The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

Abstract

Acetate as the major by-product in industrial-scale bioprocesses with Escherichia coli is found to decrease process efficiency as well as to be toxic to cells, which has several effects like a significant induction of cellular stress responses. However, the underlying phenomena are poorly explored. Therefore, we studied time-resolved population heterogeneity of the E. coli growth reporter strain MG1655/pGS20PrrnBGFPAAV expressing destabilized green fluorescent protein during batch growth on acetate and glucose as sole carbon sources. Additionally, we applied five fluorescent stains targeting different cellular properties (viability as well as metabolic and respiratory activity). Quantitative analysis of flow cytometry data verified that bacterial populations in the bioreactor are more heterogeneous in growth as well as stronger metabolically challenged during growth on acetate as sole carbon source, compared to growth on glucose or acetate after diauxic shift. Interestingly, with acetate as sole carbon source, significant subpopulations were found with some cells that seem to be more robust than the rest of the population. In conclusion, following batch cultures population heterogeneity was evident in all measured parameters.Our approach enabled a deeper study of heterogeneity during growth on the favored substrate glucose as well as on the toxic by-product acetate. Using a combination of activity fluorescent dyes proved to be an accurate and fast alternative as well as a supplement to the use of a reporter strain. However, the choice of combination of stains should be well considered depending on which population traits to aim for. K E Y W O R D Sflow cytometry, growth on acetate, metabolic activity, population heterogeneity, reporter strain, viability

Quantitative Flow Cytometry to Understand Population Heterogeneity in Response to Changes in Substrate Availability in Escherichia coli and Saccharomyces cerevisiae Chemostats

“…This might be connected to acetate growth which was accompanied by a decrease in slope (Figure 5G). Possibly, the cells used noise in gene expression while growing on acetate, as also found earlier (Heins et al, 2019). Consequently, the reporter strain could capture both phases, however, with a less dynamic response for the lowest D. For D = 0.1 h −1 only a slight decrease in slope was seen after glucose depletion.…”

Advances in automated real‐time flow cytometry for monitoring of bioreactor processes

“…For both tools standardized functions to load FC data as FCS files exist. For MATLAB the fcs_read and fcs_readfcs algorithms are popular [ 19 , 26 , 52 , 84 , 85 ]. For R, the Bioconductor platform exists, that hosts the largest collection of open source FCM software covering data analysis and visualization of FCM data [ 86 ].…”

“…Width at baseline level, skewness and the slope of the cumulative distribution function plot can further quantify the shape of fluorescence distributions [ 26 ]. Where significant subpopulations appear, its percentage can be computed [ 19 ]. Calculations can be performed by MATLAB and R‐routines and the results automatically displayed in real‐time [ 26 , 46 , 54 , 64 ].…”

“…Thus, single cells in a bioprocess expressing fluorescent proteins as reporters for different cellular characteristics or alternatively single cells stained with one or several fluorescent dyes can be monitored simultaneously [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. Also, the combination of reporter strain and dyes or cell size characteristics is applied [ 18 , 19 ]. Its ability to measure single cell characteristics makes FCM also a suitable tool to monitor population heterogeneity in bioreactors as well as to investigate the cells’ physiological state under process‐related conditions [ 2 , 7 ].…”