As Terras Pretas de Índio (TPIs) na Amazônia Brasileira são altamente férteis e o seu conteúdo químico parece não exaurir mesmo em condições de floresta tropical. Por essa razão, são frequentemente procuradas pelas populações locais para o cultivo de subsistência. A importância das comunidades microbianas tem aumentado o interesse em compreender a relação entre o uso da terra, as comunidades de plantas, os micro-organismos e os processos do ecossistema. Portanto, o objetivo principal desta pesquisa foi investigar as comunidades bacterianas sob a influência da cobertura vegetal em sistemas de uso da terra (floresta secundária e plantio de mandioca) e na rizosfera de plantas leguminonas nativas em comunidades de bactéria das TPIs. Além disso, investigou-se também as bactérias desnitrificantes nesses solos. A área de estudo está localizada na Estação Experimental do Caldeirão, pertencente à Embrapa Amazônia Ocidental, no município de Iranduba-AM. A funcionalidade da comunidade bacteriana foi determinada pela Análise de Perfil Fisiológico da Comunidade Microbiana (CLPP), a estrutura da comunidade bacteriana foi acessada por Polimorfismo do Tamanho do Fragmento de Restrição Terminal (T-RFLP), a composição e distribuição das comunidades bacterianas foram determinadas por sequenciamento em larga escala (pirosequenciamento), e para quantificar as bactérias desnitrificantes foi utilizada a técnica de PCR quantitativa (qPCR). Os estudos foram realizados no laboratório de Biologia Celular e Molecular (CENA / USP) e no departamento de Biogeoquímica (Max Planck Institute for Terrestrial Microbiology). A análise de T-RFLP mostrou que o uso da terra e a sazonalidade afetaram as comunidades bacterianas na TPI, e mostrou também um claro efeito da rizosfera nas comunidades bacterianas. CLPP demonstrou que a atividade funcional da TPI não foi afetada pela sazonalidade. Além disso, a tecnologia de pirosequenciamento foi uma ferramenta importante para diferenciar filotipos raros. Diferenças distintas de alguns filos bacterianos da rizosfera foram observadas, indicando que a zona de raiz contribui para moldar essas comunidades. A abundância relativa do gene nirK não foi afetada pelo uso da terra nos dois tipos de solos. Alterações na estrutura das comunidades dos genes nirK e nosZ foram observadas em ambos os tipos de solos. As comunidades desnitrificantes na TPI pareceram ser mais influenciadas pelo uso da terra do que pela sazonalidade, e ACH foi mais influenciada pelas variações de sazonalidade. / Amazonian Dark Earths (ADEs) in the Brazilian Amazon are highly fertile and its chemical content seems not to get depleted even under tropical humid conditions. For this reason, these soils are frequently searched by local population for subsistence farming. The importance of microbial communities has grown the interest in understanding the relationship between land use, plant communities, microorganisms, and ecosystem processes. Therefore, the main objective of this research was to investigate the effect of vegetation cover in land use systems (secondary forest and cassava plantation) and rhizosphere of native leguminous plants on bacterial communities of ADEs. Furthermore, it was also aimed to investigate denitrifying bacteria in these soils. The study area is located at the Experimental Station of Caldeirão, belonging to Embrapa Amazônia Ocidental, Iranduba, AM. The bacterial community function was determined by Community Level Physiological Profile (CLPP), the bacterial community structure was assessed by Terminal Restriction Fragment Length Polymorphism (T-RFLP), the bacterial community composition and distribution by high-throughput sequencing (pyrosequencing), and the quantification of denitrifier bacteria by Quantitative PCR (qPCR). The studies were performed in the Laboratory of Cell and Molecular Biology (CENA/USP) and the Deparment of Biogeochemistry (Max Planck Institute for Terrestrial Microbiology). T-RFLP analysis showed that land use and seasonality affected bacterial communities in ADE, and also showed a clear rhizosphere effect on bacterial communities. CLPP have shown that ADE functional activity was not affected by seasonality. Furthermore, pyrosequencing technology was an important tool to differentiate rare phylotypes. Distinct differences of some rhizosphere bacterial phyla were also observed, indicating that the root zone contributed to shape these communities. The relative abundance of nirK gene was not affected by land use in both studied soils. Alterations in the community structure of nirK and nosZ genes were observed for both soils. ADE denitrifying communities seemed to be more affected by land use than seasonality, and ACH was more influenced by seasonal variations.

O objetivo deste estudo foi avaliar a remoção do surfactante não iônico álcool etoxilado de cadeia não ramificada (AE) em reator anaeróbio de leito fluidificado preenchido com areia como material suporte, em escala de bancada (1,2 L) com TDH de 18 horas, recirculação e fluxo contínuo. O reator foi inoculado com lodo proveniente de reator UASB utilizado no tratamento de dejetos de suinocultura e alimentado com substrato sintético acrescido de surfactante não iônico GENAPOL® C-100 (Sigma-Aldrich®) como fonte de (AE). Análises de monitoramento da concentração do surfactante não iônico AE e matéria orgânica, bem como, dos parâmetros físico-químicos foram realizadas para observar e quantificar a estabilidade do reator, na remoção e degradação do surfactante. A operação do reator foi dividida em cinco etapas: inoculação (535±121 mg/L de DQO), adaptação da biomassa (600±70 mg/L de DQO), Fase I (4,7 mg/L de AE e 623±65 mg/L de DQO), Fase II (22,5 mg/L de AE e 735±87 mg/L de DQO), Fase III (51,4 mg/L de AE e 697±68 mg/L de DQO), Fase IV (107,4 mg/L de AE e 845±87 mg/L de DQO) e Fase V (97,9 mg/L de AE e 882±126 mg/L de DQO). Aplicação das técnicas de PCR/DGGE e pirosequenciamento da região do rRNA 16S foi realizada para constatar a diversidade microbiana nas fases operacionais IV e V. A eficiência média de remoção de matéria orgânica e AE foi de 88% e 99%, respectivamente, durante a operação do reator. A similaridade das populações dos Domínios Archaea e Bacteria foi de 74% e 59%, respectivamente, para as amostras da Fase IV (com sacarose) e Fase V (sem sacarose). A sacarose não alterou o comportamento físico-químico do reator de leito fluidificado, mas este co-substrato influenciou, tanto, na produção de ácidos orgânicos voláteis, quanto, na diversidade dos microrganismos envolvidos na degradação do AE. Por meio da análise de pirosequenciamento das amostras das Fases IV e V do material suporte e separador de fases do reator foram identificados 83 gêneros dos quais 18 foram relacionados com a degradação de surfactante não iônico, bem como, seus subprodutos. Obteve-se maior abundância relativa para os seguintes gêneros: Sporomusa, Geobacter, Desulfobulbus, Synergistes, Sedimentibacter, Holophaga, Serpens e Azonexus. Observou-se elevada diversidade filogenética e similaridade com sequências de bactérias relacionadas com a degradação de surfactante não iônico AE. / The aim of this study was to evaluate the removal of nonionic alcohol ethoxylate non-branched (AE) in anaerobic fluidized bed reactor filled with sand as support material, on a bench scale (1.2 L) with 18 hours of TDH, recirculation and continuous flow. The reactor was inoculated with sludge from a UASB reactor used in the treatment of swine manure and fed with synthetic substrate plus nonionic GENAPOL® C-100 (Sigma-Aldrich®) as a source of AE. Monitoring analysis of the nonionic surfactant AE concentration and organic matter, as well as the physicochemical parameters were performed to observe and quantify the reactor stability, in the removal and degradation of the surfactant. The reactor operation was divided into five phases: inoculation (535±121 mg/L of COD), biomass adaptation (600±70 mg/L of COD), Phase I (4,7 mg/L of AE and 623±65 mg/L of COD), Phase II (22,5 mg/L of AE and 735±87 mg/L of COD), Phase III (51,4 mg/L of AE and 697±68 mg/L of COD), Phase IV (107,4 mg/L of AE and 845±87 mg/L of COD) and Phase V (97,9 mg/L of AE and 882±126 mg/L of COD). Application of the techniques PCR/DGGE and pyrosequencing of the 16S rRNA region were performed to observe the microbial diversity in the operational phases IV and V. The average removal efficiency of organic matter and AE was 88% and 99%, respectively, during the reactor operation. The populations similarity of the Archaea and Bacteria Domains were 74% and 59%, respectively, for samples from Phase IV (with sucrose) and Phase V (without sucrose). Sucrose did not alter the physical-chemical behavior of the fluidized bed reactor, but this co-substrate influenced both in the volatile fatty acids production, as in the diversity of microorganisms involved in the AE degradation. Through the pyrosequencing analysis of samples from Phases IV and V of the reactor (support material and phase separator), 83 genera were identified of which 18 were related to the nonionic surfactant degradation, as well as its byproducts. Highest relative abundance values were obtained for the following genera: Sporomusa, Geobacter, Desulfobulbus, Synergistes, Sedimentibacter, Holophaga, Serpens and Azonexus. A high phylogenetic diversity and similarity to sequences of bacteria related to the degradation of nonionic AE surfactant were observed.

Nesse trabalho avaliaram-se quatro hipóteses sobre a remoção do alquilbenzeno linear sulfonado (LAS) em reator EGSB (expanded granular sludge bed) alimentado ora com água residuária de lavanderia comercial, ora como meio sintético acrescido de LAS Padrão, com e sem suplementação de Fe(III) afluente. Para tanto, em todas as hipóteses utilizou-se reator EGSB (1,4 L) com tempo de detenção hidráulica (TDH) de 36h, condição mesofílica (30ºC) e carga de LAS específica aplicada (CLEA) variando de 1,0 - 2,7 mgLAS.gSTV-1.d-1. DGGE e sequenciamento massivo do gene rRNA 16S (Plataforma 454-Pirosequenciamento e Ion Torrent) foram utilizados para caracterização microbiana. Em relação à Hipótese A avaliou-se o efeito da adaptação prévia da biomassa na remoção do LAS em água residuária. Para tanto, o EGSB-BA (biomassa adaptada) teve uma etapa prévia com LAS padrão e meio sintético (Etapa I), seguida da Etapa II com água residuária; e o EGSB-BNA (biomassa não adaptada) teve etapa única e alimentação diretamente com água residuária. Para a Hipótese B avaliou-se o efeito da suplementação com meio sintético na remoção de LAS em água residuária. Para tanto, o EGSB-Ag.Lav foi alimentado apenas com água residuária e bicarbonato de sódio e duas CLE (Etapa II - 1,0 e Etapa III - 2,7 mg LAS.gSTV-1.d-1). Em relação às Hipóteses C e D, avaliou-se o efeito da suplementação de Fe(III) na remoção de LAS Padrão em meio sintético e LAS em água residuária, respectivamente. A Hipótese A foi refutada uma vez que as remoções de LAS em EGSB-BA-Etapa II (76%) e EGSB-BNA-Etapa I (78%) foram similares (ambas com água residuária). A remoção de LAS foi maior quando foi adicionada água residuária (EGSB-BA-Etapa II-76%) do que com LAS Padrão (EGSB-BA-Etapa I-63%). A Hipótese B foi aceita, uma vez que a alimentação do EGSB apenas com água residuária de lavanderia (CLE 1,0 mg LAS.gSTV-1.d-1) mais bicarbonato de sódio resultou em remoções do surfactante de 93%, ou seja, 15-17% maior que nos reatores suplementados com meio sintético (EGSB-BA Etapa II e EGSB-BNA Etapa I). Na Etapa III verificou-se diminuição da remoção em 30%. A Hipótese C foi aceita uma vez que se notou 20% de aumento na remoção de LAS quando comparado com reator não suplementado com Fe(III) (EGSB-Fe - 84,3% e EGSB-BA Etapa I - 63,5%). A Hipótese D foi refutada, uma vez que embora tenha sido obtida alta remoção de LAS (91,2%), esta não foi acompanhada pela redução férrica. Por meio do DGGE (domínio Bactéria) notou-se estratificação microbiana ao longo do reator na Etapa III (Hipótese B), provavelmente, em função do tamanho do grânulo que variou ao longo do reator. Por meio do sequenciamento massivo identificou-se bactérias semelhantes à Geobacter na amostra proveniente do reator EGSBFe da Hipótese C (17% da abundância relativa), portanto, as condições impostas favoreceram esse gênero. Fato este não observado para o reator EGSB-Fe-Ag.Lav. da Hipótese D. A comparação da análise filogenética das bactérias para os diferentes reatores permitiu identificar gêneros em comum relacionados com a degradação de LAS, a saber: Desulfobulbus, Geobacter, Syntrophorhabdus, Sporomusa, Comamonas, Holophaga, Mycobacterium, Pseudomonas, Stenotrophomonas e Synergistes. / This study evaluated four hypotheses about the removal of linear alkylbenzene sulfonate (LAS) in EGSB reactor (expanded granular sludge bed) fed sometimes with commercial laundry wastewater, sometimes with synthetic medium more Standard LAS, with and without Fe(III) influent supplementation. Therefore, in all hypotheses were used an EGSB reactor (1.4 L) with a hydraulic retention time (HRT) of 36 h, mesophilic condition (30°C) and load specific LAS (CLE) ranging from 1,0 to 2,7 mgLAS.gSTV-1.d-1. DGGE and massive sequencing of 16S rRNA gene (454-pyrosequencing and Ion Torrent platform) were used for microbial characterization. Regarding the Hypothesis A, it was evaluated the effect of biomass pre-adaptation for removal of LAS in wastewater. Then, the EGSBBA (adapted biomass) had a previous step with standard LAS and synthetic medium (Phase I), followed by Stage II with wastewater; and EGSB-BNA (not adapted biomass) had single step and feeding directly with wastewater. Regarding the Hypothesis B, it was evaluated the effect of synthetic medium supplementation in the removal of LAS in wastewater. Then, the EGSB-Ag.Lav was fed only with wastewater and sodium bicarbonate with two CLE (Stage II - 1,0 e Stage III - 2,7 mg LAS.gSTV-1.d-1). Regarding the Hypothesis C and D, it was evaluated the effect of Fe(III) supplementation in the removal of standard LAS and LAS in wastewater, respectively. The Hypothesis A was refuted since the LAS removal in EGSB-BA-Stage II (76%) and EGSB-BNA-Step I (78%) were similar (both with wastewater). The LAS removal was highest when wastewater was added (EGSB-BA-Stage-II 76%) than with standard LAS (EGSB-BAStage- I 63%). The Hypothesis B was accepted, since the feed of the EGSB only with wastewater from laundry (CLE 1,0 mg LAS.gSTV-1.d-1) more sodium bicarbonate resulted in removal of 93% of surfactant, in other words, 15-17% higher than in the reactors supplemented with synthetic medium (EGSB-BA Stage II e EGSB-BNA Stage I). In the Stage III, there was a decrease by 30% of LAS removal. The Hypothesis C was accepted, since there was an increase of 20% in the removal of LAS as compared to unsupplemented reactor with Fe (III) (EGSB-Fe - 84,3% e EGSB-BA Stage I - 63,5%). The Hypothesis D was refuted since although high LAS removal was obtained (91,2%), this was not accompanied by ferric reduction. By means of DGGE (Bacteria domain) was noted a microbial stratification along the reactor in the Stage III (Hypothesis B), probably in function of granule size along the reactor. By means of massive sequencing were identified bacteria similar to Geobacter in the sample from the reactor EGSB-Fe Hypothesis C (relative abundance 17%), therefore, the conditions favored this genre. This fact was not observed in the reactor EGSB-Fe-Ag.Lav. hypothesis D. A comparison of phylogenetic analysis of bacteria for different reactors allowed to identify common genera related to LAS degradation, namely: Desulfobulbus, Geobacter, Syntrophorhabdus, Sporomusa, Comamonas, Holophaga, Mycobacterium, Pseudomonas, Stenotrophomonas and Synergistes.

Abstract Disturbance may shift microbial communities from one state to another. However, species differ in their ecological characteristics and their abilities to withstand disturbance. No single species or individuals of a species exist alone, but they are parts of complex interaction networks including species above- and belowground. In boreal and subarctic forests, almost all plants and a high number of fungi form mycorrhizas at the plant roots. In mycorrhiza, the fungal partner harvests nutrients for the host plant and, in return, gains carbon from the plant. In general, these common associations benefit both partners, but as heterotrophs, fungi are dependent on carbon photosynthesized by plants, whereas plants can survive alone as autotrophs. In addition to mycorrhizal fungi, also other fungi, such as endophytes, saprotrophs and pathogens, live in and on plant roots. This thesis concerns the impacts of disturbance on fungi living in plant roots and in soil near the roots. I hypothesized that i) root-associated fungal (RAF) and soil fungal communities and colonization types change after disturbance, that ii) the observed shifts relate to disturbance intensity and that iii) they co-occur with changes in soil conditions and vegetation. Changes in RAF were studied as changes in root fungal colonization, or in fungal community composition. The latter were detected with next-generation sequencing methods. The responses of RAF to disturbance seemed to be context dependent and related to sources of fungal communities (e.g. soil, RAF networks), environmental conditions (e.g. soil pH and nutrients) and host performance. It seems that abundances of those RAF species, which are present in the roots first (priority effect), may be increased by disturbance. Research produced new information related to ecological roles of the genera Phialocephala and Meliniomyces. Altogether, the results indicate connections between both abiotic and biotic environments and RAF, and host species viability and RAF. / Tiivistelmä Häiriöt voivat siirtää eliöyhteisön tilasta toiseen. Lajien ominaisuudet ja häiriönsietokyvyt eroavat toisistaan. Mikään laji tai yksilö ei elä yksin, vaan lajit ovat osa maan ylä- ja alapuolelle ulottuvia monimutkaisia vuorovaikutusverkostoja. Boreaalisissa ja subarktisissa metsissä lähes kaikki kasvit ja useat sienet muodostavat sienijuuren eli mykorritsan. Mykorritsassa sieniosakas hankkii isäntäkasville ravinteita ja saa vastavuoroisesti kasvilta hiiltä. Tavallisesti nämä vuorovaikutussuhteet hyödyttävät molempia sienijuuren osakkaita, mutta toisenvaraisina (heterotrofeina) sienet ovat riippuvaisia kasvien yhteyttämästä hiilestä, kun taas tuottajina (autotrofeina) kasvit voivat elää itsenäisesti. Mykorritsasienten lisäksi kasvien juurissa elää yleisesti myös muita sieniä kuten endofyyttejä, saprotrofeja ja patogeeneja. Tämä väitöskirja käsittelee häiriön vaikutuksia sieniin, jotka elävät kasvien juurissa ja juuria ympäröivässä maassa. Hypoteesieni mukaan i) juurissa ja maassa elävien sienten yhteisöt ja kolonisaatiotyypit muuttuvat häiriön jälkeen, ii) muutokset liittyvät häiriön voimakkuuteen ja iii) muutokset tapahtuvat samanaikaisesti maan olosuhteiden ja kasvillisuuden muutoksien kanssa. Juurissa elävien sienten esiintymisen muutokset tutkittiin sienten kolonisaation tai yhteisörakenteen muutoksina. Sieniyhteisöt selvitettiin NGS-menetelmien avulla. Juurissa elävien sienten vasteet häiriöön vaikuttavat olevan tilannesidonnaisia ja liittyvän sienilajien lähteisiin (esim. maa, juurisieniverkostot), ympäristömuuttujiin (esim. maan pH, ravinteet) ja isäntäkasvin menestymiseen. Häiriö voi vahvistaa juurissa ensimmäisenä läsnä olevien sienilajien menestymistä (prioriteettivaikutus). Uutta tietoa tuotettiin Phialocephala ja Meliniomyces –sienisukujen ekologiasta, jota tunnetaan huonosti. Kaiken kaikkiaan, tulokset osoittavat yhteydet sekä elottoman ja elollisen ympäristön ja juurten sieniyhteisön että isäntäkasvin elinkyvyn ja juurten sieniyhteisön välillä.

Les chloroacétanilides sont une famille d'herbicides largement utilisée en agriculture, et contribuent de ce fait à la pollution environnementale. Leur devenir, y compris dans les écosystèmes rédox-dynamiques récepteurs comme les zones humides, est encore mal compris. Dans ce travail, la dégradation microbienne de chloroacétanilides (métolachlore, acétochlore et l'alachlore) a été étudiée par des approches innovantes de chimie analytique et de biologie moléculaire, à l'échelle du laboratoire en utilisant des microcosmes en colonnes, et in situ dans des zones humides construites à ciel ouvert et conçues pour traiter les intrants chimiques issus de l'agriculture.Une nouvelle méthode d’analyse isotopique composés-spécifiques a été développée. Les résultats indiquent la biodégradation des chloroacétanilides dans les zones humides, également suggérée par la détection des produits de dégradation correspondants (acides éthane sulfonique et oxanilique). Dans les expériences en microcosme de laboratoire, les chloroacétanilides ont principalement été dégradés dans les zones anoxiques de la rhizosphère, suggèrant un rôle prépondérant des processus anaérobies. L'analyse par chromatographie chirale du métolachlore a en outre révélé la dégradation préférentielle de l'énantiomère S du métolachlore, confirmant l'importance des processus biologiques dans la dissipation des chloroacétanilides. Les corrélations qui ont pu être observées entre les changements de variables hydrochimiques et de conditions hydrauliques et des différences de composition bactérienne détectées par génotypage par polymorphisme de longueur des fragments de restriction (T-RFLP) et par pyroséquençage du gène ARNr 16S confirme le potentiel de bio-indicateurs basés sur l'ADN pour suivre le fonctionnement des écosystèmes.Sur la base de ce travail, la détection et l'identification des micro-organismes et des voies biochimiques responsables de la dégradation de chloroacétanilides dans les zones humides, ainsi que l'élaboration d'indicateurs génétiques bactériens pour le suivi de la dégradation de chloroacétanilides en zones humides, émergent comme autant d’objectifs de recherche à court-terme. / Chloroacetanilide herbicides are widely used in agriculture, and thereby contribute to environmental pollution. Their fate, including in redox-dynamic receptor ecosystems such as wetlands, remains poorly understood. In this work, microbial degradation of chloroacetanilides (metolachlor, acetochlor and alachlor) was investigated by emerging chemical and molecular biological approaches, at the lab-scale using microcosm columns, and in situ, in outdoor constructed wetlands designed for the treatment of chemical pollutants originating from agriculture.A novel compound-specific isotope analysis (CSIA) method was developed, and the results indicated biodegradation of chloroacetanilides in wetlands, which was also suggested by detection of ethane sulfonic acid and oxanilic acid degradation products. In lab-scale wetland microcosms, chloroacetanilides were mainly degraded in anoxic rhizosphere zones, suggesting a predominant role of anaerobic processes. Chiral chromatographic analysis of metolachlor revealed preferential degradation of the (S) enantiomer of metolachlor, and further confirmed the role of biological processes in chloroacetanilide dissipation. Changes in hydrochemical variables and hydraulic conditions correlated with differences in wetland bacterial composition detected by terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing analyses of the bacterial 16S rRNA gene, confirming the potential of DNA-based bioindicators for follow-up of ecosystem functioning.On the basis of this work, detecting and identifying the microorganisms and biochemical pathways responsible for chloroacetanilide degradation in wetlands, as well as developing bacterial gene-based indicators of wetland functioning, emerge as research objectives for the near future.

Les effets de différentes activités anthropiques (vignoble, cuverie) sur les populations fongiques du raisin au vin ont été étudiés. Afin de caractériser ces effets, il était nécessaire d’avoir accès à la diversité globale des populations (pyroséquençage et IR-TF) mais également à la diversité intraspécifique (IR-TF). La spectroscopie a ainsi été validée pour sa capacité à caractériser la population globale et à discriminer les souches pour trois espèces de levures non-Saccharomyces (NS). Pour la première fois, il est démontré que la baie de raisin constitue une source limitée pour les levures NS alors que la cuverie semble constituer une source importante; l’air étant un vecteur important de dissémination de ces levures. De plus, la persistance et la réimplantation des levures NS dans le moût l’année suivante ont été démontrées. Les activités anthropiques étudiées modifient la diversité fongique. Une biodiversité plus faible pour les raisins de la modalité biologique a été mesurée pour les 3 millésimes considérés. Les populations fongiques sont ensuite fortement remaniées par l’étape de pressurage/clarification et l’influence de la flore de cuverie est confirmée. L’ajout de SO2 modifie les dynamiques des populations et favorise la domination de l’espèce S. cerevisiae. L’analyse chimique non-ciblée des vins montre pour la première fois que ces derniers peuvent être discriminés à la fin de la fermentation alcoolique en fonction de la protection phytosanitaire, que ces fermentations aient eu lieu en présence ou non de SO2. Ainsi, l'existence dans les vins de signatures de diversité chimique et microbiologique liées au mode de protection au vignoble est mise en évidence. / The effects of different anthropogenic activities (vineyard, winery) on fungal populations from grape to wine were studied. To characterize these effects, it was necessary to access to the overall diversity of populations (pyrosequencing and spectroscopy FT-IR) but also to intra-specific diversity (FT-IR). Spectroscopy FT-IR has been validated for their ability to characterize the global population and to discriminate the strains for three species of non-Saccharomyces yeasts (NS). For the first time, it is shown that the grape berry is a limited source for NS yeasts while the winery seems to be a significant source; the air is an important vector for dissemination of these yeasts. In addition, persistence of NS yeast strains from year to year in the winery has been demonstrated. The studied anthropogenic activities modify the fungal diversity. Thus, lower biodiversity of grapes from organic modality was measured for the three vintages considered. The pressing / clarification step revises strongly fungal populations and the influence of the winery flora is confirmed. The addition of SO2 changes the population dynamics and favors the dominance of the species S. cerevisiae. The non-targeted chemical analysis shows, for the first time, that these wines can be distinguished at the end of the alcoholic fermentation (with or without SO2) depending on plant protection. Thus, the existence in wines of chemical and microbiological signatures associated with vineyard protection mode is highlighted.

La serpentinisation est un processus d’altération des roches ultramafiques entrainant la formation de fluides alcalins chauds, riches en dihydrogène et méthane. Elle fournit l'énergie et la matière pour soutenir des communautés microbiennes chimiosynthétiques mais crée des conditions extrêmes en termes de pH et de disponibilité limitée en accepteurs d’électrons. Ce travail porte sur l’étude des communautés procaryotiques du système hydrothermal serpentinisé côtier de la baie de Prony en Nouvelle-Calédonie. Il a permis de déterminer leur composition taxonomique, leur structure et leur diversité. Le « noyau stable » des communautés indigènes déterminé par des techniques moléculaires (DGGE, SSCP, séquençage de banques de clones) se compose de Methanosarcinales, Thaumarchaeota, Chloroflexi, Alpha-, Gamma-, Delta, Beta-proteobacteria et Firmicutes. Ces communautés sont alimentées par des réactions d’oxydoréduction impliquant l’hydrogène, le méthane et les composés soufrés. Elles sont caractérisées par une faible diversité archéenne composée principalement de Methanosarcinales utilisant ou produisant le méthane. Le séquençage haut-débit de l’ADNr 16S bactérien et archéen a permis de mettre en évidence la présence de très nombreux taxons rares, certains jouant potentiellement un rôle dans les cycles biogéochimiques de Prony. Une nouvelle espèce de bactérie fermentaire, Acetoanaerobium pronyense ST07-YE, appartenant aux Firmicutes a été isolée et caractérisée par une approche culturale. Cette bactérie pourrait jouer un rôle important dans le maintien de certaines populations microbiennes, comme les méthanogènes, dans l’écosystème par la production de composés comme l’acétate. / Serpentinization is an alteration process of ultramafic rocks resulting in hydrogen- and methane-rich alkaline hot fluids formation. Serpentinization can provide energy and matter to support chemosynthetic microbial communities but also creates extreme living conditions in terms of pH and limited availability of electron acceptors. This work consisted in the study of the microbial communities of the coastal serpentinized hydrothermal system of the Bay of Prony in New Caledonia. It allowed to determine the taxonomic composition, diversity and structure of prokaryotic communities. The "stable core" of indigenous communities identified by molecular methods (DGGE, SSCP, Sanger sequencing of clone libraries) consisted of Methanosarcinales, Thaumarchaeota, Chloroflexi, Alpha-, Gamma-, Delta, Beta-proteobacteria and Firmicutes. These communities are fueled by redox reactions involving hydrogen, methane and sulfur compounds. The low archaeal diversity is mainly composed of Methanosarcinales who are potential primary producers using or producing methane. High throughput sequencing of the bacterial and archaeal 16S rDNA highlighted numerous rare taxa. Some potentially play a role in the biogeochemical cycles of Prony. The fermentative Acetoanaerobium pronyense ST07-YE sp. nov., belonging to Firmicutes, was isolated and characterized by cultivation approach. This bacterium capable of acetate and other substrates production may play an important role in this ecosystem, especially in the maintenance methanogens.

L'objectif principal de ce travail etait d'explorer les bactéries pathogènes que recèle le tube digestif des gorilles. Pour ce faire, un total de 48 échantillons de selles, provenant du Cameroun, appartenant à 21 gorilles ont été analysés. D'abord la culturomique et le pyroséquençage ont été utilisés pour évaluer exhaustivement la diversité bactérienne. En appliquant la culturomique, 86 conditions de culture dont des milieux fabriqués à base de plantes tropicales, sur un échantillon de selles de gorille, 12 800 colonies microbiennes ont été isolées et testées, et 147 espèces bactériennes identifiées. De nombreux pathogènes opportunistes ont été observés, dont 8 qui sont fréquemment associés à des maladies chez l'homme: Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus et Clostridium botulinum. En utilisant la PCR en temps réel pour cribler des pathogènes bactériens dans les 48 échantillons de selles de gorilles, des bactéries fastidieuses telles que Bartonella spp. Borrelia spp., Coxiella burnetii, Tropheryma whipplei ont été observées. Nous avons estimé la prévalence de ces agents pathogènes qui varie entre 4,76% et 85,7%. Ce travail a permis de savoir que l'homme et le gorille ont en commun plusieurs espèces bactériennes dont des pathogènes émergents. Par conséquent, les gorilles sauvages peuvent servir de réservoir et de source pour l'émergence et/ou la réémergence des bactéries pathogènes pour l'homme. / The main objective of this work is to explore the gorilla's potential role as a reservoir for pathogenic bacteria. We used both microbial culturomics and pyrosequencing to analyze the gorilla gut bacteria. By applying culturomics to one index gorilla, we tested 12,800 colonies and identified 147 different bacterial species, including 5 new species. Many opportunistic human pathogens were observed, including 8 frequently associated with human disease: Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus and Clostridium botulinum. Using specific real-time PCR on 48 gorilla fecal samples, we also observed the fastidious pathogens Bartonella spp. Borrelia spp., Coxiella burnetii, Tropheryma whipplei. Using microsatellite analysis of the gorilla samples, we estimated that the prevalence of these pathogens was between 4.76% and 85.7%. Therefore, the gorilla shares many bacterial pathogens with humans, which suggests that wild gorillas might be a reservoir for the emergence and/or reemergence of these pathogens, especially in areas where human and gorilla habitats overlap and because of the increasing presence of humans in the African equatorial forests.

Les sols forestiers sont des habitats hétérogènes, véritables réservoirs de microorganismes. Parmi ces microorganismes, les eucaryotes filamenteux (champignons et oomycètes) sont très divers et jouent un rôle important dans le fonctionnement et la durabilité des écosystèmes forestiers. Leur diversité et leur répartition spatiale à différentes échelles sont encore peu connues et les facteurs qui sous-tendent cette dispersion sont encore peu étudiés. Aussi, les objectifs étaient (i) d'exploiter le séquençage haut-débit pour des études d'écologie microbienne à large échelle et valider son application aux communautés d'oomycètes pathogènes en milieu forestier, (ii) de décrire ces communautés microbiennes, en termes de diversité et de structure, à différentes échelles spatiales (locale, régionale et continentale), (iii) de caractériser les variables biotiques et abiotiques structurant ces communautés et (iv) d'évaluer la réponse éventuelle des communautés aux variations climatiques. Une première étude pilote à l'échelle de la parcelle a été suivi de deux études à grande échelle spatiale le long de gradients environnementaux. Des gradients d'altitude et un gradient latitudinal, à l'échelle continentale, ont été utilisés comme gradient climatique. L'étude préliminaire a donc validé l'utilisation du pyroséquençage pour les communautés fongiques, et en particulier pour les espèces ectomycorhiziennes, et apporté des éléments pour établir une méthodologie d'échantillonnage couplée à cette technique. L'application de ces outils moléculaires à l'étude des communautés oomycètes pathogènes reste à optimiser. Les résultats obtenus sur les communautés fongiques telluriques suggèrent que dans l'hypothèse d'un réchauffement climatique, la richesse fongique ne serait pas directement affectée mais la composition des communautés le serait. La composition des communautés fongiques est également fortement liée au pH du sol. Ces résultats sont à affiner en étudiant plus en détail divers groupes taxonomiques et écologiques en lien avec des variables climatiques plus précises. Par ailleurs, de nombreuses perspectives sont envisageables pour améliorer la détection des oomycètes dans les sols forestiers, qui reste un challenge en écologie microbienne / Forest soils are heterogeneous habitats full of microorganisms. In particular, the filamentous eukaryotes (fungi and oomycetes) exhibit a huge diversity and play essential functions in the dynamic and sustainable growth of the forest ecosystem. However, their diversity and their distribution are poorly known; thus, so are the structuring factors of these microbial communities. The main goals were: (i) use a high-throughput pyrosequencing to study soil microbial communities at a broad geographical scale, and particularly to validate its use for the study of soil forest pathogenic oomycete communities, (ii) study the diversity and structure of fungal and pathogenic oomycetes communities at several spatial scales, (iii) identify possible climatic and edaphic variables structuring these communities and (iv) estimate the possible response of these microbial communities to climatic variation. A pilot study was undertaken at the stand scale. Then, two additional studies were carried out along environmental gradients at the regional and continental scales. The use of the pyrosequencing technique was found appropriate for the fungal communities, but difficulties arose in studying the pathogenic oomycete community. At the stand scale, results suggested the soil to be a valuable substitute for the roots to access the ectomycorrhizal richness and composition using pyrosequencing. The results along the broad scale gradients suggested that fungal richness may not be affected by climate warming but that the composition would be. Moreover, our work indicated that soil pH is a major factor explaining fungal community composition. The main conclusions are still to be confirmed and deeper knowledge of the response of different fungal phylum, or family, would be required. The detection and thus the diversity estimation of the pathogenic oomycetes in forest soil remains a current challenge

La phyllosphère est l’habitat fourni par la partie foliaire des plantes. De nombreuses espèces microbiennes - pathogènes, saprophytes ou mutualistes des plantes - peuplent cet environnement. Ce compartiment microbien influence donc la dynamique et la structure des communautés végétales. L’objectif principal de cette thèse était d’étudier les effets potentiels du changement climatique sur la structure des assemblages fongiques de la phyllosphère des arbres forestiers, et sur la niche écologique des espèces fongiques pathogènes des arbres forestiers. Nous avons pour cela utilisé deux approches, i) l’étude de gradients altitudinaux et ii) la construction de modèles de niche bioclimatique.Les assemblages fongiques de la phyllosphère des arbres forestiers étant encore peu connus, nous avons dans un premier temps décrit leur diversité et quantifié leur variabilité spatiale à l’échelle d’une parcelle forestière.Nos résultats montrent que la phyllosphère d’un arbre forestier abrite quelques centaines d’espèces fongiques, avec quelques espèces dominantes et beaucoup d’espèces rares. Les facteurs structurant ces assemblages incluent à la fois des facteurs abiotiques et biotiques : la température apparaît comme la variable climatique la plus explicative le long d’un gradient altitudinal ; à l’échelle d’une parcelle, la proximité génétique entre arbres est plus déterminante que leur distance géographique.L’analyse des modèles de niche des champignons pathogènes forestiers à l’échelle de la France met en évidence des limitations climatiques, les pluies estivales étant une variable explicative importante.Toutefois, plusieurs espèces introduites occupent déjà la plus grande part de la distribution de leur hôte,sans limitation apparente par le climat. Les effets du changement climatique sur la plupart des pathogènes s’exerceront d’abord indirectement par des effets dépressifs très importants sur l’abondance de leurs arbres-hôtes. Seuls les pathogènes adaptés au biotope méditerranéen verraient leur impact s’accroitre. / Phyllosphere is the habitat provided by the leaves of living plants. Many microbial species -pathogens, saprophytes or mutualists of plants - inhabit this environment. These microbes therefore influence the dynamics and structure of plant communities. The main objective was to study the potential effects of climate change on the structure of phyllosphere fungal assemblages, and on the ecological niche of pathogenic fungal species of forest trees. We used two approaches, i) the study of altitudinal gradients and ii) the construction of bioclimatic niche models. Since phyllosphere fungal assemblages of forest trees are still poorly known, we first described their diversity and quantified their spatial variability at the scale of a forest stand.Our results show that the phyllosphere of a forest tree houses hundreds of fungal species, with few dominant species and many rare species. Factors structuring these assemblages include both abiotic and biotic factors: the temperature appears as the most explanatory variable along an elevation algradient. At the scale of a forest stand, the genetic proximity between trees is more important than the geographic distance. Analysis of the bioclimatic niche models of pathogenic fungi forest at the French scale highlights some climatic limitations, and the summer rainfall is an important explanatory variable. However, many introduced species already occupy the distribution of their host, without apparent climatic limitation. The effects of climate change on most pathogens will be exercised indirectly by very important depressive effects on the abundance of their host trees. Only pathogens adapted to the Mediterranean biotope would increase their impact.