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Caractérisation Des Communautés Procaryotiques Impliquées Dans La Bioremédiation D'un Sol Pollué Par Des Hydrocarbures

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Caractérisation Des Communautés Procaryotiques Impliquées Dans La Bioremédiation D'un Sol Pollué Par Des Hydrocarbures Caract´erisationdes communaut´esprocaryotiques impliqu´eesdans la biorem´ediationd'un sol pollu´epar des hydrocarbures et d´eveloppement d'outils d'analyse `a haut d´ebit C´ecileMiliton To cite this version: C´ecile Militon. Caract´erisation des communaut´es procaryotiques impliqu´ees dans la biorem´ediationd'un sol pollu´epar des hydrocarbures et d´eveloppement d'outils d'analyse `ahaut d´ebit. Protistologie. Universit´eBlaise Pascal - Clermont-Ferrand II; Universit´ed'Auvergne - Clermont-Ferrand I, 2007. Fran¸cais. <NNT : 2007CLF21810>. <tel-00718551> HAL Id: tel-00718551 https://tel.archives-ouvertes.fr/tel-00718551 Submitted on 17 Jul 2012 HAL is a multi-disciplinary open access L'archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destin´eeau d´ep^otet `ala diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publi´esou non, lished or not. The documents may come from ´emanant des ´etablissements d'enseignement et de teaching and research institutions in France or recherche fran¸caisou ´etrangers,des laboratoires abroad, or from public or private research centers. publics ou priv´es. UNIVERSITE BLAISE PASCAL UNIVERSITE D’AUVERGNE N° D.U. 1810 ANNEE 2007 ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE N° d’ordre 474 THESE Présentée à l’Université Blaise Pascal pour l’obtention du grade de DOCTEUR D’UNIVERSITE Spécialité : GENOMIQUE ET ECOLOGIE MICROBIENNE Présentée et soutenue publiquement par Cécile MILITON le 17 décembre 2007 CARACTERISATION DES COMMUNAUTES PROCARYOTIQUES IMPLIQUEES DANS LA BIOREMEDIATION D’UN SOL POLLUE PAR DES HYDROCARBURES ET DEVELOPPEMENT D’OUTILS D'ANALYSE A HAUT DEBIT: LES BIOPUCES ADN. JURY Présidente : Arlette DARFEUILLE-MICHAUD (Pr., Université d’Auvergne, Clermont-Ferrand) Rapporteurs : Pascale BAUDA (Pr., Université Paul Verlaine, Metz) Pascal SIMONET (DR CNRS, Ecole Centrale, Ecully) Examinateur : Robert DURAN (Pr., Université de Pau et des Pays de l'Adour, Pau) Directeurs : Pierre PEYRET (Pr., UMR CNRS 6023, Université d’Auvergne, Clermont-Ferrand) Eric PEYRETAILLADE (Dr., UMR CNRS 6023, Université d’Auvergne, Clermont-Ferrand) Laboratoire Biologie des Protistes - UMR CNRS 6023 - Equipe Génomique Intégrée des Interactions Microbiennes, Université Blaise Pascal, Clermont-Ferrand. REMERCIEMENTS Je souhaite exprimer mes remerciements à Christian Amblard, directeur du laboratoire de Biologie des Protistes (UMR CNRS 6023), pour m’avoir accueilli au sein de son laboratoire et permis de réaliser cette thèse dans de bonnes conditions. Mes remerciements vont ensuite à mon directeur de thèse, Pierre Peyret, pour avoir encadré mes travaux jusqu’à leurs aboutissements mais surtout pour m’avoir fait partager sa passion pour la recherche, son ouverture d’esprit et sa rigueur scientifique durant ces années passées à ses côtés. Merci de m’avoir donné une grande liberté et des conditions de travail idéales qui m'ont permis de m’épanouir scientifiquement. Merci enfin de m’avoir fait confiance en me permettant de présenter mes travaux au cours de différents congrès et de m’avoir soutenue jusqu’en Autriche (dont je garde de très bons souvenirs). Que vous trouviez ici l’expression de ma profonde gratitude pour tout ce que vous avez fait pour moi. Mes remerciements vont aussi à Eric Peyretaillade pour l’intérêt qu’il a porté à ce travail en acceptant de co-diriger cette thèse, pour sa disponibilité, ses orientations et ses remarques fructueuses. Merci sincèrement Eric pour ton soutien scientifique mais aussi pour la bonne humeur que tu as le don de transmettre autour de toi. Je souhaite exprimer mes remerciements les plus sincères à Pascale Bauda et à Pascal Simonet pour m’avoir fait l’honneur d’accepter d’être rapporteurs de cette thèse ainsi qu’à Arlette Darfeuille-Michaud et à Robert Duran pour avoir accepté d’examiner ce travail et de faire partie du jury. Un grand merci à Anne Moné pour sa précieuse collaboration. Je tiens à lui rendre hommage pour son courage et pour le soutien qu’elle m’a offert depuis le premier jour. Anne, ce fut un véritable plaisir de travailler à tes côtés et de partager un neurone. Merci aussi à Delphine Boucher et à Corinne Petit pour leur aide dans la rédaction de ce mémoire ainsi que pour toutes les connaissances qu’elles m’ont fait partager durant ces années. Merci à Sébastien Rimour et Geneviève Gagne pour leur contribution aux travaux bioinformatiques et biologiques ainsi que pour la rédaction des publications. Merci à tous les membres de l’équipe GIIM qui ont été pour moi une seconde famille durant cette thèse. Un merci particulier à Brigitte Chebance pour son éternelle bonne humeur, sa disponibilité, son efficacité et sa gentillesse. Merci de m’avoir rendu plus douces ces années difficiles. Je remercie Julien Troquet, directeur de Biobasic Environnement, et Cédric Vachelard pour le matériel biologique fourni et les données associées dans le cadre d’un projet commun. Merci à Valérie et Abdel de m’avoir prise sous leur aile les premières années, de m’avoir offert leur précieuse amitié et pour toutes ces soirées passées ensemble. Un grand merci aussi à Cécile pour sa gentillesse et sa lucidité qu’elle m’a fait partager. Merci à Stéphanie et Isabelle, pour leur amitié sans faille. Merci aussi à Carine, Sylvie, Thomas, Antoine, Benoît, Maider, Marc-Antoine, Sigolène, Jean-Philippe, Olivier, Simon, Jean-Denis, Denis, Fabien et Ingrid pour leurs amitiés qui dure depuis le lycée (ou presque). Pour terminer, j’aimerais dédier cette thèse à ma famille. A mon père, qui m’a permis d’être ce que je suis aujourd’hui, à ma mère pour m’avoir toujours soutenue et pour m’avoir permis de faire mes études dans les meilleures conditions, à Séverine, à Loïc, à mes neveux Lison, Eliot et Nine. Je n’oublie pas non plus ma belle-famille et Philippe pour leur soutien. A Anthony… CARACTERISATION DES COMMUNAUTES PROCARYOTIQUES IMPLIQUEES DANS LA BIOREMEDIATION D’UN SOL POLLUE PAR DES HYDROCARBURES ET DEVELOPPEMENT D’OUTILS D'ANALYSE A HAUT DEBIT: LES BIOPUCES ADN. Résumé : Les activités humaines sont à l’origine de nombreuses pollutions par des hydrocarbures au niveau des écosystèmes et plus particulièrement au niveau des sols. Afin de préserver la santé humaine et environnementale, il est nécessaire de réduire les risques d’exposition et de contamination en dégradant les polluants en produit moins toxiques voire même inoffensifs. Dans ce but, les techniques de bioremédiation apparaissent aujourd’hui comme de réelles alternatives aux techniques classiques pouvant être très invasives et onéreuses. Cependant, la mise en place et/ou l’optimisation de tels procédés nécessitent une meilleure connaissance des communautés microbiennes impliquées dans la biodégradation de ces polluants. Ainsi, après avoir fait un état des lieux des outils développés pour isoler, identifier et caractériser ces populations, nous avons étudié la structure et la dynamique des communautés bactériennes au cours d’un procédé de biostimulation d’un sol pollué aux hydrocarbures aliphatiques. Outre la caractérisation d’un « core-set » bactérien présent tout au long du procédé de bioremédiation, cette étude nous a permis d’identifier des phylotypes potentiellement impliqués dans la biodégradation de ces polluants. Enfin, nous nous sommes attachés à mettre au point des outils moléculaires à haut débit, afin d’étudier plus globalement ces communautés bactériennes environnementales, et ce par le développement d’un logiciel de sélection de sondes sensibles, spécifiques et exploratoires pour biopuces ADN phylogénétiques (PhylArray). CHARACTERIZATION OF THE PROKARYOTIC COMMUNITIES IMPLIED IN THE HYDROCARBON-POLLUTED SOIL BIOREMEDIATION AND DEVELOPMENT OF HIGH-THROUGHPUT ANALYZE TOOLS : THE DNA MICROARRAYS. Abstract : Environmental ecosystems are sensitive to damage from human activities and there is an increasing need to develop better methods for removing pollutants from soils. The removal of pollutants or their transformation to less toxic products by bioremediation is a less invasive and expensive process than classical decontamination. However, the use and optimization of bioremediation treatments requires knowledge on the microbial communities directly and indirectly involved in the degradation of the pollutants. Therefore, after the creation of a synthesis describing the microbial diversity and the available tools for the isolation and the identification of microorganisms, we have studied the structure and the dynamic of the bacterial communities during the bioremediation of an aliphatic hydrocarbon-polluted soil. Beyond the characterization of a bacterial “core-set” present throughout the decontamination process, we have identified several phylotypes potentially involved in degradation. In order to globally monitor these bacterial communities, we also have developed a probe design software (PhylArray) generating efficient and explorative probes for high-throughput molecular tools: the DNA microarrays. Discipline : Ecologie et Génomique Microbienne. Mots-clés : Diversité bactérienne, ADNr 16S et ARNr 16S, Biopuces phylogénétique, Sélection de sondes, Sols pollués, Hydrocarbures aliphatiques, Bioremédiation. Laboratoire : Biologie des Protistes, UMR CNRS 6023, 24 av. des Landais, 63177 Aubière. SOMMAIRE INTRODUCTION GENERALE .....................................................................................1 PARTIE I : SYNTHESE BIBLIOGRAPHIQUE ..........................................................7 Chapitre I : Les sols pollués
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