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.