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Pdf/6-2-4 Fiche Ferme.Pdf DEVELOPPEMENT D'OUTILS MOLECULAIRES POUR DETERMINER LES NIVEAUX D'ACTIVITE DES GROUPES BACTERIENS PRESENTS DANS LE LISIER DE PORC par Caroline Roy memoire presents au Departement de biologie en vue de l'obtention du grade de maitre es sciences (M.Sc.) FACULTE DES SCIENCES UNIVERSITE DE SHERBROOKE Sherbrooke, Quebec, Canada, novembre 2007 771 -I 2 tf Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-49578-0 Our file Notre reference ISBN: 978-0-494-49578-0 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. •*• Canada Le 30 novembre 2007 lejury a accepte le memoire de Mme Caroline Roy dans sa version finale. Membres dujury Mme Carole Beaulieu Directrice Departement de biologie Madame Caroline Talbot Codirectrice Departement de biologie Mme Marie-France Palin Membre Departement de biologie M. SebastienRoy President-rapporteur Departement de biologie SOMMAIRE La production porcine represente une part importante de l'economie au Quebec. Bien que Ton note une diminution du nombre de fermes possedant des pores, celles qui demeurent se specialised et augmentent leur volume. La bonne gestion du lisier produit est par consequent imperative pour le maintien de la sante des sols et des cours d'eau en plus d'une bonne qualite de Fair. Les odeurs et autres composes nuisibles sont produits lors de la degradation anaerobie de la matiere organique par les microorganismes indigenes au systeme digestif. Les etudes portant sur les communautes microbiennes du systeme digestif et du lisier de pore sont encore peu nombreuses. La connaissance de cette population microbienne et la comprehension de leurs interactions sont fondamentales dans Pelaboration des techniques de traitement. L'une de ces methodes de traitement est la digestion anaerobie effectuee par un bioreacteur. Pour etudier les communautes mibrobiennes, Putilisation de methodes moleculaires, telles Pheterogeneite des longueurs d'amplicon PCR (LH-PCR) et le polymorphisme des longueurs de fragment terminal de restriction (T-RFLP), permet la detection d'organismes non-cultivables en laboratoire. Le projet presente dans ce memoire vise a demontrer Pefficacite de ces deux techniques d'empreintes a ADN (profils de phylotypes) dans Petude des dynamiques de communautes microbiennes dans le lisier de pore, ainsi que la quantification de certains groupes microbiens par PCR en temps reel. II vise egalement a caracteriser les microorganismes metaboliquement actifs via Panalyse de PARN ribosomal (ARNr) 16S. De plus, ce travail veut montrer Putilite de differentes analyses statistiques permettant le traitement des nombreuses donnees produites de maniere a en degager des interpretations sur le dynamisme microbien. Pour atteindre ces objectifs, deux approches experimentales ont ete utilisees. La premiere approche consistait a Petude de la dynamique des communautes microbiennes d'un bioreacteur a huit compartiments a ecoulement piston. La seconde visait a determiner les differences de population entre des lisiers bruts preleves a la ferme. Les resultats obtenus du traitement par bioreacteur ont montre une specialisation des compartiments dans la digestion de la matiere organique. Durant les six mois d'operation, chaque compartiment 1 s'est specialise et a favorise le developpement d'une population qui lui etait propre et presentait differents niveaux d'acides gras volatils (AGV) et de degagement de methane. Des phylotypes qui pourraient expliquer la difference entre les profils obtenus ont ete identifies. De plus, des profils semblables ont ete correles a la production d'acides gras volatils ou de methane. La seconde approche experimentale portait sur la caracterisation de trois lisiers bruts preleves a un mois d'intervalle et provenant des operations de finition, ainsi que d'un melange de lisiers de finition et de maternite. Les analyses des profils de phylotypes obtenus ont mene a la conclusion que la structure des communautes bacteriennes et archaebacteriennes des trois lisiers de pore provenant des operations de finition etaient semblables mais se distinguaient de celle du lisier mixte provenant des operations de finition et de maternite. De plus, ces analyses ont permis de faire la detection de changements subtils entre les echantillons. Dans ce travail, il a ete montre que l'etude au niveau de PARNr des bacteries etait plus appropriee qu'au niveau de l'ADNr. En effet, les resultats obtenus pour ces deux cibles etaient differents. L'utilisation de l'ARNr a permis d'observer plus precisement les changements des bacteries metaboliquement actives. Par contre, la difference entre les resultats de l'ADNr et l'ARNr pour les archaebacteries etait beaucoup moins marquee. Finalement, l'utilisation d'outils statistiques tels que les indices de diversite, l'analyse en composantes principales (PCA), le positionnement multidimensionnel non-metrique (NMS), l'UPGMA (unweighted pair group method with arithmetic mean) ainsi que l'analyse des especes indicatrices (ISA) a confirme leur utilite dans l'analyse des donnees complexes et la visualisation des resultats obtenus. n REMERCIEMENTS Je remercie ma directrice de recherche Guylaine Talbot pour m'avoir offert cette maitrise et d'avoir cm en mes capacites. Elle a su rendre l'experience agreable et enrichissante tout en m'accordant toujours sa grande disponibilite. Elle est pour moi plus qu'un guide mais une amie. De plus, je remercie ma co-directrice Carole Beaulieu pour ses conseils et ses encouragements. De merae, je remercie Claude Dery pour son incroyable disponibilite et l'interet qu'il porte a tous les etudiants. Je remercie egalement Sebastien Roy pour le temps et Penergie portes a la correction et a revaluation de ce memoire. Je desire egalement remercier la Federation des Producteurs de Pore du Quebec pour son important apport financier. Merci d'avoir cru en ce projet. De plus, je remercie Daniel Masse pour l'opportunite offerte ainsi que toute son equipe d'experts. Je remercie tous les membres du laboratoire 211 du centre de recherche d'AAC de Lennoxville pour leur accueil, leur amitie, leurs conseils et leur comprehension. J'ai grandement apprecie l'atmosphere de camaraderie qu'ils ont si naturellement inspiree. Un gros merci a Marie-France Palin et Daniele Beaudry qui m'ont consideree comme un membre a part entiere de leur equipe. Je souligne aussi l'important apport de Benoit Labrecque par son ecoute, ses idees et surtout son amitie. Je tiens aussi a remercier toute ma famille qui m'a soutenue et encouragee durant toute cette aventure mais surtout ma mere qui a toujours eu notre education a cceur. Un merci special a Paul-Aime Roy pour son aide financiere et a mes neveux qui m'ont laisse faire mon tres long devoir. iii TABLE DES MATIERES SOMMAIRE i REMERCIEMENTS iii TABLE DES MATIERES iv LISTE DES ABREVIATIONS viii LISTE DES TABLEAUX x LISTE DES FIGURES xii INTRODUCTION 1 1.1. La production porcine et sa problematique environnementale 1 1.2. La degradation anaerobie du lisier 3 1.3. Les odeurs 5 1.3.1. Les acides gras volatils 6 1.3.1.1. Catabolisme desproteines 6 1.3.1.2. Catabolisme des hydrates de carbone 9 1.3.1.3. Catabolisme des lipides 9 1.3.2. Indicateur d'odeur 9 1.3.3. Microorganismes indigenes au pore 10 1.4. Methodes de microbiologie classique et moleculaire 13 1.5. Le gene de l'ARN ribosomal 16S 14 1.5.1. Activite metabolique 15 1.5.2. L'ARNr et les genes fonctionnels 15 1.5.3. Methodes de biologie moleculaire 16 1.5.3.1. LH-PCR 18 1.5.3.2. T-RFLP 18 1.5.3.3. DGGE 19 1.5.3.4. Hybridation de fluorescence in-situ (FISH) 20 1.5.3.5. PCR en temps reel 21 1.6. Approche experimentale 23 iv CHAPITRE 1 DYNAMIQUE DES COMMUNITES BACTERIENNES ET ARCHAEBACTERIENNES D'UN BIOREACTEUR DE TYPE ECOULEMENT PISTON TRAITANT LE LISIER DEPORC 26 Bacterial and archaeal community dynamics from an anaerobic plug
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