*4.3. Fermentative Limonene Production in a Stirred-Tank Reactor*

Two-liquid phase fermentations at the bioreactor scale were carried out in a 3.1 L stirred-tank reactor (KLF 2000, Bioengineering AG, Wald, Switzerland). The tank reactor was equipped with two Rushton turbine stirrers. An M9 preculture was used to inoculate an initial batch cultivation, which was performed in 1 L of M9 minimal medium containing a 1.5% *w*/*v* carbon source at a starting OD600 of 0.11. The pH was set to 7.2 and controlled by the automatic addition of either 30% *v*/*v* phosphoric acid or 25% *v*/*v* ammonia hydroxide solution. Batch cultures were incubated at 30 ◦C, with shaking at 1800 rpm and an aeration rate of 1 vvm, until the batch phase was finished after 13 to 17 h (indicated by a steep pO2 increase). Before the start of the carbon-limited fed-batch fermentation, 1 mL of US\* trace element solution was added. To keep the exponential growth rates between 0.18 and 0.2 h<sup>−</sup>1, a carbon-limited and controlled exponential feed with a 73% *<sup>w</sup>*/*<sup>v</sup>* carbon source and 19.6 g·L−<sup>1</sup> MgSO4 · 7 H2O was started. The pO2 value was kept above 30% by adjusting the stirrer speed and the aeration rate. If desired, recombinant gene expression was induced by adding 0.1 mM IPTG after 2 h of exponential feeding, and 0.5 L of DINP were added subsequently. Antifoam A was added only in cases of excessive foaming. Regular sampling was carried out as described above.

## *4.4. Quantification of Limonene*

The quantification of limonene in DINP was carried out with GC using a TRACE GC Ultra (Thermo Fisher Scientific Inc., Waltham, MA, USA), equipped with a FactorFour-5ms column (30 m, 0.25 mm, 0.25 μm, Varian, Inc., Palo Alto, CA, USA) and a flame ionization detector. Nitrogen was used as a carrier gas and the injection volume was set to 1 <sup>μ</sup>L (80 ◦C, 5 min; 80–140 ◦C, 7.5 ◦C·min<sup>−</sup>1; 140–300 ◦C, 40 ◦C·min<sup>−</sup>1; 300 ◦C, 5 min). Samples of the organic phase were prepared in diethyl ether containing 0.2 mM dodecane as an internal standard. The quantification of limonene was performed using a standard curve of the ratio between commercial (S)-limonene (Merck KGaA, Darmstadt, Germany) and the internal standard.
