EPPENDORF_Nov2021_UpstreamBioprocessingImprovingEfficiency - 14

PID controlled Constant RQ Fermentation of Pichia pastoris in the DASbox® Mini Bioreactor System
which managed to fully convert glucose and additionally,
avoid ethanol formation by controlling
RQ ≥ 0.9 (Tippmann et al., 2016). The publication has
shown additionally a clear improvement in the
productivity of the cells due to this feeding strategy.
The intention of this study is to demonstrate the
feasibility of constant RQ based feeding for protein
production-oriented yeast fermentation using the
DASbox Mini Bioreactor System.
The Respiratory quotient (RQ)
The respiratory quotient is the quotient of carbon
dioxide produced and oxygen consumed by a
culture, expressed with the carbon dioxide transfer
rate (CTR) and the oxygen uptake rate (OUR). Under
conditions of a constant dissolved oxygen concentration
the OUR equals the oxygen transfer rate (OTR)
(Clarke, 2013). Therefore, the following calculations
were done to determine the RQ:
1) Oxygen transfer rate (OTR):
OTR = c × vvm (XO2, in
-
Fout
Fin
produced under aerobic conditions in respiro-fermentative
yeasts once the tricarboxylic acid (TCA) cycle is
overflown by glucose.
As ethanol is more reduced than glucose, the use
of ethanol as substrate results in a RQ value below 1.
Thus, the RQ value can serve as an inline parameter
indicating which substrate is consumed by the culture,
and a constant RQ process can be used to optimize
fermentation based on specific carbon source.
× XO2, out
2) Carbon dioxide transfer rate (CTR):
CTR = c × vvm (XCO2, in
-
3) Respiratory quotient (RQ):
RQ =
CTR
OTR
The RQ for the metabolization of glucose is 1.
This can be explained by the fact that per mol
glucose, 6 mol O2
are needed and 6 mol CO2
produced. Which means that the O2
the CO2
are
uptake equals
transmission and thus their quotient is 1.
Once glucose is fully metabolized the culture
starts to consume fermentation by-products. These
by-products mainly consist of ethanol which is
14 | GENengnews.com
Fout
Fin
)
× XCO2, out
)
Materials and Methods
Yeast strain and glycerol stocks
P. pastoris has been taxonomically reclassified as
Komagataella pastoris (Peña et al., 2018). To simplify
the matter, we will continue to refer to it as Pichia
pastoris further on.
In this study we used the Pichia pastoris strain
DSMZ 70382 obtained from the Leibniz Institute
DSMZ - German Collection of Microorganisms and
Cell Cultures GmbH.
We reactivated the freeze-dried cells in 10 mL YPD
medium and incubated them in a 500 mL Erlenmeyer
flask with baffles over night at 30 °C, 200 rpm using a
New Brunswick Innova® 42 incubator. The next
morning, we transferred the culture into a 1000 mL
Erlenmeyer flask with baffles and added 100 mL
sterile YPD-medium. After an incubation period of
6 h at the previous mentioned settings, we checked
the culture to be contamination-free using a Zeiss®
Primo star microscope.
Next, we measured the OD600
of the culture and
mixed 800 μL broth with 200 μL 70 % (w/v) glycerol
in previously sterilized cryovials to create a Pichia
working cell bank with OD600
of 0.3.
We stored the cryovials at -20 °C and individually
thawed at room temperature before the run.
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EPPENDORF_Nov2021_UpstreamBioprocessingImprovingEfficiency

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