2.3 Fed-batch cultivations
For fed-batch fermentation, cells were grown in a 1.5 L (1.2 L working volume, 0.4 L minimal volume) DASGIP® Parallel Bioreactor System (Eppendorf AG, Hamburg, DE) equipped with standard control units. The pH was maintained at 7.0 ± 0.05 by the addition of 12.5% ammonia solution (Thermo Fisher Scientific, Waltham, USA). The temperature was maintained at 37 ± 0.5°C during the batch phase and decreased to 30 ± 0.5°C during the feed phase. The dissolved oxygen (O2) level was stabilized at > 30% saturation by controlling the stirrer speed and aeration rate. Foaming was suppressed by the addition of antifoam suspension (Glanapon, 2000, Bussetti, AT). For inoculation, a deep-frozen (-80°C) WCB vial was thawed and 1 mL transferred aseptically to a 250 mL pre-culture shake flask containing 25 mL M9ZB [30] for cultivation for at least 8 hours at 37°C. Subsequently, a volume equivalent to 25 OD600 units (25/OD600 = volume in mL) was transferred aseptically to the bioreactors.
Feeding was initiated when the culture, grown to 10 g/L CDM in 0.6 L batch medium, entered the stationary phase. A fed-batch regimen with exponential carbon-limited substrate feed was used to provide a constant growth rate of µ = 0.1/h over 19 hours or 2.74 doublings. The substrate feed was controlled by increasing the pump speed according to the exponential growth algorithm, \(x=x_{0}e^{\text{µt}}\), with superimposed feedback control of weight loss in the substrate bottle. The CDM yield coefficient on glucose was 0.33 g/g. Therefore, the feed medium provided 66 g/L glucose and sufficient components to yield a final CDM concentration of 30 g/L in 1.2 L. The expression system was induced by adding IPTG to the reactor to yield a concentration of 10 µmol/g CDM. The minimal medium was prepared as previously described.[15]