Analyses physiologiques, biochimiques et génétiques pour appréhender la diveristé fonctionnelle des bactéries dénitrifiantes en milieu marin côtier

La dénitrification est un processus respiratoire réalisé par des bactéries hétérotrophes anaérobies facultatives dites dénitrifiantes. En absence d'oxygène, des oxydes d'azotes (nitrate ou nitrite) sont alors utilisés comme accepteurs terminaux d'électrons alternatifs et sont réduits en composés gazeux (oxyde nitrique, oxyde nitreux et azote moléculaire). La dénitrification est un processus clef du cycle de l'azote dans les sédiments marins puisqu'elle diminue la quantité d'azote disponible pour la production primaire par diffusion dans l'atmosphère. Cependant, dans zones cotières qui reçoivent d'importantes quantités d'azote d'origine anthropique, la dénitrification permet d'éliminer l'excès d'azote et par conséquent de limiter l'eutrophisation de l'écosystème. [...] La première partie de ce travail a permis de développer une méthode originale de DGGE (Denaturing Gradient Gel Electrophoresis) pour analyser la diversité fonctionnelle des bactéries dénitrifiantes via l'étude des gènes codant pour les nitrite réductases. Nous avons comparé la diversité structurale et fonctionnelle de 89 bactéries dénitrifiantes isolées à partir de sédiments et associé cette diversité aux caractéristiques biochmiques et physiologiques de ces isolats. Les résultats révèlent qu'il n'existe pas de correspondance entre les arbres phylogénétiques construits à partir des séquences du gène nirS et du gène codant pour l'ARNr 16s. Des souches taxonomiquement proches peuvent aussi présenter une physiologie et un métabolisme azoté différent. [...]Denitrification is a respiratory process mediated by heterotrophic facultative anaerobes called denitrifiers.In anaerobiosis, nitrogen oxides (nitrate or nitrite) are used as alternative electron acceptors and are reduced in gaseous compounds (nitric oxide, nitrous oxide and dinitrogen). In marin sediments, denitrification is a key process of nitrogen cycle since it limits the quantity of nitrogen available for primary production by diffusion in the atmosphere. However, in coastal zones receiving high levels of anthropogenic nitrogen, denitrification allows the elimination of nitrogen overload and consequently reduces ecosystem eutrophication. [...] The first part of this work presented an original DGGE method (Denaturing Gradient Gel Electrophoresis) developped to assess the functional biodiversity of denitrifying bacteria through the sudy of nitrite reductase encoding genes. We compared the structural and the functional diversity of 89 denitrifers isolated from sediments and we associated this diversity of 89 denitrifiers isolated from sediments and we associated this diversity with the biochemical and physiological characteristics of these isolates. Results showed that it didn' exist any relation between nirS and 16s rRNA phylogenetic trees. Moreover, some taxonomically closed strains presented different physiology and different nitrogen metabolism [...]AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Comparison of Methods for Quantification of Cytochrome cd(1)-Denitrifying Bacteria in Environmental Marine Samples

Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F–cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting

Flux d'azote dans les sédiments marins sous influence de la conchyliculture et processus microbiens associés

L excès de nitrate et d ammonium dans le sédiment côtier peut être la cause de développement phytoplanctonique important, avec potentiellement une production de toxine causant l arrêt des ventes des produits de la conchyliculture du fait de risques sanitaires et ainsi avoir des répercussions économiques négatives. L étude des processus microbiens permettant l entrée ou l élimination de l azote des écosystèmes a donc été entreprise dans deux zones côtières présentant des activités conchylicoles : le golfe de Fos et le bassin d Arcachon. Les processus impliqués dans les flux d azote ont été déterminés en utilisant une nouvelle méthode d appariement isotopique mise au point dans cette étude et prenant en compte non seulement la dénitrification mais également la nitrification et le processus d Anammox. Les calculs théoriques ont été validés par des données expérimentales. L application de cette méthode sur des sédiments de l anse de Carteau (Golfe de Fos) étudiés au cours d un cycle annuel nous a permis de montrer que la dénitrification, processus principal d élimination de l azote dans cette zone, a vu son activité multipliée par 20 au cours des dix dernières années pour atteindre, à l heure actuelle, sa potentialité maximale. A l heure actuelle, les flux sortant d azote restent supérieurs à la fixation du N2, ce qui permet l élimination de nitrate du sédiment. La nitrification et l Anammox n étant détectés qu occasionnellement, l ammonium du sédiment n est quasiment pas éliminé par les processus bactériens. Dans le bassin d Arcachon, zone conchylicole soumise à la marée, des études ont été menées au cours de deux missions, l Anammox peut constituer jusqu à 58% de la production de N2 même si le processus de dénitrification reste majoritaire dans la plupart des cas. Un impact de la marée et de l année sur la production de N2 a été remarqué sans que soit notée une variation de la structure quantitative et qualitative de la communauté dénitrifiante. De même les effectifs des procaryotes nitrifiants et fixateurs d azote n ont pas présenté de variation importante. Dans cette zone l activité fixatrice d azote est plus importante et a entraîné des flux positifs d azote en 2005. Enfin une production d oxyde nitreux intermédiaire de la dénitrification a été démontré, montrant que ce type de sédiment pouvait dans certaines conditions, être une source non négligeable de biogaz.The excess of nitrate and ammonium in coastal sediment can have as consequences an important phytoplanctonic development with potential production of toxin causing the stopping of shellfish sales due to health risks and so can have adverse economic repercussions. The study of the microbial processes allowing the entrance or the elimination of nitrogen from ecosystems was therefore undertaken in two coastal shellfish zones: Fos and Arcachon Gulf. Processes implicated in nitrogen fluxes were determined by using a new method of isotope paring developed in this study and taking into account not only denitrification but also nitrification and Anammox process. Theoretical data was validated by experiments. The application of this method on the sediment of the Carteau gulfe (Gulf of Fos) studied in the course of an annual cycle allowed us to show that the denitrification, that is the main process of elimination of nitrogen in this zone, saw its activity multiplying by 20 in the course of last ten years to reach its maximum potentiality, in present time. In present time, out going nitrogen fluxes remain higher than N2 fixation rate, having as consequence the elimination of nitrate from sediment. The nitrification and Anammox being only occasionally detected, the ammonium of sediment is not efficiently eliminated by bacterial processes. In the basin of Arcachon, shellfish zone subjected to the tide, studied in the course of two missions, Anammox could constitute up to 58 % of the production of N2 although denitrification process remained majority in most cases. An effect of the tide and of the year on the N2 production has been noticed without notable variation of qualitative or quantitative structure of the denitrifying community. In the same way, the number of nitrifying or N2-fixing prokaryotes did not show important variation. In this area, the N2-fixation activity was more intense and has lead to positive flux in 2005. A Nitrous oxide production, a by product of denitrification, has been highlighted showing this kind area, under certain condition, could emit this green house gas.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Bacterial diversity in relatively pristine and anthropogenically-influenced mangrove ecosystems (Goa, India)

To appreciate differences in benthic bacterial community composition at the relatively pristine Tuvem and the anthropogenically-influenced Divar mangrove ecosystems in Goa, India, parallel tag sequencing of the V6 region of 16S rDNA was carried out. We hypothesize that availability of extraneously-derived anthropogenic substrates could act as a stimulatant but not a deterrent to promote higher bacterial diversity at Divar. Our observations revealed that the phylum Proteobacteria was dominant at both locations comprising 43-46&#37; of total tags. The Tuvem ecosystem was characterized by an abundance of members belonging to the class Deltaproteobacteria (21&#37;), ~ 2100 phylotypes and 1561 operational taxonomic units (OTUs) sharing &gt; 97&#37; similarity. At Divar, the Gammaproteobacteria were ~ 2x higher (17&#37;) than at Tuvem. A more diverse bacterial community with &gt; 3300 phylotypes and &gt; 2000 OTUs mostly belonging to Gammaproteobacteria and a significantly higher DNT (n = 9, p < 0.001, df = 1) were recorded at Divar. These findings suggest that the quantity and quality of pollutants at Divar are perhaps still at a level to maintain high diversity. Using this technique we could show higher diversity at Divar with the possibility of Gammaproteobacteria contributing to modulating excess nitrate

Morphological and Molecular Characters Differentiate Common Morphotypes of Atlantic Holopelagic Sargassum

International audienceSince 2011, massive new strandings of holopelagic Sargassum have been reported on the coasts of the Caribbean, northern Brazil, Guiana, and West Africa, causing severe economic and ecological damage. Three common morphotypes (S. fluitans III, S. natans I, and S. natans VIII) were identified as responsible for these catastrophic events, with dominance shifts between them over time. However, the taxonomic status of these holopelagic Sargassum morphotypes remains unclear. Using an integrative taxonomy framework, combining a morphological study and molecular analyses, this study aimed to clarify their taxonomic status. Morphological analyses of 54 characters revealed no intermediate form between the three morphotypes, with the overall shape, nature of the axis, and size and shape of blades and vesicles being the most discriminating. An analysis of mitochondrial (IGS, cox2, cox3, mt16S rRNA, and nad6) and plastid (rbcL) markers confirmed the genetic divergence among the three morphotypes, with a lower level of divergence between the two S. natans morphotypes. Without additional molecular characterization, these morphotypes cannot be classified as three distinct species. However, due to their distinct morphological characteristics and sympatry within drifting aggregations, a revision of holopelagic species names is proposed, with Sargassum fluitans var. fluitans (for S. fluitans III), Sargassum natans var. natans (for S. natans I), and S. natans var. wingei (for S. natans VIII). This revision provides necessary clarity on the species involved in inundations of the tropical Atlantic

Genome sequence of the marine bacterium Marinobacter hydrocarbonoclasticus SP17, which forms biofilms on hydrophobic organic compounds

cited By 20International audienceMarinobacter hydrocarbonoclasticus SP17 forms biofilms specifically at the interface between water and hydrophobic organic compounds (HOCs) that are used as carbon and energy sources. Biofilm formation at the HOC-water interface has been recognized as a strategy to overcome the low availability of these nearly water-insoluble substrates. Here, we present the genome sequence of SP17, which could provide further insights into the mechanisms of enhancement of HOCs assimilation through biofilm formation

Assumptions and model structure for the growth and survival of the holopelagic brown macroalga Sargassum spp. based on DEB (Dynamic Energy Budget) theory

Understanding and forecasting the proliferation of holopelagic Sargassum in the tropical Atlantic Ocean requires knowledge on the biology and transport of these macroalgae. Forecasting the biomass, proliferation and distribution of Sargassum rafts at time scales greater than a few weeks needs improved modeling. Recent modeling efforts focused mostly on transport and less on the biology of Sargassum. Three morphotypes in varying proportions compose these Sargassum rafts. Better understanding and modeling the specificities of each of the three morphotypes is the current challenge.The main objectives of the BIOMAS project (BIOenergetic Modeling Approach for Sargassum dynamics) are to acquire the necessary knowledge to build an individual morphotype based model of Sargassum growth, to include this model into a drift model, and finally perform simulations of the integrated drift-growth model in order to forecast Sargassum morphotypes proliferation at seasonal scale. A strong synergy between laboratory experiments and modeling will allow us to build the first DEB (Dynamic Energy Budget) model applied to Sargassum and apply it to the three morphotypes. In-situ monitoring of Sargassum morphotypes in the Western and Eastern Atlantic will allow us to validate the integrated model.We here focus on the description of the assumptions and structure of the DEB model for Sargassum growth and survival. Specific assumptions to differentiate each morphotype are discussed and model simulation will be used to contribute to the design of optimal protocols for experiments so that the parameters are accurately estimated

Assumptions and model structure for the growth and survival of the holopelagic brown macroalga Sargassum spp. based on DEB (Dynamic Energy Budget) theory

Understanding and forecasting the proliferation of holopelagic Sargassum in the tropical Atlantic Ocean requires knowledge on the biology and transport of these macroalgae. Forecasting the biomass, proliferation and distribution of Sargassum rafts at time scales greater than a few weeks needs improved modeling. Recent modeling efforts focused mostly on transport and less on the biology of Sargassum. Three morphotypes in varying proportions compose these Sargassum rafts. Better understanding and modeling the specificities of each of the three morphotypes is the current challenge.The main objectives of the BIOMAS project (BIOenergetic Modeling Approach for Sargassum dynamics) are to acquire the necessary knowledge to build an individual morphotype based model of Sargassum growth, to include this model into a drift model, and finally perform simulations of the integrated drift-growth model in order to forecast Sargassum morphotypes proliferation at seasonal scale. A strong synergy between laboratory experiments and modeling will allow us to build the first DEB (Dynamic Energy Budget) model applied to Sargassum and apply it to the three morphotypes. In-situ monitoring of Sargassum morphotypes in the Western and Eastern Atlantic will allow us to validate the integrated model.We here focus on the description of the assumptions and structure of the DEB model for Sargassum growth and survival. Specific assumptions to differentiate each morphotype are discussed and model simulation will be used to contribute to the design of optimal protocols for experiments so that the parameters are accurately estimated