Physiological heterogeneities in microbial populations and implications for physical stress tolerance

Abstract

Background: Traditionally average values of the whole population are considered when analysing microbial cell cultivations. However, a typical microbial population in a bioreactor is heterogeneous in most phenotypes measurable at a single-cell level. There are indications that such heterogeneity may be unfavourable on the one hand (reduces yields and productivities), but also beneficial on the other hand (facilitates quick adaptation to new conditions--i.e. increases the robustness of the fermentation process). Understanding and control of microbial population heterogeneity is thus of major importance for improving microbial cell factory processes.

Results: In this work, a dual reporter system was developed and applied to map growth and cell fitness heterogeneities within budding yeast populations during aerobic cultivation in well-mixed bioreactors. The reporter strain, which was based on the expression of green fluorescent protein (GFP) under the control of the ribosomal protein RPL22a promoter, made it possible to distinguish cell growth phases by the level of fluorescence intensity. Furthermore, by exploiting the strong correlation of intracellular GFP level and cell membrane integrity it was possible to distinguish subpopulations with high and low cell membrane robustness and hence ability to withstand freeze-thaw stress. A strong inverse correlation between growth and cell membrane robustness was observed, which further supports the hypothesis that cellular resources are limited and need to be distributed as a trade-off between two functions: growth and robustness. In addition, the trade-off was shown to vary within the population, and the occurrence of two distinct subpopulations shifting between these two antagonistic modes of cell operation could be distinguished.

Conclusions: The reporter strain enabled mapping of population heterogeneities in growth and cell membrane robustness towards freeze-thaw stress at different phases of cell cultivation. The described reporter system is a valuable tool for understanding the effect of environmental conditions on population heterogeneity of microbial cells and thereby to understand cell responses during industrial process-like conditions. It may be applied to identify more robust subpopulations, and for developing novel strategies for strain improvement and process design for more effective bioprocessing.

Figure 1

Mean fluorescence, biomass formation, substrate utilisation and product formation during aerobic batch cultivation of theS. cerevisiaereporter strain in defined mineral media. Symbols: Mean GFP fluorescence (snowflakes); OD₆₀₀ (open diamonds); Glucose (open circles); Acetate (open triangle pointing upwards); Ethanol (open squares); Glycerol (open triangles pointing downwards). The vertical line marks the time point glucose depletion was observed.

Figure 2

Mean GFP level as a function of mean FSC for each percentile during aerobic batch cultivation. For each time point there is a line (in a different colour) with markers for each percentile. Details of the percentile analysis can be found in the methods section and the Additional file 1: Figure S1-2.

Figure 3

Distribution of PI-stained cells prior to and after freeze-thaw stress exposure plotted against point of harvest during aerobic batch cultivation. Symbols: PI negative cells prior to freeze-thaw stress (circles), PI negative cells (triangles) and PI positive cells (squares) after freeze-thaw stress.

Figure 4

Inverse correlation between growth and cell membrane robustness. Mean GFP level of the whole population prior to freeze-thaw stress (empty symbols) as a function of the percentage of PI negative cells after freeze-thaw stress; and the mean GFP of the PI negative cells after freeze-thaw stress (full symbols) as a function of the percentage of PI negative cells after freeze-thaw stress. ^a These outlying data points are from the first sample of aerobic batch cultivation.

Figure 5

Effect of freeze-thaw stress on GFP signal. Mean GFP level of the entire population prior to freeze-thaw stress (circles) and mean GFP level of PI negative cells (triangles) and PI positive cells (squares) after freeze-thaw stress plotted against point of harvest during aerobic batch cultivation. The GFP signal was significantly decreased in cells that have been permeabilised by the stress exposure, hence making PI staining redundant to estimate number of cells with intact membranes.

Figure 6

Dynamic responses of subpopulations and metabolites in glucose gradient experiments mimicking large-scale cultivation conditions. An aerobic glucose-limited chemostat was perturbed with a low concentration glucose pulse. Cells were sampled from the bioreactor and exposed to freeze-thaw stress and subsequently analysed with flow cytometry. Symbols: Subpopulation P1 (high GFP level, intact cell membranes) (crosses); Subpopulation P2 (low GFP level, permeabilised cell membranes) (snowflakes); Glucose (open circles); Acetate (open diamonds); Ethanol (open squares); Glycerol (open triangles).

Figure 7

GFP histograms illustrating the distribution of subpopulations with different degree of cell membrane robustness following the glucose pulse. GFP histograms (grey area). FI = Fluorescence intensity. The black lines correspond to the fitted mixture of two Gaussian distributions. The vertical line corresponds to the subpopulation classification threshold.