Potential use of GFP microbial biosensors for the detection of mixing imperfections and cell viability in bioreactors

The dynamics of microbial stress response in intensive cultivation conditions remains misunderstood. In this work, two green fluorescent protein (GFP) transcriptional reporters have been used as biosensors of the heterogeneities generated in a two-compartment scale-down reactor. The stress promoters have been chosen for their responsiveness to carbon limitation corresponding to the global substrate profiles encountered in intensive fed-batch cultures. From our results, it can be concluded that the exposure of microbial cells to substrates heterogeneities tends to decrease the GFP expression level in fed-batch mode. Fluorescence intensities have been monitored at the single cell level by using flow cytometry. During the course of the fed-batch culture, a drop at the level of the intracellular GFP content has been observed for the two scale-down operating conditions and for the two promoters sensitive to substrate limitation (rpoS and csiE). The fluorescence drop can be attributed to the repression of these promoters but also to the release of GFP to the extracellular medium according to the increase of the fluorescence level of the supernatant. This leakage has been observed for all the operating conditions, i.e. the scale-down reactors and the culture operating in the normal mode. Interestingly, GFP leakage is more pronounced in the case of the cultures operated in the normal mode. Indeed, staining by propidium iodide (PI) tends to be more elevated for the microbial cells cultured under the normal mode by comparison with those cultured in scale-down conditions, indicating a higher permeability of the membrane. These results are in accordance with previously published ones (Hewitt and co-workers) suggesting that microbial cells cultivated in heterogeneous bioreactors (scale-down and large-scale bioreactors) exhibits a higher viability level. These results suggest that GFP microbial biosensors could be used to detect simultaneously mixing imperfections and their impact on the viability of microorganisms.