## 4 Discussion  
### Cultivation  
The present study demonstrates that a SCO production process showing high productivity, SCO content, and biomass concentration, and relying solely on HAc and HPr as carbon sources, can be realized with a pH-stat fed-batch approach. Furthermore, it was shown that the developed process is robust, can be used for different organisms with only minor strain-specific modifications, and can readily be adapted to different scales. In comparison to other VFA-based processes reported in the literature [8; 20; 41], both strains showed high DCW concentrations, with the DCWs reached in the present work being more than twice as high. The high DCW concentration is mainly attributed to the cultivation mode, since many values reported in the literature relate to batch cultivation, and the cytotoxic effect of VFAs prevents the use of high initial VFA concentrations, thus limiting the amount of available carbon [20]. When using various modes of fed-batch cultivation, DCWs similar to or above the DCWs reported in this work could be achieved [14; 42; 43]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Condition of** & **Extracted Lipids** & **Recovery 1** \\
**Biomass** & **Pretreatment Method** & **[g/100 g DCW]** & **[\%]** \\ \hline \multirow{3}{*}{lyophilized} & - & 17.80 \(\pm\) 0.49 & 36.82 \(\pm\) 1.02 \\ \cline{2-4}  & bead milling & 22.98 \(\pm\) 0.11 & 47.54 \(\pm\) 0.22 \\ \cline{2-4}  & bead milling + & & \\  & induced autolysis & 38.05 \(\pm\) 0.14 & 78.72 \(\pm\) 0.29 \\ \hline wet & acid hydrolysis & 35.13 \(\pm\) 0.25 & 72.68 \(\pm\) 0.52 \\ \hline \hline \end{tabular}  
* Calculated from the extracted lipid amount and the lipid content of the biomass according to analysis (48.3 \(\pm\) 0.5 g/100 g DCW).  
\end{table} 
Table 2: Comparison of different biomass pretreatment methods prior to ASE in laboratory scale. Results for laboratory scale are reported as mean values from three extractions each \(\pm\) standard deviation (SD) values. The different pretreatments form homogeneous groups that are significantly different from each other at the 95% confidence level (LSD). Due to high SCO contents and short fermentation durations, the volumetric productivities reported in the present work are above those reported by [PERSON] et al. [14] and comparable to the ones reported by [PERSON] et al. [42]. With 0.51 g/Lh, only [PERSON] et al. [43] were able to achieve substantially higher volumetric productivities than those reported in this work. However, in contrast to the present work, in which a mixture of HAc and HPr was used as a carbon source, the reported productivities were achieved by using only HAc as a carbon source. This is relevant, since the metabolization of HPr is less effective than the metabolization of HAc [19], and the presence of HPr has a substantial effect on the composition of the produced lipids [44]. HPr directly reacts with CoA forming propionyl-CoA that is subsequently used for the synthesis of odd-numbered FAs [19]. Thus, the presence of unusual odd-numbered FAs in the produced SCO is a direct result of the use of HPr as carbon source. Scale-up to the small pilot scale for both strains resulted in values for DCW, SCO content, and SCO yield (g SCO/g VFA) that were comparable to the laboratory scale results. In contrast, lipid productivity was reduced in both strains, due to longer fermentation times. The process durations were observed to be approximately 23.5 h longer in small pilot scale than in laboratory scale. This increase can be attributed to three factors. For one, the inoculation volume was reduced from 10% of the starting volume in laboratory scale to 4% in the small pilot scale due to technical reasons. For another, the OD\({}_{600}\) reached in the pre-culture in laboratory scale could not be achieved in small-pilot scale. This effect is assumed to be a result of a lower oxygen transfer rate (OTR) in the pre-culture due to the geometry of the bigger shake flasks used in the inoculum preparation.