References of "Delvigne, Frank"
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See detailEvolution de la viabilité cellulaire dans les procédés de production de ferments lactiques : effet du type de bactérie sur l'évaluation par cytométrie en flux
Delvigne, Frank ULg; Thiry, Christophe ULg; Pierart, Céline ULg et al

Conference (2011, November 29)

La cytométrie en flux est une technique permettant d'effectuer des analyses au niveau des cellules individuelles. Cette technique a été développée à l'origine pour l'analyse de cellules animales dans des ... [more ▼]

La cytométrie en flux est une technique permettant d'effectuer des analyses au niveau des cellules individuelles. Cette technique a été développée à l'origine pour l'analyse de cellules animales dans des applications à finalité médicale. Ce n'est que dans les années 90 que cette technique a été considérée dans le cadre des cellules microbiennes procaryotes et eucaryotes. Grâce à l'évolution des technologies (laser à l'état solide, progrès dans la maîtrise des micro-écoulements,…), plusieurs développeurs proposent actuellement des cytomètres en flux pour des budgets abordables (environ 30.000 euros). Cette évolution entraîne actuellement un engouement des industriels pour la mise en oeuvre de cette technique pour le suivi de l'évolution des populations microbiennes dans divers procédés : brasserie, vinaigrerie, production de starters pour la boulangerie, production de starters lactiques,… L'intérêt manifesté pour cette technique est simple à comprendre si on considère les techniques actuelles pour l'estimation de la viabilité cellulaire. En effet, celles-ci reposent souvent sur la revification des cellules sur milieu gélosé et comptage des colonies après un temps d'incubation. Le problème fondamental de cette technique repose sur l'utilisation de conditions de culture qui ne sont pas forcément identiques à celle rencontrée au niveau du processus de production. Ce phénomène entraîne une sous-estimation des cellules microbiennes actives rassemblées sous le terme de "viables mais non cultivables". A cela s'ajoutent des problèmes techniques imposés par le temps de remise en culture et l'obtention des résultats bien après que le processus de production soit terminé, ainsi que par des procédures de laboratoire coûteuses en personnel et en consommables. La cytométrie s'impose donc comme une technique de choix qui permet d'analyser les cellules microbiennes individuelles sans étape de remise en culture et avec un débit expérimental élevé. En effet, 30.000 cellules peuvent être analysées en 30 secondes immédiatement après la prise d'échantillon, ce qui permet éventuellement de corriger les conditions de procédés en fonction de l'état des micro-organismes. L'utilisation de fluorochromes spécifiques permet l'analyse de caractéristiques cellulaires. Dans le cas des bactéries lactiques, l'utilisation du couple de colorant "carboxyfluorescéine diacétate" / "iodure de propidium" (cFDA/PI) est mis en oeuvre en routine pour la détermination de la viabilité cellulaire. L'iodure de propidium pénètre dans les cellules ayant une membrane endommagée et les colore en rouge, tandis que le cFDA est un composé non fluorescent qui diffuse au travers de toutes les membranes cellulaires et est hydrolysées par les activités estérases intracellulaires pour donner un composé fluorescent vert. Ces deux composantes de fluorescence peuvent être facilement caractérisées par analyse multi paramétrique au niveau d'un cytomètre en flux. La difficulté majeure que rencontre l'application de la cytométrie en flux au niveau industriel réside dans l'interprétation des résultats. Dans ce travail, trois souches de bactéries lactiques ayant des sensibilités différentes au stress de procédé ont été mise en oeuvre dans des schémas de production industriels faisant intervenir les étapes suivantes : production en bioréacteur agité de 2m³, récolte par centrifugation continue, congélation et lyophilisation. L'analyse par cytométrie en flux montre des tendances fondamentalement différentes pour les trois types de micro-organismes. Les deux souches microbiennes plus sensibles au stress de procédé du fait de leur caractère anaérobie strict ou microaérophile montrent des sous-populations bien distinctes au niveau des cytogrammes, tandis que la souche plus résistante ne montre qu'une seule population évoluant suivant les étapes du procédé. Ces observations sont utilisées pour une meilleure interprétation des cytogrammes pour l'estimation de la viabilité cellulaire en conditions de procédé. Une meilleure interprétation peut également être obtenue en considérant la possibilité de relargage des produits de dégradation fluorescents provenant de l'hydrolyse du cFDA en fonction de l'état de la membrane cellulaire. [less ▲]

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See detailPotential use of GFP microbial biosensors for the detection of mixing imperfections and cell viability in bioreactors
Delvigne, Frank ULg; Delafosse, Angélique ULg; Collignon, Marie-Laure ULg et al

Conference (2011, September 25)

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 ... [more ▼]

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. [less ▲]

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See detailPotentialités d’application des technologies biologiques pour la depollution des sols en Wallonie
Aldric, Jean-Marc ULg; Druart, P.; Maesen, Philippe ULg et al

in Journal des Ingénieurs (Le) (2011), 132

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See detailCharacterization of the response of GFP microbial biosensors sensitive to substrate limitation in scale-down bioreactors
Delvigne, Frank ULg; Brognaux, Alison ULg; Gorret, Nathalie et al

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 ▲]

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See detailDimensionnement et extrapolation des bioréacteurs sur base de paramètres physiologiques : cas de la production de lipase par Yarrowia lipolytica
Kar, Tambi ULg; Delvigne, Frank ULg; Destain, Jacqueline ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2011), 15(4), 585-595

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See detailApplicability of GFP Microbial Whole Cell Biosensors to Bioreactor Operations : Mathematical Modeling and Related Experimental Tools
Delvigne, Frank ULg; Brognaux, Alison ULg; Gorret, Nathalie et al

in Biosensors : emerging materials and applications (2011)

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See detailDynamic analysis of microbial behaviour face to envrionmental heterogeneities encountered in large-scale bioreactors
Sunya, Sirichai; Bideaux, Carine; Uribellarea, Jean-Louis et al

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

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See detailE.coli prpoS::gfp strain as biosensor of glucose heterogeneity inside industrial bioreactors
Brognaux, Alison ULg; Delvigne, Frank ULg; Thonart, Philippe ULg

Poster (2010, December 08)

• OBJECTIVE: Escherichia coli is a microorganism widely used in the industry for the production of recombinant proteins. The performances obtained at the laboratory level are not reproducible at a large ... [more ▼]

• OBJECTIVE: Escherichia coli is a microorganism widely used in the industry for the production of recombinant proteins. The performances obtained at the laboratory level are not reproducible at a large scale. Actually, the mixing operation is not efficient enough: gradients of glucose and oxygen appear when operating in fed-batch mode (addition of glucose during the culture). These gradients cause adverse impacts on the production of biomass and recombinant protein. The aim of this work is to use the microbial population as biocaptor of the encoutered stress inside heterogeneous industrial bioreactors to better scale-up and regulate these reactors. • MATERIALS AND METHODS: A plasmid carrying a stress promoter followed by the coding sequence of the Green Fluorescent Protein (GFP) is introduced in the bacterial host (in our case, the strain E. coli K12 will be considered as a model organism). When the cell is submitted to given stress condition, GFP synthesis is induced and accumulated into the cytoplasm, leading to the increase of the cell's fluorescence. Flow cytometry detection is used in order to quantify the fluorescence at the single cell level. Obtained results are frequency histograms of fluorescenceintensity in the microbial population • RESULTS: The rpoS gene is a gene of the general stress response, mainly induced at the entrance to stationary phase (during a lack of glucose). The tracking of the GFP fluorescence linked to the activation / repression of the rpoS promoter gives good results. Indeed, there is appearance of a segregation at the level of the GFP content among the microbial population. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency. • CONCLUSION: prpoS::gfp strains can be used as biosensors of the heterogeneity of glucose encountered inside industrial reactors. • POTENTIAL APPLICATIONS & KEY BENEFITS: These strains could be used to validate a fed-batch regulation (addition of glucose) at the industrial level. [less ▲]

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See detailBioprocesses scale-up : Interactions between physico-chemical and biological parameters
Delvigne, Frank ULg

Scientific conference (2010, March 15)

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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 detailBioprocédés et biotechnologies
Delvigne, Frank ULg

Scientific conference (2010)

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See detailDevelopment of an original approach to evaluate effects of surfactants, biomass and pollutants on the scaling-up of a two-phase partitioning bioreactor
Aldric, Jean-Marc ULg; Gillet, Sébastien ULg; Delvigne, Frank ULg et al

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 ▲]

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