*2.2. Glycerol as the Sole Carbon Source for Fermentative Limonene Production*

A prolonged growth phase of *E. coli* and higher product concentrations with glycerol as the sole carbon source were described by Willrodt et al. in an aforementioned study [13]. In order to investigate if these influences can also be observed with *E. coli* BL21 (DE3) pJBEI-6410, shake flask experiments were carried out using either glucose or glycerol as the sole carbon source. In the case of glucose, the substrate was consumed completely after 10 h, whereas glycerol was still present in the fermentation medium after 11 h. Finally, the carbon source was fully consumed in both cultivations. The growth curves were also similar (Appendix, Figure A1). Using glucose for the carbon supply resulted in a final limonene concentration of 121 <sup>±</sup> 1 mg·Lorg−<sup>1</sup> in the organic phase (Figure 3A). By comparison, the fermentation with glycerol showed a prolonged production phase, resulting in a

final limonene concentration of 184 <sup>±</sup> 11 mg·Lorg<sup>−</sup>1. The limonene yields relative to the carbon source were 9.3 <sup>±</sup> 0.1 g·C-mol−<sup>1</sup> and 14.2 <sup>±</sup> 0.8 g·C-mol<sup>−</sup>1, for glucose and glycerol, respectively (Figure 3B). These results confirm previous observations that glycerol is the better choice as a carbon source for fermentative limonene production with *E. coli*.

**Figure 3.** Two-liquid phase shake flask fermentations with *E. coli* BL21 (DE3) pJBEI-6410 in M9 minimal medium with either 0.5% *w*/*v* glucose (closed symbols) or glycerol (open symbols) as the sole carbon source. (**A**) Limonene concentrations (-, ) in the organic phase and carbon source (, ) concentrations were determined at regular intervals. (**B**) Carbon specific limonene yields after 26 h of cultivation. The error bars relate to biological duplicates.
