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Université De Pau Et Des Pays De L'adour Pour M’Avoir Confié Ce Travail De Recherche Et Pour Ses Précieux Conseils Au Cours De Ces Trois Années

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Université De Pau Et Des Pays De L'adour Pour M’Avoir Confié Ce Travail De Recherche Et Pour Ses Précieux Conseils Au Cours De Ces Trois Années THÈSE Présentée à UNIVERSITÉ DE PAU ET DES PAYS DE L’ADOUR École Doctorale des Sciences Exactes et de leurs Applications Par Yannick COLIN Pour obtenir le grade de DOCTEUR Spécialité : Microbiologie __________________________________________________________________ DEVELOPPEMENT METHODOLOGIQUE POUR L'ETUDE DE LA DIVERSITE DES MICRO-ORGANISMES SULFATO-REDUCTEURS ET LEUR ROLE DANS LA METHYLATION DU MERCURE __________________________________________________________________ Soutenue publiquement le 19 décembre 2012 Après avis de : M. Rutger DE WIT DR CNRS, Université de Montpelier 2 Rapporteur Mme Agnès HIRSCHLER-REA MC-HDR, Université de la Méditerranée Rapporteur Devant la comission d’examen formée de : Mme Karine ALAIN CR CNRS, Université de Bretagne Occidentale Examinateur M. Robert DURAN Pr, Université de Pau et des Pays de l’Adour Examinateur M. Rémy GUYONEAUD Pr, Université de Pau et des Pays de l’Adour Co-directeur de thèse M. Pierre CAUMETTE Pr, Université de Pau et des Pays de l’Adour Directeur de Thèse REMERCIEMENTS Je tiens dans un premier temps à remercier Monsieur Pierre Caumette, Professeur à l'Université de Pau et des Pays de l'Adour pour m’avoir confié ce travail de recherche et pour ses précieux conseils au cours de ces trois années. Je remercie également et Monsieur Rémy Guyoneaud, Professeur à l'Université de Pau et des Pays de l'Adour, co-encadrant de ce travail de thèse, pour sa sympathie, sa disponibilité, ses idées et ses conseils. Enfin, merci à Monsieur Robert Duran, Directeur de l'Equipe Environement et Microbiologie, qui m’a ouvert les portes du laboratoire. Je tiens à remercier Monsieur Rutger DE WIT, Directeur de Recherche à l'Université de Montpelier 2, et Madame Agnès HIRSCHLER-REA, Maître de Conférences à l'Université d’Aix-Marseille, d’avoir accepté d’être les rapporteurs de ce travail. Je remercie également Madame Karine Alain, Chargée de Recherche au laboratoire de Microbiologie des Environnements Extrêmes de Brest, d’avoir accepté de participer à ce jury, ainsi que pour sa disponibilité et son soutien durant mon Master à l’Univeristé de Bretagne Occidentale. Ce travail est le résultat d’étroites collaborations avec le Laboratoire de Chimie Analytique Bio-inorganique et Environnement (CNRS UMR 5254). Je remercie David amouroux, Mathilde Monperrus, Emmanuel Tessier et Abu Sharif pour leurs collaborations. J’aimerai adresser un remerciement particulier à Monsieur Phillipe Goulas pour m’avoir permis d’effectuer mon enseignement de monitorat à l'Univeristé de Pau et des Pays de l'Adour. et pour sa confiance dès mon entrée en fonction. Je remercie les autres enseignants avec qui j’ai eu le plaisir de travailler (Beatrice Lauga, Christine Cagnon, Pierre Sivadon et Jérome Gury) Ce travail n’aurait pu aboutir sans l’aide de nombreuses personnes. J’adresse une pensée particulière à Marisol Goni, Jerome Gury, Claire Gassie, FLorence Hakil et Sophie Gentès. Je tiens à remercier mes amis qui m’ont soutenu, m’ont fait décompresser, et avec qui j'ai passé de supers moments. Nombreux parmi eux ont fortement contribué à la création du CUB 2, I on peut dire que c'est une belle réussite. Un grand merci à vous: Saussicon, Mich, Mitch, Maité, Karine & Seb, Mu & Ben, Maxou; Vévert, Ludo, Evan, Gaetan, Rich, JB, Jerome, Matthieu, Vince, … Une dédicace particulière à Jean-Phi & Judith: En souvenir des nombreuses soirées jeux (7 wonders, les aventuriers du rail, King of Tokyo, …) et autres moments très sympathiques… et ce n'est pas terminé! Finalement, je garde une place toute particulière pour mes parents et à ma sœur, Célia. Je les remercie pour leur soutien et les encouragements qui m’ont été très précieux durant les années de ma thèse. C’est l’éducation que vous m’avez offert qui m’a permis d’arriver là où je suis aujourd’hui, merci. Ma vie personnelle a beaucoup compté pendant mon doctorat. On ne peut travailler bien que si l’on se sent bien, merci à toi Hélène pour ta patience, ton aide et la joie que tu m'as apporté. II SOMMAIRE REMERCIEMENTS ..................................................................................................... I SOMMAIRE ............................................................................................................ III LISTE DES FIGURES ................................................................................................VI LISTE DES TABLES ................................................................................................... X LISTE DES ABBREVIATIONS ...................................................................................... XI INTRODUCTION GENERALE ........................................................................................ 1 CHAPITRE I - SYNTHESE BIBLIOGRAPHIQUE .................................................................. 5 1. LES MICRO -ORGANISMES SULFATO -REDUCTEURS ...................................................... 6 1.1 Taxonomie des BSR .................................................................................... 6 1.2 Ecologie des BSR........................................................................................ 8 i. Réduction dissimilatrice du sulfate .......................................................................................... 8 ii. Autres accepteurs terminaux d’électrons ............................................................................... 9 iii. Donneurs d’électrons et source de carbone ........................................................................ 10 1.3 Marqueurs moléculaires des BSR.............................................................. 11 i. ADNr 16S................................................................................................................................ 12 ii. Gènes dsrAB ......................................................................................................................... 12 1.4 Distribution des BSR dans l’écosystème côtier.......................................... 14 i. Les sédiments ........................................................................................................................ 14 ii. La colonne d’eau ................................................................................................................... 15 2. METHYLATION DU MERCURE DANS LES ECOSYSTEMES AQUATIQUES ............................ 16 2.1 Le mercure dans l'environnement ............................................................. 16 2.2 Spéciation du mercure dans les sédiments anoxiques ............................... 17 i. Méthylation du mercure.......................................................................................................... 18 ii. Déméthylation du mercure .................................................................................................... 19 iii. Facteurs environnementaux influençant la méthylation du mercure .................................... 19 2.3 Métabolisme des BSR et méthylation du mercure ...................................... 22 2.4 Taxonomie des BSR et méthylation du mercure ......................................... 23 3. MISE EN CULTURE DES MICRO -ORGANISMES ........................................................... 26 3.1 Composition d'un milieu de culture............................................................ 26 3.2 Limites de l'approche culturale ................................................................. 26 3.3 Amélioration des méthodes de culture ...................................................... 27 i. Concentration des substrats .................................................................................................. 27 ii. Maintien de la communication cellulaire in vitro.................................................................... 28 iii. Temps d'incubation et taille de l'inoculum ............................................................................ 29 iv. Procédures d'enrichissement............................................................................................... 31 v. Techniques de culture haut débit .......................................................................................... 31 3.4 Microbiologie en conditions d'anaérobiose................................................ 32 i. Création et maintien de l'anaérobiose des milieux de culture................................................. 32 ii. Techniques traditionnelles d'isolements des micro-organismes sulfato-réducteurs............. 34 CHAPITRE II - PROCEDURES EXPERIMENTALES ............................................................ 37 1. SITE D 'ETUDE .................................................................................................... 38 1.1 Description de l'estuaire de l'Adour........................................................... 38 1.2. Stratégies d’échantillonnage ................................................................... 39 i. Station B20............................................................................................................................. 39 ii. Campagne METADOUR III ..................................................................................................... 40 2. APPROCHE CULTURALE DES BSR......................................................................... 42 2.1 Milieux de culture pour l'isolement de BSR ................................................ 42 2.2 Enrichissements des sédiments de la station B20...................................... 43 2.3 Isolement haut- débit des BSR..................................................................
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