Substrate Gradients In fed-batch cultures, the existence of excess substrate zones
in the broth defeats the purpose of this tight control for the fraction of the culture that
comes into contact with these zones. The exposure of the culture to zones of higher
substrate concentrations has direct consequences on the uptake capacities of the cells
for this substrate [51, 53]. As a result, the excess substrate zones may cause the cells
to grow at the maximum specific growth rate, which may plunge organisms such as
E. coli and Saccharomyces cerevisiae into overflow metabolic states as reported in
numerous studies [51, 54–56]. The high metabolic flux of glucose through the
glycolysis which is favored by high affinity uptake systems, i.e. low K S values,
also leads to the accumulation of NADH-H
+
, and thus to a higher rate of respiration.
As a consequence, the high metabolic activity in the feeding zone can also lead to
oxygen limitation if the biochemical reduction of oxygen by the cells is faster than
the limited diffusion of oxygen into the cultivation medium. It is likely that the
uneven distribution of the substrate due to feeding is the main cause for the dissolved
oxygen gradients, besides the uneven fluid-dynamic distribution of the gas bubbles.
The dissolved oxygen problem which is basically caused by the inherently low
solubility of oxygen in fermentation broths [49] becomes even greater in processes
with pellet forming organisms (oxygen gradient in the pellet) or shear-sensitive cells
(limited sparging to prevent shear stress caused by the bursting of gas bubbles) [57].
Temperature Gradients Temperature gradients are among the least studied scaleup effects in bioprocess development. Although it is clear from a microbiological
point of view that small temperature fluctuations of a few degrees have a major
impact on cellular reactions and that, from a process engineering perspective, precise
temperature control in industrial bioreactors is a serious problem, to the best of our
knowledge, there is no information about local temperature profiles in industrial
bioreactors, nor have experiments been performed in scale-down simulators to
simulate the effect of perturbing temperatures on a process.
pH Gradients pH gradients are recently gaining attention in the bioprocess
research community. Simen et al. investigated the effect of ammonia pulses (shifts
in pH) in E. coli cultivations and observed a higher maintenance energy and the
activation of over 400 genes in response to the pH gradients [58]. pH gradients are
also relevant in industrial-scale batch cultivations of lactic acid bacteria. This has
been revealed by combined approach by the use of multiple pH probes and a
computational fluid dynamic model coupled with a kinetic model for a process of
Streptococcus thermophilus in a 700 L pilot scale bioreactor [59]. Recently, we also
could demonstrate by two- and three-compartment bioreactor cultivations that such
pH oscillations affect the cocci chain length and decrease the growth rate in
S. thermophilus cultures (manuscript in preparation). Also in CHO fed-batch
bioprocesses pH perturbations decrease the cell viability and increase lactate accumulation [60]. Also pH oscillations have been recently demonstrated to affect
product accumulation in a cell line specific manner [61].
Carbon Dioxide Gradients In microbial cultivations, a recent study of CO 2 /
HCO 3
À gradients in Corynebacterium glutamicum showed no significant impact
Potential of Integrating Model-Based Design of Experiments Approaches and. . .
9
in the broth defeats the purpose of this tight control for the fraction of the culture that
comes into contact with these zones. The exposure of the culture to zones of higher
substrate concentrations has direct consequences on the uptake capacities of the cells
for this substrate [51, 53]. As a result, the excess substrate zones may cause the cells
to grow at the maximum specific growth rate, which may plunge organisms such as
E. coli and Saccharomyces cerevisiae into overflow metabolic states as reported in
numerous studies [51, 54–56]. The high metabolic flux of glucose through the
glycolysis which is favored by high affinity uptake systems, i.e. low K S values,
also leads to the accumulation of NADH-H
+
, and thus to a higher rate of respiration.
As a consequence, the high metabolic activity in the feeding zone can also lead to
oxygen limitation if the biochemical reduction of oxygen by the cells is faster than
the limited diffusion of oxygen into the cultivation medium. It is likely that the
uneven distribution of the substrate due to feeding is the main cause for the dissolved
oxygen gradients, besides the uneven fluid-dynamic distribution of the gas bubbles.
The dissolved oxygen problem which is basically caused by the inherently low
solubility of oxygen in fermentation broths [49] becomes even greater in processes
with pellet forming organisms (oxygen gradient in the pellet) or shear-sensitive cells
(limited sparging to prevent shear stress caused by the bursting of gas bubbles) [57].
Temperature Gradients Temperature gradients are among the least studied scaleup effects in bioprocess development. Although it is clear from a microbiological
point of view that small temperature fluctuations of a few degrees have a major
impact on cellular reactions and that, from a process engineering perspective, precise
temperature control in industrial bioreactors is a serious problem, to the best of our
knowledge, there is no information about local temperature profiles in industrial
bioreactors, nor have experiments been performed in scale-down simulators to
simulate the effect of perturbing temperatures on a process.
pH Gradients pH gradients are recently gaining attention in the bioprocess
research community. Simen et al. investigated the effect of ammonia pulses (shifts
in pH) in E. coli cultivations and observed a higher maintenance energy and the
activation of over 400 genes in response to the pH gradients [58]. pH gradients are
also relevant in industrial-scale batch cultivations of lactic acid bacteria. This has
been revealed by combined approach by the use of multiple pH probes and a
computational fluid dynamic model coupled with a kinetic model for a process of
Streptococcus thermophilus in a 700 L pilot scale bioreactor [59]. Recently, we also
could demonstrate by two- and three-compartment bioreactor cultivations that such
pH oscillations affect the cocci chain length and decrease the growth rate in
S. thermophilus cultures (manuscript in preparation). Also in CHO fed-batch
bioprocesses pH perturbations decrease the cell viability and increase lactate accumulation [60]. Also pH oscillations have been recently demonstrated to affect
product accumulation in a cell line specific manner [61].
Carbon Dioxide Gradients In microbial cultivations, a recent study of CO 2 /
HCO 3
À gradients in Corynebacterium glutamicum showed no significant impact
Potential of Integrating Model-Based Design of Experiments Approaches and. . .
9