2.4 Bioreactor fed-batch cultivations
Bioreactor fed-batch mode cultivations were carried out in biological triplicates on initial YPG medium (50 g/L glycerol, 10g/L yeast extract (Carl Roth), 10 g/L peptone) in controlled stirred bioreactor vessels with 1.0 L total aerated end working volume (Infors Minifors 2 systems, Infors AG, Switzerland) at 28°C. All reactors were equipped with pH and optical dissolved oxygen probes (Hamilton AG, Switzerland) and off-gas analyzers (BlueSens GmbH, Germany). pH was controlled at 4.5±0.1 with automated addition of a 4 M solution of NaOH. The dissolved oxygen control was set at a minimum threshold of 20% with a cascade control by gradually increasing the stirrer speed of two six-blade Rushton turbines and the aeration rate using Eve fermentation control software (Infors AG, Switzerland). The initial aeration rate was set to 1 L/min, with stirring at 400 rpm. The CO2 (%) and O2(%) in the off-gas were monitored continuously during the fed-batch cultivations. Bioreactor cultures of strain Y. lipolytica ST9253 were inoculated from shake flask precultures in the exponential growth phase (250 mL baffled shake flasks, 40 mL cultivation volume, YPG 40 g/L glycerol, 10 g/L yeast extract, 10 g/L peptone) to a starter fermentation volume of 620 mL. After the depletion of the initially supplied glycerol carbon source, the cultures were fed with a nutrient-rich feed solution (700 g/L glycerol, 10 g/L yeast extract, 10 g/L (NH4)2SO4). The composition of the feed solution was set to facilitate the production of the target compounds after an initial biomass buildup phase. Off-line samples were taken regularly to analyze residual glycerol concentration (Megazyme Inc. assay kit), optical density at 600 nm (Genesys photometer, Thermo Fischer Scientific), cell dry weight, and fatty alcohol concentrations.