The consortia of microorganisms responsible for the hydrolysis of cellulose in situ are at present poorly characterized. Nonetheless, the importance of these communities is underscored by their capacity for converting biomass to greenhouse gases such as carbon dioxide and methane. The metabolic capacities of these organisms is particularly alarming considering the volume of biomass that is projected to re-enter the carbon cycle in Arctic tundra soil environments as a result of a warming climate. Novel cold-adapted cellulase enzymes also present enormous opportunities for a broad range of industries. DNA stable-isotope probing (DNA-SIP) is a powerful tool for linking the phylogenetic identity and function of cellulolytic microorganisms by the incorporation of isotopically labelled substrate into nucleic acids. By providing 13C-enriched glucose and cellulose to soil microcosms, it was possible to characterize the communities of microorganisms involved in the metabolism of these substrates in an Arctic tundra soil sample from Resolute Bay, Canada. A protocol for generating 13C-enriched cellulose was developed as part of this thesis, and a visual DNA-SIP protocol was generated to demonstrate the experimental outline. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone libraries were used to visualize changes in community structure and to identify prevalent, active phylotypes in the SIP incubations. Notably, predominant phylotypes changed over time and clustered based on substrate metabolism. Labelled nucleic acids identified by sequenced DGGE bands and 16S rRNA gene clone libraries provided converging evidence indicating the predominance of Clostridium and Sporolactobacillus in the 13C-glucose microcosms, and Betaproteobacteria, Bacteroidetes, and Gammaproteobacteria in the 13C-cellulose microcosms. Active populations consuming glucose and cellulose were distinct based on principle coordinate analysis of “light” and “heavy” DNA. A large portion of the recovered sequences possessed no close matches in the GenBank database, reflecting the paucity of data on these communities of microorganisms.

The consortia of microorganisms responsible for the hydrolysis of cellulose in situ are at present poorly characterized. Nonetheless, the importance of these communities is underscored by their capacity for converting biomass to greenhouse gases such as carbon dioxide and methane. The metabolic capacities of these organisms is particularly alarming considering the volume of biomass that is projected to re-enter the carbon cycle in Arctic tundra soil environments as a result of a warming climate. Novel cold-adapted cellulase enzymes also present enormous opportunities for a broad range of industries. DNA stable-isotope probing (DNA-SIP) is a powerful tool for linking the phylogenetic identity and function of cellulolytic microorganisms by the incorporation of isotopically labelled substrate into nucleic acids. By providing 13C-enriched glucose and cellulose to soil microcosms, it was possible to characterize the communities of microorganisms involved in the metabolism of these substrates in an Arctic tundra soil sample from Resolute Bay, Canada. A protocol for generating 13C-enriched cellulose was developed as part of this thesis, and a visual DNA-SIP protocol was generated to demonstrate the experimental outline. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone libraries were used to visualize changes in community structure and to identify prevalent, active phylotypes in the SIP incubations. Notably, predominant phylotypes changed over time and clustered based on substrate metabolism. Labelled nucleic acids identified by sequenced DGGE bands and 16S rRNA gene clone libraries provided converging evidence indicating the predominance of Clostridium and Sporolactobacillus in the 13C-glucose microcosms, and Betaproteobacteria, Bacteroidetes, and Gammaproteobacteria in the 13C-cellulose microcosms. Active populations consuming glucose and cellulose were distinct based on principle coordinate analysis of “light” and “heavy” DNA. A large portion of the recovered sequences possessed no close matches in the GenBank database, reflecting the paucity of data on these communities of microorganisms.

A cana-de-açúcar é uma cultura importante no contexto agrário brasileiro, sobretudo com relação a manutenção e sustentabilidade dos agroecossistemas e da biodiversidade do solo. As comunidades microbianas associadas à canade- açúcar são participantes da manutenção dos ciclos biogeoquímicos, podendo ter sua estrutura e diversidade alteradas por mudanças no manejo da cultura e nas condições climáticas. Esse estudo teve como objetivo avaliar a diversidade microbiana associada à rizosfera de diferentes genótipos de cana-de-açúcar e empregar a metodologia de Stable Isotope Probing (DNA-SIP) para se avaliar a estrutura dos grupos responsivos a este ambiente. Para tanto, variedades de cana-de-açúcar foram selecionadas, extraindo-se o DNA total rizosférico e do bulk soil para análise por PCR-DGGE das regiões do gene 16S rDNA de bactérias, selecionando-se amostras representativas para o sequenciamento da região V6 do gene 16S rDNA através da plataforma Ion Torrent(TM). Os resultados demonstraram diferenças entre a diversidade das comunidades microbianas da rizosfera e do bulk soil, havendo a predominância dos grupos Actinobacteria, Proteobacteria e Acidobateria. Para o estudo da estrutura dos grupos responsivos na rizosfera, plantas da variedade RB86-7515 foram cultivadas sob duas concentrações de CO2 (350 e 700 ppm), realizando-se o enriquecimento com 13CO2, e posteriormente realizando a extração do DNA rizosférico para aplicação na técnica de DNA-SIP. A eficiência desta técnica foi avaliada por meio da técnica de PCR-DGGE para as regiões 16S rDNA de bactérias e ITS de fungos, onde foi verificado que após 48 horas já ocorre a incorporação de 13C pelas comunidades microbianas, havendo diferença entre os grupos que incorporaram o 13C. Diferenças foram também observadas para as distintas concentrações de CO2, indicando o DNA-SIP como uma poderosa ferramenta de estudos da ecologia das comunidades microbianas na rizosfera de cana-deaçúcar. / The sugarcane is an important crop in Brazilian agrarian context, especially in respect to maintenance and sustainability of agroecosystems and soil biodiversity. The microbial communities associated to sugarcane are involved biogeochemical cycles processes and it may have their structure and diversity changed due to crop management and climatic conditions. The aim of this study was to evaluate the microbial diversity associated to the rhizosphere of different sugarcane\'s genotypes and employ the Stable Isotope Probing tecnique (DNASIP) to evaluate the structure of the groups that are responding to this environment attributes. Therefore, some sugarcane varieties were selected and the total DNA in bulksoil and rhizosphere for analysis by PCR-DGGE of 16S rDNA gene regions of bacteria was extracted, selecting representative samples for sequencing the 16S rDNA gene of V6 region by Ion Torrent (TM) platform. The results showed differences between the diversity of microbial communities in the rhizosphere and bulk soil, with the predominance of Actinobacteria, Proteobacteria and Acidobateria groups. To study the structure of the responsive rhizosphere groups, the genotype RB86-7515 were grown under two CO2 concentrations (350 and 700 ppm), performing the 13CO2 enrichment. Afterwards, was performed the extraction of DNA for application of the SIP-rhizosphere DNA technique. The efficiency of this technique was assessed by PCR-DGGE over the regions of bacteria 16S rDNA and fungi ITS, which of these showed that occurs after 48 hours the incorporation of 13C by microbial communities, and it elucidate differences between the groups that incorporate the 13C. These differences were also observed for those different CO2 concentrations, indicating that the DNA-SIP is a powerful tool for studies of the ecology of microbial communities in the rhizosphere of sugarcane.

A cana-de-açúcar é uma cultura importante no contexto agrário brasileiro, sobretudo com relação a manutenção e sustentabilidade dos agroecossistemas e da biodiversidade do solo. As comunidades microbianas associadas à canade- açúcar são participantes da manutenção dos ciclos biogeoquímicos, podendo ter sua estrutura e diversidade alteradas por mudanças no manejo da cultura e nas condições climáticas. Esse estudo teve como objetivo avaliar a diversidade microbiana associada à rizosfera de diferentes genótipos de cana-de-açúcar e empregar a metodologia de Stable Isotope Probing (DNA-SIP) para se avaliar a estrutura dos grupos responsivos a este ambiente. Para tanto, variedades de cana-de-açúcar foram selecionadas, extraindo-se o DNA total rizosférico e do bulk soil para análise por PCR-DGGE das regiões do gene 16S rDNA de bactérias, selecionando-se amostras representativas para o sequenciamento da região V6 do gene 16S rDNA através da plataforma Ion Torrent(TM). Os resultados demonstraram diferenças entre a diversidade das comunidades microbianas da rizosfera e do bulk soil, havendo a predominância dos grupos Actinobacteria, Proteobacteria e Acidobateria. Para o estudo da estrutura dos grupos responsivos na rizosfera, plantas da variedade RB86-7515 foram cultivadas sob duas concentrações de CO2 (350 e 700 ppm), realizando-se o enriquecimento com 13CO2, e posteriormente realizando a extração do DNA rizosférico para aplicação na técnica de DNA-SIP. A eficiência desta técnica foi avaliada por meio da técnica de PCR-DGGE para as regiões 16S rDNA de bactérias e ITS de fungos, onde foi verificado que após 48 horas já ocorre a incorporação de 13C pelas comunidades microbianas, havendo diferença entre os grupos que incorporaram o 13C. Diferenças foram também observadas para as distintas concentrações de CO2, indicando o DNA-SIP como uma poderosa ferramenta de estudos da ecologia das comunidades microbianas na rizosfera de cana-deaçúcar. / The sugarcane is an important crop in Brazilian agrarian context, especially in respect to maintenance and sustainability of agroecosystems and soil biodiversity. The microbial communities associated to sugarcane are involved biogeochemical cycles processes and it may have their structure and diversity changed due to crop management and climatic conditions. The aim of this study was to evaluate the microbial diversity associated to the rhizosphere of different sugarcane\'s genotypes and employ the Stable Isotope Probing tecnique (DNASIP) to evaluate the structure of the groups that are responding to this environment attributes. Therefore, some sugarcane varieties were selected and the total DNA in bulksoil and rhizosphere for analysis by PCR-DGGE of 16S rDNA gene regions of bacteria was extracted, selecting representative samples for sequencing the 16S rDNA gene of V6 region by Ion Torrent (TM) platform. The results showed differences between the diversity of microbial communities in the rhizosphere and bulk soil, with the predominance of Actinobacteria, Proteobacteria and Acidobateria groups. To study the structure of the responsive rhizosphere groups, the genotype RB86-7515 were grown under two CO2 concentrations (350 and 700 ppm), performing the 13CO2 enrichment. Afterwards, was performed the extraction of DNA for application of the SIP-rhizosphere DNA technique. The efficiency of this technique was assessed by PCR-DGGE over the regions of bacteria 16S rDNA and fungi ITS, which of these showed that occurs after 48 hours the incorporation of 13C by microbial communities, and it elucidate differences between the groups that incorporate the 13C. These differences were also observed for those different CO2 concentrations, indicating that the DNA-SIP is a powerful tool for studies of the ecology of microbial communities in the rhizosphere of sugarcane.