which a defined pH value is set in each of the reactors [68]. When using plug-flow
reactors, a gradient is established within the system. Then, the sampling in different
positions along the plug-flow reactor also allows an insight into the time course of
the cellular reactions [69, 70]. In contrast to multi-compartment systems, scale-down
simulators with a pulse-based feeding are easier to establish and to run in parallel.
Parameter control, however, may be more difficult to achieve due to the restriction of
the feeding profiles. Since the feeding profile is easy to change (e.g., distance
between the feed pulses), pulse-based systems also seem to be well applicable for
robustness analyses. Alternative approaches, in which installations (e.g., plates
between the different stirrers to restrict the tangential flow [71]) are realized in a
laboratory reactor to extend the mixing time to the order as it is measured in the large
reactor, can, in individual cases, reproduce the industrial process quite well, but are
technically more complex.
4.1.1 Monitoring of the Cellular State Across Different Scales
The most successful scale-down methodology will maintain the physiological state
of cells across lab and industrial scales. Naturally, it is the most suitable pre-requisite
to obtain similar results, and should be considered as scaling parameter, although the
examination of the physiological cell status is not easy to quantify with suitable
measures. The impact of gradient formation on physiology has to be investigated
with the measurement of sensitive parameters, e.g. the energy charge, stress response
factors, and the respiratory activity, among others [68, 72–74]. Additionally, the
physiological state may vary from cell to cell, which demands the consideration of
population heterogeneity. It has been observed several times that gradient formation
in fed-batch cultivation mode has an impact on population heterogeneity [75]. It
adds additional parameters that lead to different phenotypes in culture (Fig. 3).
In natural habitats, mainly the cell cycle, cell ageing, and epigenetic regulation are
known to have a great impact on the evolvement of phenotype diversity [77]. Stochastically asymmetric growth and mutation events drive the formation of subpopulations, which might be even better adapted to a previous change in an
environment. Nevertheless, these events usually lead to lower yields in processes,
which are conducted in bioreactors [78]. The role of cell cycling on the development
of subpopulations in industrial bioprocesses, however, is not clear yet, while it was
found out that the dominant driver for different protein concentrations, and thus
various metabolic activity, is the growth rate in Pseudomonas putida [79].
In particular, singe-cell based and sensitive volumetric measurement techniques
can provide new information about the impact of gradients on the cellular viability
and metabolic activity and the formation of subpopulations independently of the
scale-down system. Suitable monitoring technologies in combination with a physiological understanding of stress responses support the identification of the suitable
scale-down conditions, as it puts the cell in the center of the investigation of
consequences of gradient formation in the liquid phase. Such technologies, including
proper accompanying off-line measurements, allow one to properly model the stress
12
P. Neubauer et al.
reactors, a gradient is established within the system. Then, the sampling in different
positions along the plug-flow reactor also allows an insight into the time course of
the cellular reactions [69, 70]. In contrast to multi-compartment systems, scale-down
simulators with a pulse-based feeding are easier to establish and to run in parallel.
Parameter control, however, may be more difficult to achieve due to the restriction of
the feeding profiles. Since the feeding profile is easy to change (e.g., distance
between the feed pulses), pulse-based systems also seem to be well applicable for
robustness analyses. Alternative approaches, in which installations (e.g., plates
between the different stirrers to restrict the tangential flow [71]) are realized in a
laboratory reactor to extend the mixing time to the order as it is measured in the large
reactor, can, in individual cases, reproduce the industrial process quite well, but are
technically more complex.
4.1.1 Monitoring of the Cellular State Across Different Scales
The most successful scale-down methodology will maintain the physiological state
of cells across lab and industrial scales. Naturally, it is the most suitable pre-requisite
to obtain similar results, and should be considered as scaling parameter, although the
examination of the physiological cell status is not easy to quantify with suitable
measures. The impact of gradient formation on physiology has to be investigated
with the measurement of sensitive parameters, e.g. the energy charge, stress response
factors, and the respiratory activity, among others [68, 72–74]. Additionally, the
physiological state may vary from cell to cell, which demands the consideration of
population heterogeneity. It has been observed several times that gradient formation
in fed-batch cultivation mode has an impact on population heterogeneity [75]. It
adds additional parameters that lead to different phenotypes in culture (Fig. 3).
In natural habitats, mainly the cell cycle, cell ageing, and epigenetic regulation are
known to have a great impact on the evolvement of phenotype diversity [77]. Stochastically asymmetric growth and mutation events drive the formation of subpopulations, which might be even better adapted to a previous change in an
environment. Nevertheless, these events usually lead to lower yields in processes,
which are conducted in bioreactors [78]. The role of cell cycling on the development
of subpopulations in industrial bioprocesses, however, is not clear yet, while it was
found out that the dominant driver for different protein concentrations, and thus
various metabolic activity, is the growth rate in Pseudomonas putida [79].
In particular, singe-cell based and sensitive volumetric measurement techniques
can provide new information about the impact of gradients on the cellular viability
and metabolic activity and the formation of subpopulations independently of the
scale-down system. Suitable monitoring technologies in combination with a physiological understanding of stress responses support the identification of the suitable
scale-down conditions, as it puts the cell in the center of the investigation of
consequences of gradient formation in the liquid phase. Such technologies, including
proper accompanying off-line measurements, allow one to properly model the stress
12
P. Neubauer et al.