## 3 Results and Discussion
### Single-Cell Oil (SCO)
#### 3.4.2 PCA Analysis  
PCA was employed to further investigate the effect of the different VFAs on the fatty acid composition of SCO. PCA biplots are shown in Figure 9A for _C. curvatus_ and in Figure 9B for _R. toruloides_. The biplots occur as a combination of the loading plot together with the score plot. The plots represent the two-dimensional projection of the samples which contain the main principal components (PCs) as the newly constructed axes. The PCs are a new set of orthogonal axes that are uncorrelated but display the variation present in the original variables since they occur as a linear combination of the latter. For instance, the first two PCs capture the maximum variation of the original variables and are usually enough for descriptive purposes. For _C. curvatus_, PC1 retained 50.3% of the variance, while PC2 16.8%, accounting for a total of 67.1% variance. For _R. toruloides_, PC1 retained 45.6% of the variance, while PC2 27.3%, resulted again in a total of 72.9% variance. The correlation between the variables is described by the loading factors. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & & _C. curvatus_ & & & \\ \hline
**Concentration (g/L)** & **Substrate** & **SFA (\%)** & **MUFA (\%)** & **PUFA (\%)** & **OCFAs/Total Lipids (\%)** & **OCFAs (g/L)** \\ \hline  & GC & 40.2 & 47.7 & 12.1 & 0.4 & 0.02 \\
40 & C2 & 45.6 & 39.9 & 14.5 & 0.4 & 0.02 \\  & C3 & 54.4 & 20.0 & 25.6 & 35.4 & 2.17 \\  & C4 & 39.0 & 35.0 & 26.0 & 1.5 & 0.14 \\ \hline
60 (C/N 50) & GC & 37.3 & 50.7 & 12.0 & 0.4 & 0.03 \\  & C2 & 43.9 & 40.0 & 16.1 & 0.4 & 0.02 \\ \hline
60 (C/N 100) & GC & 40.4 & 45.7 & 13.9 & 2.0 & 0.18 \\  & C2 & 45.0 & 40.6 & 15.6 & 2.1 & 0.11 \\ \hline (10.1 C2; 0.8 C3; 1.2 C4) & BSG VFAs & 45.1 & 37.5 & 17.4 & 10.0 & 0.15 \\ \hline \hline \multicolumn{7}{c}{_R. toruloides_} \\ \hline
**Concentration (g/L)** & **Substrate** & **SFA (\%)** & **MUFA (\%)** & **PUFA (\%)** & **OCFAs/Total Lipids (\%)** & **OCFAs (g/L)** \\ \hline  & GC & 38.2 & 43.3 & 18.5 & 0.0 & 0.00 \\
10 & C2 & 38.9 & 43.8 & 17.3 & 0.0 & 0.00 \\  & C3 & 61.9 & 16.6 & 21.4 & 24.6 & 0.18 \\  & C4 & 47.9 & 30.5 & 21.6 & 0.0 & 0.00 \\ \hline  & GC & 35.3 & 49.9 & 14.8 & 0.0 & 0.00 \\
20 & C2 & 41.2 & 44.3 & 14.5 & 0.0 & 0.00 \\  & C3 & 65.9 & 16.0 & 18.1 & 30.8 & 0.16 \\  & C4 & 46.6 & 29.7 & 23.7 & 1.4 & 0.01 \\ \hline
30 (C/N 50) & GC & 38.1 & 49.1 & 12.7 & 0.0 & 0.00 \\  & C2 & 38.8 & 51.0 & 10.2 & 0.8 & 0.03 \\ \hline
30 (C/N 100) & GC & 38.9 & 47.2 & 13.9 & 1.2 & 0.07 \\  & C2 & 39.9 & 49.5 & 10.6 & 0.5 & 0.02 \\ \hline (10.1 C2; 0.8 C3; 1.2 C4) & BSG VFAs & 23.6 & 57.2 & 16.0 & 5.7 & 0.10 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: _Cont._Figure 9: PCA biplots of PC1-PC2 for _C. curvatus_ (**A**) and _R. toruloides_ (**B**). The scores (dots) signify the substrates that were used for cultivation (glucose, acetic acid, propionic acid, butyric acid, BSG VFAs) and the loading plots (arrows) refer to the fatty acids of the SCO. For instance, PC1 for _C. curvatus_ better differentiated the samples according to the following fatty acids (factor loadings higher than 0.7): positive direction; C18:0, C18:1, C16:0, C20:0, negative direction; C17:1, C19:0, C15:0, C17:0, C19:1. On the other hand, PC2 better separated the samples according to the following fatty acids (factor loadings higher than 0.5): positive direction; C22:1, C18:2, C25:0, negative direction; C20:0. The positive or negative direction of the factor loadings indicates that a factor will be higher in the respective axis of that PC. For example, C18:0 has a high positive loading on PC1, therefore the samples located in that part of PC1, like glucose, acetic acid, and butyric acid (pink, green, and orange scores, respectively) will contain higher amounts of C18.0, while propionic acid (blue scores) will contain lower amounts of C18.0, but higher amounts of C17:1, which has a high negative loading factor on PC1. Additionally, based on the cosine of the angle from the center between two loading plots, an assumption on their correlation can be made.