## 3 Results and Discussion
### Evaluation of the Yeasts' Growth on VFAs Derived from the Acidogenic Fermentation of BSG  
VFAs produced from BSG acidogenic fermentation were tested as a cost-competitive raw material for the production of lipids from oleaginous yeasts. The VFAs effluent contained in total 12.1 g/L of short-chain VFAs (10.1 g/L C2, 0.8 g/L C3, 1.2 g/L C4). The higher amount of acetic acid as compared to the low amounts of propionic and butyric acid is beneficial for the growth of the yeasts, while the absence of long-chain VFAs reduces any potential inhibitory effect [51]. Both _C. curvatus_ (Figure 7A) and _R. toruloides_ (Figure 7C) could utilize the VFAs mixture, with acetic acid being consumed at a faster rate than propionic and butyric acid, as expected by the results when individual VFAs were tested. Biomass production reached a maximum concentration at 72 h, which was 7.4 g/L for _C. curvatus_ (Figure 7B) and 5.7 g/L for _R. toruloides_ (Figure 7D). It is worth noting that for both yeasts, higher biomass concentration was achieved on the acidogenic effluent as compared to the synthetic media with 10 g/L of acetic acid. Specifically, the increase in the DCW was 54% for _C. curvatus_ and 21% for _R. toruloides_. The above observation suggests that the VFA effluent has very good potential for the growth of the yeasts. One explanation as to why the increase was higher for _C. curvatus_ can be due to the higher preference of this strain to produce high amounts of biomass with butyric acid as a substrate compared to _R. toruloides_. Therefore, the combination of acetic together with butyric acid, even in low concentration, leads to an improvement on the conversion towards biomass accumulation for this yeast species. The lipid concentration reached its highest value in 24 h of cultivation for both yeasts (1.8 g/L for _RT_ and 1.5 g/L for CC) (Figure 7B,D). The same is observed for the lipid content, with _R. toruloides_ having 42.8% \(w/w\) and _C. curvatus_ with 39.3% \(w/w\). After this point, the lipid concentration declined, despite a steady increase in DCW being observed. That can be attributed to the rapid consumption of the VFAs within 24 h of cultivation. Moreover, when VFA effluent was used, the lipid accumulation and lipid content for both yeasts improved compared to 10 g/L acetic acid as synthetic media. Specifically, an increase in the lipid concentration was observed for both yeasts, however, this was considerably higher for _R. toruloides_ (4.2% for _CC_ and 57% for _RT_). The lipid content increased 56% for _C. curvatus_ and 54% for _R. toruloides_, respectively. This can be explained again due to the different substrate preferences of the two strains. As indicated earlier, _R. toruloides_ has higher lipid yield when acetic acid is used as a substrate, while for _C. curvatus_, this is the case when butyric acid is used. Therefore, since the VFA effluent contains higher amounts of acetic acid, the produced lipids for _C. curvatus_ are not expected to have such a significant improvement. _C. curvatus_ would benefit more regarding lipids formation by a higher content of butyric acid in the composition of the VFA effluent. Figure 6: (**A**) Biomass (Y\({}_{\text{X/S}}\)) and (**B**) lipid (Y\({}_{\text{L/S}}\)) yields for _C. curvatus_ when grown on different concentrations of synthetic VFAs and glucose (GC). As supported by other studies [3; 52; 53; 54], it can be concluded that the use of VFAs derived from the digestion of a variety of waste sources, containing a mixture of different VFAs in varying ratios, can support the biomass and lipid accumulation of the yeasts. In this study, the effect of VFAs derived from BSG, which contained a mixture of acetic, propionic, and butyric acid was tested and the acquired results were compared to previous studies using synthetic or waste-derived VFAs as carbon sources for the growth of _C. curvatus_ and _R. toruloides_ and are summarized in Tables 1 and 2.