<p>Ancient DNA has the power to elucidate ecological and evolutionary relationships that were previously only quantifiable by proxy. This work details both a metagenetic and a targeted metagenomic study of ancient sedimentary DNA. By using DNA to investigate the plants and animals present in twelve different time points, we describe the nature of the ecological change over the Pleistocene-Holocene transition. We show that as the stability of the habitat degraded due to climate change, the dominant plant communities exhibited a shift from functional groups such as forbs to shrubs and trees. As this cascading change consequently affected the animal communities, we demonstrate the decline, extinction, and replacement of a variety of megafaunal species and mammoths. As well, we provide a proof-ofconcept for the targeted oligonucleotide enrichment for ancient sedimentary DNA. By processing the same DNA extracts with targeted enrichment, we show that metagenomic soil DNA can provide the same taxonomic fingerprint unique to each time period even using different genetic loci. This unique pattern can be used as a reference in future studies. Although the oligonucleotide baits did not yield the composition of taxa that we expected, the oligonucleotide baits did increase the eukaryotic fraction of DNA extracts by up to 50%. Overall, this technique is open to further study and has fantastic potential to redefine the metagenomic work of ancient DNA soil cores.</p> / Master of Science (MSc)

L’avènement du séquençage métagénomique aléatoire a révolutionné la microbiologie en permettant la caractérisation sans culture préalable de communautés microbiennes complexes telles que le microbiote intestinal humain. Des outils bioinformatiques récemment développés atteignent une résolution au niveau de la souche en recensant des gènes accessoires ou en capturant des variants nucléotidiques (SNPs). Toutefois, ces outils sont limités par l’étendue des génomes de référence disponibles qui sont loin de couvrir toute la variabilité microbienne. En effet, de nombreuses espèces n’ont pas encore été séquencées ou sont représentées par seulement quelques génomes.La création de catalogues de gènes non redondants par assemblage de novo suivie du regroupement des gènes co-abondants révèlent une partie de la matière noire microbienne en reconstituant le répertoire de gènes d’espèces potentiellement inconnues. Bien que les méthodes existantes identifient avec précision les gènes core présents dans toutes les souches d’une espèce, elles omettent de nombreux gènes accessoires ou les divisent en petits groupes de gènes qui ne sont pas associés aux core génomes. Or, capturer ces gènes accessoires est indispensable en recherche clinique et épidémiologique car ces derniers assurent des fonctions spécifiques à certaines souches telles que la pathogénicité ou la résistance aux antibiotiques.Lors de cette thèse, nous avons développé MSPminer, un logiciel performant qui reconstitue et structure des pan-génomes d’espèces métagénomiques (ou MSPs pour Metagenomic Species Pan-genomes) en regroupant les gènes co-abondants dans un ensemble d’échantillons métagénomiques. MSPminer s’appuie sur une nouvelle mesure robuste de la proportionnalité couplée à un classificateur empirique pour regrouper et distinguer les gènes core mais aussi les gènes accessoires des espèces microbiennes.Grâce à MSPminer, nous avons structuré un catalogue de 9,9 millions de gènes du microbiote intestinal humain en 1 661 MSPs. L’homogénéité de l’annotation taxonomique, de la composition nucléotidique ainsi que la présence de gènes essentiels indiquent que les MSPs ne correspondent pas à des chimères mais à des objets biologiquement cohérents regroupant des gènes provenant de la même espèce. Parmi ces MSPs, 1 301 (78%) n’ont pas pu être annotées au niveau espèce montrant que de nombreux microorganismes colonisant l’intestin humain demeurent inconnus malgré les progrès substantiels des techniques de culture microbienne. Remarquablement, les MSPs capturent bien plus de gènes que les clusters générés par les outils existants tout en garantissant une spécificité élevée.Cet ensemble de MSPs peut d’ores et déjà être utilisé pour le profilage taxonomique et la découverte de biomarqueurs dans des échantillons de selles humaines. Ainsi, nous tirons parti des MSPs pour comparer l’impact sur le microbiote intestinal des deux principaux types de chirurgie bariatrique, la gastrectomie par laparoscopie (LSG) et la dérivation gastrique de Roux-en-Y (LRYGB). Enfin, les MSPs ouvrent la voie à des analyses au niveau souche. Dans une autre cohorte, nous avons mis en évidence l’existence de sous-espèces associées à l’origine géographique de l’hôte en étudiant les profils de présence/absence des gènes accessoires groupés dans les MSPs. / The advent of shotgun metagenomic sequencing has revolutionized microbiology by allowing culture-independent characterization of complex microbial communities such as the human gut microbiota. Recently developed bioinformatics tools achieved strain-level resolution by making a census of accessory genes or by capturing nucleotide variants (SNPs). Yet, these tools are hampered by the extent of available reference genomes which are far from covering all the microbial variability. Indeed, many species are still not sequenced or are represented by only few genomes.Building of non-redundant gene catalogs followed by the binning of co-abundant genes reveals a part of the microbial dark matter by reconstituting the gene repertoire of species potentially unknown. While existing methods accurately identify core genes present in all the strains of a species, they miss many accessory genes or split them into small gene groups that remain unassociated to core genomes. However, capturing these accessory genes is essential in clinical research and epidemiology because they provide functions specific to certain strains such as pathogenicity or antibiotic resistance.In this thesis, we developed MSPminer, a computationally efficient software tool that reconstitutes Metagenomic Species Pan-genomes (MSPs) by binning co-abundant genes across metagenomic samples. MSPminer relies on a new robust measure of proportionality coupled with an empirical classifier to group and distinguish not only species core genes but accessory genes also.With MSPminer, we structured a catalog made up of 9.9 million genes of the human gut microbiota in 1 661 MSPs. The homogeneity of the taxonomic annotation, of the nucleotide composition as well as the presence of essential genes indicate that the MSPs do not correspond to chimeras but to biologically consistent objects grouping genes from the same species. Among these MSPs, 1 301 (78%) could not be annotated at species level showing that many microorganisms colonizing the human intestinal tract are still unknown despite the substantial improvements of microbial culture techniques. Remarkably, MSPs capture more genes than clusters generated by existing tools while ensuring high specificity.This set of MSPs can be readily used for taxonomic profiling and biomarkers discovery in human gut metagenomic samples. In this way, we take advantage of the MSPs to compare the impact of two main types of surgeries, the laparoscopic sleeve gastrectomy (LSG) and the Roux-En-Y gastric bypass (LRYGB). Finally, the MSPs open the way to strain-level analyses. In another cohort, we identified subspecies associated the host geographical origin by studying presence/absence patterns of the accessory genes grouped in the MSPs.

This dissertation examines the microbial and functional diversity in Kartchner Caverns, a limestone cave in Arizona, USA. Kartchner is highly oligotrophic due to the lack of photosynthesis and the limited inputs of organic material from the surface. This characteristic poses a challenge for microbial life in the cave. The first objective of this work was to evaluate the bacterial richness, diversity and taxonomic composition of speleothems surfaces within Kartchner Caverns in order to gain insight into the distribution patterns associated with these communities. Secondly, the metabolic strategies used by cave communities to survive harsh cave conditions were investigated based on phylogenetic associations and metagenomics. Both objectives were directed toward answering the questions "who are there?" and "what are they doing?". The 454-pyrotag analysis of the V6 region of the 16S rRNA gene revealed an unexpectedly high bacterial diversity with each speleothem supporting a unique bacterial community profile. A focused study on one room of the cave revealed three community types: Type 1 was dominated by the phylum Proteobacteria; Type 2 by Actinobacteria; and Type 3 by Acidobacteria. Phylogenetic associations of the sequences generated by the 454 sequencing and by a Sanger clone library suggested cave microbial communities are supported by chemoautotrophic activities such as nitrite and iron oxidation. Results from the phylogenetic associations guided the metagenomic analysis which supports the presence of chemoautotrophic activities in the cave. Genes for two complete CO2 fixation mechanisms, the Calvin-Benson-Bashan and the rTCA cycles were identified in the cave metagenome, as well as genes for ammonia and nitrite oxidation. These genes are associated with both Bacteria and Archaea suggesting members of both domains are acting as primary producers in the cave ecosystem. Comparative analysis of cave samples to other environments suggests an overabundance of DNA repair mechanisms which could be potentially used by cave communities to overcome the toxicity due to high concentrations of calcium on the speleothem surfaces. This work provides the first comprehensive analysis of the microbial diversity and potential strategies used by microbial communities to survive under the extreme conditions found in a semi-arid limestone cave environment.

O processamento do café via úmida inclui as etapas de despolpamento, fermentação, lavagem e separação dos grãos, no qual gera-se grande quantidade de resíduos sólidos (casca e polpa) e água residuária. O estudo dos aspectos microbiológicos na produção de hidrogênio a partir de resíduos do processamento do café é pouco explorado, o que torna esse resíduo atrativo e foco de pesquisas. Neste cenário, buscou-se avaliar o potencial uso destes resíduos como substrato e fonte de bactérias e fungos fermentativos para produção de H2. Adicionalmente, estudou-se a homoacetogênese, processo que afeta a produção de H2, uma vez que o H2/CO2 pode ser consumido para a síntese de ácido acético. Para isso, ensaios em reatores em batelada foram conduzidos para seleção da melhor condição de pré-tratamento hidrotérmico (severidade), dos fatores da fermentação (pH, temperatura, agitação, volume do headspace, concentração de bioaumentação do consórcio microbiano, resíduo de polpa e casca, água residuária e extrato de levedura) que afetam a produção de H2 (Planejamento Plackett-Burman) e homoacetogênese (planejamento fracionado 24-1), seguido do delineamento composto central rotacional (DCCR) para otimização da produção de hidrogênio. Verificou-se que a severidade de pré-tratamento hidrotérmico de 3,53 (180 °C, 15 minutos), resultaram no aumento da concentração de açúcares fermentáveis, com baixa concentração de lignina, fenol, furfural e 5-hidroximentil furfural, obtendo valores de potencial máximo de produção de H2 (P) de 1,8 mL H2. Em relação ao efeito dos fatores da fermentação sobre a produção de H2, obteve-se o maior P de 82 mL H2 em pH 7,0, 30 g DQO L-1 de água residuária, 6 g L-1 de polpa e casca, 30 ºC, 50% de headspace, 180 rpm, sem bioaumentação do consórcio microbiano e 2 g L-1 de extrato de levedura. Por outro lado, as condições que conduziram a consumo significativo de H2 por homoacetogênese foram em pH inicial entre 5,5 e 7,5, headspace entre 40 e 60%, concentração de água residuária entre 10 e 30 g DQO L-1 e concentração de casca e polpa entre 3 e 9 g L-1. Em relação às variáveis estatisticamente significativas (pH, concentração de polpa e casca e volume do headspace), as condições operacionais ótimas para obtenção de P de 240 mL H2, estimadas via planejamento DCCR, foram em pH 7,0, concentração de polpa e casca 7 g L-1 e volume de headspace 30%. No consórcio microbiano, houve predominância de bactérias semelhantes a Lactobacillus sp. e Clostridium sp. e de fungos semelhantes a Saccharomyces sp. e Kazachstania sp. Tais microrganismos foram associados a produção de ácido lático, H2, etanol e ácido acético nos reatores, identificando genes relacionados a enzimas para a degradação de lignina, fenol, celulose, hemicelulose e pectina. Observou-se alta abundância relativa de Clostridium sp. tanto nos ensaios de produção de H2 quanto nos ensaios com consumo de hidrogênio, sendo seus genes associados a homoacetogênese, produção de ácido propiônico e butanol. / Wet coffee processing includes pulping, fermentation, washing and grain separation, in which large amounts of solid waste (husk and pulp) and wastewater are generated. The study of microbiological aspects in the hydrogen production from coffee waste was little explored, which makes this residue attractive and the focus of research. In this scenario, we evaluate the potential use of coffee waste as substrate and source of fermentative bacteria and fungi for H2 production. In addition, homoacetogenesis, a process, that negatively affects H2 production, was studied since H2/CO2 can be consumed for acetic acid production. Assays in batch reactors were conducted to select the best hydrothermal pretreatment condition (severity), fermentation factors affecting the H2 production (pH, temperature, agitation, headspace, bioaugmentation of microbial consortium, concentration of pulp and husk, wastewater and yeast extract) (Plackett and Burman design) and homoacetogenesis (fractional factorial design 24-1), followed by the Rotational Central Composite Design (RCCD) to optimize the H2 production. It was verified that the hydrothermal pretreatment severity of 3,53 (180 °C, 15 minutes), resulted in the increase of fermentable sugars, with low concentration of lignin, phenol, furfural and 5-hydroxyximethyl furfural, obtaining values of the maximum H2 production potential (P) of 1,8 mL H2. The fermentation factors conducing to highest P of 82 mL H2 were pH 7,0, wastewater 30 g COD L-1, 6 g L-1 pulp and husk, 30 °C, 50% headspace, 180 rpm, without bioaugmentation of the microbial consortium and 2 g L-1 yeast extract. On the other hand, the conditions leading to significant H2 consumption by homoacetogenesis were initial pH between 5,5 and 7,5, headspace between 40 and 60%, wastewater concentration between 10 and 30 g COD L-1 and concentration of husk and pulp between 3 and 9 g L-1. In relation to the fermentation factors statistically significant in the H2 production (pH, pulp and husk concentration and headspace), the optimal operational conditions to obtain P of 240 mL H2, estimated through RCCD design, were at pH 7,0, 7 g L-1 pulp and husk concentration and 30% headspace volume. In the microbial consortium, there was a predominance of bacteria similar to Lactobacillus sp. and Clostridium sp. and fungi similar to Saccharomyces sp. and Kazachstania sp. These microorganisms were associated with the production of H2, lactic acid, ethanol and acetic acid in the reactors, identifying genes related to enzymes for the degradation of lignin, phenol, cellulose, hemicellulose and pectin. There was a high relative abundance of Clostridium sp. both in the H2 production assays and in the H2 consumption assays, with genes associated to homoacetogenesis, production of propionic acid and butanol.

Nessa pesquisa foram testados separadamente dois inóculos solo/compostagem e lodo termofílico de reator anaeróbio de manta de lodo e fluxo ascendente (UASB) do tratamento de vinhaça em relação ao potencial de produção de compostos de valor agregado a partir do bagaço de cana-de-açúcar (BCA) em reatores em batelada em condição mesofílica e termofílica, respectivamente. Pré-tratamentos térmico, ácido e diluição seriada foram aplicados ao inóculo termofílico, como tentativa de inibir as arqueias metanogênicas. A diluição seriada foi o pré-tratamento aplicado em ambos os inóculos, mesofílico e termofílico, para obtenção de consórcio de bactérias fermentadoras. Cinco meios de culturas foram testados como fonte de nutrientes para o crescimento de bactérias celulolíticas e fermentadoras. Dentre esses foi selecionado para os ensaios mesofílicos e termofílicos, o meio de cultura mais complexo suplementado com extrato de levedura. O BCA foi submetido à pré-tratamento biológico, térmico, explosão a vapor, deslignificação alcalina e hidrotérmico sendo o último utilizado nos ensaios do planejamento fatorial. O efeito das variáveis independentes de concentração de extrato de levedura e temperatura foi avaliado na produção de hidrogênio, metano e ácidos orgânicos a partir do inóculo termofílico. O efeito da concentração de extrato de levedura e substrato (BCA) foi avaliado na produção de bioprodutos a partir do inóculo mesofílico (37ºC). A maior produção de hidrogênio foi de 17,30 mmol/L à 60ºC e 3,42 g/L de extrato de levedura para a condição termofílica. Em relação aos ensaios mesofílicos observou-se 1,53 mmol/L com 3,42 e 5,00 g/L de extrato de levedura e BCA, respectivamente. Caracterização taxonômica e funcional dos microrganismos dos reatores de melhor desempenho de produção de hidrogênio dos planejamentos fatoriais termofílico e mesofílico foi realizada por análise Metagenômica (Illumina HiSeq). Nestas condições foram identificados microrganismos dos domínios Archaea, Bacteria, Eukarya, além de vírus. Para o domínio Bacteria foram identificados microrganismos celulolíticos e fermentadores como Coprothermobacter e Clostridium, enquanto para o domínio Archaea, foram identificadas metanogênicas hidrogenotróficas e acetoclásticas, como Methanothermobacter e Methanosarcina. Foram ainda identificados genes codificantes de enzimas catalisadoras da degradação de celulose, hemicelulose e lignina, constituintes principais da biomassa lignocelulósica utilizada como substrato, tais como celulase, carboxylesterase e 2-ácido hidroxi oxidase, respectivamente. Microrganismos aderidos no BCA in natura foram observados por microscopia eletrônica de varredura (MEV) e identificados por sequenciamento do RNAr 16S, e semelhantes a Streptomyces, Paenibacillus, Stenotrophomonas, Sphingomonas. Os microrganismos do lodo termofílico, solo e compostagem foram caracterizados por sequenciamento do RNAr 16S, e foram semelhantes a Clostridium, Thermoanaerobacter, Caloramator, Anaerobaculum, Tatlockia, Coprothermobacter, Dysgonomonas, Coprococcus, Sporomusa, Methanobacterium, Methanothermobacter, Methanosaeta, dentre outros. / In the present study, two inoculum (soil/compost and thermophilic sludge from upflow anaerobic sludge blanket from vinasse treatment) were separately evaluated as potential to production of value-added products from sugarcane bagasse in mesophilic and thermophilic conditions, respectively. Thermal, acid and serial dilution pretreatments were performed in the thermophilic inoculum to inhibition of methanogenic archaea. Serial dilution was applied into the mesophilic inoculm. Five culture medium were evaluated as nutritional source to enrichment of cellulolytic and fermenters bacteria; between then, the most complex one, supplemented with yeast extract was selected for the mesophilic and thermophilic bioassays. The sugarcane bagasse (SCB) was submitted to biological, thermal, stem explosion alkaline delignification and hydrothermal pretreatments, and the last one was used as substrate for the factorial designs. The effect of independent variables, such as yeast extract and temperature were evaluated on the hydrogen, methane and organic acids production from the thermophilic inoculum. The effect of yeast extract and substrate (SCB) concentrations were evaluated on the bioproducts generation from the mesophilic inoculum. The highest hydrogen production of 17.3 mmol/L was obtained at 60ºC and 3.42 g/L of yeast extract, on the thermophilic factorial design. In relation to the mesophilic factorial design, obtained 1.53 mmol/L of hydrogen with 3.42 and 5.00 g/L of yeast extract and SCB, respectively. Taxonomical and functional characterizations from the microorganisms were performed in the reactors with highest hydrogen production on the factorial designs using Metagenomics analysis (Illumina HiSeq). In both condition, mesophilic and thermophilic were found microorganisms from Archaea, Bacteria, Eukarya domin, besides Viruses. Concerning the Bacteria domain were found cellulolytic and fermenters microorganisms similar to Coprothermobacter and Clostridium, whiles for Archaea domain were identified hydrogenotrophic and acetoclastic methanogenic similar to Methanothermobacter and Methanosarcina. There were obtained genes coding to enzymes related to the cellulose, hemicellulose and lignin degradation such as carboxylesterase e 2-acid-hydroxioxidase, respectively. Microorganisms adhered into the in natura SCB fiber were observed by scanning electronic microscopy (SEM) and identified by 16 S rRNA sequencing, mainly as similar to Streptomyces, Paenibacillus, Stenotrophomonas, Sphingomonas. The microorganisms from the thermophilic sludge, soil and composting were also characterized by 16S RNAr sequencing and were similar to Clostridium, Thermoanaerobacter, Caloramator, Anaerobaculum, Tatlockia, Coprothermobacter, Dysgonomonas, Coprococcus, Sporomusa, Methanobacterium, Methanothermobacter, Methanosaeta, among others.

Metagenômica é a técnica que permite analisar os genomas de microorganismos que habitam determinados nichos do ambiente sem a necessidade de isolar e cultivar cada um separadamente. Ao conjunto de microorganismos que habita um determinado nicho se dá o nome de microbi- oma. Análises do perfil da diversidade taxonômica e funcional de comunidades microbianas em microbiomas são comuns em estudos de metagenômica. No entanto, atualmente as plata- formas de uso geral (como MG-RAST e IMG/M) tendem a separar as análises baseadas em reads (sequências não montadas) das baseadas em contigs (sequências montadas), isto dificulta as análises destes dados. Motivado por esta separação, desenvolvemos uma plataforma web, batizada de CARAVELA, que facilita a conexão entre os resultados de análises de diversidade taxonômica e funcional baseadas em reads e contigs respectivamente. Uma das principais fun- ções da plataforma CARAVELA é associar a identificação taxonômica de cada read com o contig que este read faz parte e, anotações funcionais do contig, quando existirem. Essa função deve permitir a rápida identificação de contigs potencialmente quiméricos bem como contigs taxonomicamente bem resolvidos. Também é possvel fazer buscas, tais como: listar todos os contigs que tenham um ou mais reads classificados como Pseudoxanthomonas suwonensis em sua composição e ainda, é possvel navegar nos contigs de maneira similar a navegadores de metagenomas tradicionais. Podem ser utilizados como arquivos de entrada a sada de outros programas, desde que o formato atenda certos padrões. A plataforma CARAVELA foi desenvol- vida com Java, HTML, CSS, Javascript e Mysql, e com o fim de testar a ferramenta, utilizamos o conjunto de dados metagnômicos obtidos a partir da operação de compostagem do Parque Zoológico de São Paulo. / The taxonomic diversity analysis (read-based) and functional analysis (contig / gene-based) from metagenomic studies usually generate information that is complementary. However, the tools that produce gene annotations (eg IMG / M) and taxonomic assignments (eg MyTaxa) do not allow easy integration of these results. Motivated by this split, we are develop a web platform called Caravela to facilitate the integration, search and visualization of information provided by read-based analyzes and contig / gene-based analyzes. The tool is able to display the list of contigs and for each contig, it displays annotated genes, reads participating in its composition and rate associated with each read (when such association exists). Such a capability enable manual / automated curation of assembly as well as taxonomic assignments (detection of possible mis-assignments). The platform able to accept output files from a variety of tools, as long as the file formats follow certain standards. The tests was performed on a dataset of metagenomic reads obtained from the composting operation of the São Paulo Zoological Park. The tool was implemented using Java technology, HTML, CSS and Javascript. Information was stored in a MySQL database.

Considerando a dieta como um fator modulador do microbioma ruminal, neste trabalho objetivou-se investigar o impacto do bagaço da cana-de-açúcar sobre a composição e funcionalidade das espécies microbianas residentes no rúmen de carneiros (Ovis aries). Foram utilizados seis animais machos fistulados de O. aries, dos quais três foram alimentados com uma dieta composta por 70% de volumoso e 30% de concentrado (tratamento controle) e outros três animais alimentados com uma dieta similar a anterior, mas com 14% do volumoso substituído por bagaço de cana-de-açúcar (tratamento bagaço). O conteúdo ruminal (líquido e fibra) foram amostrados quinzenalmente durante 60 dias. A partir dessas amostras foram acessadas a estrutura e a composição da comunidade microbiana pela extração de DNA total e amplificação das regiões V3 e V6-V7 do gene 16S rRNA bacteriano e a região intergênica fúngica (ITS2). Além disso, foram feitas análises metagenômicas e metatranscriptômicas de comunidade microbianas enriquecidas em fibra ruminal para identificar enzimas lignocelulolíticas expressas. As frações líquida e fibrosa do conteúdo ruminal de O. aries revelaram uma comunidade bacteriana dominada principalmente por Bacteroidetes e Firmicutes ao longo de todo período experimental. Dois gêneros, Prevotella e Ruminococcus representaram 20% e 4% da comunidade bacteriana ruminal, respectivamente. Para a comunidade fúngica o filo Neocallimastigomycota representou 91% das sequências e os principais gêneros deste filo foram Piromyces, Neocallimastix, Orpinomyces, Anaeromyces, Caecomyces e Cyllamyces aderidos a fibra ruminal. O gênero Caecomyces, foi significativamente mais abundante na fibra ruminal de animais que se alimentaram de bagaço de cana-de açúcar. Além disso, foi observado um aumento significativo na frequência de enzimas como, por exemplo, 1,4-&alpha;-glucano, &alpha;-galactosidase, endo 1,4-&beta;-xilanase, &beta;- xilosidase, xilose isomerase, celobiose fosforilase e &alpha;-N-arabinofuranosidase no tratamento com bagaço de cana-de-açúcar. Considerando que a recuperação de enzimas a partir de comunidades microbianas naturalmente selecionadas para a degradação de biomassa é uma estratégia promissora para superar a atual ineficiência da ação enzimática na produção industrial de biocombustíveis, os resultados deste trabalho representam a possibilidade de aumentar a capacidade de recuperação ou descoberta de enzimas a partir de ruminantes, ou ainda, a possibilidade de manipular a estrutura do microbioma do rúmen para usá-lo como fonte de inóculo enriquecido em processos industriais de degradação de biomassa. / Considering the diet as a modulator of ruminal microbiome, this work aimed to investigate the impact of sugarcane bagasse on the composition and function of microbial species residents in the sheep (Ovis aries) rumen. Six cannulated male animals were used in the experiment, where three individuals were fed on a diet consisting of 70% forage and 30% concentrate (control treatment), and three were fed on a similar diet, but with sugarcane bagasse replacing 14% of the forage portion (bagasse treatment). The ruminal content (i.e., liquid and fiber) were sampled every two weeks during 60 days. From these samples, the structure and composition of the microbial community were assessed by total DNA extraction and amplification of V3 and V6-V7 regions of 16S rRNA gene from bacteria and the fungal intergenic region (ITS2). Furthermore, metagenomics and metatranscriptomics approaches were used to evaluate the enrichment of specific members of the microbial community in the ruminal fiber and genes related to lignocellulolytic enzymes. The liquid and fiber fractions of the O. aries rumen revealed a microbial community dominated mainly by Firmicutes and Bacteroidetes throughout the experimental period. The genera Prevotella and Ruminococcus accounted for 20% and 4% of the bacterial community of rumen, respectively. In the fungal community, the phylum Neocallimastigomycota accounted for 91% of sequences and its main genera adhered on the ruminal fiber were Piromyces, Neocallimastix, Orpinomyces, Anaeromyces, Caecomyces and Cyllamyces. The genus Caecomyces was significantly more abundant in the ruminal fiber in animals fed on sugarcane bagasse. Furthermore, there was a significant increase in the frequency of enzymes, such as &alpha;-1,4-glucan, &alpha;-galactosidase, endo- 1,4-&beta;-xylanase, &beta;-xylosidase, xylose isomerase, cellobiose phosphorylase and &alpha;- Narabinofuranosidase in the bagasse treatment. Considering that the recovery of enzymes from ecosystems naturally evolved for degradation of biomass is a promising strategy to overcome the current inefficient enzymatic action in industrial production of biofuels, the results of this study bring great possibilities to increase the discovery and or recovery of enzymes from ruminants, as well as the possibility of the ruminal microbiome structure manipulation to be used as source of an enriched inoculum for biomass degradation in industrial processes.

A construção de bibliotecas metagenômicas tornou possível o acesso ao potencial biotecnológico de micro-organismos não cultiváveis. O solo é um habitat pouco explorado, com elevada diversidade bacteriana que possuem genes codificadores de enzimas de interesse industrial, chamadas de biocatalisadores naturais. Dentre estas, destacam-se as enzimas lipolíticas, lipases e esterases, por promoverem a hidrólise de matérias-primas de alto valor agregado e com muitas aplicações biotecnológica. Devido a necessidade, existe uma constante busca para isolar novos genes com diferentes especificações. Neste trabalho, o gene lip4, isolado de biblioteca metagenômica de solo, foi superexpresso, purificado e identificado como membro da família V das lipases bacterianas. Tem atividade ótima hidrolisando triacilglicerol de cadeia média em pH alcalino sem presença de íons. A estrutura cristalográfica obtida identificou o dobramento conservado das &alpha;/&beta; hidrolases e a tríade catalítica, Ser94-Asp217-His245, além da presença do domínio CAP, comum nas esterases. / Metagenomic libraries construction make possible the access to the biotechnological potential of not cultivated microorganisms. Soil environment has a big bacterial diversity with genes encoding industrial interest enzymes, named natural biocatalysts. Among these, lipolytic enzymes, that includes lipases and estarases, are able to catalyse different biochemistry reaction and promoting raw material hydrolysis with added values. In this overview, a search for new genes with different specifications, allowed isolation by functional screening in tributyrin agar, a gene lip4 from a soil metagenomic library. These gene was overexpressed, purified and identified as a member to family V of bacterial lipases. Presents better activity in alkaline pH with medium chain triacyglycerol without ions. Three dimensional structure of Lip4 identified a conserved &alpha;/&beta; hydrolase backbone and the catalytic triad Ser94-Asp217-His245, besides the presence of CAP domain, common structure in esterase.

La crevette hydrothermale Rimicaris exoculata est une espèce endémique des sites hydrothermaux de la ride médio-atlantique (MAR). Cette espèce vit en agrégats denses sur les parois des cheminées hydrothermales actives et domine la mégafaune de la plupart de ces sites. Cette crevette abrite deux communautés microbiennes distinctes dont les rôles demeurent partiellement élucidés. Une de ces communautés est associée à la cavité céphalothoracique et aux pièces buccales hypertrophiées. Celles-ci sont recouvertes par une importante communauté épibionte correspondant à des bactéries chimioautotrophes associées à des dépôts minéraux. Afin d’approfondir notre connaissance de cette communauté symbiotique et dans le cadre de la thèse, une approche de métagénomique a été entreprise.Celle-ci a permis de mettre en évidence la diversité microbienne présente, avec un phylum dominant, celui des Protéobactéries. Au sein de ce phylum, deux classes sont dominantes, les Gamma- et les Epsilonproteobacteria, d’autres classes sont également présentes, les Alpha-, Delta- et Beta-proteobacteria.Une nouvelle classe de Protéobactéries, les Zetaproteobacteria, très récemment décrite comme bactéries libres dans l'environnement hydrothermal, a été mise en évidence. Les types métaboliques bactériens ont également pu être étudiés de façon plus approfondie permettant d’avancer encore davantage dans la compréhension de la symbiose entre la crevette et les microorganismes s’y développant. Il a notamment été possible de reconstituer des voies métaboliques entières pour chaque épibionte principal (à savoir Epsilon-et Gammaproteobacteria) ainsi que d'étudier des gènes impliqués notamment dans la réponse aux fluctuations environnementales, dans la communication cellulaire et le quorum-sensing. Le groupe des Zetaproteobacteria a également été étudié, tout d'abord dans le cadre de l'analyse métagénomique, mais également par des approches moléculaires sur des spécimens provenant de plusieurs sites hydrothermaux de la MAR, et ce, afin de mieux définir l'association possible entre ces bactéries ferro-oxydantes et R. exoculata. La seconde communauté associée à R. exoculata réside dans le tractus digestif. Des analyses complémentaires aux précédentes études ont été menées lors de ce travail de thèse combinant notamment approche métagénomique et culturale. / The hydrothermal shrimp Rimicaris exoculata is an endemic species of the Mid-Atlantic Ridge (MAR).This species lives in dense aggregates on the walls of active chimneys and dominates the megafauna of most of these sites. This shrimp has two distinct microbial communities whose roles remain almost unknown. One of these communities is associated with the cephalothorax and hypertrophied mouthparts.These are covered by a dense community of bacteria affiliated to chemoautotrophic epibionts associated with mineral deposits. To deepen our knowledge on the symbiotic community in the context of the thesis, a metagenomic approach was undertaken. This helped highlighting the microbial diversity present with a dominant phylum, the Proteobacteria. Two classes are dominant, Gamma-and Epsilonproteobacteria, other classes are also present. A new class of Proteobacteria, the Zetaproteobacteria very recently described as free living in hydrothermal environments, was identified. Bacterial metabolic types have also been studied in more detail via the metagenome to advance further in the understanding of the symbiosis. It has been possible to reconstruct metabolic pathways for each main epibiont (i.e. Epsilon- and Gammaproteobacteria), and to study genes in response to environmental fluctuations, in cell communication and quorum-sensing. The Zetaproteobacteria group has also been studied, first in the context of metagenomic analysis, but also using molecular approaches on several specimens from the MAR hydrothermal sites, to better define the possible association between R. exoculata and iron-oxidizing bacteria. The second community associated with R. exoculata is located in the digestive tract. Additional analyzes to previous studies have been conducted in this thesis including a metagenomic approach and cultures.

Falhas reprodutivas são importante causa de prejuízos econômicos na suinocultura. Elas implicam na diminuição do número de leitões nascidos vivos e aumentam o descarte de animais e as taxas de reposição de matrizes, levando à redução da produtividade do rebanho. Embora a maioria dos casos de natimortalidade sejam associados a fatores não infecciosos, os agentes infecciosos possuem um papel importante e ainda pouco conhecido na etiologia deste quadro. Até o presente, nenhum trabalho foi realizado visando o estudo do conjunto de vírus que possam estar presentes em matrizes com eventos de natimortalidade por ocasião do parto. Em função disso, o presente trabalho teve por objetivo examinar o viroma do soro de matrizes suínas com e sem casos de natimortalidade. Foram coletadas 94 amostras de soro de matrizes de seis granjas distribuídas em cinco municípios do Rio Grande do Sul. Em cada granja foram formados dois pools de soros: um composto por matrizes que pariram (um ou mais) natimortos e outro por matrizes que pariram leitegadas sem natimortos. Os pools foram submetidos à extração de ácido nucleico viral, enriquecimento e sequenciamento de alto desempenho, buscando a identificação de agentes que possam representar um fator de risco à natimortalidade em suínos Não foi possível identificar diferenças significativas nos viromas de matrizes correlacionadas à ocorrência de natimortalidade. Não obstante, foi possível identificar uma ampla variedade de genomas virais, a maioria deles correspondendo a vírus das famílias Anelloviridae. Este estudo permitiu ainda identificar 20 genomas completos de três espécies de vírus: torque teno vírus suíno 1a e 1b, circovírus suíno tipo 3 (PCV3) e vírus circulares DNA fita simples codificantes de replicase (CRESS), seis dos quais até o presente ainda não reportados em suínos. Em duas granjas, em matrizes que apresentaram natimortalidade, foram identificados genomas de PCV3, cuja participação como potencial causador de problemas reprodutivos precisa ser futuramente investigada. Não foram identificados vírus com genoma de RNA. Este estudo traz uma contribuição ao conhecimento do viroma em soros de matrizes suínas e, paralelamente, busca contribuir para o esclarecimento das possíveis causas de natimortalidade de origem infecciosa em suínos. / Reproductive failure in swine herds is an important cause of economic losses. It leads to a decrease in the number of piglets reared per sow and may imply in the need for replacement of sows, reducing the productivity in a herd. Although the majority of cases of stillbirths have been attributed to non-infectious causes, several infectious agents have been implicated in the etiology of such condition. Nevertheless, other as yet unknown agents may be involved in the pathogenesis of stillbirths. The aim of this work was to investigate the virome in sera of sows without and with one or more cases of stillbirth in the litter. Sera were collected from 94 sows of six commercial farms in five municipalities in the state of Rio Grande do Sul, Brazil. Two pools of sera were collected from each farm: one representative of sows that had at least on stillbirth in the last litter and another composed by sera of sows that had litters with no stillbirths. The pools were subjected to nucleic acid extraction, enrichment and high throughput sequencing. No significant differences were detected in the serum viromes of sows with or without stillbirth Nevertheless, it was possible to identify a wide variety of viral genomes, most of these representing viruses of Anelloviridae family. In addition, the present work allowed the identification of 20 complete genome sequences including torque teno sus virus 1a and 1b, porcine circovirus 3 (PCV3) and circular rep-encoding ssDNA viruses (CRESS), including six species not previously reported in swine. In two farms, PCV3 genomes were identified in the serum pools of sows which had cases of stillbirth. The role for this virus as a potential cause of reproductive failure needs additional investigations. No genomes of viruses with RNA genomes were identified. This study provides a contribution to the knowledge on the serum virome of pregnant sows. In addition, it is expected to aid in the identification of possible causes of stillbirth in swine.