Couplage hydrodynamique-biomasse dans les procédés de dépollution. Approche locale des mécanismes de croissance et d'adhésion/détachement de micro-organismes sur substrats solides

Cette thèse a pour objectif d'apporter une meilleure compréhension des mécanismes qui gouvernent le bon fonctionnement et les performances des procédés de dépollution à biomasse fixée et d'en développer leur modélisation. Ces procédés pourraient voir leur efficacité intensifiée si les couplages entre les divers mécanismes locaux qui les gouvernent étaient mieux compris. L'interaction forte écoulement / biofilm dans ces procédés rend très difficile leur modélisation sans des progrès drastiques dans la compréhension de phénomènes intervenant à diverses échelles (biofilm, pore, bioréacteur). En conséquence, un des premiers verrous à lever est d'apporter une meilleure compréhension des mécanismes locaux responsables de l'adhésion, du détachement et de la croissance de micro-organismes sous écoulement. Dans ce but une chambre d'écoulement a été mise au point pour permettre l'observation microscopique et la caractérisation in-situ de ces phénomènes sous conditions hydrodynamiques contrôlées. Le système étudié est une bactérie de Pseudomonas putida et le polluant modèle est du phénol. En conditions non limitantes, nous montrons que les paramètres de la loi de Monod, pour les instants initiaux de croissance du biofilm et les conditions hydrodynamiques en régime très diffusif, sont dépendants du cisaillement imposé, ce qui n'est pas pris en compte dans la plupart des modèles. Des expériences mettant en œuvre l'observation de la croissance du biofilm sous écoulement (à bas Reynolds) ont ensuite permis de montrer la nature hétérogène de la structure du biofilm (structures filamenteuses, distribution de protubérances sur le support solide). Ces structures pourraient entre autre expliquer comment la croissance du biofilm influence le frottement. Pour étudier l'influence de la microstructure sur cette quantité, une technique de reconstruction 3D du biofilm a été développée et mise en œuvre en complément de la microscopie optique directe. La simulation de l'écoulement dans la microstructure ainsi reconstituée et l'ordre de grandeur des perméabilités calculées montrent bien l'importance de la distribution locale de la biomasse sur ce paramètre.The biofilms, mainly composed of micro-organisms and exopolymers, develop themself on nonsterile wet surfaces. They are of considerable importance in many industrial and environmental applications, among which biofilters used in water treatment. The strong interaction between the flow and the biofilm development in this type of processes returns very difficult their modelling without drastic progress in the comprehension of phenomena appearing on various scales (biofilm, pore, biofilter). This thesis aims to bring a better comprehension of the mechanisms which control the biofilm growth on a local scale. A flow chamber characterized by a laminar flow profile was developed to allow the in-situ observation and the analysis of cell adhesion, detachment and the growth of P. putida bacteria under sheared flow. The results also showed that the growth kinetics measured in batch was not applied, for low Reynolds number in the case of a biomass fixed to solid support and subjected to a shear stress. The study revealed also, as already shown before in certain research tasks, the biofilms organization in response to the sheared flow. The technique of 3d-reconstruction developed and implemented in complement to the direct optical microscopy allowed a better interpretation of global biofilm architecture and have explained how the microstructure can influence the biofilm friction toward fluid flow. We have simulated the distribution of the local velocity profiles in biofilm microstructure and our estimation of permeability has highlighed the importance of local distribution of biomass in this parameter.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Couplage hydrodynamique - biofilm en milieu poreux (application aux biofiltres)

Les biofiltres sont des milieux poreux saturés contenant un matériau support permettant la croissance d'une biomasse épuratrice sous forme de biofilm. La croissance du biofilm conduit à un colmatage progressif du biofiltre, entraînant des modifications de la structure interne du milieu poreux. Ceci entraine des variations de performances du bioréacteur. En vue de la compréhension et de l'optimisation de ces procédés, les couplages entre l'hydrodynamique et la croissance de la biomasse ont été étudiés expérimentalement dans cette thèse. Un pilote expérimental et des méthodes d'analyses originales ont ainsi été mis au point, permettant la reconstruction de l'historique du fonctionnement du système à partir de l'état stationnaire. Les résultats expérimentaux ont montré, entre autre, que la microstructuration jouait un rôle dans l'évolution de la perméabilité du milieu. Toutefois, des modèles de type Vandevivere permettent de s'affranchir de la connaissance complète de cette microstructure : testé dans un modèle 1D "simple", on montre qu'un tel modèle de perméabilité permet de rendre compte de manière satisfaisante des couplages et de prédire les mesures faites sur le pilote.Biotilter are saturated porous media fi lied with a materiaL which allows biomass growth as a biotilm. This growth leads to a graduai clogging of the bioreactor, modification of its internai structure and loss of performances. '1'0 get a better understanding and optimize these processes, the coupling between hydrodynamic and biomass growth was studied du ring this thesis. An experimental setup was bui]d and an original data analysis methodology was deve]oped : in particular, a complete know]edge of the temporal behaviour of the bioreactor cou]d be reconstructed fl'om the steady state. Among ail the experimental results, it was shown that the biomass microstructuration played a great raIe in the permeabi]ity loss and that the Vandevivere model was the most adapted to account for the experimenta] results. The Vandevivere model was tested in a very simple 10 dimensional mode! to simulate the bioreactor. The numerical results proved to predict tàirly weil the experÎ"1nenta] data. This mode] paves the way for the design of efficient research and engineering too]s to account for these comp]ex multiphysic systems behaviour without a detailed knowledge of the biomass structuration at the pore scale.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Natural molecules induce and synergize to boost expression of the human antimicrobial peptide β-defensin-3

International audienceAntimicrobial peptides (AMPs) are mucosal defense effectors of the human innate immune response. In the intestine, AMPs are produced and secreted by epithelial cells to protect the host against pathogens and to support homeostasis with commensals. The inducible nature of AMPs suggests that potent inducers could be used to increase their endogenous expression for the prevention or treatment of diseases. Here we aimed at identifying molecules from the natural pharmacopoeia that induce expression of human β-defensin-3 (HBD3), one of the most efficient AMPs, without modifying the production of proinflammatory cytokines. By screening, we identified three molecules isolated from medicinal plants, andrographolide, oridonin, and isoliquiritigenin, which induced HBD3 production in human colonic epithelial cells. This effect was observed without activation of the NF-κB pathway or the expression of associated proinflammatory cytokines. We identified the EGF receptor as the target of these compounds and characterized the downstream-activated MAPK pathways. At the chromatin level, molecules increased phosphorylation of histone H3 on serine S10 and recruitment of the c-Fos, c-Jun, and Elk1 or c-Myc transcription factors at the HBD3 promoter. Interestingly, stimulating cells with a combination of andrographolide and isoliquiritigenin synergistically enhanced HBD3 induction 10-fold more than observed with each molecule alone. Finally, we investigated the molecular basis governing the synergistic effect, confirmed our findings in human colonic primary cells, and demonstrated that synergism increased cellular antimicrobial activity. This work shows the capability of small molecules to achieve induction of epithelial antimicrobial defenses while simultaneously avoiding the deleterious risks of an inflammatory response

Alexandrium ostenfeldii growth and spirolide production in batch culture and photobioreactor

International audienceGrowth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 degrees C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 degrees C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 degrees C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied. This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 degrees C than at 22 C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell(-1)) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x(-0.858). Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29-46%), followed by SPX-D (21-28%), 13-desMeC (21-23%), and 13-desMeD (17-21%). Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 x 10(4) cells mL(-1). As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x(-0.8986). In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60-87%) and lower proportions of SPX-D (3-12%) and 13-desMeD (1.6-10%) as compared to batch culture

Measurement accuracy of a mono-fiber optical probe in a bubbly flow

The measurement accuracy of a mono-fiber optical probe is studied experimentally using isolated bubbles rising freely in a still liquid. The dwell time of the probe tip within the gas phase, which is obtained from both the optical probe signal and high-speed visualization, is compared with the value expected for a non-perturbed bubble. The difference originates mainly from the intrusive nature of the optical probe, which modifies the bubble behavior when it comes into contact with the probe tip. This interaction increases the dwell time if the bubble is pierced by the probe near its pole, and shortens it for piercing near the equator. The mean dwell time, obtained by averaging for various piercing locations, is shortened and the local void fraction indicated by the probe is thus underestimated. It is shown that the void fraction error can be correlated with a modified Weber number, and this correlation is helpful for sensor selection and for uncertainty estimate. In addition, the distribution of gas dwell time usually differs from the response expected for an ideal probe. This deviation results from the dependence of the dwell time error on the piercing location. The dwell time distribution can be used to infer the dependence of the dwell time on the piercing location. Finally, the deformation of long fibers during the bubble-probe interaction significantly increases the measurement error. Observed results are consistent with data of Andreotti (2009), which were measured in an airlift flow, suggesting that present results are applicable also to the case of moving liquid. Conclusions of this study could be applied also to conductivity probes or more generally to the interaction of a bubble with any kind of thin, intrusive sensor or fiber

Spirolide uptake and detoxification by Crassostrea gigas exposed to the toxic dinoflagellate Alexandrium ostenfeldii

International audienceOysters (Crassostrea gigas) were exposed 4 days to cultures of the toxic dinoflagellate Alexandrium ostenfeldii (strain CCMP1773) that produces spirolides belonging to fast acting toxins (FAT) and let depurate for 7 days. During depuration, oysters were either fed the non-toxic algae Isochrysis galbana Tahitian clone (T. Iso) or starved. The objectives of this experiment were to evaluate (i) spirolide uptake and depuration by oysters (ii) spirolide effects on oysters and (iii) oyster recovery according to food supply during depuration. A. ostenfeldii cells were filtered and ingested by oysters while faeces contained numerous intact cells of the toxic diet. This suggested that ingested cells were not totally digested by oysters. Contents of spirolides (SPX) in digestive gland and remaining tissues during contamination and detoxification periods were measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Four different SPX analogues (13,19-didesMeC, 13-desMeC, 13-desMeD and trace of SPX-D) were detected. The 13,19-didesMeC-SPX dominated in both digestive gland and remaining tissues. After four days exposure, digestive gland (DG) contained 83% of the total initial spirolide concentration, whereas remaining tissues contained only 17%. During detoxification, spirolide content in DG was lower in fed than in unfed oysters but similar in remaining tissues. Exposure to A. ostenfeldii resulted in an inflammatory response consisting of hemocyte infiltration and diapedesis into the intestinal tract of the oysters. Percentage of active digestive tubules in oysters fed A. ostenfeldii was significantly lower than in control (prior exposition) oysters (36% and 61%, respectively). At the end of the detoxification period, there was a significant difference in the percentage of active digestive tubules (P < 0.001) between fed and unfed oysters. When oysters were fed T-Iso following the A. ostenfeldii exposure, 80% of digestive tubules were active, thus revealing a rapid recovery after toxic algae exposure. Overall, both spirolide detoxification and recovery from their toxic effects are almost complete within 7 days after exposure to spirolide producers. Such information may help to resume faster oyster sales after toxic events involving FAT implying thus more frequent chemical analysis

Dinophag. Programme de recherche sur Dinophysis dans les eaux littorales des Pays de la Loire

Ce document résume les conclusions du programme de recherche Dinophag réalisé par l’Ifremer avec l’aide de la Région Pays de la Loire de janvier 2011 à décembre 2012. Il est destiné à tous ceux qui se sont interrogés sur la place que tient la microalgue toxique Dinophysis dans l’économie littorale de notre Région. Les lecteurs y découvriront aussi que Dinophysis n’est pas vraiment une microalgue comme les autres et qu’il reste encore pour les chercheurs de nombreuses interrogations sur le déterminisme de son développement et de sa toxicité

Histone deacetylase inhibition enhances antimicrobial peptide but not inflammatory cytokine expression upon bacterial challenge

International audienceSignificanceAntimicrobial peptides exert antimicrobial, antifungal, antiviral, and antiprotozoan activity. They are expressed at high concentrations at the intestinal mucosal surface, where they play a crucial role in intestinal homeostasis. Therefore, approaches aiming to boost expression of antimicrobial peptides represent a future therapeutic strategy to treat infections and dysbiosis-driven diseases in humans at a time of increasing incidence of antibiotic resistance.AbstractAntimicrobial peptides (AMP) are defense effectors of the innate immunity playing a crucial role in the intestinal homeostasis with commensals and protection against pathogens. Herein we aimed to investigate AMP gene regulation by deciphering specific characteristics allowing their enhanced expression among innate immune genes, particularly those encoding proinflammatory mediators. Our emphasis was on epigenetic regulation of the gene encoding the AMP β-defensin 2 (HBD2), taken as a model of possibly specific induction, upon challenge with a commensal bacterium, compared with the proinflammatory cytokine IL-8. Using an in vitro model of colonic epithelial cells challenged with Escherichia coli K12, we showed that inhibition of histone deacetylases (HDAC) by trichostatin A dramatically enhanced induction of HBD2 expression, without affecting expression of IL-8. This mechanism was supported by an increased phosphorylation of histone H3 on serine S10, preferentially at the HBD2 promoter. This process occurred through activation of the IκB kinase complex, which also led to activation of NF-κB. Moreover, we demonstrated that NF-κB was modified by acetylation upon HDAC inhibition, partly by the histone acetyltransferase p300, and that both NF-κB and p300 supported enhanced induction of HBD2 expression. Furthermore, we identified additional genes belonging to antimicrobial defense and epithelial restitution pathways that showed a similar pattern of epigenetic control. Finally, we confirmed our finding in human colonic primary cells using an ex vivo organoid model. This work opens the way to use epigenetic pharmacology to achieve induction of epithelial antimicrobial defenses, while limiting the deleterious risk of an inflammatory response

Expression of the human antimicrobial peptide β-defensin-1 is repressed by the EGFR-ERK-MYC axis in colonic epithelial cells

Abstract The human β-defensin-1 (HBD1) is an antimicrobial peptide constitutively expressed by epithelial cells at mucosal surfaces. In addition to its microbicidal properties, the loss of HBD1 expression in several cancers suggests that it may also have an anti-tumor activity. Here, we investigated the link between HBD1 expression and cancer signaling pathways in the human colon cancer cell lines TC7 and HT-29, and in normal human colonic primary cells, using a mini-gut organoid model. Using available datasets from patient cohorts, we found that HBD1 transcription is decreased in colorectal cancer. We demonstrated that inhibiting the Epidermal Growth Factor Receptor (EGFR) increased HBD1 expression, whereas activating EGFR repressed HBD1 expression, through the MEKK1/2-ERK1/2 pathway that ultimately regulates MYC. We finally present evidences supporting a role of MYC, together with the MIZ1 coregulator, in HBD1 regulation. Our work uncovers the role and deciphers the function of the EGFR-ERK-MYC axis as a repressor of HBD1 expression and contributes to the understanding of HBD1 suppression observed in colorectal cancer

Histone deacetylase inhibition enhances antimicrobial peptide but not inflammatory cytokine expression upon bacterial challenge

Antimicrobial peptides (AMP) are defense effectors of the innate immunity playing a crucial role in the intestinal homeostasis with commensals and protection against pathogens. Herein we aimed to investigate AMP gene regulation by deciphering specific characteristics allowing their enhanced expression among innate immune genes, particularly those encoding proinflammatory mediators. Our emphasis was on epigenetic regulation of the gene encoding the AMP β-defensin 2 (HBD2), taken as a model of possibly specific induction, upon challenge with a commensal bacterium, compared with the proinflammatory cytokine IL-8. Using an in vitro model of colonic epithelial cells challenged with Escherichia coli K12, we showed that inhibition of histone deacetylases (HDAC) by trichostatin A dramatically enhanced induction of HBD2 expression, without affecting expression of IL-8. This mechanism was supported by an increased phosphorylation of histone H3 on serine S10, preferentially at the HBD2 promoter. This process occurred through activation of the IκB kinase complex, which also led to activation of NF-κB. Moreover, we demonstrated that NF-κB was modified by acetylation upon HDAC inhibition, partly by the histone acetyltransferase p300, and that both NF-κB and p300 supported enhanced induction of HBD2 expression. Furthermore, we identified additional genes belonging to antimicrobial defense and epithelial restitution pathways that showed a similar pattern of epigenetic control. Finally, we confirmed our finding in human colonic primary cells using an ex vivo organoid model. This work opens the way to use epigenetic pharmacology to achieve induction of epithelial antimicrobial defenses, while limiting the deleterious risk of an inflammatory response