2.4 Pilot-scale bioreactor operation
The pilot-scale cultivation of transgenic rice cell suspension cultures was conducted in a 40-L in situ sterilizable STB with i-ControlXP control software (Applikon Biotechnology Inc., Foster City, CA) with a H/D of 3. The vessel is equipped with an upward-pumping pitched blade impeller (10.2 cm diameter corresponding to Di to Dt ratio of 0.38) placed at 23 cm from the base of the impeller shaft that is magnetically driven at the bottom of the vessel. A 24-hole ring sparger (1.5 mm hole diameter) is placed 10 cm above the bottom of the vessel around the impeller shaft. The bioreactor containing DI water was sterilized using the sterilize-in-place (SIP) system at 122˚C for 20 min. Then 5x concentrated medium was sterile filtered into the bioreactor through a sterile push valve using a 0.2 µm sterilizing filter capsule (Sartobran® P, Sartorius Stedim North America Inc, Bohemia, NY) connected to a dispensing pressured vessel (Amicon, Millipore-Sigma, Burlington, MA), a liquid holder for filtration, and a compressed air source (10 psig). The amount of concentrated medium filtered into the bioreactor was calculated and prepared to obtain 1x medium after mixing with sterile DI water in the bioreactor. The medium was then agitated at 150 rpm, aerated with sterile compressed air at 0.2 vvm, and the temperature was maintained at 27˚C overnight to verify sterility of the medium prior to inoculation.
The plastic tubing connected to the sampling tube in the 5-L STB was aseptically cut and fused to the tubing attached to a push valve inserted into the headplate of the 40-L STB using a sterile tube fuser (Cytiva; formerly GE Healthcare Life Science, Marlborough, MA). Then, the inoculum was transferred from the 5-L STB to the 40-L STB by flowing compressed pure O­2 at 10 psig through a 0.22-µm vent filter connected to the 5-L sparger ring to push the culture into the 40-L STB. The inoculation corresponded to 15-18% v/v (volume of inoculating culture to final working volume of culture). Single-stage operations were used in this study. The first bioreactor run was operated in a cyclical semicontinuous mode where the media exchange to start a new cycle was performed at day 4 since extracellular sugar depletion by cellular uptake. The second bioreactor run was operated in a batch mode. Table 1 summarizes the conditions and bioreactor parameters used in the runs as well as the initial working volume after inoculation and the final working volume in which the culture volume was reduced due to sampling and evaporation. In both runs, the aeration rate of 0.2-0.4 vvm was maintained during the growth phase and 0.2 vvm during the induction phase, while the temperature was maintained at 27˚C throughout the operation. The pressure inside the bioreactor was controlled at 0.2 psig. The oxygen uptake rate (OUR) was routinely measured by momentarily halting the aeration and recording the declining rate of DO. The online culture pH was monitored using Applikon pH sensor but was not controlled.
The media exchange was performed to start a new cycle of the semicontinuous operation in following order. The pressurization, agitation, and aeration were halted to allow rice cell aggregates to settle. A sterile tube fuser (Cytiva; formerly GE Healthcare Life Science, Marlborough, MA) was used to aseptically connect the sterile tubing attached to carboys and the tubing attached to the sampling assembly at the bottom of the bioreactor. Then the spent medium was drained out into sterile carboys through the sampling assembly. Freshly prepared NB+S or NB+0.5xS medium (Table 1) was 0.22-µm filtered into the bioreactor as described earlier. The agitation, aeration, and pressurization were then resumed.