Use of on-line flow cytometry for the characterization of microbial stress dynamics during the bioprocess

Microbial cell population heterogeneity is now recognized as a major source of issues for the development and optimization of bioprocesses. Flow cytometry is a very powerful tool for the follow up of physiological properties of microbial cells in process-related conditions at the single cell level, and can be used to study the dynamics of segregation directly in bioreactors. In this context, specific interfaces have been developed in order to connect flow cytometer (FC) directly on bioreactor for automated analyses. In this work, we propose a simplified version of such interface and demonstrated its usefulness for multiplexed experiments.
This automated FC system has been tested for the follow up of the dynamics of an E. coli pfis::gfpAAV fluorescent bio-reporter and its PI uptake, correlated with membrane permeability. This bioreporter is composed of a fis promoter, a growth dependent promoter-indicator of the nutrient status of cells, fused to a gene expressing an unstable variant of GFP. The results obtained showed that the dynamics of the GFP synthesis is complex and can be attributed to a complex set of biological parameters. Segregation in the membrane permeability has been noticed.
This work demonstrates that a simplified version of on-line FC can be used at the process level for the investigation of the dynamics of complex physiological mechanisms.