Scale-up of Bioprocesses
509
formation in
S. cerevisiae
will rapidly occur if oxygen is depleted. The intracellular response in
NADH levels to a high glucose concentration has been found to occur within a few seconds.
Microbial kinetics is, furthermore, not only determined by the local concentrations, but also by
the time history of the organisms. Cells will experience a changing environment as they circulate
in a large-scale reactor. This periodic change in extracellular conditions may trigger regulatory
phenomena,
e.g.
gene expression turn-on or turn-off (see Fig. 2.2). It is for example well known
that both oxygen and glucose trigger several regulatory responses. The response time will be
determined by the rate of mRNA polymerization and the rate of translation (Konz
et al.,
1998).
In a study by Schweder
et al.
1999, seven different mRNA levels in
E. coli
were studied in a
scaled-down system. It was found that the transcription of several genes, such as the
proU
gene,
which is involved in osmoregulation, responded within 15 s of exposure to high glucose
concentration. The rate of protein synthesis is probably somewhat lower. The peptide elongation
rate has been estimated to be between 13-16 amino acids per second in
E. coli
(Einsele et al.,
1978, see also Note 7.6). For this reason, a single “dip” in oxygen concentration of short duration
does not necessarily result in a change in the overall protein synthesis pattern.
. However, the
effects of repeated depletions, as occuring for a cell circulating in a large-scale bioreactor, are
indeed difficult to predict and need to be studied experimentally.
It is clearly expensive to do the experiments in the actual large-scale bioreactor, and
scale-down
experiments
are therefore made. Concentration gradients can be created by making step-change
experiments in a laboratory scale stirred tank reactor, or by connecting two small reactors. Step-
change experiments in one reactor can be used to study dynamic effects of a sudden increase in
glucose concentration or aerobic/anaerobic transitions. In a sense one can say that an
experimental “compartment system” is used to mimic the large-scale reactor. A compilation of
such experimental studies is given by Liden (2001). A very rapid mixing of glucose can be
obtained in a laboratory reactor. However, the oxygen transfer rate is not sufficiently high to
enable studies of fast dynamics related to aerobic/anaerobic transitions. A combination of a
stirred tank reactor and a plug flow reactor may in this case be a better option (George et al.,
1993).
The
a priori
expectation is probably that gradients will have a negative effect on process
performance. This has also been reported for
e.g.
biomass yield. However, not all results show a
decreased performance in large-scale. The leavening capacity of Baker’s yeast was found to be
higher in both a scaled-down model system and a large-scale process compared to an ideally
mixed system (George et al., 1998). Furthermore, the stability of a heterologous protein was
found to increase in a non-ideally mixed system (Bylund et al, 2000).
Also shear rates may be a concern for shear sensitive organisms. This normally does not apply to
yeast or bacteria, whereas mammalian cells or plant cells are shear sensitive (Tanaka, 1981). The
situation is complicated by the fact that the average shear rate normally decreases during scale-
up, whereas the maximum shear rate normally increases. Again, an “oscillating environment”
with respect to shear will be sensed by the organisms during circulation.
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