2.3 Inoculum preparation
Combined shake flasks of 7-day-old rice cell suspensions were inoculated
at ~20% v/v (volume of inoculum per total working
volume) into a sterile 5-L STB with a height-to-diameter (H/D) ratio of
2 (BioFlo 3000, formerly New Brunswick Scientific, Eppendorf Inc.,
Hauppauge, NY) containing 4 L of sterile NB+S corresponding to initial
inoculation density of 1-2 g DW/L. The bioreactor was operated under
conditions similar to those previously described (Macharoen et al.,
2020). The agitation of an upward-pumping pitched blade impeller (10.2
cm diameter corresponding to impeller diameter (Di) to
tank diameter (Dt) ratio of 0.60) was kept constant at
75 rpm. The temperature and the aeration rate were maintained at 27˚C
and 0.2-0.4 vvm (volume of gas sparged per bioreactor working volume per
minute), respectively. The cultures were grown until mid-to-late
exponential growth phase was reached, typically around day 9-12 of
cultivation depending on initial biomass concentration, and ready for
the 40-L STB inoculation.