2.2 Microbioreactor cultivations
The strains were cultivated in the BioLector® microfermentation system in 48-well Flowerplates® (m2p-labs, Baesweiler, Germany) as described by Török et al..[29] Synthetic feed in time (FIT) fed-batch medium containing 1 g/L glucose and 16.5 g/L dextran as carbon sources (m2p-labs GmbH, Baesweiler, Germany) was used with the following additions (g/L): 27.40 MOPS, 6.54 (NH4)2SO4, 1.96 K2HPO4, 1.96 trisodium citrate·2H2O, 1.31 Na2SO4, 0.65 NH4Cl, 0.33 MgSO4·7H2O, and 0.0065 Thiamin·HCl.
The trace element solution contained (mg/L): 0.36 ZnSO4·7H2O, 0.33 CuSO4·5H20, 0.20 MnSO4·H2O, 27.30 FeCl3·6H2O, 21.84 Titriplex III, 0.36 CoCl2·6H2O, and 1.31 CaCl2·2H2O. Immediately prior to inoculation, 0.6% (v/v) of the glucose releasing enzyme mix (EnzMix) was added. The GFPmut3.1 expression level was monitored at an excitation of 488 nm and emission of 520 nm. The signal is given in relative fluorescence units (rfu). The cell dry matter (CDM, given in g/L) was calculated from light scatter signals using calibration settings obtained by linear regression analysis. The cycle time for all parameters was 20 min.
The initial cell density was equivalent to an optical density of OD600 = 0.3. For inoculation of passage 1, a deep-frozen (-80°C) working cell bank (WCB; OD600 = 3.5) was thawed and biomass harvested by centrifugation (7500 rpm, 5 min). Cells were washed with 500 μL of the corresponding medium to remove residual glycerol and centrifuged. Pellets were then re-suspended in the total cultivation medium. All subsequent passages were inoculated with induced cells from the previous passage, but without adding batch glucose again, keeping the cells in carbon-limited conditions. All cultivations were performed at 30°C. Recombinant gene expression was induced with IPTG at a final concentration of 0.5 mM.