## 3 Results  
### Cultivation  
Using a pH-stat fed-batch approach and phosphate limitation (see Figure 1a), DCWs of approximately 30 g/L and an SCO content of approximately 58 g/100 g DCW could be reached in both strains (see Table 1). During the first 18 h of fermentations, phosphate limitation occurred (see Figure 1), inducing lipid production and resulting in volumetric lipid productivities of 0.296 g/Lh (strain V194) and 0.268 g/Lh (V134). The lipid yields (Y\({}_{\text{P/S}}\)) reached were approximately 0.17 g SCO produced per g VFA used. In the course of the fermentation, HAc concentrations reached a maximum of 0.9 g/L, while the maximum HPr concentration was 1.3 g/L (Figures S1-S4; Table S1).  
Based on the findings in laboratory scale, the process was adapted to the small pilot scale. While DCWs, SCO contents, and lipid yields were comparable to the laboratory scale results, the volumetric lipid productivity dropped to 0.200 g/Lh in both strains (see Table 1)  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Strain**} & \multirow{2}{*}{**Scale**} & **DCW** & **SCO** & **SCO** & **Y\({}_{\text{P/S}}\)** & **Productivity** \\  & & **[g/L]** & **[g/100 g DCW]** & **[g/L]** & **[gSCO/gVFA]** & **[gSCO/Lh]** \\ \hline \multirow{2}{*}{V194} & laboratory scale & 33.2 \(\pm\) 0.1 & 57.9 \(\pm\) 2.2 & 19.2 \(\pm\) 0.7 & 0.162 \(\pm\) 0.005 & 0.296 \(\pm\) 0.010 \\ \cline{2-6}  & small pilot scale & 31.1 & 57.1 & 17.8 & 0.167 & 0.201 \\ \hline \multirow{3}{*}{V134} & laboratory scale & 31.1 \(\pm\) 0.6 & 56.9 \(\pm\) 0.9 & 17.7 \(\pm\) 0.6 & 0.174 \(\pm\) 0.005 & 0.268 \(\pm\) 0.009 \\ \cline{2-6}  & small pilot scale & 32.6 & 56.4 & 18.4 & 0.176 & 0.205 \\ \cline{2-6}  & pilot scale & 36.9 & 57.8 & 21.3 & 0.179 & 0.152 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Overview of key process parameters for strains _A. brassicae_ V134 and _P. kudriavzevii_ V194 in laboratory scale (400 mL starting volume), small pilot scale (45 L starting volume), and pilot scale (500 L starting volume). Results for laboratory scale are reported as mean values of two fermentation runs \(\pm\) standard deviation (SD). Pilot scale results each relate to a single fermentation run.  
Figure 1: Key parameters for cultivation of A. brassicae V134. Dry cell weight (DCW, \(-\)\(-\)\(-\)) and single cell oil (SCO, \(-\)\(-\)\(-\)) are given as concentrations in g/L; the concentration of phosphate (\(-\)\(-\)\(-\)) is given as concentration in mg P/L. (**a**) shows the course of the fermentation process in laboratory scale over 72 h. Cultivations were performed in duplicate. (**b**) shows the course of a fermentation process in small pilot scale over 96 h.  
due to a longer fermentation duration (see Figure 1). HAc concentrations in small pilot scale varied in a range of 0.1 g/L to 2.1 g/L, and HPr concentrations varied in a range of 0.4 g/L to 3.2 g/L (Figures S5 and S6, Table S1).  
Given a similar productivity and a slightly higher lipid yield in _A. brassicae_ V134, this strain was chosen for a subsequent scale-up to the pilot scale. HAc concentrations in pilot scale varied in a range of 0.2 g/L to 0.9 g/L, and HPr concentrations varied in a range of 1.5 g/L to 2.4 g/L (Figures S7-S9, Table 1). The lipid yield and SCO content of the biomass were comparable to the smaller scale experiments, reaching 0.18 g/g VFA and 58 g/100 g DCW (see Table 1); however, the SCO concentration was higher, while the volumetric lipid productivity dropped further. The former is explained by the use of concentrated VFAs, eliminating the diluting effect feed addition has on the cell suspension in the smaller scale reactors. The decreased volumetric productivity is again attributed to an increase in fermentation duration by 51 h compared to small pilot scale.