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See detailDevelopment of optical trajectography device for the lagangian study of turbulent flow inside a stirred tank used in pharmaceutical industry
Collignon, Marie-Laure ULg; Delafosse, Angélique ULg; Delvigne, Frank ULg et al

Poster (2012, September 10)

Stirred tanks are devices that are widely used in various process steps of the pharmaceutical industry. The performance of these processes (animal cell culture, crystallization, flocculation …) strongly ... [more ▼]

Stirred tanks are devices that are widely used in various process steps of the pharmaceutical industry. The performance of these processes (animal cell culture, crystallization, flocculation …) strongly depend on the physico-chemical and hydrodynamic environment present locally in these tanks. To fully describe this local environment, an Eulerian - Lagrangian approach must be adopted in order to establish history of conditions met by a particle as a cell, a crystal or a floc. This approach implies to determine the trajectory followed by the particle. To this aim, the Chemical Laboratory of Liege University has developed a prototype of optical trajectography device. The objective of this paper is to present the device, developments that were necessary for its use and the results obtained. The device is composed of two cameras modeled by a pinhole model which record the position of a bead that has a size equal to 490 µm and that perfectly follows local flow structures. The measured trajectory has been validated by comparing average time velocity fields deduced from it to those measured, in the same operating conditions, by particle image velocimetry (P.I.V.). [less ▲]

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See detailImprovement of the cellulose hydrolysis yields and hydrolysate concentration by management of enzymes and substrate input
Jacquet, Nicolas ULg; Vanderghem, Caroline ULg; Blecker, Christophe ULg et al

in Cerevisia : Belgian Journal of Brewing and Biotechnology (2012), 37

In order to improve the hydrolysis of cellulose fiber and to obtain highly concentrated hydrolysate, two methods based on successive addition of enzyme and substrate were assessed. The first method, which ... [more ▼]

In order to improve the hydrolysis of cellulose fiber and to obtain highly concentrated hydrolysate, two methods based on successive addition of enzyme and substrate were assessed. The first method, which required only substrate addition, allowed to increase by 50% the hydrolysate concentration and to decrease by 30% enzyme units needed. The second method highlighted the ability to reach very high concentrated hydrolysate (up to 170 g/l) by simultaneous addition of enzyme and substrate. In parallel, relationships between some limiting factors and the yields of hydrolysis were investigated. In conclusion, viscosity evolution of cellulose suspension during hydrolysis step was investigated with an aim to improve the management of enzyme and substrate addition. [less ▲]

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See detailPathogenicity test of the fungus Aspergillus clavatus on aphid Acyrthosiphon pisum (Aphididae)
Seye, Fawrou; Bawin, Thomas ULg; Delvigne, Frank ULg et al

Poster (2012, August 19)

Pea aphid is a pest of many cultivated and wild plants, but also a vector of several viral diseases. To control this pest, the most widely used methods are physical, chemical and more recently an ... [more ▼]

Pea aphid is a pest of many cultivated and wild plants, but also a vector of several viral diseases. To control this pest, the most widely used methods are physical, chemical and more recently an integrated approach that includes biological control. With the use of pathogenic agents against insects, the use of entomopathogenic fungi is one of the most promising. The present study demonstrated the possibility of using an entomopathogenic fungus Aspergillus clavatus against aphids. In laboratory conditions (8/16 photoperiod, average temperature 25°C), the insects were in contact with different concentrations ranging from 10^2 to 10^4 spores/cm2 deposited on filter paper in Petri dishes, or applied directly to young plants with doses ranging from 10^4 to 10^6 spores/ml. In 24 hours, mortality was 0 to 31.5% in Petri dishes. For treatment plants, the cumulative mortality in 5 days was 55 to 79%. Microscopic observations showed that the aphids were infected by contact and fungus has a mycosis effect. From these preliminary results, investigations should be made to study the action of the fungus on the reproduction of aphids. Therefore, A. clavatus could be introduced along with other fungi found in the literature as a biological control agent against aphids. [less ▲]

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See detailDevelopment of mini scale-down platform based on the response of GFP microbial biosensors
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

Poster (2012, May 18)

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor ... [more ▼]

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor). Indeed, such heterogeneities cause a lowering of the biomass yield and an increase of by-products concentration. In our previous works, green fluorescent protein reporters have been used as biosensors of the heterogeneities generated in a two compartment scale-down reactor. As there is a huge variety of available whole cell biosensor to characterize the impact of such heterogeneities at the biological level, there is a need for high-throughput cultivation tools in order to investigate the usefulness of a given microbial biosensor among a library comprising several thousands of clones. This work is based on this statement and aims to investigate the potentialities of a mini scale-down platform. Four green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli: rpoS::gfp, uspA::gfp, csiE::gfp and yciG::gfp. The promoters rpoS and uspA are induced in response to a variety of stresses whereas the two other promoters, csiE and yciG, are supposed to be more specific in front of a glucose limitation. First, the response of these biosensors has been assessed in chemostat reactors. These kinds of experiments allow easier interpretation of responses of stress gene related to a glucose limitation since the extracellular conditions are constants and cells are renewed. Biosensors carrying the csiE and yciG promoters have exhibited an induction in function of the glucose limitation. Secondly, a scale-down platform has been tested with the same biosensors and two kinds of glucose addition mode. This scale-down platform involves high-throughput cultivation tools, i.e. in our case shake flask, equipped with non-invasive optical sensors for the monitoring of the dissolved oxygen profile in front of the glucose addition mode. The first system is based on a commercial package (Enbase) based on the enzymatic release of glucose in the medium. The Enbase system allows the generation of a very smooth glucose profile without any perturbations. For comparison purpose, we have also used an intermittent feeding that induces strong fluctuation at the level of the glucose and the dissolved oxygen concentration. The intermittent addition of glucose induces a slow down at the level of the GFP synthesis, suggesting that temporal accumulation of glucose inhibits the activity of the yciG and csiE promoters. In conclusion, the scale-down platform is able to reproduce the same kind of glucose fluctuations that encounters the cells in large-scale processes but not allows studying the impact of high-cell density culture on gene expression. [less ▲]

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See detailExtrapolation des bioréacteurs/Bioreactor scale-up
Delvigne, Frank ULg

Scientific conference (2012, May 06)

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See detailUse of microbial biosensors to detect substrate heterogeneities at the single cell level and assess microbial viability: Validation of a mini-bioreactor platform
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

Conference (2012, March 15)

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor ... [more ▼]

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor). Indeed, such heterogeneities cause a lowering of the biomass yield and an increase of by-products concentration. In this work, we have used these biosensors for the elaboration 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 of substrate limitation and exhibit a strong fluorescence attenuation when cultivated 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 has been proposed as a high throughput tool to investigate rapidly the usefulness of a given microbial biosensor. This platform is composed of shake flask able to operate in fed-batch mode either by using the slow release or the intermittent feeding principle. The first system is based on a commercial package (Enbase) based on the enzymatic release of glucose in the medium. The Enbase system allows the generation of a very smooth glucose profile without any perturbations. For comparison purpose, we have also used an intermittent feeding that induces strong fluctuation at the level of the glucose and the dissolved oxygen concentration. Local heterogeneities have thus been reproduced at the level of these mini-bioreactors and these one have caused a decrease of GFP expression, as in conventional scale-down reactor. The presence of GFP in supernatants has also been noticed 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 the membrane permeability. [less ▲]

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See detailImpact of glycerol and storage temperature on gluatathione concentration and physiological state of Pseudomonas fluorescens BTP1 freeze-dried
Mputu Kanyinda, Jean-Noël ULg; Pierart, C.; Delvigne, Frank ULg et al

Poster (2012, February 15)

Pseudomonas fluorescens is commonly used as bio-fungicides in agriculture. For this use it requires formulations as either liquid or powder. Formulations have two advantages, storage and transport. Freeze ... [more ▼]

Pseudomonas fluorescens is commonly used as bio-fungicides in agriculture. For this use it requires formulations as either liquid or powder. Formulations have two advantages, storage and transport. Freeze-drying is a commonly used method to preserve bacteria. However, freeze-drying damages the cells, which results in loss of viability. Protective compounds are used to reduce loss of viability during process (freeze-drying and storage). In our study we used flow cytometry analysis to assess the physiological state in which cells are at the end of freeze-drying and Glutathione (GSH) was measured before and during storage. [less ▲]

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See detailDesign of a biofilm reactor comprising a metal structured packing for the production of lipopeptides by B. subtilis
Zune, Quentin ULg; Ongena, Marc ULg; Toye, Dominique ULg et al

Poster (2012, February 08)

Abstract : The design of a new single species biofilm bioreactor has been investigated. Bacillus subtilis S499 has been chosen as a model organism for the production of lipopetides. Nevertheless ... [more ▼]

Abstract : The design of a new single species biofilm bioreactor has been investigated. Bacillus subtilis S499 has been chosen as a model organism for the production of lipopetides. Nevertheless, considering the surface active properties for this kind of metabolite, processes based on submerged culture in stirred-tank bioreactor involve the use of important amount of antifoam and therefore downstream processes are tedious. In this work, an original process was developed with an experimental setting leading to the suppression of foam formation during the culture. B. subtilis S499 makes a biofilm on a stainless steel structured packing in the top of a bioreactor, nutrient and oxygen supply being carried out by the media recirculation as liquid film on the packing. Lipopeptides secreted by biofilm are accumulated in the liquid phase under the packing and can reach concentrations as high as 800 mg/l. The colonization of the packing by the biofilm has been monitored by X-ray tomography. [less ▲]

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See detailEffect of the intensity and frequency of glucose pulse perturbation on transient E. coli behavior : a step toward the large-scale bioreactor
Gorret, Nathalie; Sunya, Sirichai; Uribellarea, Jean-Louis et al

Conference (2012)

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See detailImpact of mixing imperfections on yeast bioreactor performances: Scale-down reactor concept and related experimental tools
Delvigne, Frank ULg; Blaise, Yannick ULg; Destain, Jacqueline ULg et al

in Cerevisia and Biotechnology (2012), 37

A method combining environmental data extracted from the dissolved oxygen profile of a fed-batch bioreactor and a dynamic discrete Markov chain model has been presented in order to give more insight about ... [more ▼]

A method combining environmental data extracted from the dissolved oxygen profile of a fed-batch bioreactor and a dynamic discrete Markov chain model has been presented in order to give more insight about the glucose and dissolved oxygen fluctuations experienced by the microorganisms during cultivation in heterogeneous bioreactor. The fed-batch cultivation of Saccharomyces cerevisiae has been performed in a well-mixed and a partitioned scale-down reactor (SDR). The analysis of the environmental sequences has shown extended time lengths for the glucose availability and depletion sequences in the case of the SDR under a DO-controlled fed-batch culture. The Markov chain model developed in this work is able to capture the stochastic environmental events, i.e. in our case the environmental states experienced by the microorganisms crossing the tubular part of the SDR. The simulation results show clearly an extension of the starvation periods in the case of the culture performed in the SDR. The simulations have been performed at the single cells level allowing future improvements of our model and notably in the context of the population segregation phenomena occurring in fed-batch cultures. As a perspective, flow cytometry has been presented as a high-throughput analytical tool for the investigation of yeast physiology at the single cell level and in process-related conditions. [less ▲]

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See detailComparison of the transient responses of Escherichia coli to a glucose pulse of various intensities
Sunya, Sirichai; Delvigne, Frank ULg; Uribellarea, Jean-Louis et al

in Applied Microbiology & Biotechnology (2012), 95(4), 1021-1034

tDynamic stimulus-responses of Escherichia coli DPD2085, yciG::LuxCDABE reporter strain, to glucose pulses of different intensities (0.08, 0.4 and 1 gL −1) were compared using glucose-limited chemostat ... [more ▼]

tDynamic stimulus-responses of Escherichia coli DPD2085, yciG::LuxCDABE reporter strain, to glucose pulses of different intensities (0.08, 0.4 and 1 gL −1) were compared using glucose-limited chemostat cultures at dilution rate close to 0.15 h −1. After at least five residence times, the steady-state cultures were disturbed by a pulse of glucose, engendering conditions of glucose excess with concomitant oxygen limitation. In all conditions, glucose consumption, acetate and formate accumulations followed a linear relationship with time. The resulting specific uptake and production rates as well as respiratory rates were rapidly increased within the first seconds, which revealed a high ability of E. coli strain to modulate its metabolism to a new environment. For transition from glucose-excess to glucoselimited conditions, the cells rapidly re-established its pseudo-steady state. The dynamics of transient responses at the macroscopic viewpoint were shown to be independent on the glucose pulse intensity in the tested range. On the contrary, the E. coli biosensor yciG::luxCDABE revealed a transcriptional induction of yciG gene promoter depending on the quantities of the glucose added, through in situ and online monitoring of the bioluminescence emitted by the cells. Despite many studies describing the dynamics of the transient response of E. coli to glucose perturbations, it is the first time that a direct comparison is reported, using the same experimental design (strain, medium and experimental set up), to study the impact of the glucose pulse intensity on the dynamics of microbial behaviour regarding growth, respiration and metabolite productions. [less ▲]

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See detailPhysical and physiological impacts of different foam control strategies during a process involving hydrophobic substrate for the lipase production by Yarrowia lipolytica
Kar, Tambi ULg; Destain, Jacqueline ULg; Thonart, Philippe ULg et al

in Bioprocess and Biosystems Engineering (2012), 35(4), 483-492

The potentialities for the intensification of the process of lipase production by the yeast Yarrowia lipolytica on a renewable hydrophobic substrate (methyloleate) have been investigated. The key factor ... [more ▼]

The potentialities for the intensification of the process of lipase production by the yeast Yarrowia lipolytica on a renewable hydrophobic substrate (methyloleate) have been investigated. The key factor governing the lipase yield is the intensification of the oxygen transfer rate, considering the fact that Y. lipolytica is a strict aerobe. However, considering the nature of the substrate and the capacity for protein excretion and biosurfactant production of Y. lipolytica, intensification of oxygen transfer rate is accompanied by an excessive formation of foam. Two different foam control strategies have thus been implemented: a classical chemical foam control strategy (CFM) and a mechanical foam control (MFM) based on the Stirring As Foam Disruption (SAFD) principle. The second strategy allows foam control without any modifications of the physico-chemical properties of the broth. However, the MFM system design induced the formation of a persistent foam layer in the bioreactor. This phenomenon has led to the segregation of microbial cells between the foam phase and the liquid phase in the case of the bioreactors operated with MFM control, and induced a reduction at the level of the lipase yield. More interestingly, flow cytometry experiments have shown that residence time of microbial cells in the foam phase tends to induce a dimorphic transition which could potentially explain the reduction of lipase excretion. [less ▲]

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See detailReal-time monitoring of metabolic shift and transcriptional induction of yciG::luxCDABE E. coli reporter strain to a glucose pulse of different concentrations
Sunya, Sirichai; Gorret, Nathalie; Delvigne, Frank ULg et al

in Journal of Biotechnology (2012), 157(3), 379-390

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See detailPotentiality of using microbial biosensors for the detection of substrate heterogeneities and the assessment of microbial viability in industrial bioreactors: a complete set of experiments in chemostat and scale-down reactors, and elaboration of a mini scale-down platform
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

in Communications in Agricultural and Applied Biological Sciences (2012), 77(1), 3-7

Substrate limitation responsive biosensors have been used in order to detect spatial substrate heterogeneities, , inside industrial bioreactors (whole-cell biosensor). Three green fluorescent protein (GFP ... [more ▼]

Substrate limitation responsive biosensors have been used in order to detect spatial substrate heterogeneities, , inside industrial bioreactors (whole-cell biosensor). Three green fluorescent protein (GFP) transcriptional reporters have been chosen in E.coli, i.e. uspA::gfp, csiE::gfp and yciG::gfp. The promoter uspA is induced in response to a variety of stresses whereas the two other promoters, csiE and yciG, are supposed to be more specific in front of a substrate limitation. The responsiveness of these biosensors has been assessed in chemostat reactor. Secondly, the same biosensors have been tested in well-mixed laboratory reactors and in scale-down reactors able to reproduce industrial conditions. Finally, a mini scale-down platform has been proposed as a high throughput tool to investigate rapidly the usefulness of a given microbial biosensor. Local heterogeneities in mini-bioreactor have caused a decrease of GFP expression, as in scale-down reactor. The presence of GFP in supernatants was noticed and this leakage seems to be correlated with the membrane permeability. [less ▲]

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See detailModèle hybride Euler-Lagrange pour la description des hétérogénéités dans les bioréacteurs.
Delafosse, Angélique ULg; Delvigne, Frank ULg; Collignon, Marie-Laure ULg et al

in SFGP (Ed.) Récents Progrès en Génie des Procédés - N° 101 - Des procédés au service du produit au coeur de l'Europe - Actes du XIIIème Congrès de la société Française de Génie des Procédés - Du 29 Nov. au 1er Décembre 2011 - Lille Grand Palais, FRANCE (2011, November 29)

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See detailStratégies de production de 6-amyl-α-pyrone produit par Trichoderma spp. par culture en milieu semi-solide aspergé
Musoni, Michel ULg; Delvigne, Frank ULg; Destain, Jacqueline ULg et al

Conference (2011, November 29)

La biosynthèse de la 6-amyl-α-pyrone (arôme de noix de coco) à partir de l'espèce de Trichoderma a été étudiée dans différents bioréacteurs. L’étude compare la production du volatile dans un réacteur ... [more ▼]

La biosynthèse de la 6-amyl-α-pyrone (arôme de noix de coco) à partir de l'espèce de Trichoderma a été étudiée dans différents bioréacteurs. L’étude compare la production du volatile dans un réacteur classique submergé et un réacteur adapté avec plateau aspergé. La source de carbone était le glucose et l’huile de ricin, ce dernier et reconnu être le précurseur de la formation des lactones dans la biotransformation. Les milieux seront submergé et semi-solide, le volume de travail de 6, 12 et 16 litres. Il ressort des résultats obtenus au cours de l’étude que le composé aromatique est produit par la souche utilisé à partir du deuxième (133.8 mg/l) jour dans l’espace de tête et dans le milieu quand la culture est réalisé avec du glucose jusqu’au quatrième jour, alors qu’avec l’huile de ricin il est retrouvé dans le milieu uniquement (342,23 mg/l). La biomasse produite dans le réacteur de 6 l avec l’huile de ricin est de 279,6 g/l alors que pour le glucose est de 139,75, dans celui de 12 l il de 61,71 g/l avec l’huile de ricin et de 6,37 g/l avec le glucose, et celui de 16 l, 115,66 g/l et le glucose 7,4 g/l, ainsi, plus le volume est petit plus la production est meilleure. Il en va de même pour la concentration du volatile qui était de 2,42 g/l avec l’huile de ricin sur plateau et de 0,28 g/l avec le glucose. Dès lors, Il convient de noter que le système de production du volatile par le réacteur adapté avec plateau aspergé permet l’augmentation de la production de celui-ci, il présente la facilité d’aménagement, avec les contours possible, il offre la possibilité d’être extrapolable. En se référant à la possibilité de renouveler le milieu de culture en faisant circuler le milieu frais, compte tenue du fait que la biomasse est déposée sur les plateaux et qu’à la fin de culture la solution est translucide, retirable après un certain temps, il y découlerait l’amélioration de la productivité. [less ▲]

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See detailPrise en compte l'état de la membrane cellulaire sur la réponse de biocapteurs fluorescents pour la détection de défaut d'écoulement au niveau des bioréacteurs : vers une intégration du "sécrétome" dans la problématique de l'extrapolation
Delvigne, Frank ULg; Brognaux, Alison ULg; Gorret, Nathalie et al

Conference (2011, November 29)

Malgré les nombreuses approches envisagées à ce jour, la dynamique du stress microbien en conditions de culture intensive (procédé fed-batch) est encore un aspect mal maîtrisé. Au cours de ce travail ... [more ▼]

Malgré les nombreuses approches envisagées à ce jour, la dynamique du stress microbien en conditions de culture intensive (procédé fed-batch) est encore un aspect mal maîtrisé. Au cours de ce travail, deux biocapteurs microbiens basés sur le principe de la protéine fluorescente verte (Green Fluorescent Protein ou GFP) ont été mis en œuvre dans des réacteurs scale-down permettant de reproduire les défauts d'écoulement rencontrés dans les bioréacteurs industriels. Les promoteurs de stress associés à la synthèse de la GFP ont été choisis suivant leur sensibilité à la limitation en source de carbone, condition standard qui est rencontrée dans les processus fed-batch ou l'apport en substrat carboné est limité afin d'éviter des déviations du métabolisme microbien (promoteurs rpoS et csiE). Les résultats obtenus montrent clairement que l'exposition des biocapteurs à des hétérogénéités locales de substrat entraîne une diminution de l'expression de la GFP. L'intensité de fluorescence a été mesurée à l'échelle cellulaire par cytométrie en flux. Durant les cultures, une chute significative du niveau de GFP intracellulaire a été observée pour les deux conditions scale-down considérées et pour les deux types de promoteur. Cette chute de fluorescence peut être attribuée à des phénomènes de répression des promoteurs suite à la levée locale de la limitation en carbone, mais également au relargage de la GFP dans le milieu extracellulaire. Ce relargage a été observé dans toutes les conditions opératoires considérées, comme le montrent les analyses des surnageants de culture par SDS-PAGE. L'intensité du relargage est néanmoins plus forte dans les conditions standard de culture (c'est-à-dire dans des réacteurs classiques sans approche scale-down). En effet, la coloration par l'iodure de propidium des biocapteurs cultivés en conditions standards est plus élevée que dans les conditions scale-down, suggérant une perméabilité membranaire plus élevée. Ces résultats offrent des potentialités intéressantes pour l'analyse simultanée de la viabilité cellulaire et de la détection des défauts d'écoulement par l'emploi de biocapteurs microbiens. De plus, les analyses par SDS-PAGE ont montre une grande diversité de protéines relargée en cours de procédé. Cette observation est étonnante du fait de l'emploi de milieux minéraux définis dans toutes les conditions étudiées. L'analyse de ce sécrétome offre des potentialités intéressantes pour la caractérisation des conditions de stress encourues par les micro-organismes au cours des procédés. [less ▲]

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