EPPENDORF_Nov2021_UpstreamBioprocessingImprovingEfficiency - 16

PID controlled Constant RQ Fermentation of Pichia pastoris in the DASbox® Mini Bioreactor System
with 10 % NH3 which additionally serves as nitrogen
source during fermentation. We then added 0.45 mL
PTM1 solution per vessel which equals an amount of
4.5 mL per liter of basal salts medium. After we did all
additions to the medium, the total volume reached
ca. 110 mL.
Feed medium
As feed medium a 33 % (w/v) glucose solution was
used (Table 4).
the second pre-culture for another 7 h at 30 °C and
200 rpm to archive a threshold growth of 15-20 OD600
initial OD600
We inoculated each of the fermenters to reach an
of 1.
Vessel setup and sterilization
In this study, we did all liquid additions to the bioreactor
submersed and separate from each other via an
additionally installed triple port. We installed pH and
DO sensors in the Pg13.5 threads of the head plate
and used the sample port of the bioreactor for inoculation.
After we did these installations, we filled the
bioreactor with medium and autoclaved it for 20 min,
using 121 °C liquid cycle setting.
Inoculum preparation
Before inoculum preparation, we took one cryovial of
the previous prepared glycerol stocks to thaw at room
temperature. We allocated 10 mL sterile YNB-medium
to two sterile 125 mL Corning® Erlenmeyer flasks with
baffles. We inoculated one of the Erlenmeyer flasks
using the 1 mL glycerol stock to an OD600
of 0.03.
Whereas we used the other Erlenmeyer flask,
containing only sterile YNB-medium, as sterility
control. We incubated both flasks in an Innova 42
shaker for 17 h at 30 °C, 200 rpm. After that, we
started a second pre-culture by transferring 10 mL of
the grown culture to 40 mL fresh YNB-medium in a
sterile 125 mL Corning Erlenmeyer flask. We grew
16 | GENengnews.com
Sensor calibration
We calibrated the pH sensors in parallel outside of
the vessel, before sterilization, following the 2-point
calibration procedure of DASware control 5 professional
software. We used a pH buffer 7.0 to set the
sensors' offset and a pH buffer 4.01 to set the slope.
Before we calibrated the DO sensors and before autoclaving
the bioreactors, we filled the sensor cap with
fresh electrolyte solution. We calibrated the DO
sensors in parallel inside the vessel after sterilization
and addition of the medium supplements. A period
of 6 hours is needed to polarize the sensors by
connecting them to the control system. The control
system provides a polarization voltage to establish
an anode and a cathode within the sensor. It is
recommended to set the reactor conditions during
DO calibration to the initial set points of the fermentation.
In our case, that equals a stirring rate of
400 rpm, a gas flow of 3 sL/h and a temperature of
30 °C. Additionally, we added all the sensitive media
after autocaving and before the pH was set to 5.0.
We did the 2-point calibration by sparging in pure
nitrogen first to set the offset (0 % DO) followed by
sparging in air to calibrate the slope (100 % DO).
Pump calibration
Before connecting the vessels to the fermentation
system, we performed the pump calibration following
the procedure of the DASware control 5 professional
software. We calibrated the feed pumps in parallel by
using the feed medium and setting the density to
1.12 g/mL. We calibrated the base addition- pumps in
parallel using water as calibration solution.
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