Abstract
Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.
Highlights
Optimization of industrial cultivation processes requires comprehensive analysis and understanding of host strain physiology throughout the cultivation
Investigation of population heterogeneity in bioprocesses employing flow cytometry analysis often results in multiple fluorescence distribution data for e.g., a fluorescence marker and FSC collected from consecutive samples following the process or responses to environmental changes that have to be quantitatively compared in shape and fluorescence intensity (Heins and Weuster-Botz, 2018)
To demonstrate the applicability of the combination of these parameters for interpretation of flow cytometry data collected in bioprocesses, we investigated the influence of growth rate and glucose excess on microbial population heterogeneity assessed through green fluorescent protein (GFP) fluorescence expressed from ribosomal-related promoters, and cell membrane robustness, measured as response to freeze-thaw treatment
Summary
Optimization of industrial cultivation processes requires comprehensive analysis and understanding of host strain physiology throughout the cultivation. Microbial cells exhibit intrinsic cell-to-cell variability that is influenced by the cultivation parameters and in itself affects overall process performance (Müller et al, 2010; Delvigne et al, 2017; Lemoine et al, 2017). To this omnipresent intrinsic heterogeneity, that even arises in stable environments and can lead to e.g., metabolic specialization (Nikolic et al, 2017), heterogeneity originating from extrinsic sources can evolve. Averaged response values fail to describe the influence of different subpopulations and may even mask important characteristics of single cells in a bioprocess (Díaz et al, 2010; Fernandes et al, 2011; Tibayrenc et al, 2011; Gonzalez-Cabaleiro et al, 2017)
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