EPPENDORF_Nov2021_UpstreamBioprocessingImprovingEfficiency - 13

we grew the respiro-fermentative yeast Pichia
pastoris in the DASbox® Mini Bioreactor System.
Further, we compared the results to a standard
DO-spike triggered feeding done in the same set up.
The objectives of this study are (1) to demonstrate
the feasibility of a constant RQ based feeding using
the DASware control 5 professional software and the
DASbox Mini Bioreactor System; (2) to show the
benefits of constant RQ control in P. pastoris fermentation
as well as its effect on the amount of feed used
and the microbial growth; (3) to explain the measurements
necessary for RQ determination as well as the
corresponding calculations automatically done by
the Eppendorf GA4 gas analyzer module; and (4) to
give background information of the RQ, showcasing
its potential for usage.
Figure 1: DASGIP® GA4 gas analyzer
Pichia pastoris is a methylotrophic yeast largely used
for heterologous protein production in biotechnology.
As an expression system, the main advantages
of P. pastoris over E. coli is the ability to carry out
post-translational modifications such as glycosylation.
Compared to mammalian cell culture, P. pastoris
displays a much faster growth and requires less
expensive growth medium, making it ideal for
protein production in bioreactors. For optimizing the
production yield of a P. pastoris culture, one of the
most important factors is the feeding strategy.
A convenient and well-established approach for
feeding is a strategy based on the amount of
dissolved oxygen (DO) in the medium. In this approach
the feeding start is triggered by a DO-spike. After
that, the DO set point is generally set to a fixed value
of e.g. 30 %. However, it is not guaranteed that these
conditions lead to full metabolization of the carbon
source, and a further optimization of this feeding
strategy would be necessary (Hang et al., 2009).
One of the leading strategies for protein productionoriented
feeding is based on respiratory quotient
(RQ). Constant RQ based feeding ensures that the
respiratory metabolism of glucose/glycerol are optimized
for protein production purposes and the
formation of by-products are limited (Xiong et al., 2010).
In a recent publication, a strategy was introduced
GENengnews.com | 13


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