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.

Pelagic aggregates function as biological carbon pumps for transporting fixed organic carbon to sediments. In iron-rich (ferruginous) lakes, photoferrotrophic and chemolithoautotrophic bacteria contribute to CO2 fixation by oxidizing reduced iron, leading to the formation of iron-rich pelagic aggregates (iron snow). The significance of iron oxidizers in carbon fixation, their general role in iron snow functioning and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis of iron snow collected from an acidic lake with protein-based stable isotope probing to determine general metabolic activities and to trace 13CO2 incorporation in iron snow over time under oxic and anoxic conditions. mRNA-derived metatranscriptome of iron snow identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6–85.7%) encoding ecologically relevant pathways, including carbon fixation and polysaccharide biosynthesis. No transcriptional activity for carbon fixation from archaea or eukaryotes was detected. 13CO2 incorporation studies identified active chemolithoautotroph Ferrovum under both conditions. Only 1.0–5.3% relative 13C abundances were found in heterotrophic Acidiphilium and Acidocella under oxic conditions. These data show that iron oxidizers play an important role in CO2 fixation, but the majority of fixed C will be directly transported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.

Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial ???indicator species??? for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.

Metabolic heterogeneity (MH) occurs when isogenic microbial populations display cell-to-cell differences in metabolic traits, albeit exposed to homogeneous conditions. Despite the increasing focus on MH, its triggering factors remain largely unknown. In the present thesis, I used stable isotope probing and chemical imaging with nanoscale Secondary Ion Mass Spectrometry (nanoSIMS) to study MH at single-cell level, in model organisms, synthetic and natural communities, to understand i) how abiotic factors, biotic interactions and antibiotics exposure influence MH and ii) its potential ecological role. Moreover, I optimized sample preparation for chemical and high-resolution imaging and suggested two different indices as ‘unit measure’ of MH. As results, I have shown for the first time that MH is displayed by microorganisms under favorable growth conditions, although none of the tested abiotic factors prevailed as the main trigger of MH. I brought insights on how biotic interactions play a role in the functional heterogeneity using bacteria pseudo-fungi co-cultures. I found that antibiotics reduce Carbon and Nitrogen assimilation rates of targeted phylogenetic groups in river-water communities, while increasing their MH, pointing to its ecological importance in natural environments. To conclude, I provided novel insights on the phenomenon of MH and its dynamics at different ecological scales.:Abbreviation list Summary Introduction Knowledge gaps Results and discussion - Optimization of sample preparation - Validation of quantitation methods - Abiotic factors shaping metabolic heterogeneity in bacterial populations - Influence of biotic factors in shaping heterogeneity - Metabolic Heterogeneity and ecophysiology of natural microbial populations influenced by emerging contaminants Conclusions Outlook Bibliography Appendix Acknowledgments Curriculum Vitae List of publications

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