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.