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Analysis of E. coli cell to cell stress response in function of the intensity of the extracellular perturbations : investigation at the level of different scale-down configurations

Frank Delvigne1,2, Nathalie Gorret3, Sirichai Sunya3, Sophie Ingels1, Philippe Thonart1

1 Faculté Universitaire des Sciences Agronomiques, Unité de Bio-industries/CWBI, Passage des Déportés 2 , 5030 Gembloux, Belgium

2 Fond de la recherche scientifique (FRNS-FRS), Rue d'Egmont 5, 1000 Bruxelles, Belgium

3 Laboratoire d'Ingénierie des Systèmes Biologiques et Procédés CNRS UMR 5504 INRA UMAR 792, INSA Toulouse, 135 Avenue de Rangueil 31077,Toulouse Cedex 4 France

135 Avenue de Rangueil 31077 Toulouse Cedex 4. France

Numerous studies have shown that the up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of the bioreactor, the appearance of concentration gradients induced by a loss of the global mixing efficiency with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. At this time, these modifications are not fully understood since they involve complex physiological mechanisms. In this work, we propose to investigate, at the single cell level, the expression of a prpoS::gfpmut2 reporter gene associated with the stress response of E. coli. The cultures of the green fluorescent protein (GFP) reporter strain have been performed in a small scale reactor as well as in a series of scaled-down bioreactors able to induce extracellular perturbations with increasing level of magnitude.

By following the rpoS expression level for different reactor configurations, a significant fluorescence drop has been correlated to the reactor mixing efficiency. The higher fluorescence drop is attributed to a partitioned bioreactor with a low recirculation flow rate between the mixed and the plug-flow part. By examining the GFP flow cytometric profile, fluorescence drop has been attributed to the appearance of a segregation at the level of the GFP content among the microbial population, with a major sub-population exhibiting a low expression level and a minor sub-population keeping its initial elevated expression level. It has been shown that the segregation phenomena is reversible when cells are put in fresh medium without perturbations, suggesting that cell viability is not involved in the segregation. This result has been subsequently validated by propidium iodide (PI) staining of the cells and flow cytometry analysis.

This work highlights the potentialities of a fluorescent reporter strain in order to estimate the level of heterogeneities in different reactor configurations, and also highlights the need to obtain data at a single cell resolution, considering the segregation phenomena that have been reported. The principal limitation of this method is its relatively high response time (depending on gene induction and subsequent GFP synthesis and folding) compared with the time constants associated with bioreactor hydrodynamics.

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