on growth and secretion profiles. Up to now, however, the change of
macromorphology due to scaling effects, e.g. an oscillating shear force regime, has
not been investigated thoroughly. Nevertheless, it can be assumed that the
macromorphology of filamentous organisms will change in comparison with the
lab scale if the exposure time to high-shear forces is diminished, like it most
probably is in large-scale cultivations at high cell densities and elevated viscosities,
where large residence times exist in different compartments. The knowledge that
exists so far about how a changed shear force regime influences the process
performance [91, 92] leads to the assumption that macromorphology is an important
parameter to consider while choosing a suitable scale-down system. Alternating
shear forces can be achieved by interrupted stirring, which usually couples low-shear
stress to oxygen limitation in stirred tank reactors, or in multi-compartment reactors,
in which low-shear and high-shear regimes are applied at similar gas mass transfer
rates. The application of other reactor systems beyond stirred tanks can support the
investigation of consequences of low-shear forces on the macromorphology, physiology, and overall process performance as recently described for clavulanic acid
production with Streptomyces clavuligerus in shaken bioreactors. Secretion of
clavulanic acid was strongly diminished while thicker filaments were observed
[93]. Consequently, morphological monitoring in an automated manner [94] is a
promising technique to identify crucial characteristics for growth and product
formation under specific environmental regimes.
Finally, macromorphological heterogeneity can be modeled to describe the
response of a cell to environmental perturbations. In silico prediction of physiological population heterogeneity was conducted by a combination of computational
fluid dynamics (CFD) and a cell cycle model of P. putida [95]. It was observed that
72% of the cells switched between standard and multifork replication and 52.9%
showed higher than average adenosine triphosphate (ATP) maintenance demands
(12.2%, up to 1.5 fold). Such an approach, however, requires sufficient knowledge
of the interaction between gradient formation and consequences for the
macromorphology of a population. This still represents a bottleneck as the time
frequency with which morphological changes are measured might be inadequate to
achieve a sufficient accuracy while correlating the response to specific regulatory
events in a cell. In recent years, however, many more techniques like in situ
microscopy and others are being developed rapidly. It is hoped that they become
more applicable in biotechnological processes operating at elevated cell densities.
The impact of gradients in the liquid phase on the formation of phenotypic heterogeneity can be investigated also if spectroscopic methods are coupled to microfluidic
devices, when the growth of single cells can be monitored constantly [96]. The
aforementioned methodologies will increase the possibility for the consideration of
heterogeneity in population balance models and their integration in the description of
consequences of gradient formation. So far, the few attempts rely on physiological
measures, e.g. the adaptation to substrate excess [97, 98], but investigations will
benefit from the additional consideration of macromorphological characterization
data in future.
Potential of Integrating Model-Based Design of Experiments Approaches and. . .
15
macromorphology due to scaling effects, e.g. an oscillating shear force regime, has
not been investigated thoroughly. Nevertheless, it can be assumed that the
macromorphology of filamentous organisms will change in comparison with the
lab scale if the exposure time to high-shear forces is diminished, like it most
probably is in large-scale cultivations at high cell densities and elevated viscosities,
where large residence times exist in different compartments. The knowledge that
exists so far about how a changed shear force regime influences the process
performance [91, 92] leads to the assumption that macromorphology is an important
parameter to consider while choosing a suitable scale-down system. Alternating
shear forces can be achieved by interrupted stirring, which usually couples low-shear
stress to oxygen limitation in stirred tank reactors, or in multi-compartment reactors,
in which low-shear and high-shear regimes are applied at similar gas mass transfer
rates. The application of other reactor systems beyond stirred tanks can support the
investigation of consequences of low-shear forces on the macromorphology, physiology, and overall process performance as recently described for clavulanic acid
production with Streptomyces clavuligerus in shaken bioreactors. Secretion of
clavulanic acid was strongly diminished while thicker filaments were observed
[93]. Consequently, morphological monitoring in an automated manner [94] is a
promising technique to identify crucial characteristics for growth and product
formation under specific environmental regimes.
Finally, macromorphological heterogeneity can be modeled to describe the
response of a cell to environmental perturbations. In silico prediction of physiological population heterogeneity was conducted by a combination of computational
fluid dynamics (CFD) and a cell cycle model of P. putida [95]. It was observed that
72% of the cells switched between standard and multifork replication and 52.9%
showed higher than average adenosine triphosphate (ATP) maintenance demands
(12.2%, up to 1.5 fold). Such an approach, however, requires sufficient knowledge
of the interaction between gradient formation and consequences for the
macromorphology of a population. This still represents a bottleneck as the time
frequency with which morphological changes are measured might be inadequate to
achieve a sufficient accuracy while correlating the response to specific regulatory
events in a cell. In recent years, however, many more techniques like in situ
microscopy and others are being developed rapidly. It is hoped that they become
more applicable in biotechnological processes operating at elevated cell densities.
The impact of gradients in the liquid phase on the formation of phenotypic heterogeneity can be investigated also if spectroscopic methods are coupled to microfluidic
devices, when the growth of single cells can be monitored constantly [96]. The
aforementioned methodologies will increase the possibility for the consideration of
heterogeneity in population balance models and their integration in the description of
consequences of gradient formation. So far, the few attempts rely on physiological
measures, e.g. the adaptation to substrate excess [97, 98], but investigations will
benefit from the additional consideration of macromorphological characterization
data in future.
Potential of Integrating Model-Based Design of Experiments Approaches and. . .
15