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]