Characterization of the response of GFP microbial biosensors sensitive to substrate limitation in scale-down bioreactors

in Biochemical Engineering Journal (2011), 55(2), 131-139

The dynamics of microbial stress response in intensive cultivation conditions remains not completely understood. In this work, two green fluorescent protein (GFP) transcriptional reporters have been used ... [more ▼]

The dynamics of microbial stress response in intensive cultivation conditions remains not completely understood. 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, i.e. in a well-mixed bioreactor. Interestingly, GFP leakage is more pronounced in the case of the cultures operated in the normal mode. Indeed, staining by propidium iodide 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 suggest that GFP microbial biosensors could be used to detect simultaneously mixing imperfections and their impact on the viability of microorganisms. [less ▲]

Applicability of GFP Microbial Whole Cell Biosensors to Bioreactor Operations : Mathematical Modeling and Related Experimental Tools

in Biosensors : emerging materials and applications (2011)

Strategies of improving the production of 6-amyl-1 α-pyrone obtained 2 with Trichoderma spp. through the bioreactor structure and substrate

in Récents Progrès en Génie des Procédés (2011), 101

Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect

in Biotechnology Journal (2011), 6

Dynamic analysis of microbial behaviour face to envrionmental heterogeneities encountered in large-scale bioreactors

in Delvigne, Frank (Ed.) BASE (2011)

Production of gamma-decalactone by the psychrophilic yeast Rhodotorula aurantiaca A19

Poster (2010, September)

Evaluation of a set of E. coli reporter strains as physiological tracer for estimating bioreactor hydrodynamic efficiency

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 ▲]

Development of an original approach to evaluate effects of surfactants, biomass and pollutants on the scaling-up of a two-phase partitioning bioreactor

in Journal of Chemical Technology & Biotechnology (2010), 84

BACKGROUND: Two-phase partitioning bioreactors (TPPBs) are considered as a new technology for xenobiotic degradation in gaseous effluents. However, there is still a need for more knowledge on how to ... [more ▼]

BACKGROUND: Two-phase partitioning bioreactors (TPPBs) are considered as a new technology for xenobiotic degradation in gaseous effluents. However, there is still a need for more knowledge on how to design and scale-up TPPBs. The partitioning of the two phases remains a misunderstood way of research. In particular, the impact of pollutant (isopropylbenzene), biomass and surfactant extract needs to be better evaluated. RESULTS:. An adaptated scale-down apparatus has been developed in order to quantify the speed of phase partitioning (SPP) into a plug flow section. Firstly, it was shown that isopropylbenzene (IPB) doesn’t destabilize more significantly the system. Secondly, respectively 0.5 g.L-1 and 0.05 g.L-1 of biomass and surfactant extract, separately or in mixture, were sufficient to ensure the stability of the two-phase system. Finally, a 100 m3 limit of scaling-up was suggested on the basis of the circulation time comparison. CONCLUSION: The scaling-up of an aqueous/silicone-oil TPPB was found to be definitely conceivable when the presence of biotic compounds were considered. However, further considerations are needed to verify our assumptions, in particular by taking into account the velocity field pattern in full-scale bioreactors and reproduce it in lab-scale apparatus. [less ▲]

L'extrapolation des bioréacteurs : un problème de génie des procédés ou de physiologie microbienne ?

in Chimie Nouvelle (2010), 104

Bioreactor scale-up often pose a serious issue during the industrial development of a bioprocess considering the numerous physical and biological phenomena occurring in the reacting volume. The basic ... [more ▼]

Bioreactor scale-up often pose a serious issue during the industrial development of a bioprocess considering the numerous physical and biological phenomena occurring in the reacting volume. The basic principles of scale-up coming from the traditional chemical and process engineering approaches will be first reviewed and will be then compared to a new one involving recent development at the level of microbial strain manipulation. This "physiological" approach of scale-up involves directly a biological component of the system (by comparison with the traditional approach for scaling-up involving physical parameters indirectly linked to the physiological phenomena occurring in the bioreactor), i.e. the synthesis of a reporter fluorescent protein when microbial cells are exposed to stress. It will be shown how this principle can be used for a better understanding of the relationship between bioreactor hydrodynamics and microbial stress. [less ▲]

Microbial bioprocesses : current state and future prospect

Scientific conference (2010)