## 3 Results and Discussion  
### Evaluation of R. toruloides Growth on Different Concentrations of VFAs  
Batch cultivations were carried out to study the tolerance, growth, and lipid accumulation of the yeast on different VFAs (Acetic acid; Propionic acid; Butyric acid), while glucose (GC) was used as a positive control. Figure 1 shows the consumption of VFAs at different starting concentrations (10, 20, and 30 g/L), while biomass and lipid accumulation are depicted in Figure 2.  
Acetic acid appeared to be the most favorable VFA as a carbon source, with substrate consumption similar to glucose. Acetic acid consumption decreased with an increasing concentration, with 30 g/L being the upper limit that the yeast could fully utilize. [PERSON] et al. (2016) also reported growth of _R. toruloides_ (strains ATCC 10788 and Y4) at a concentration of up to 30 g/L acetic acid. In our study, when 40 g/L acetic was used, the growth of _R. toruloides_ started to get inhibited, with the consumption of acetic acid dropping to 76.1% (data not shown). Similar findings were reported in the study of [PERSON] et al. (2016), where acetic acid at 40 g/L inhibited the growth of the yeast. In the same study, it was found that the maximum concentration of acetic acid that the yeast could completely consume was 10 g/L, while [PERSON] et al. (2016) found that the inhibiting effect on yeast growth appears in concentrations higher than 20 g/L. However, to the best of our knowledge, no studies have been carried out to study the growth limit of _R. toruloides_ on other VFAs, like propionic and butyric acid. In our study, it was found that propionic and butyric acid inhibited the growth of _R. toruloides_ at much lower concentrations. At 10 g/L initial concentration, the yeast could consume 94.3% of propionic acid and 87.9% of butyric acid, which dropped to 65.6% and 62.1%, respectively, for 20 g/L, while at 40 g/L, the consumption of propionic acid was only 30.8% (data not shown) and no growth was observed with butyric acid. Figure 1: Consumption of VFAs and glucose (GC) throughout cultivation in 10 g/L (**A**), 20 g/L (**B**), and 30 g/L (**C**) initial concentration for _R. toruloides_. As shown in Figure 2, when glucose was used, the biomass and the lipid concentration were the highest, however, the highest lipid content was attained with acetic acid. Specifically, at an initial concentration of 10 g/L (Figure 2A), the biomass accumulation was similar for all VFAs (4.7, 4.7, 4.8 g/L for C2, C3, C4, respectively) while for glucose it was 5.5 g/L. The lipid concentration was 1.3 g/L on glucose (24.2% \(w/w\) lipid content), followed by acetic acid with 1.1 g/L (27.9% \(w/w\) lipid content). For propionic and butyric acid, lipid accumulation had very similar values (0.7 g/L for C3 and 0.8 g/L for C4) and the same was observed for the lipid content (9.3% \(w/w\) for C3 and 8.8% \(w/w\) for C4). When the concentration increased to 20 g/L (Figure 2B), the observed trend for biomass and lipid accumulation was the same for glucose and acetic acid. The DCW and lipid concentration was higher for glucose with 9.3 g/L and 3.0 g/L, respectively, followed by acetic acid with 5.2 g/L and 2.1 g/L. On the contrary, the lipid content was lower for glucose (32.8% \(w/w\)) than for acetic acid (40.3% \(w/w\)). The same trend was found by [PERSON] et al., [46] where biomass accumulation is favored with glucose, while the lipid content is higher with acetic acid. In the case of propionic and butyric acid, a slightly different trend was observed at 20 g/L. Specifically, the DCW was higher for butyric acid (6.4 g/L) than for propionic acid (5.7 g/L), as well as the lipid accumulation (0.9 g/L for C4 and 0.5 g/L for C3) and the lipid content (13.3% \(w/w\) for C4 and 9.2% \(w/w\) for C3). At 30 g/L (Figure 2C) and C/N 50, the DCW for glucose increased to 12.6 g/L and for acetic acid to 7.1 g/L. The lipid  
Figure 2: DCW (g/L), total lipids (g/L), lipid content (% \(w/w\)), and substrate consumption (% \(w/w\)) on the last day of cultivation of \(R\).