EPPENDORF_Nov2021_UpstreamBioprocessingImprovingEfficiency - 17

Feed start
Feed process triggered by DO-spike
The end of the batch phase of a fed-batch culture is
often indicated by a DO-spike. At this point the
carbon source within the initial culture medium is
consumed, the metabolic activity of the culture and
therefore, its oxygen demand drops rapidly, resulting
in a sharp increase of DO in the medium.
We took advantage of this DO-spike to trigger an
automatic start of the feed pump using an automatic
programming script.
Therefore, we implemented a so-called reactor
script, based on visual basics, which starts with the inoculation
of the fermenter. After a delay time of 12 hours,
a low threshold of 30 % DO is anticipated followed by
a high DO threshold of 38 % which starts the feed
pump, following a step wise exponential-like feed
(Table 5). We established this feed profile based on
trial and error during the previous runs with P. pastoris.
Automatic RQ control by programmed feeding
We implemented a reactor script which starts automatically
after inoculation. After a delay time of
12 hours, the RQ value of 1 is used to start the
feeding pump, and further feeding is automatically
controlled based on the RQ value. As stated before,
the RQ value can serve as indicator for which
substrate is used by the culture. An RQ of 1 indicates
the consumption of glucose. A lower RQ indicates
the consumption of reduced fermentation by-products.
For creation of a constant RQ based feeding, we
programmed a script with built-in PID function to
start the feed pump once the RQ value drops below
1, indicating that the glucose has been consumed.
We used a script to set the feeding pump to its
maximum speed (40 mL/h), when RQ drops to 0.8.
Once the RQ value rose to 1 again, the script pauses
the feeding automatically. To reduce the oscillation of
the RQ value, we implemented a PID controller
within the script with the following PI values
(p = 3.33, Ti = 360 s, I = 0.0027 s-1
Process parameter setup
We kept the P. pastoris cultures at 30 °C, pH 5 and
30 % DO. Both setups used the process parameters
listed in Table 6.
We designed the DO cascade to maintain the
After a total feeding time of 32 h, the pump is
automatically turned off. Additionally, we activated
the overflow protection of the reactor which is a
safety feature of the DASware control 5 professional
software. The overflow protection works as follows:
During the course of the experiment, the volume
which is added by the pumps is accumulated to the
reactor's initial volume. Once the maximal working
volume of the reactor is reached, the pumps stop
automatically to prevent the reactor from overflowing.
dissolved oxygen at 30 % by first accelerating the
agitation from 400 to 1600 rpm, then increasing the air
flow rate from 3 sL/h to 9 sL/h (0.5 - 1.5 vvm for 100 mL
initial volume), and finally enriching the oxygen
concentration in the gas flow from 21 % to 100 %.
These three steps corresponded to the DO output of
0 - 40 %, 40 - 80 % and 80 - 100 %. We lead the exhaust
gases into 250 mL Schott bottles which served as foam
traps, and then further to the respective channels of
the GA4 gas analyzer. The GA4 gas analyzer has a
built-in function to calculate RQ using the equations
shown in the introduction section of this work.
GENengnews.com | 17


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EPPENDORF_Nov2021_UpstreamBioprocessingImprovingEfficiency - Contents
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