may result in the stimulation of cells for growth [45]. The observations highlight the importance of the cellular oxygen demand in relation to growth, but also to the process of cellular lipid accumulation. Despite the negligible increase in biomass yield, the yeast still efficiently used oxygen from the substrate, which could be related to the intensively occurring process of lipid biosynthesis. Interestingly, it may be reasonably assumed that the cells should be exposed to a sufficiently high concentration of oxygen in the medium. Nevertheless, too high a concentration of dissolved oxygen may cause inhibition of cellular metabolism resulting from oxidative stress. Given the need to maintain an appropriate degree of oxygenation of the culture medium, agitation rate is an important parameter. Under conditions of increased cellular oxygen demand, high agitation rate is used. As is widely known, this kind of approach may have negative consequences. High shear forces and mechanical stress may make culture development impossible [46]. Therefore, despite the increase in cellular oxygen demand in bioreactor cultures, the set amplitude of the agitator rotation was not able to meet the demand of the yeast. According to [PERSON] et al. [47], high dissolved oxygen (1.5 mg/dm\({}^{3}\)) in the culture media stimulates cellular lipid synthesis. Upregulation of ATP-citrate lyase (ATP-CL) and malic enzyme (ME), which are involved in microbial lipid synthesis, was observed under such conditions. The importance of malic enzyme for cellular metabolism of oleaginous microorganisms including _Y. lipolytica_ yeast has been recognized as crucial. The product of reaction catalyzed by ME gives the product necessary for fatty acid synthesis, namely, coenzyme NADPH. Under conditions of limited access of cells to nitrogenous compounds, NADPH is produced only by enzymatic reactions involving ME. Conversely, increasing the dissolved oxygen concentration resulted in a decrease in NAD-dependent isocitrate dehydrogenase enzyme activity. When the Krebs cycle is inhibited due to the depletion of nitrogen sources by the cells and allosteric AMP (adenosinemonophosphate) activator concentrations being reduced, ICDH is deactivated. This phenomenon is considered to be characteristic of the de novo biosynthesis pathway [48]. When yeast cultures were conducted in glucose medium, it was observed that limited cellular access to phosphorus, as with nitrogen, results in a reduction in AMP levels to activate cellular phosphorus reserves. There are two strategies to maximize cellular lipid accumulation. The first one, by deleting genes encoding for acyl-oxidase activity, assumes the prevention of the degradation of storage lipids [49]. The second one aims to support the production via the overexpression of genes encoding for ATP-CL and ME activity [50; 51]. [PERSON] et al. [52] using 13C-Metabolic Flux Analysis singled the oxidative pentose-phosphate pathway out as the main source of lipogenic coenzyme NADPH rather than malic enzyme activity. In view of the above, the purpose for research on the role of phosphorus as a coenzyme component in processes related to cellular respiration and provision of energy necessary for SCO biosynthesis has been justified.