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.