conditions, but constantly present across scales. The results, however, would be
biased by the noise, if the samples measured are not enough to ensure a statistically
valid distribution. Nevertheless, the methodology is suitable to understand the
effects of gradients on cell viability and vitality while a large amount of cells can
be examined in a considerably short time. While the possibility of the application of
fluorescent markers represents an approach mostly on the expression level, cell
staining can be used to investigate several cellular components, including the
quantification of metabolite concentrations. If accumulated intracellularly, metabolite concentrations can be used to assess population heterogeneity as well, if the
metabolite can be quantified with sufficient accuracy in individual cells. Flow
cytometry is able to quantify the accumulation of intracellular lipids in microbial
cells, and thus identify subpopulations of different lipid contents [85], e.g. with Nile
Red or Bodipy
® stains. The accumulation of lipids and also other components might
correlate with changes in macromorphology of organisms. The measurement of lipid
content with optical methods can lead to conclusions about metabolic activity of
individual cells. This has been shown for heterotrophic algae, which accumulate to
large extent polyunsaturated fatty acids in lipid droplets. While using light microscopy and 3-dimensional holographic microscopy, the individual lipid storage in cells
was measured based on their individual cell size [86]. With rapid image analysis
using trained software, image acquisition can be performed in flow cells that are
connected to a cultivation. Automated workflows that offer considerably fast analysis of populations similar to flow cytometry are feasible, without the requirement of
staining.
Besides intracellular product accumulation, the macromorphology can provide
suitable information about the cell status and the impact of gradient formation on
it. It was examined that the cell size of S. cerevisiae cultures changed with the degree
of environmental heterogeneity in a three-compartment scale-down reactor
[87]. This happened in parallel to growth reduction and side metabolite
accumulation with a concomitant change of the sterol content, in comparison with
homogeneous growth conditions. Cells showed a diverse macromorphology under
scale-down cultivations, which supports the hypothesis that population heterogeneity is rather increased under growth in gradients. A morphologic response of cells to
scale-down cultivation conditions can also lead to agglomeration due to stress
response. Although the macromorphology of individual cells may stay unchanged,
the secretion of side products or proteins supports the agglomeration of cells.
Observations with laser-light back-reflection for cell particle size measurement
indicated the formation of clumps of C. glutamicum, when exposed to oscillatory
oxygen supply, either in a scale-down reactor concept (three-compartment reactor)
or in shake flask cultures with interrupted shaking [88]. Agglomeration under
fluctuating oxygen availability was postulated to be a result of increased secretion
of biofilm forming metabolites, e.g. in Mycobacterium tuberculosis and E. coli
cultures in connection with oxidative stress response [89, 90].
In case of filamentous organisms, macromorphological similarity across scales is
often achieved only if the shear force regime is maintained. Mechanical shear forces
as they appear close to the stirrer can lead to filament disruption, with consequences
14
P. Neubauer et al.
biased by the noise, if the samples measured are not enough to ensure a statistically
valid distribution. Nevertheless, the methodology is suitable to understand the
effects of gradients on cell viability and vitality while a large amount of cells can
be examined in a considerably short time. While the possibility of the application of
fluorescent markers represents an approach mostly on the expression level, cell
staining can be used to investigate several cellular components, including the
quantification of metabolite concentrations. If accumulated intracellularly, metabolite concentrations can be used to assess population heterogeneity as well, if the
metabolite can be quantified with sufficient accuracy in individual cells. Flow
cytometry is able to quantify the accumulation of intracellular lipids in microbial
cells, and thus identify subpopulations of different lipid contents [85], e.g. with Nile
Red or Bodipy
® stains. The accumulation of lipids and also other components might
correlate with changes in macromorphology of organisms. The measurement of lipid
content with optical methods can lead to conclusions about metabolic activity of
individual cells. This has been shown for heterotrophic algae, which accumulate to
large extent polyunsaturated fatty acids in lipid droplets. While using light microscopy and 3-dimensional holographic microscopy, the individual lipid storage in cells
was measured based on their individual cell size [86]. With rapid image analysis
using trained software, image acquisition can be performed in flow cells that are
connected to a cultivation. Automated workflows that offer considerably fast analysis of populations similar to flow cytometry are feasible, without the requirement of
staining.
Besides intracellular product accumulation, the macromorphology can provide
suitable information about the cell status and the impact of gradient formation on
it. It was examined that the cell size of S. cerevisiae cultures changed with the degree
of environmental heterogeneity in a three-compartment scale-down reactor
[87]. This happened in parallel to growth reduction and side metabolite
accumulation with a concomitant change of the sterol content, in comparison with
homogeneous growth conditions. Cells showed a diverse macromorphology under
scale-down cultivations, which supports the hypothesis that population heterogeneity is rather increased under growth in gradients. A morphologic response of cells to
scale-down cultivation conditions can also lead to agglomeration due to stress
response. Although the macromorphology of individual cells may stay unchanged,
the secretion of side products or proteins supports the agglomeration of cells.
Observations with laser-light back-reflection for cell particle size measurement
indicated the formation of clumps of C. glutamicum, when exposed to oscillatory
oxygen supply, either in a scale-down reactor concept (three-compartment reactor)
or in shake flask cultures with interrupted shaking [88]. Agglomeration under
fluctuating oxygen availability was postulated to be a result of increased secretion
of biofilm forming metabolites, e.g. in Mycobacterium tuberculosis and E. coli
cultures in connection with oxidative stress response [89, 90].
In case of filamentous organisms, macromorphological similarity across scales is
often achieved only if the shear force regime is maintained. Mechanical shear forces
as they appear close to the stirrer can lead to filament disruption, with consequences
14
P. Neubauer et al.