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International audience; The hollow fiber membrane bioreactor (HFMB) has been investigated for the cultivation of mammalian Chinese hamster ovary cell expansion. The experiments were carried out in Petri’s dishes and in the hollow fiber membrane bioreactor having 20 fibers (S2025 from FiberCell Systems). The approach to HFMB modelling which combines the model of cell growth kinetics and hydrodynamics has been proposed. The hydrodynamic model is made using ANSYS Fluent software. The mathematical model of HFMB was developed, allowing the study of the hydrodynamics into the lumen and the extracapillary spaces, the filtration through the membrane fiber with the cell expansion on outer membrane surface. The direct nutrient medium flow variant into the extracapillary space was suggested. Based on the numerical simulations, the optimal parameters were selected for daily changes in the medium flow-rate into the lumen space. The HFMB scaling up was performed for the larger size HFMB (60 fibers).

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The feasibility of baker's yeast production using fruits and peels of Opuntia ficus indica (OFI) as carbohydrate feedstock was investigated. Two response surface methodologies involving central composite face centered design (CCFD) were successfully applied. The effects of four independent variables on baker's yeast production from OFI fruit juice was evaluated using the first CCFD. The best results were obtained with 24 H of inoculum age, 30 degrees C temperature, 200 rpm of agitation, and 10% inoculum size. At the maximum point, the biomass concentration reached 9.29 g/L. A second CCFD was performed to optimize the sugar extraction from OFI fruit peels. The potential of these latter as a fermentation substrate was determined. From the experimental results, the OFI fruit peel is an appropriate carbon source for the production of baker's yeast. The maximum biomass concentration was 12.51 g/L. Different nitrogen supplements were added to promote the yields of baker's yeast. Corn steep liquor was found to be the best alternative nutrient source of casein hydrolysate and yeast extract for baker's yeast production. info:eu-repo/semantics/publishedVersion

National audience; Les aidants familiaux jouent un rôle central auprès des patients présentant un cancer de stade avancé. Parmi les proches susceptibles d’apporter une aide aux personnes malades, les conjoints, en raison des liens qui les unissent et parce qu’ils partagent leur vie quotidienne, sont sans doute les plus confrontés à la maladie. Ils sont également majoritairement désignés comme étant l’aidant principal. L’irruption de la maladie et le besoin d’accompagnement qu’elle nécessite, viennent bouleverser en profondeur les différents équilibres structurant leur quotidien et leur organisation. Ils viennent aussi questionner la nature des relations qu’ils entretiennent, dans une diversité d’espace-temps soumise à une régulation précise et rigoureuse permettant la stabilité des activités sociales de ses membres. L’aide à apporter n’étant jamais entièrement prévisible ni planifiable, les conjoints doivent constamment adapter leurs comportements, modifier leur(s) rôle(s) et leur aide en fonction des évolutions de la maladie, de l’état de santé et du vécu du proche malade. Ils doivent donc apprendre à devenir « compétents » et tenter de tirer parti de combinaisons appropriées de ressources, qu’elles soient internes ou externe au couple. S’interroger, à partir d’une enquête qualitative, sur ces aspects organisationnels et structurels de l’aide apportée par les conjoints nous a semblé important dans cette étude car la nature de ces choix et leurs conséquences peuvent considérablement venir renforcer, ou au contraire affaiblir, le degré d’implication des aidants, leur potentielle reconnaissance vis-à-vis de leur rôle d’accompagnement ainsi que leur propre état de santé et leur qualité de vie. L’objectif de notre analyse était donc d'identifier les compétences mobilisées, développées ou réactualisés par les conjoints de patients présentant un cancer du côlon, ainsi que les ressources associées dans lesquelles ils choisissent ou non de puiser.

IF 5.008 (2015/2016); International audience; Until recently, the molecular diagnosis of hereditary breast and ovarian cancer (HBOC) was mostly based on BRCA1/2 testing. Next generation sequencing and the recent discovery of new genes involved in HBOC now permit the transfer of genomic capture targeting multiple candidate genes from research to clinical use. However, the implications for the management of patients and their families have not been extensively studied, in particular since some of these genes are not well-established cancer predisposing genes. We studied 583 consecutive patients from Burgundy (France) fulfilling the criteria for BRCA testing using a next generation sequencing 25-genes panel including 20 well-established high-risk cancer genes as well as more recently identified predisposing HBOC cancer. A pathogenic BRCA1/2 mutation was found in 51 patients (9%). Besides, we found 37 pathogenic or likely pathogenic mutations in 10 different high to low-risk genes in 34 patients (6%). The most frequently mutated genes were CHEK2 (n = 12; 2%), ATM (n = 9; 1.5%), and PALB2 (n = 4; 0.6%). Three patients had a mutation in two different predisposing genes. The analysis of clinical actionability conducted in mutation-positive individuals revealed that additional disease-specific screening and/or prevention measures beyond those based on personal and family history alone had been recommended in 69% of cases. In conclusion, multigene panel testing is a powerful tool to identifying high to low-risk HBOC susceptibility genes. The penetrance and spectrum of cancers with these other genes are sometimes undefined, and further collaborative work is crucial to address this question.

International audience; Waste valorization practices have attracted a significant amount of attention in recent years with the aim of managing waste in the most sustainable way. Food waste constitutes a largely under-exploited residue from which a variety of valuable chemicals can be derived. The operation in global yoghurt market provokes frequently the reject and withdrawn of yoghurt derivates out of shelf life. This work comprises a contribution on the valorisation of this high polluting waste of the dairy industry, based on the production of lactic acid using residual yoghurt whey as raw material. Response surface methodology (RSM) based on central composite design (CCD) was used to evaluate the effects of fermentation parameters for lactic acid production by Lactococcus lactis subsp. in batch experiments. The critical factors selected for the investigation were: sugars concentration (X1), yeast extract concentration (X2) and inoculum size (X3). The experimental results were fitted with a second order polynomial equation by a regression analysis and 93% of the variation could be predicted by the model. A maximum lactic acid production (6.80 g/l) was obtained under following optimal conditions: sugars concentration 57 g/l, yeast extract concentration 10.8 g/l and inoculum size 10 %. Moreover, the maximum of lactic acid production predicted by the model was 6.61 g/l. These results confirmed the validity of the model, and the experimental values were quite close to the predicted values

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International audience; o improve glutamic acid production from Corynebacterium glutamicum 2262 using date juice, a culture medium was screened and optimized using the statistical experimental designs of Plackett-Burman and response-surface methodology. In the first step, a two-level Plackett-Burman design was adopted to select the most important nutrients influencing the glutamic acid production, which showed that the date juice sugars, urea, peptone, and glycine betaine were the most significant ingredients (P < 0.05). Finally, response surface Box-Behnken design was employed to develop a mathematical model to identify the optimum concentrations of key components for higher glutamic acid production, which revealed the following: date juice (45 g/L), urea (16.9 g/L), peptone (15 g/L), and glycine betaine (12 g/L). The high correlation between the predicted and observed values indicated the validity of the model. Glutamic acid concentration increased significantly with optimized medium (33.2 g/L) when compared with non-optimized medium (12 g/L).

International audience; Two hundred years ago, Henri Braconnot described a polysaccharide containing a substantial percent of nitrogen, later to be called chitin: that discovery stemmed from investigations on the composition of edible mushrooms and their nutritional value. The present interdisciplinary article reviews the major research topics explored by Braconnot, and assesses their importance in the light of our most advanced knowledge. Thus, the value of fungi, seafoods and insects is described in connection with the significance of the presence of chitin itself in foods, and chitinases in the human digestive system. The capacity of chitin/chitosan to depress the development of microbial pathogens, is discussed in terms of crop protection and food preservation. Other topics cherished by Braconnot, such as the isolation of pectin from a large number of plants, and inulin from the Helianthus tubers, are presented in up-to-date terms. Acids isolated from plants at that early time, led to enormous scientific advancements, in particular the glyoxylic acid and levulinic acid used for the preparation of soluble chitosan derivatives that paved the way to a number of applications. An opportunity to trace the origins of the carbohydrate polymers science, and to appreciate the European scientific heritage.

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International audience; This study describes the biodegradation of phenanthrene in aqueous media in the presence and in the absence of a surfactant, Brij 30. Biodegradations were performed using either Pseudomonas putida DSMZ 8368 or a bacterial consortium Pyr01 isolated from one PAHs-polluted site. P. putida degraded phenanthrene to form 1-hydroxy-2-naphthoic acid (1H2Na) as the major metabolite. LC-MS analysis revealed the production of complementary intermediates in the presence of Brij 30, showing intense ions at mass-to-charge ratios (m/z) 97 and 195. Higher phenanthrene biodegradation rate was obtained in the presence of Brij 30. Conversely, in the case of Pyr01consortium, the addition of Brij 30 (0.5 g L-1) had a negative effect on biodegradation: no phenanthrene biodegradation products were detected in the medium, whereas a production of several intermediates (m/z 97, 195 and 293) was obtained without surfactant. New results on phenanthrene metabolism by P. putida DSMZ 8368 and Pyr01 consortium in the presence and in the absence of Brij 30 we obtained. They confirm that the knowledge of the effect of a surfactant on bacterial cultures is crucial for the optimization of surfactant-enhanced PAHs biodegradation.

Introduction: Notre étude avait pour but d'évaluer la politique de préservation laryngée pratiquée à Nancy depuis 1995.Patients et méthodes: notre étude porte sur l'analyse rétrospective de 116 patients. Nous avons voulu comparer 3 stratégies de traitement: deux stratégies séquentielles par chimiothérapie d'induction type cisplatine-5FU (PF) ou docétaxel-cisplatine-5FU (TPF) suivie d'une radiothérapie (associée ou non a unechimiothérapie ou à du cetuximab de facon concomitante) chez les bons répondeurs et une troisième stratégie de radiothérapie et de chimiothérapie (PF) alternées. Les patients mauvais répondeurs lors du bilan d'évaluation devaient alors bénéficier d'une (pharyngo-)Iaryngectomie totale suivie d'une radiothérapie postopératoire. L'objectif primaire était le taux de survie sans dysfonctionnement laryngo-cesophagien (SDLO) et parmi les objectifs secondaires nous avons étudié le taux de préservation laryngée et la survie globale.Résultats: Le groupe TPF a compris 21 patients, le groupe PF 75 patients et le groupe radiothérapie et chimiothérapie alternées 20 patients. La SDLO concernant toute la population a été de 35,6% à 5 ans. Elle a été meilleure dans le groupe TPF avec 68,7% de SDLO à 2 ans versus 44,5% dans le groupe PF et 27,5% dans le groupe radiothérapie et chimiothérapie alternées avec une différence significative entre les 3 groupes (p=0,009). Le taux de préservation laryngée a été de 59% à 3 ans pour toute notre population et semble meilleur dans le groupe TPF, mais sans différence statistiquement significative. Il n'y a pas de différence statistiquement significative entre les 3 groupes concernant la survie globale.Conclusion: Les résultats de notre étude sont superposables à ceux de la littérature. Le schéma d'induction par TPF suivi chez les bons répondeurs d'une radiothérapie exclusive peut être considéré comme le meilleur dans un objectif de préservation laryngée

International audience; In the present study surfactant addition with the help of either a mechanical dispersion or a thermal treatment was applied in order to increase the solubility and the bioavailability of phenanthrene in aqueous media, and therefore to promote its biodegradation. Among four tested surfactants (Tween 80, Brij 30, sodium dodecyl sulphate and rhamnolipids), Brij 30 (0.5 gL(-1)) showed the best results allowing us to attain about 20 mgL(-1) of soluble phenanthrene. An additional thermal treatment at 60°C for 24h, 200 rpm permitted to increase the solubility of phenanthrene in the presence of Brij 30 (0.5 gL(-1)) to about 30 mgL(-1). Higher dispersions of phenanthrene particles as well as the reduction of their size were obtained using Ultra-Turrax and French press. The biodegradation of phenanthrene by Pseudomonas putida was then investigated. The reduction of size of phenanthrene particles by mechanical dispersion did not influence its biodegradation, suggesting that P. putida consumed only soluble phenanthrene. The addition of Brij 30 (0.5 gL(-1)) permitted to obtain more phenanthrene metabolized. The use of Brij 30 coupled with a transitory heating of phenanthrene-containing medium at 60°C led to an even more complete biodegradation. This might be a promising way to enhance biodegradation of PAHs.

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The galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) catalyzes the transfer of glucuronic acid from UDP-alpha-D-glucuronic acid onto the terminal galactose of the trisaccharide glycosaminoglycan-protein linker region of proteoglycans. This enzyme plays a key role in the process of proteoglycan assembly since the completion of the linkage region is essential for the conversion of a core protein into a functional proteoglycan. To investigate the enzymatic properties of human GlcAT-I, we established an expression system for producing a soluble form of enzyme in the methylotrophic yeast Pichia pastoris and developed a three-step purification procedure using a combination of anion exchange, cation exchange and heparin chromatographies. This procedure yielded 1.6 mg homogeneous enzyme from 200 ml yeast cell culture, with a specific activity value of 1.5 micromol/min/mg protein. Analysis of the specificity of GlcAT-I towards Galbeta1-3Gal and Galbeta1-4GlcNAc derivatives known as substrates of the beta1,3-glucuronosyltransferases, showed that the enzyme exhibited a strict selectivity towards Galbeta1-3Gal structures. Thus, the large source of purified active enzyme allowed the determination of the kinetic parameters of GlcAT-I towards the donor substrate UDP-GlcA and the acceptor substrate digalactoside Galbeta1-3Gal.

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Alkaline phosphatases (APs) are non-specific phosphomonoesterases which are of interests for basic research as well as for practical applications. So far, Pyrococcus abyssi AP is the only AP which was isolated from a hyperthermophilic archaeon. In the present work, expression of this enzyme using mesophilic hosts was investigated. When P. abyssi AP was produced by Escherichia coli using the regulation of the T7 promoter, only 0.6 mg of recombinant enzyme per liter of culture were obtained. Using the same system with the co-expression of tRNAs related to the rare codons AGA, AGG and ATA, the expression level was 14-fold higher. The methylotrophic yeast Pichia pastoris was also used to produce P. abyssi AP. A recombinant pPICZαA was constructed in order to have the AP secreted in the culture medium. A Pc. pastoris cell line producing P. abyssi AP was studied in 1.2 l working volume fermentor. Secretion was achieved with low expression yield but the recombinant enzyme appeared more active than when produced in E. coli .

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Date-coat sugar extract and date-seed hydrolysate were utilized as the main constituents of a medium for the production of a thermophilic dairy starter strain. Date-coat sugar extract was used as the carbon source, while date-seed hydrolysate was used as the nitrogen source. A suitable concentration of date-coat sugar was in the range of 50 mg sugar/ml. Addition of various amounts of date-seed hydrolysate as the sole nitrogen source in the medium showed that, in spite of a nitrogen insufficiency found in the hydrolysate, the production of the starter strain increased with date-seed hydrolysate (nitrogen) concentration, but the maximum production of biomass was less than that observed with other nitrogen sources. Therefore, various amounts of urea were added and a mixture of urea (6 mg/ml) and of date-seed hydrolysate (4.0 mg/ml) allowed an increase in the concentration of the biomass. The addition of date-seed ash as a mineral source, at a concentration of 1.0 mg/ml in the medium containing date-coat sugar extract, date-seed hydrolysate, and urea could substitute for MgSO4, and MnSO4 of the usual medium. This medium gave the maximum production of the thermophilic lactic acid bacteria (0.57 mg/ml) and lactic acid (2 mg/ml), very close to what was obtained with the Elliker broth medium.

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This work was an approach to waste date products valorization through biomass production with the yeast Saccharomyces cerevisiae. The carbon and nitrogen sources of a semi-synthetic fermentation medium were substituted by date-coat (fleshy part) sugar extract, date-seed hydrolysate, and ammonium nitrate. This modified medium was enriched with date-seed ash and date-seed lipid. Date-coat sugar extract as a carbon source was found to be satisfactory at a concentration of 25 g/l (expressed as its glucose concentration) and date-seed hydrolysate as a nitrogen source was equally suitable at 25 g/l. The addition to the medium of 1·0 g/l ammonium nitrate increased the efficiency of yeast biomass formation, as did phosphorus, which produced a maximum when the medium was supplemented with about 6·0 g/l KH 2 PO 4 . The presence of 1 g/l date-seed lipid in the medium also increased the efficiency of biomass formation. Finally, the addition of date-seed ash (0·6 g/l), as a mineral source, to the fermentation medium could substitute for MgSO 4 and CaCl 2 of the semi-synthetic medium.

Analysis of amino acids on samples of a semi-synthetic medium containing casamino acids, on which an Escherichia coli strain (a genetically engineered one) has been cultivated, allowed the quantification of the amino acid utilization by this strain during batch and continuous cultures. In batch culture the main results were that the observed utilizations corroborated what is known about the transport of amino acids by E. coli . Indeed, amino acids are transported by common or specific carriers as described in the literature. In continuous cultures the amino acid utilization in terms of probable destination, either constitutive or energy source is studied. The following results were obtained. Amino acid utilizations (and corresponding rates) vary with glucose availability. Amino acids are differently distributed in the cell. Three classes exist: mainly protein amino acids, mainly energy amino acids, and slightly imported amino acids. Glucose availability has an influence on this distribution. When it increases, the energy utilization ratio decreases. An optimum for glucose availability conditions with regard to the recombinant protein produced by the E. coli strain used is confirmed. The results obtained during the continuous cultivations corroborate the hypothesis concerning the amino acid carriers made during the batch cultivations and confirm the fact that the mainly energy amino acids are the most rapidly utilized.

The present results concern the recombinant bacteriaEscherichia coli HB101(GAPDH) which produces glyceraldehyde 3-phosphate dehydrogenase. An unusual phenomenon was noticed concerning the plasmid stability of this strain growing in batch culture. The determination of sensitivity to ampicillin, whose resistance is carried by the plasmid, has been tested as a function of time on Petri dishes containing increasing concentrations of ampicillin in a batch culture in a complex medium without any selection pressure. A transitory decrease in the percentage of resistant cells has been noted during the exponential phase of growth. This phenomenon corresponds to a momentary plasmid instability probably due to a transitory gap between the growth rate of the cell and the duplication rate of the plasmid.

The kinetics of growth, substrate consumption and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) production by Escherichia coli C600 galK (GAPDH) have been studied. Using the corresponding observations, an unstructured physiological model has allowed description of the behaviour of the recombinant strain in batch culture on a selective complex medium. This model is characterized by two physiological states (glucose and acetic acid consumption) and is based on the Monod equation applied to the growth rate combined with inhibition by acetate. Since the strain used is genetically engineered, it also incorporates the probability of plasmid loss which has been modelled previously. The model has been applied to a set of batch cultures at varying initial glucose concentrations. According to the simulation results, the model provides an excellent fit to the experimental results.

Experimental results were obtained withEscherichia coli C600 galK (GAPDH), a genetically engineered strain that synthetizes a large quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (80 % of the soluble proteins). Data concerning the stability of plasmid-containing cells and gene expression as a function of dilution rate have been obtained in continuous cultures. Contrary to other studies, our results show a clear indication that the rate of the recombinant activity was dependent on dilution rate. The results support the finding that the apparent stability of the plasmid decreases with dilution rate.

A numerical method to process experimental data concerning plasmid stability of a recombinant bacteria during continuous cultures with nonselective media is proposed here. This method differs from previous ones in that it uses the derivatve form of the state equation of the Imanaka-Aiba model for recombinant cultures. The methodology proposed here allows one to estimate values for the two model parameters without forcing them to be constant. Until now, this could not be done using classical analytical techniques because these parameters have been considered invariable because of the integration used in the evaluation of the model. These parameters are (1) the difference in the specific growth rates between plasmid-carrying cells and plasmid-free cells (deltamu), and (2) the probability of plasmid loss by plasmid-containing cells (rho(r) mu(+)). The derivative technique used here is completed by mathematical treatments involving data filtering and smoothing. The values of the two parameters are in agreement with those already published. The current technique does not impose preconditions and permit us to further study related phenomena.

The influence of complex compounds on the growth of a recombinant strain of Escherichia coli containing the gene encoding glyceraldehyde 3-phosphate dehydrogenase, as well as the production of this enzyme have been studied. Batchwise cultures led to an accumulation of acetate, which was not utilized in a yeast extract-free medium. After glucose exhaustion, growth stopped and enzyme activity decreased. Whereas yeast extract allowed acetate assimilation and growth, peptone stabilized the enzymatic activity. The addition of both compounds resulted in optimal performances for enzyme production.

Four control techniques applied to fed-batch fermentations of glucose by Candida utilis CBS 621 are presented. Respiratory quotient (RQ), residual glucose concentration (RGC) and glucose feed rate (GFR) have been used as control tools. The best control strategy, to optimize the growth of C. utilis , was obtained after taking into account RQ, RGC and GFR simultaneously.

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Enzymes were immobilized on a magnetic support by a cocross-linking method with an inactive protein. Active porous particles obtained were used in a fluidized bed reactor. Outlet product concentration was measured as a function of a flow rate and inlet substrate concentration. No enzyme leakage was observed and a circular magnet surrounding the column was shown to be an efficient device for the retention of fine particles.

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Due to the loss of enzymatic activity as a function of time, an alkaline protease, selected for the continuous preparation of protein hydrolysates (J. Boudrant and C. Cheftel, Biotechnol. Bioeng., 18, 1735, 1976), was chemically stabilized by a simple treatment with glutaraldehyde. Two fractions, soluble and insoluble, were obtained. The activities of these two fractions were measured with casein and N-benzoyl-L-arginine ethyl ester (BAEE) as a function of glutaraldehyde concentration used. It was noted that the insoluble fraction was practically inactive with the first substrate and that the heat stability of the soluble form was likewise enhanced. Molecular weights of these two forms were unchanged, but the uv-spectrum of the soluble form was modified. From amino acid analysis, it appears that this treatment mainly provokes a decrease in lysine content.

Summary Continuous hydrolysis of sucrose solutions by yeast invertase has been studied in a continuous stirred tank reactor (C.S.T.R.) limited by an ultrafiltration membrane capable of selectively retaining the enzyme. o 1. Initially, studies were made of the kinetics of batch sucrose hydrolysis and of invertase stability, under conditions applicable to a continuous process, in particular at high sucrose concentrations. 2. Ultrafiltration studies showed that various membranes retain invertase selectively and remain permeable to solutions of hydrolysed sucrose. The flux of the products of hydrolysis was found to be maximum when the concentration of the solution was close to 50 p. cent. 3. Studies of continuous hydrolysis without enzyme retention showed that the steady state ratio of sucrose hydrolysis may be predicted from the kinetics of the batch enzymatic reaction and from the substrate material balance in a C.S.T.R. 4. Studies of continuous hydrolysis with enzyme retention through ultrafiltration showed that invertase reutilisation for 5 days, at 50°C, does not lead to any detectable loss of enzyme activity. Under the conditions adopted, the ratio of sucrose hydrolysis remained constant, and close to 90 p. cent.

Accès restreint aux membres de l'Université de Lorraine jusqu'au 2014-12-12; The objective of this work is the study of Corynebacterium glutamicum metabolism, and of its potentialities, during an aero-anaerobic process. After a first phase during which the oxygen was supplied to favor the bacterial growth, the anaerobic phase was induced by the stopping of the oxygen supply and the decreasing of the agitation speed. In these culture conditions, lactate was the main metabolite produced. The production of this organic acid has been increased by modifying the transition time between the aerobic and the anaerobic phases. C. glutamicum 2262 was able to produce up to 27 g/l lactate during a batch process and up to 55 g/l during a fed batch process. To exploit the lactic acid synthesis pathway of C. glutamicum for ethanol production, the PDC and ADH genes from Zymomonas mobilis were expressed under the control of the endogenous promoter of ldhA, in the wild-type strain and in a ldhA-disrupted strain of C. glutamicum 2262. Although the ethanol productivities of these engineered strains were relatively low, the depletion of ldhA resulted in the increases of ethanol final concentration up to 15 times. A similar strategy was applied for the production of itaconate. As previously for the ethanol production, the final concentration of itaconate remained very low despite of some modifications of the process; L'objectif de cette thèse est l'étude du métabolisme de Corynebacterium glutamicum, et de ses potentialités, au cours de procédés aéro-anaérobies. Après une première phase avec apport d'oxygène pour permettre la croissance bactérienne, une phase anaérobie est induite par arrêt de l'aération et réduction de la vitesse d'agitation. Dans ces conditions, le lactate est le principal métabolite produit. La synthèse de ce dernier a été améliorée en jouant, essentiellement, sur le moment de la transition entre les 2 phases. C. glutamicum 2262 peut ainsi produire 27 g/l de lactate en mode discontinu et 55 g/l en mode semi-continu, suite à un arrêt de l'aération lorsque la concentration en biomasse est d'environ 2,6 g/l. Afin d'exploiter la voie de synthèse d'acide lactique chez C. glutamicum pour la production d'éthanol, les gènes PDC et ADH de Zymomonas mobilis ont été exprimés sous le contrôle du promoteur ldhA endogène de C. glutamicum 2262 et d'une souche de C. glutamicum 2262 sans ldhA. Bien que les productivités en éthanol de ces souches aient été relativement faibles, la suppression de ldhA a entraîné des augmentations de la concentration en éthanol d'environ 15 fois. Une stratégie similaire a été utilisée pour la production d'itaconate. Comme dans le cas de l'éthanol, la concentration en itaconate obtenue est demeurée très faible malgré des essais d'amélioration du procédé de mise en oeuvre de la souche productrice d'itaconate

The objective of this work is the study of Corynebacterium glutamicum metabolism, and of its potentialities, during an aero-anaerobic process. After a first phase during which the oxygen was supplied to favor the bacterial growth, the anaerobic phase was induced by the stopping of the oxygen supply and the decreasing of the agitation speed. In these culture conditions, lactate was the main metabolite produced. The production of this organic acid has been increased by modifying the transition time between the aerobic and the anaerobic phases. C. glutamicum 2262 was able to produce up to 27 g/l lactate during a batch process and up to 55 g/l during a fed batch process. To exploit the lactic acid synthesis pathway of C. glutamicum for ethanol production, the PDC and ADH genes from Zymomonas mobilis were expressed under the control of the endogenous promoter of ldhA, in the wild-type strain and in a ldhA-disrupted strain of C. glutamicum 2262. Although the ethanol productivities of these engineered strains were relatively low, the depletion of ldhA resulted in the increases of ethanol final concentration up to 15 times. A similar strategy was applied for the production of itaconate. As previously for the ethanol production, the final concentration of itaconate remained very low despite of some modifications of the process, L'objectif de cette thèse est l'étude du métabolisme de Corynebacterium glutamicum, et de ses potentialités, au cours de procédés aéro-anaérobies. Après une première phase avec apport d'oxygène pour permettre la croissance bactérienne, une phase anaérobie est induite par arrêt de l'aération et réduction de la vitesse d'agitation. Dans ces conditions, le lactate est le principal métabolite produit. La synthèse de ce dernier a été améliorée en jouant, essentiellement, sur le moment de la transition entre les 2 phases. C. glutamicum 2262 peut ainsi produire 27 g/l de lactate en mode discontinu et 55 g/l en mode semi-continu, suite à un arrêt de l'aération lorsque la concentration en biomasse est d'environ 2,6 g/l. Afin d'exploiter la voie de synthèse d'acide lactique chez C. glutamicum pour la production d'éthanol, les gènes PDC et ADH de Zymomonas mobilis ont été exprimés sous le contrôle du promoteur ldhA endogène de C. glutamicum 2262 et d'une souche de C. glutamicum 2262 sans ldhA. Bien que les productivités en éthanol de ces souches aient été relativement faibles, la suppression de ldhA a entraîné des augmentations de la concentration en éthanol d'environ 15 fois. Une stratégie similaire a été utilisée pour la production d'itaconate. Comme dans le cas de l'éthanol, la concentration en itaconate obtenue est demeurée très faible malgré des essais d'amélioration du procédé de mise en oeuvre de la souche productrice d'itaconate

Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants widely represented in ecosystems all over the world. The utilization of microorganisms for degradation of PAHs is an economically effective method having a great potential. The aim of the present work was to study the dissolution and the biodegradation of phenanthrene (model PAH) in the presence of surfactants. The chosen microorganisms were Pseudomonas putida DSMZ 8368 and a microbial consortium Pyr01.The first part of the thesis concerns the study of the effect of surfactants, dispersion and a thermal treatment on solubility and biodegradation of phenanthrene by P. putida DSMZ 8368. The best results were obtained by combining the use of Brij 30 surfactant with a preliminary thermal treatment. The second part presents results on the degradation of phenanthrene by Pyr01 consortium. The comparison between the biodegradation of phenanthrene by P. putida DSMZ 8368 and by Pyr01 consortium was done. Contrary to what has been observed in the case of the biodegradation by P. putida DSMZ 8368, the presence of Brij 30 (0.5 g L-1) had a negative effect on the biodegradation of phenanthrene by Pyr01 consortium. The third part of this work consisted in the study of phenanthrene solubilisation at cloud point of Brij 30. The effect of lowering the temperature to that of the culture conditions for P. putida DSMZ 8368 on the solubilization of phenanthrene was also studied.; Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants persistants très répandus dans les écosystèmes du monde entier. L'utilisation des microorganismes pour la dégradation des HAP est une méthode économique et ayant un grand potentiel. Ce travail avait pour objectif d'étudier la dissolution et la biodégradation du phénanthrène (HAP modèle) en présence de tensioactifs. Les microorganismes choisis dans ce travail sont Pseudomonas putida DSMZ 8368 et un consortium microbien Pyr01. La première partie de la thèse concerne l'étude de l'effet des tensioactifs, de la dispersion et du traitement thermique sur la solubilité et la biodégradation du phénanthrène par P. putida DSMZ 8368. Les meilleurs résultats ont été obtenus en combinant l'utilisation d'un tensioactif Brij 30 (0,5 g L-1) avec un traitement thermique préliminaire. La deuxième partie présente les résultats sur la dégradation du phénanthrène par le consortium Pyr01. La comparaison entre la biodégradation du phénanthrène par P. putida DSMZ 8368 et le consortium Pyr01 a été réalisée. Contrairement à ce qui a été observé dans le cas de la biodégradation par P. putida DSMZ 8368, la présence du Brij 30 (0,5 g L-1) a eu un effet négatif sur la biodégradation du phénanthrène par le consortium Pyr01. La troisième partie de ce travail a consisté en l'étude de la solubilisation du phénanthrène au point de trouble du Brij 30. L'effet de l'abaissement de la température jusqu'à la valeur de la température de culture de P. putida DSMZ 8368 sur la solubilisation du phénanthrène a été également étudié.

Communication présentée au Colloque Visualisation, Image, Modélisation, Nancy, France, 15-17 mai 2001; National audience; L'acquisition d'images en microscopie 3D de fluorescence repose sur l'utilisation d'un dispositif effectuant un "découpage optique" de spécimens biologiques transparents. Les déformations dues aux singularités de la fonction de transfert optique rendent indispensable une restauration des images avant toute mesure quantitative 3D. L'identification de la réponse impulsionnelle permet de préciser les protocoles d'acquisition. La connaissance du modèle de formation d'image est nécessaire à l'optimisation de la déconvolution. Différents algorithmes de déconvolution ont été implémentés, le choix des paramètres de régularisation a été automatisé. L'enjeu de ce travail est de proposer des conditions analytiques reproductibles en biologie, avec un exemple d'application sur des cellules d'adénocarcinome mammaire.

The aim of this work was to develop an efficient process of lactic acid production by Lactobacillus casei subsp. rhamnosus on date juice In a first part, the batch cultures were realized to carry out the nutritional requirement of the strain. These studies allowed us to describe the effects of carbon substrates, nitrogen substrates and vitamins on the lactic acid production. We showed that ammonium sulphate is a satisfying economic alternative and we have determined feasibility of minimizing the addition of the yeast extract by the partial use of yeast extract combined with the ammonium sulphate plus the addition of vitamins of the group B. Pure and mixed cultures of Lactobacillus casei and Lactococcus lactis were carried out. The mixed system gives better results concerning the lactic acid production and the use of the sugars compared with those obtained in pure cultures of Lactobacillus casei or Lactococcus lactis. The effect of pure sugars (glucose and fructose) and mixed sugars (glucose/fructose) on the lactic acid production was studied. The lactic acid production is more important on mixed sugars than on pure sugars what explains the performances of fermentation on date juice. In a second part, a mode of fed batch operations was defined to improve the performances of the fermentation. The two factors influencing the process are the feeding rate and the concentration of the feeding medium. In a third part, kinetic study in continuous culture was developed. We studied the influence of dilution rate on growth, substrate utilization and lactic acid production. The productivity of the continuous process was considerably increased in comparison with the batch process. Finally, a tentative model has been established for the fermentation process. The corresponding model, although not perfect, is apparently able to simulate growth rate, substrates uptake and lactic acid production in batch culture; L'objectif de ce travail a été de développer un procédé performant de production d'acide lactique par Lactobacillus casei subsp. rhamnosus sur jus de datte. Dans une première partie, des cultures en mode discontinu ont été réalisées afin d'étudier les besoins nutritionnels de la souche. Ces études ont pour but de décrire les effets des sources carbonées, azotées et les vitamines sur la production d'acide lactique. Nous avons montré que le sulfate d'ammonium est une bonne alternative économique et nous avons pu déterminer la faisabilité de minimiser l'ajout de l'extrait de levure par l'utilisation partielle de l'extrait de levure combinée avec du sulfate d'ammonium plus l'ajout de vitamines du groupe B. Des cultures pures et mixtes de Lactobacillus casei et Lactococcus lactis ont été réalisées. Le système de culture mixte donne de meilleurs résultats concernant la production d'acide lactique et l'utilisation des sucres comparés à ceux obtenus en cultures pures de Lactobacillus casei ou Lactococcus lactis. L'effet des sucres purs (glucose et fructose) et mixtes (glucose/fructose) sur la production d'acide lactique a été étudié. La production d'acide lactique est plus importante avec un mélange de sucres qu'avec des sucres non mélangés ce qui explique les performances de fermentation sur jus de datte. Dans une seconde partie, une stratégie d'alimentation du réacteur en culture semi-continue a été mise en oeuvre, permettant d'améliorer les performances de la fermentation. Les deux facteurs influençant le bon fonctionnement sont le débit et la concentration du milieu d'alimentation. Dans une troisième partie, une étude cinétique a été développée en réacteurs continus. Nous avons étudié l'influence du taux de dilution sur la croissance, l'utilisation du substrat et la production d'acide lactique. La productivité du procédé continu a été considérablement augmentée en comparaison avec le procédé discontinu. Enfin, dans une dernière partie, un modèle a été établi. Ce modèle, bien qu'imparfait, permet apparemment de simuler la croissance, la consommation des sucres de jus de datte et la production d'acide lactique en culture discontinue

Alkaline phosphatases (E.C. 3.1.3.1.) are defined as aspecific phosphomonoesterases, optimally active under alkaline conditions. Despite these enzymes are considered as ubiquitous, no alkaline phosphatase have been studied from a hyperthermophilic archeaon. The first step of the present work was to isolate the first alkaline phosphatase from such an organism: Pyrococcus abyssi, an euryarchaeon discovered in a deep-sea hydrothermal vent which grows optimally at 96°C. The phoA gene was identified on P abyssi genome, cloned and the recombinant protein was expressed in Escherichia coli. This protein was purified, confirming phoA product was an alkaline phosphatase. Optimum temperature and pH were found to be 70°C and 11.0, respectively. The enzyme showed a high thermostability with half-lives at 100°C and 105°C of 18 hours and hours, respectively. Metal dependancy was investigated, revealing its metalloenzyme nature, which is a general property of alkaline phosphatases. The second part of this project consisted in determining the quaternary structure of P. abyssi alkaline phosphatase. Several experiments, showed that the homodimer is the only active form. This quaternary structure, two subunits of 52 kDa each, appeared to be dependant of pH, buffer composition and enzyme metal content. The last part of this thesis deals with the expressipn of recombinant P. abyssi alkaline phosphatase. Investigation was performed about the expression of the enzyme by the Escherichia coli system using the T7 promoter regulation, with or without the co-expression of tRNA, related to rare codons. Recombinant P. abyssi alkaline phosphatase was also performed using the methylotrophic yeast Pichia pastoris by secretion, in a 1.2 liters working volume fermentor.; Les phosphatases alcalines (E.C. 3.1.3.1.) sont par définition des phosphomonoestérases aspécifiques, actives de façon optimale à pH alcalin. Décrites comme ubiquitaires, elles n'ont cependant jamais été étudiées chez les Archaea hyperthermophiles. La première étape de ces travaux a été l'isolement de la première phosphatage alcaline d'un tel organisme: Pyrococcus abyssi, Euryarchaeote issu d'une source hydrothermale, dont la température optimale de croissance est de 96°C. Le gène phoA, identifié sur génome de P. abyssi, a été cloné et la protéine recombinante exprimée chez Escherichia coli. Celle-ci a été purifiée, permettant de confirmer l'activité phosphatase alcaline codée par ce gène. La température et le pH permettant son activité optimale sont respectivement de 70°C et 11,0. L'enzyme a montré une grande thermostabilité, avec des demi-vies respectives à 100°C et 105°C de 18 heures et 5 heures. Une étude sur l'influence d'effecteurs métalliques a révélé sa nature de métallo-enzyme, caractéristique des phosphatases alcalines. Dans un second temps, ces travaux ont consisté à déterminer la structure quaternaire de la phosphatase alcaline de P. abyssi. Plusieurs tests ont permis d'identifier la forme homodimérique comme unique forme active. Cette structure quaternaire, deux sous-unités de 52 kDa chacune, s'est avérée dépendante du pH, de la composition du tampon et du contenu métallique de l'enzyme. Enfin, la troisième partie de cette thèse concerne l'étude de l'expression de la phosphatase alcaline recombinante de P. abyssi. Une étude a été réalisée en utilisant le système Escherichia coli et la régulation du promoteur T7, avec ou sans co-expression des ARNt relatifs aux codons rares. La protéine recombinante a également été exprimée dans la levure méthylotrophe Pichia pasto ris par sécrétion, dans un fermenteur d' 1,2 litres.

Alkaline phosphatases (E.C. 3.1.3.1.) are defined as aspecific phosphomonoesterases, optimally active under alkaline conditions. Despite these enzymes are considered as ubiquitous, no alkaline phosphatase have been studied from a hyperthermophilic archeaon. The first step of the present work was to isolate the first alkaline phosphatase from such an organism: Pyrococcus abyssi, an euryarchaeon discovered in a deep-sea hydrothermal vent which grows optimally at 96°C. The phoA gene was identified on P abyssi genome, cloned and the recombinant protein was expressed in Escherichia coli. This protein was purified, confirming phoA product was an alkaline phosphatase. Optimum temperature and pH were found to be 70°C and 11.0, respectively. The enzyme showed a high thermostability with half-lives at 100°C and 105°C of 18 hours and hours, respectively. Metal dependancy was investigated, revealing its metalloenzyme nature, which is a general property of alkaline phosphatases. The second part of this project consisted in determining the quaternary structure of P. abyssi alkaline phosphatase. Several experiments, showed that the homodimer is the only active form. This quaternary structure, two subunits of 52 kDa each, appeared to be dependant of pH, buffer composition and enzyme metal content. The last part of this thesis deals with the expressipn of recombinant P. abyssi alkaline phosphatase. Investigation was performed about the expression of the enzyme by the Escherichia coli system using the T7 promoter regulation, with or without the co-expression of tRNA, related to rare codons. Recombinant P. abyssi alkaline phosphatase was also performed using the methylotrophic yeast Pichia pastoris by secretion, in a 1.2 liters working volume fermentor., Les phosphatases alcalines (E.C. 3.1.3.1.) sont par définition des phosphomonoestérases aspécifiques, actives de façon optimale à pH alcalin. Décrites comme ubiquitaires, elles n'ont cependant jamais été étudiées chez les Archaea hyperthermophiles. La première étape de ces travaux a été l'isolement de la première phosphatage alcaline d'un tel organisme: Pyrococcus abyssi, Euryarchaeote issu d'une source hydrothermale, dont la température optimale de croissance est de 96°C. Le gène phoA, identifié sur génome de P. abyssi, a été cloné et la protéine recombinante exprimée chez Escherichia coli. Celle-ci a été purifiée, permettant de confirmer l'activité phosphatase alcaline codée par ce gène. La température et le pH permettant son activité optimale sont respectivement de 70°C et 11,0. L'enzyme a montré une grande thermostabilité, avec des demi-vies respectives à 100°C et 105°C de 18 heures et 5 heures. Une étude sur l'influence d'effecteurs métalliques a révélé sa nature de métallo-enzyme, caractéristique des phosphatases alcalines. Dans un second temps, ces travaux ont consisté à déterminer la structure quaternaire de la phosphatase alcaline de P. abyssi. Plusieurs tests ont permis d'identifier la forme homodimérique comme unique forme active. Cette structure quaternaire, deux sous-unités de 52 kDa chacune, s'est avérée dépendante du pH, de la composition du tampon et du contenu métallique de l'enzyme. Enfin, la troisième partie de cette thèse concerne l'étude de l'expression de la phosphatase alcaline recombinante de P. abyssi. Une étude a été réalisée en utilisant le système Escherichia coli et la régulation du promoteur T7, avec ou sans co-expression des ARNt relatifs aux codons rares. La protéine recombinante a également été exprimée dans la levure méthylotrophe Pichia pasto ris par sécrétion, dans un fermenteur d' 1,2 litres.

International audience; Dans cet article, nous présentons nos premiers résultats concernant la caractérisation du mouillage de la semoule de blé dur par l'eau. Les deux méthodes expérimentales appliquées à la semoule sont décrites successivement : l'ascension capillaire et la méthode de la goutte posée. Les résultats obtenus mettent en évidence que le contact de la semoule de blé et l'eau s'accompagne d'une absorption rapide sans réel étalement de surface. Ces travaux montrent également les limites de ces méthodes pour l'étude des poudres qui absorbent le liquide de mouillage. Si ce phénomène d'absorption (par sa cinétique) est important, il génère une difficulté d'analyse dans les deux cas de figure.

Parmi les méthodes de construction de plans fractionnaires, avec éventuellement un ou plusieurs systèmes de blocs, la méthode algébrique dite de la matrice clé est l'une des plus mal connues en France. Nous illustrons son intérêt au travers de deux exemples. Le premier est un plan en blocs relatif à l'optimisation de la qualité du malt, Le second est une fraction de résolution 4 pour l'étude de la nettoyabilité des surfaces utilisées dans l'industrie agro-alimentaire

Not available, L’objectif général de ce travail est l'étude du comportement en fermenteur d'une souche d'E. coli recombinée surproductrice d'une enzyme homologue, la glycéraldéhyde-3-phosphate déshydrogénase. Dans une première partie, les performances de la souche ont été testées en culture discontinue et semi-continue en fermenteur de 2 litres. Une concentration optimum de glucose pour l'expression de l'enzyme et l'action inhibitrice de l'acétate ont été mis en évidence. Dans une deuxième partie, un milieu simplifié riche en acides aminés a été utilisé. Une étude de l'utilisation de ces derniers a été réalisée en culture discontinue et continue. Les transporteurs des différents acides aminés semblent jouer un grand rôle dans leur mode d'utilisation. Les acides aminés ont été classés en plusieurs catégories et l'influence du glucose sur leur importation a été dégagée. En utilisant ce même milieu de culture nous avons montré qu'il était possible d'augmenter fortement et de façon transitoire la production de l'enzyme recombinante par des ajouts pulsés, soit de glucose, soit de serine. Ce phénomène a été étudié en détail et en particulier la transcription et l'utilisation des promoteurs. La quatrième partie est consacrée à l'étude de la stabilité plasmidique. Nous avons mis en évidence une instabilité plasmidique transitoire en culture discontinue. En culture continue, la stabilité est meilleure à de faibles taux de dilution. Enfin, dans une dernière partie, nous avons étudié l'influence du plasmide et de la production de la protéine recombinante sur le comportement de la souche hôte en culture discontinue, semi-continue et continue

chap. 6; National audience