For calculation of the process time for both process types, we added a
down time of 12 hours for media preparation, reactor setup, CIP, and SIP
to the cultivation time. In chemostat cultivations with population
collapse, we only considered the cultivation time before the decrease in
cell density, which was the case with B3 <T7-GFP>
after 48 hours.
As can be concluded from the productivity comparisons (Table 3), the
fed-batch process with the T7 expression system and the model protein
GFP were inferior to the chemostat process in terms of volumetric yield
and STY. The reason for this can be attributed to the duration of the
induction. In fed-batch cultivation, the production phase takes 19
hours. Chemostat cultivation can be extended to 48 hours because the
volume in the reactor is kept constant.
As already mentioned, a longer chemostat process would not be possible
under these circumstances because the process ultimately becomes
unstable. However, a short-term chemostat cultivation of 48 hours could
be interesting for easy-to-produce proteins. With 874 mg/L/h, the STY of
B3<T7-GFP>wt in the chemostat tripled compared to
the fed-batch cultivation. In the case of the host RNAP-dependent A1
expression system, the STY of 62.8 mg/L/h in fed-batch cultivation was
relatively low compared to the STY of 286 mg/L/h of the conventional T7
expression system. In addition to the short production time of 19 hours,
the A1 expression system was inherently weaker. However, the low titer
can be compensated by continuous production. Due to the very long stable
production time of 312 hours, the STY of the chemostat cultivation more
than quadrupled compared to the fed-batch cultivation. Nevertheless, for
the production of GFP, the conventional T7 expression system is still
superior overall, in both fed-batch and chemostat mode, especially when
considering short-term chemostat cultivation of the T7 wild-type strain
(Table 3).
The situation is different with Fab. The extraordinary strength of the
T7 expression system is a disadvantage for Fab production. In neither
fed-batch nor chemostat mode, the T7 expression system was able to
outperform the host RNAP-dependent A1 expression systems. Due to the
mutation in lacI , the volumetric yield of
BQ<A1-Fab>wt was reduced from 0.087 g/L to 0.035
g/L, as seen with derivative #E2.1. However, long-term cultivation
without population collapse was possible. Due to the long production
time of 120 h, the STY of the BQ<A1-Fab>wt was
increased from 3.00 g/L/h in fed-batch mode to 4.33 g/L/h in chemostat
mode for derivative #E2.1 when comparing the two strains to one
another.
These results indicate that, in continuous E. coli bioprocesses,
the choice of expression system depends on the recombinant protein to be
produced. In the case of an easy-to-produce protein, such as GFP, the
conventional T7 expression system is still the expression system of
choice. The duration of the chemostat cultivation is limited by the
inevitable population collapse, but this can be compensated by the
extraordinary strength of the T7 system.
However, the moderately strong host RNAP-dependent A1 system is more
suitable for the production of a challenging protein. As a result of the
large metabolic load that is triggered, a reduction in the expression
rate is advantageous. Thus, the low titer can not only be compensated,
but even increased, based on the STY. As already mentioned, the time in
which the actual fermentation occurs and recombinant protein is produced
in fed-batch cultivation is relatively low. In addition, the cost of CIP
and SIP must be included. Although these process steps must also be
carried out in a long-term chemostat cultivation, the phase in which the
actual production occurs is inevitably prolonged, which means that the
CIP and SIP frequency can be reduced.
Nevertheless, only mutants that have adapted to long-term stability
under production conditions can be cultivated in long-term chemostat
mode. In wild-type strains, the population collapse and adaptive rescue
during long-term chemostat cultivation does not allow long-term
production. This phenomenon has been observed frequently in otherE. coli chemostat cultivations and prevents the direct use of
non-adapted strains for this purpose.[5, 52, 53]