A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors

Background
Microbial cell population heterogeneity is now recognized as a major source of issues in the
development and optimization of bioprocesses. Even if single cell technologies are available
for the study of microbial population heterogeneity, only a few of these methods are available
in order to study the dynamics of segregation directly in bioreactors. In this context, specific
interfaces have been developed in order to connect a flow cytometer directly to a bioreactor
for automated analyses. In this work, we propose a simplified version of such an interface
and demonstrate its usefulness for multiplexed experiments.Results
A low-cost automated flow cytometer has been used in order to monitor the synthesis of a
destabilized Green Fluorescent Protein (GFP) under the regulation of the fis promoter and
propidium iodide (PI) uptake. The results obtained showed that the dynamics of GFP
synthesis are complex and can be attributed to a complex set of biological parameters, i.e. on
the one hand the release of protein into the extracellular medium and its uptake modifying the
activity of the fis promoter, and on the other hand the stability of the GFP molecule itself,
which can be attributed to the protease content and energy status of the cells. In this respect,
multiplexed experiments have shown a correlation between heat shock and ATP content and
the stability of the reporter molecule.
Conclusion
This work demonstrates that a simplified version of on-line FC can be used at the process
level or in a multiplexed version to investigate the dynamics of complex physiological
mechanisms. In this respect, the determination of new on-line parameters derived from
automated FC is of primary importance in order to fully integrate the power of FC in
dedicated feedback control loops.