BackgroundMicrobial 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.ResultsA 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.ConclusionThis 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.
In this work, we present the design and characterization of Green Fluorescent Protein (GFP)-based reporter systems designed to describe cellular activity in "complex," heterogeneous bioreactors. The reporter systems consist of Escherichia coli strains carrying growth dependent promoters fused to genes expressing stable and unstable variants of GFP, respectively. The response of Escherichia coli cells to transient exposure to glucose was studied in a two-compartment scale down bioreactor (SDR) consisting of a well-stirred tank reactor (STR) connected to a plug-flow reactor (PFR). Such a SDR system is employed to mimic the situation of high glucose concentration and oxygen limitation that often encountered in large-scale, fed-batch bioreactors and the response of E. coli was simulated by continuously pumping microbial cells from STR to the PFR. We found that repeated addition of concentrated glucose pulses with varied frequency at the entrance of the PFR had consequences on strain physiological behavior. The GFP expressions were significantly marked after 10 h of cultivation in STR (control reactor) and SDR, whereas, growth rates were rather similar. Additional experiments in chemostat with programmed glucose perturbation suggested that the activities of the promoters were linked with the substrate limitation signal. Taken together with immunoblot analysis, we suppose protein leakage is responsible for the overexpression of fis and the related promoters, such as rrnB in this case study, but additional works are required in order to confirm this relationship. This investigation is useful for a better understanding of the fast dynamic phenomena occurring in heterogeneous large-scale bioreactors.
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