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See detailEvaluation of a set of E. coli reporter strains as physiological tracer for estimating bioreactor hydrodynamic efficiency
Delvigne, Frank ULg; Ingels, Sophie; Thonart, Philippe ULg

in Process Biochemistry (2010), 45(11), 1769-1778

A set of different green fluorescent protein (GFP) Escherichia coli reporter strains have been evaluated in mini- and stirred bioreactors operating in fed-batch mode with different degrees of ... [more ▼]

A set of different green fluorescent protein (GFP) Escherichia coli reporter strains have been evaluated in mini- and stirred bioreactors operating in fed-batch mode with different degrees of perturbations in order to estimate their potential use as process-related stress biosensor. The mini-bioreactor platform comprises a set of parallel shake flasks operating in fed-batch mode. The advantage of this system is its high experimental throughput for the evaluation of the GFP synthesis capacity of our reporter strains. In the case of classical shake flask system, no significant evolution of GFP synthesis have been observed, considering the reduced microbial growth period allowed by the system, whereas in the case of fed-batch operated mini-bioreactors, evolution of GFP synthesis, as well as GFP distribution among the microbial population, has been observed for three preselected strains (prpoS, puspA and posmC::gfp). More interestingly, a binary mode of expression has been observed in the case of the cultures carried out with the reporter strains for which GFP synthesis is under the control of the rpoS promoter which is induced under carbon limitation conditions. However, the generation of controlled glucose perturbations is relatively limited in this system and, in a second step fully automated bioreactor with a sclae-down strategy has been used to correlate the response of a prpoS::gfp strains with extracellular glucose perturbations. In the case of the culture performed in perturbed bioreactor (glucose intermittent feeding or glucose addition at the level of the recycle loop of a two-compartment scale-down bioreactor), the slowdown of the GFP synthesis resulting in the observation of a binary repartition of GFP content among the microbial population, has been observed. This observation led to the conclusion that the prpoS::gfp can be used as a biosensor for the validation of a fed-batch profile in industrial-scale bioreactors. [less ▲]

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See detailBioreactor mixing efficiency modulates the activity of a prpoS::gfp reporter gene in E. coli
Delvigne, Frank ULg; Boxus, Mathieu ULg; Ingels, Sophie et al

in Microbial Cell Factories (2009), 8(15),

ABSTRACT: BACKGROUND: Extensive studies have shown that up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of ... [more ▼]

ABSTRACT: BACKGROUND: Extensive studies have shown that 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, concentration gradients induced by loss of the global mixing efficiency associated with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. These changes are not fully understood since they involve complex physiological mechanisms. In this work, we intend to investigate, at the single cell level, the expression of the rpoS gene associated with the stress response of E. coli. The cultures of the 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. RESULTS: The rpoS level has been monitored by the aim of a transcriptional reporter gene based on the synthesis of the green fluorescent protein (GFP). It has been observed that the level of GFP increases during the transition from batch to fed-batch phase. After this initial increase, the GFP content of the cell drops, primarily due to the dilution by cell division. However, a significant drop of the GFP content has been observed if using a partitioned bioreactor, for which the mixing conditions are very bad, leading to the exposure of the cells to cyclic and stochastic extracellular fluctuations. If considering the flow cytometric profile of the cell to cell GFP content, this drop has to be attributed to the appearance of segregation at the level of the GFP content among the microbial population. CONCLUSION: The generation of extracellular perturbations (in the present case, at the level of the sugar concentration and the dissolved oxygen level) has led to a drop at the level of the rpoS expression level. This drop has to be attributed to a segregation phenomenon in microbial population, with a major sub-population exhibiting a low expression level and a minor sub-population keeping its initial elevated expression level. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency. [less ▲]

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