## 3 Results
### Microbial Lipids Production in Two-Stage Batch Cultures
These results suggest that VFAs were channeled towards cellular maintenance, lipids biosynthesis, or other metabolites instead of cell proliferation. The accumulation of microbial lipids occurred simultaneously with yeast growth, and the lipids content was similar throughout the lipogenic phase, demonstrating that the cultures with 3 pulses of VFAs were an effective strategy to prevent lipid turnover (Figure 2c). Moreover, an approximately 2-fold improvement in lipid concentration was attained in cultures with 3 pulses, compared to those obtained in cultures with a single pulse (Table 1). As _Y. lipolytica_ demonstrated the ability to consume a total VFA concentration of 18 g\(\cdot\)L\({}^{-1}\) (divided into 3 pulses) and with a higher lipids productivity, an additional experiment was carried out with a 24 h first stage of yeast growth in glucose, followed by the addition of a single pulse of 18 g\(\cdot\)L\({}^{-1}\) VFAs (mixtures of acetate, butyrate, and propionate with 6 g\(\cdot\)L\({}^{-1}\) of each one) in the second stage. From an operational point of view, and considering the industrial production of lipids, a single addition would be more advantageous. Moreover, other _Y. lipolytica_ strains have already shown the ability to assimilate high VFA amounts without inhibitory effects [8]. _Yarrowia lipolytica_ was \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \begin{tabular}{c} **Substrate** \\ **(Growth Phase)** \\ \end{tabular} & **Pulses** & \begin{tabular}{c} **YX\({}_{\text{SS}}\)** \\ **(g\(\cdot\)g\({}^{-1}\))** \\ \end{tabular} & \begin{tabular}{c} **Lipids** \\ **(g\(\cdot\)L\({}^{-1}\))** \\ \end{tabular} & \
\begin{tabular}{c} **R\({}_{\text{SS}}\)** \\ **(g\(\cdot\)L\({}^{-1}\).h\({}^{-1}\))** \\ \end{tabular} &
\begin{tabular}{c} **R\({}_{\text{VFA}}\)** **\\ **(g\(\cdot\)L\({}^{-1}\).h\({}^{-1}\))** \\ \end{tabular} \\ \hline \multirow{3}{*}{Glucose} & \multirow{3}{*}{3} & \multirow{3}{*}{0.35 \(\pm\) 0.01  & 1 & 0.43 \(\pm\) 0.01  & 1 & 0.43 \(\pm\) 0.01  & 0.84 \(\pm\) 0.01  & 0.28 \(\pm\) 0.01  & 0.28 \(\pm\) 0.01  & 0.28 \(\pm\) 0.02  \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Values of global biomass yield (\(Y_{\text{X/S}}\)), maximum lipids concentration and substrate (glucose or glycerol) uptake rate, and VFAs (\(R_{\text{VFA}}\)) obtained in _Y. lipolytica_ W29 two-stage batch cultures. A mixture of 6 g\(\cdot\)L\({}^{-1}\) VFAs (2 g\(\cdot\)L\({}^{-1}\) acetate, 2 g\(\cdot\)L\({}^{-1}\) propionate, and 2 g\(\cdot\)L\({}^{-1}\) butyrate) was added in each pulse. Data are average \(\pm\) standard deviation of two independent replicates. Values followed by the same letter in each column do not present statistically significant differences (\(p\geq 0.05\)). able to consume 18 g\(\cdot\)L\({}^{-1}\) VFAs without inhibitory effects on cellular growth (Figure 3a) and VFA uptake rate. No statistical differences were observed in the VFA uptake rate between experiments with a pulse of 18 g\(\cdot\)L\({}^{-1}\) VFAs (0.19 g\(\cdot\)L\({}^{-1}\)\(\cdot\)h\({}^{-1}\)\(\pm\) 0.002 g\(\cdot\)L\({}^{-1}\)\(\cdot\)h\({}^{-1}\)) and with a pulse of 6 g\(\cdot\)L\({}^{-1}\) VFAs. As occurred in the previous experiments, the yeast cells demonstrated a preference for acetate, which was totally consumed 48 h after the pulse. By contrast, butyrate took 72 h to be completely assimilated, and a small amount of propionate (1.5 g\(\cdot\)L\({}^{-1}\)) remained in the medium at the end of the cultivation. Other studies have also reported that _Y. lipolytica_ strains assimilate acetic acid faster than propionate and butyrate [8; 9; 10]. The global biomass yield in these experiments (0.28 g\(\cdot\)g\({}^{-1}\)\(\pm\) 0.01 g\(\cdot\)g\({}^{-1}\)) was lower than those obtained with a single pulse of 6 g\(\cdot\)L\({}^{-1}\) VFAs and three pulses of 6 g\(\cdot\)L\({}^{-1}\) VFAs each, suggesting that VFAs were more directed to lipids synthesis than cellular growth. In fact, the lipids content (Figure 3b) was higher (\(p\) < 0.05) than that attained in the previous two-stage batch culture strategies.