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 S. cerevisiae and E. 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 transcriptional 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

Large-scale microbial industrial fermentations have significantly higher absolute pressure and dissolved CO₂ concentrations than otherwise comparable laboratory-scale processes. Yet the effect of increased dissolved CO₂ (dCO₂) levels is rarely addressed in the literature. In the current work, we have investigated the impact of industrial levels of dCO₂ (measured as the partial pressure of CO₂, pCO₂) in an Escherichia coli-based fed-batch process producing the human milk oligosaccharide 2′-fucosyllactose (2′-FL). The study evaluated the effect of high pCO₂ levels in both carbon-limited (C-limited) and carbon/nitrogen-limited (C/N-limited) fed-batch processes. High-cell density cultures were sparged with 10%, 15%, 20%, or 30% CO₂ in the inlet air to cover and exceed the levels observed in the industrial scale process. While the 10% enrichment was estimated to achieve similar or higher pCO₂ levels as the large-scale fermentation it did not impact the performance of the process. The product and biomass yields started being affected above 15% CO₂ enrichment, while 30% impaired the cultures completely. Quantitative proteomics analysis of the C-limited process showed that 15% CO₂ enrichment affected the culture on the protein level, but to a much smaller degree than expected. A more significant impact was seen in the dual C/N limited process, which likely stemmed from the effect pCO₂ had on nitrogen availability. The results demonstrated that microbial cultures can be seriously affected by elevated CO₂ levels, albeit at higher levels than expected.