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See detailDesign of growth-dependent biosensors based on destabilized GFP for the detection of physiological behavior of Escherichia coli in heterogeneous bioreactors
Han, Shanshan; Delvigne, Frank ULg; Brognaux, Alison ULg et al

in Biotechnology Progress (2013), 29(2), 553-563

In this work we present the design and characterization of GFP-based reporter systems designed in order to describe cellular activity in ‘complex’, heterogeneous bioreactors. The reporter systems consist ... [more ▼]

In this work we present the design and characterization of GFP-based reporter systems designed in order 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 stirred tank reactor (STR) connected to plug-flow reactor (PFR). Such a SDR system is employed to mimic the situation that often encountered in large-scale, fed-batch bioreactors. The response of E. coli coli to oxygen-poor and glucose-rich regions was simulated by continuously pumping E. coli cells from STR to the PFR. A concentrated glucose pulse (400 g/L) was consecutively added at the entrance of the PFR and samples were taken from PFR. The GFP expressions were significantly marked after 10 hours of culture in STR (control reactor) and SDR, whereas, growth rates were rather similar. Additional experiments in chemostat (D=0.14 h-1) with programmed glucose perturbation (30 g/L, frequency: 100/900 s) suggested that the activities of the promoter are 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. Our finding are of great interest for industrial application since the GFP signal can be detected very early during the culture and is related to relevant physiological changes. This investigation is useful for a better understanding of the fast dynamic phenomena occurring in heterogeneous large-scale bioreactors. [less ▲]

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See detailDirect and indirect use of GFP whole cell biosensors for the assessment of bioprocess performances: design of milliliter scale-down bioreactors
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

in Biotechnology Progress (2013), 29(1), 48-59

Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional ... [more ▼]

Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli, i.e., uspA::gfp, csiE::gfp and yciG::gfp. Our previous studies have shown that these kinds of promoters are induced in response to substrate limitation and are significantly repressed when cultures are carried out in heterogeneous bioreactors. This sensitivity to substrate limitation has been confirmed in the case of the csiE and yciG biosensors. A mini-scale-down platform is proposed as a high throughput tool to rapidly investigate the usefulness of a given microbial biosensor. This platform is composed of shake flasks able to operate in fed-batch mode either using the slow release or the intermittent feeding principle. Local heterogeneities were reproduced at the level of these mini-bioreactors (operating under the intermittent feeding principle) and caused a decrease in GFP expression as in conventional scale-down reactors. The presence of GFP in supernatants was also noted and seems to be correlated with the substrate limitation signal for the three cultivation systems considered in this work (i.e., chemostat, conventional and mini-bioreactors) and with membrane permeability. [less ▲]

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See detailStochastic models to study the impact of mixing on a fed-batch culture of Saccharomyces cerevisiae.
Delvigne, Frank ULg; Lejeune, Annick ULg; Destain, Jacqueline ULg et al

in Biotechnology Progress (2006), 22(1), 259-69

The mechanisms of interaction between microorganisms and their environment in a stirred bioreactor can be modeled by a stochastic approach. The procedure comprises two submodels: a classical stochastic ... [more ▼]

The mechanisms of interaction between microorganisms and their environment in a stirred bioreactor can be modeled by a stochastic approach. The procedure comprises two submodels: a classical stochastic model for the microbial cell circulation and a Markov chain model for the concentration gradient calculus. The advantage lies in the fact that the core of each submodel, i.e., the transition matrix (which contains the probabilities to shift from a perfectly mixed compartment to another in the bioreactor representation), is identical for the two cases. That means that both the particle circulation and fluid mixing process can be analyzed by use of the same modeling basis. This assumption has been validated by performing inert tracer (NaCl) and stained yeast cells dispersion experiments that have shown good agreement with simulation results. The stochastic model has been used to define a characteristic concentration profile experienced by the microorganisms during a fermentation test performed in a scale-down reactor. The concentration profiles obtained in this way can explain the scale-down effect in the case of a Saccharomyces cerevisiae fed-batch process. The simulation results are analyzed in order to give some explanations about the effect of the substrate fluctuation dynamics on S. cerevisiae. [less ▲]

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