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.