Le veau nouveau-né possède un rumen peu développé et non fonctionnel. Au cours des premiers mois, les fonctions digestives s’établissent, avec l’implantation du microbiote composé majoritairement de bactéries, archées et protozoaires. Les objectifs de ce travail étaient doubles : i) caractériser et comprendre la séquence d’implantation taxonomique des microorganismes du rumen chez le veau par des techniques de biologie moléculaire et de dénombrement, ainsi que la séquence de mise en place des paramètres fermentaires (AGV et ammoniac) et des activités principales enzymatiques chez le veau en périodes pré- et post-sevrage, ii) étudier l’effet de l’addition de levures vivantes sur la mise en place de cet écosystème ruminal en périodes pré- et post-sevrage. D’une part, nos travaux ont permis de confirmer qu’à la naissance, le rumen chez le veau, est dépourvu de micro-organismes, d’AGV, d’activité xylanasique et amylasique, avec un pH proche de la neutralité et un Eh fortement positif. La colonisation du rumen se fait dès la naissance, pendant les 15 premiers jours de la vie de l’animal par un microbiote complexe prédominé par les bactéries (phyla Proteobacteria et Bacteroidetes) et comprenant aussi des archées (majoritairement Methanobrevibacter). En même temps, le Eh devient fortement négatif. Ces communautés entraînent la production de produits fermentaires grâce à leurs activités enzymatiques. Entre 15 jours et le sevrage, avec l’ingestion d’aliments solides, la composition du microbiote du rumen évolue pour se rapprocher de celle de ruminants adultes, sans atteindre pour autant la maturité en termes de densités et abondances relatives. A cette période, le phylum Bacteroidetes est majoritaire, avec le genre Prevotella. Après le sevrage de légers changements apparaissent sur certains paramètres fermentaires comme les AGV sans doute en raison d’une évolution du microbiote qui devient moins diversifié et plus adapté à la dégradation d’aliments solides. L’apparition, à partir de 90 jours, des protozoaires ciliés dans le rumen semble conditionnée par la présence d’animaux adultes à proximité. A 4 mois d’âge, l’écosystème ruminal tend à devenir proche de celui observé chez les animaux adultes en matière de paramètres fermentaires, activités enzymatiques et composition taxonomique du microbiote. D’autre part, nos travaux ont permis de conclure que, avant sevrage, une supplémentation en levures vivantes (Saccharomyces cerevisiae) diminue l’ingestion de concentrés, conduit à une apparition plus précoce de la communauté des protozoaires et une plus grande densité d’archées, mais a peu d'effets sur la densité et la diversité de la communauté bactérienne, à l'exception de variations d’abondances de quelques taxa mineurs. L’apport de levures entraîne une diminution de la protéolyse, une augmentation de la proportion d'acétate ruminal et une diminution de la proportion de propionate. Au cours de la période post-sevrage, les veaux supplémentés en levures consomment plus de foin et la densité en archées est plus importante alors qu’une réduction de la diversité et de la densité de la communauté bactérienne est observée, mais accompagnée d’une augmentation de l’abondance relative des bactéries dégradant l’amidon, les pectines, les protéines et majoritairement les parois cellulaires en fonction des substrats présents dans le rumen. Ces changements sont probablement à relier aux augmentations de l'activité xylanasique et de la proportion d'acétate. L’ensemble des résultats acquis dans ce travail de thèse a permis d’apporter un certain nombre de connaissances et une meilleure compréhension de la mise en place de l’écosystème ruminal chez le veau de race laitière en périodes pré- et post-sevrage, ainsi que quelques pistes pour orienter ou améliorer cette implantation pour une meilleure maîtrise de l’élevage des veaux d’élevage. / The newborn calf has a little and non-fonctional rumen. During the first months of life, digestive functions establish, in relationship with the colonization by a microbiota mainly composed of bacteria, archaea and protozoa. This study had two objectves: i) characterize and understand the sequence of establishment of ruminal microbiota in calves by molecular biology and counting techniques abd describe the appearance of fermentation parameters (VFA and ammonia) and enzyme activities during the pre- and post-weaning periods, ii) define the effect of yeast supplementation on the establishment of the ruminal ecosystem in pre-and post-weaning periods. On the one hand, our work confirmed that at birth, calf rumen is devoid of micro-organisms, AGV, xylanase and amylase activities, with a pH close to neutrality and a strongly positive Eh. From 2 to 15 days of age, the rumen is colonized by a complex microbiota dominated by bacteria (Proteobacteria and Bacteroidetes phyla) and also containing archaea (with mainly the Methanobrevibacter genus), and Eh becomes strongly negative. These communities result in the production of fermentation products due to their enzymatic activities. Between 15 days and weaning, with the ingestion of solid food, the composition of rumen microbiota changes to become closer to that of adult ruminants without reaching maturity in term of densities and relative abundances. At this time, the phylum Bacteroidetes is predominant with the Prevotella genus. After weaning, slight differences occurs on some fermentative parameters such as VFA, probably related to a change in the microbiota that becomes less diversified and more adapted to the degradation of solid food. From 90 days, the establishment of ciliated protozoa in the calf rumen seems conditioned by the proximity with adult animals. From 4 months of age, considering fermentation, enzymatic and taxonomic composition of the microbiota, the ruminal ecosystem tends to be similar to that observed in adult animals. On the other hand, our work showed that during the pre-weaning period, the supplementation with live yeast (Saccharomyces cerevisiae) results in a lower concentrate intake, an earlier establishment of ciliated protozoa and a higher archaeal community density, but poorly affects the density and diversity of the bacterial community, with the exception of changes in abundances of some minors taxa. Yeast supplementation reduces proteolysis, increases the proportion of acetate and decreases that of propionate. During the post-weaning period, yeast supplemented calves consumed more hay, had a higher archaeal density, but a lower diversity and density of the bacterial community with an increased relative abundance of fiber, protein, starch, pectine degrading bacteria compared to control according to the substrates present in the rumen. These changes are probably related to increases of the xylanolytic activity and the proportion of acetate. Taken together, results obtained in his thesis have improved knowledge and understanding of the establishment of the ruminal ecosystem in the dairy calf in pre- and post-weaning periods, and carried some possibilities to orientate or improve the ruminal establishment for better control of calves rearing.

Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity. The wild-type North American firefly(Photinus pyralis)luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C. The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible. Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c7dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS). Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions. A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU. Key words:bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c7dATP, dATPαS. / <p>QCR 20161027</p>

As lagoas salino-alcalinas (salinas) da sub-região Nhecolândia do Pantanal, Mato Grosso do Sul, combinam valores de pH elevados com a presença de altas concentrações de sal, assemelhando-se aos lagos de soda da África Oriental. O entendimento atual dos mecanismos físicos, químicos e biológicos nestes ambientes extremos do Brasil é limitado. Embora os micro-organismos estejam envolvidos nos processos biogeoquímicos em ecossistemas aquáticos, investigações sobre os grupos bacterianos que contribuem para a diversidade e funções específicas nessas salinas inexistem. Assim, a presente dissertação centrou-se na avaliação da comunidade bacteriana de duas salinas (Salina Verde e Salina Preta), localizadas na sub-região da Nhecolândia. Especificamente, investigou-se a diversidade e a estrutura das comunidades bacterianas, os perfis metabólicos das lagoas e genes funcionais que codificam enzimas relacionadas a transformação do nitrogênio, mercúrio, selênio e arsênio. As amostras de água foram coletadas durante a estação seca (setembro de 2012) na Salina Verde (pH 9,5, E.C. 2575 mS cm-1), caracterizada pela presença constante de floração de cianobactérias e na Salina Preta (pH 8,9, E.C. 1500 mS cm-1), sem registro de ocorrência de floração. As amostragens foram realizadas em triplicatas em duas profundidades (superfície e fundo) e duas vezes no dia (10:00 h e 15:00 h) devido à ocorrência natural de saturação de oxigênio observada na Salina Verde. O DNA total de cada amostra ambiental foi extraído e a diversidade bacteriana e funcionalidade foram acessadas por pirosequenciamento do gene de 16S RNAr e sequenciamento metagenômico. A análise de PCR quantitativa do gene de 16S RNAr foi realizada de forma a quantificar a comunidade bacteriana. A abundância bacteriana foi maior na Salina Verde do que na Salina Preta (1010 e 109 cópias mL-1, respectivamente). As sequências parciais do gene de 16S RNAr obtidas no pirosequenciamento mostraram a dominância de táxons do gênero Anabaenopsis sp. na floração da Salina Verde, englobando até 92% do total de sequências. A comunidade bacteriana da Salina Preta apresentou os maiores índices de diversidade e riqueza, sendo dominantes os filos Proteobacteria, Bacteroidetes, Acidobacteria e Verrucomicrobia. Apenas a Salina Preta mostrou diferenças na comunidade bacteriana de acordo com as profundidades amostradas. Na superfície desta lagoa, os filos Actinobacteria e Verrucomicrobia predominaram, enquanto no fundo, prevaleceram os filos Proteobacteria e Chlamydiae. A temperatura foi detectada como o fator abiótico que influenciou a heterogeneidade espacial da Salina Preta. Por sua vez, a alcalinidade e o pH foram os fatores que impulsionaram as diferenças e variações das comunidades bacterianas em ambas as lagoas. Genes bacterianos envolvidos nos ciclos biogeoquímicos do nitrogênio, mercúrio e arsênio foram encontrados nas salinas Verde e Preta, sugerindo uma elevada redundância funcional nas transformações desses elementos. Não foram encontrados genes microbianos envolvidos no ciclo do selênio. Os dados gerados revelaram uma comunidade microbiana taxonômica e funcionalmente complexa que habita as salinas. Os resultados deste estudo fornecem uma avaliação aprofundada baseada em abordagens independentes de cultivo, sendo este um passo importante na compreensão da dinâmica funcional desses ambientes no Pantanal brasileiro. / The saline-alkaline lakes (salinas) of the Nhecolândia sub-region of the Pantanal, Mato Grosso do Sul state, combine high pH values with the presence of high salt concentrations, resembling the soda lakes of East Africa. The current understanding of physical, chemical and biological mechanisms in these extreme environments is limited. Although microorganisms are involved in biogeochemical processes in aquatic ecosystem, researches on the bacterial groups that contribute to diversity and specific functions in these salinas are scarce. This dissertation therefore focused on the evaluation of bacterial community of two salinas (Salina Verde and Salina Preta) located in the Nhecolândia subregion. Specifically, it was investigated the diversity and structure of bacterial communities, the metabolic profile of the lakes and functional genes that encode the nitrogen, mercury and arsenic-transforming enzymes. Water samples were collected during the dry season (September 2012) from Salina Verde (pH 9.5, E.C. 2575 mS cm-1), characterized by constant presence of cyanobacterial bloom, and from Salina Preta (pH 8.9, E.C. 1500 mS cm-1), with no report of bloom occurrence. Triplicate samplings were carried out in two depths (surface and bottom) and twice a day (10 AM and 3 PM) due to naturally occurrence of oxygen saturation, observed at Salina Verde. Total DNA of each environmental sample was extracted and bacterial diversity and functionality were accessed by 16S rRNA gene pyrosequencing and metagenomic sequencing. Analysis of quantitative PCR of the 16S rRNA gene was performed in order to quantify the bacterial community. Bacterial abundance was higher in the Salina Verde than in the Salina Preta (1010 and 109 copies mL-1, respectively). The partial sequences of the 16S rRNA gene obtained in the pyrosequencing revealed the genus Anabaenopsis sp. as the dominant taxa in the Salina Verde bloom, encompassing up to 92% of the total bacteria. Bacterial community of the Salina Preta showed the highest diversity and richness index, with dominant phyla Proteobacteria, Bacteroidetes, Acidobacteria and Verrucomicrobia. Only the Salina Preta showed differences in bacterial community in accordance with the depths sampled. On the surface of this lake, the phyla Actinobacteria and Verrucomicrobia predominated, while in the bottom, Proteobacteria and Chlamydiae prevailed. The temperature was detected as the abiotic factor influencing the spatial heterogeneity at Salina Preta. On the other hand, alkalinity and pH were the factors driving the differences and variation of bacterial community in both lakes. Bacterial genes involved in the biogeochemical cycles of nitrogen, mercury and arsenic were found in Salina Verde and Salina Preta, suggesting a high metabolic redundancy in the transformation these elements. No microbial genes involved in selenium cycle were found. The data showed a taxonomic and functional complex microbial community inhabiting salinas. The results of this study provide a detailed assessment based on culture-independent approaches, which is a stepping stone to understand the functional dynamics of these environments in the Brazilian Pantanal.

A aplicação da digestão anaeróbia aparece como opção para processamento da vinhaça, visto que por meio deste processo é possível aliar a recuperação de energia (hidrogênio e metano) ao enquadramento ambiental deste resíduo sem interferir em suas qualidades como biofertilizante. Nesse sentido, este trabalho avaliou a aplicação da digestão anaeróbia da vinhaça em sistema combinado acidogênico e metanogênico. Inicialmente, avaliou-se a influência de materiais suportes (argila expandida, carvão vegetal, cerâmica porosa e polietileno de baixa densidade) na produção de hidrogênio, em reatores de leito empacotado (APBR) operados em condição mesofílica (25°C) (Etapa 1). De uma forma geral, apenas traços de hidrogênio foram observados nos reatores preenchidos com partículas de carvão vegetal e cerâmica porosa (2 - 7,9 mL-H2.d-1.L-1 reator). Por outro lado, os valores para a produção volumétrica de hidrogênio (PVH) nos reatores com argila expandida e polietileno de baixa densidade foram dez vezes superior (74,3 - 84,2 mL-H2.d-1.L-1 reator), todavia, estatisticamente iguais. O critério de seleção do material suporte ocorreu com base em relatos na literatura que indicaram rebaixamento do leito e entupimento da saída de reatores APBR preenchidos com argila expandida. Portanto, o polietileno de baixa densidade foi escolhido como melhor opção dentre os suportes avaliados. Não obstante, a baixa relação C/N da vinhaça associada à microaeração do sistema (presença de microrganismos anóxicos) afetou severamente os reatores APBR com diferentes materiais suportes, levando-os à falência. Na segunda etapa, adotou-se a operação dos APBR preenchidos com polietileno de baixa densidade, em condição termofílica (55°C) a fim de diminuir o rendimento da biomassa acidogênica e a solubilidade do oxigênio. Adicionalmente, foi avaliada influência da Carga Orgânica Volumétrica aplicada (COVa - 36,4 - 108,6 kg- DQO.m-3.d-1), por meio da variação do Tempo de Detenção Hidráulica (TDH - 8 - 24 h). Produção contínua de hidrogênio foi observada em todos os reatores operados em condição termofílica (Etapa 2). Nessa etapa, estabeleceu-se a condição ótima de operação com COVa de 84,2 kg-DQO.m-3.d-1 e TDH de 10 h, resultando em PVH de valor 575,3 mL-H2.d-1.L-1 reator e rendimento de hidrogênio (Y1H2) de 1,4 mol-H2.mol-1 carboidratos totais. Na Etapa III, essas condições foram impostas na operação do APBR, resultando em aumento de 18,2% e 14,2% nos valores de PVH e Y1H2, respectivamente, em relação aos dados obtidos na Etapa II. Em paralelo, foram operados dois reatores metanogênicos do tipo manta de lodo (UASB), compondo um sistema único (UASB) e um sistema combinado (APBR/UASB). A produção de energia no sistema combinado foi 25,7% superior ao observado no sistema único. A eficiência de remoção da matéria orgânica total e solúvel aumentou de 60,7 ± 0,3% e 72,6% ± 1,2% no sistema único e 74,6 ± 0,3% e 96,1 ± 1,7% no sistema combinado, respectivamente, sob COVa de 25 kg-DQO.m-3.d-1 (calculada para os sistemas metanogênicos). / Anaerobic digestion application appears as an option for sugar cane vinasse processing, since via this process it is possible to combine the energy recovery (hydrogen and methane) to the environmental framework of this residue without interfering in their qualities as biofertilizer. In this sense, this study evaluated the application of anaerobic digestion of vinasse in two-stage system (acidogenic and methanogenic). Initially, it was evaluated the influence of support material (expanded clay, charcoal, porous ceramics, and low-density polyethylene) on hydrogen production in acidogenic packed bed reactors (APBR) operated under mesophilic condition (25°C) (Phase 1). In general, only traces of hydrogen were observed in APBR filled with charcoal and porous ceramics particles (2 - 7.9 mL-H2.d-1.L-1 reactor). On the other hand, in APBR with expanded clay and low-density polyethylene as support, the values for volumetric hydrogen production (VHP) were ten times higher (74.3 - 84.2 mL-H2.d-1.L-1 reactor), however, statistically equal. The selection criteria of support material was based on reports in the literature that indicated bed lowering and clogging in APBR outlet with expanded clay as support. Therefore, the low-density polyethylene was chosen as the best support among those evaluated. Nevertheless, the low C/N ratio of vinasse associated to microaeration condition in the systems (presence of microorganisms anoxic) severely affected the APBR with different support materials, leading them to bankruptcy. In the second phase, it was adopted the operation of APBR filled with low density polyethylene in thermophilic conditions (55°C) in order to reduce the acidogenic biomass yield and oxygen solubility. Moreover, it was evaluated the influence of organic loading rate (OLR - 36.4 to 108.6 kg-COD.m-3.d-1) by hydraulic retention time varying (HRT - 8 - 24 h). Continuous hydrogen production was observed in all reactors operated at 55°C (Phase 2). In this phase, it was established the optimum condition of operation, OLR of 84.2 kg-COD.m-3.d-1 and HRT of 10 h, resulting in values for PVH and hydrogen yield (Y1H2) of 575.3 mL-H2.d-1.L-1 reactor and 1.4 mol-H2.mol-1 total carbohydrates, respectively. In Phase III, these conditions were imposed on the operation of the APBR, resulting in an increase of 18.2% and 14.2% in the values of PVH and Y1H2, respectively, compared to the data obtained in Phase II. In parallel, two methanogenic reactors were operated (Up-flow Anaerobic Sludge Blanket UASB type), which composed a single stage system (UASB) and a two-stage system (APBR/UASB). The energy production in the two-stage system was 25.7% higher compared to the single stage system. The values for total and soluble organic matter removal were 60.7 ± 0.3% and 72.6 ± 1.2% to single stage system and 74.6 ± 0.3% and 96.1 ± 1.7% to two-stage system, respectively, at OLR of 25 kg-COD.m-3.d-1 (calculated for the methanogenic reactors).

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.

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.

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.

Xylella fastidiosa é uma bactéria Gram-negativa, do subgrupo das Gama-Proteobactérias, não-flagelada, que coloniza o xilema de diversas plantas cultivadas e silvestres, podendo ser causadora de doenças. Sua disseminação é feita por insetos conhecidos como cigarrinhas. Genomas de cepas de X. fastidiosa isoladas de distintos hospedeiros já foram sequenciados completa ou parcialmente: 9a5c de citros; Temecula-1 e GB514 de videira; Dixon, M12 e M23 de amendoeira; Ann-1 de espirradeira e EB92-1, isolada de sabugueiro e utilizada como bio-controle para Doença de Pierce de videiras. Estudos de genômica comparativa associados a abordagens de genômica funcional e de genética molecular têm possibilitado o estudo detalhado de mecanismos potencialmente relevantes tanto para a colonização de plantas e insetos por este fitopatógeno como para o desenvolvimento de sintomas associados a doenças específicas em seus respectivos hospedeiros vegetais. Exceto o genoma de 9a5c, todos os demais genomas conhecidos são de cepas isoladas na América do Norte. Neste trabalho descrevemos o pirossequenciamento dos genomas da cepa J1a12, que exibe fenótipo não-virulento em citros, e das cepas Pr8x e Hib4, isoladas, respectivamente, de ameixeira e hibisco. A cepa J1a12 possui além de seu cromossomo principal de 2.788.789 pb dois plasmídeos, pXF51 e pXF27, respectivamente de 51.180 pb e 27.268 pb. pXF51 já foi descrito também na cepa de citros 9a5c e pXF27 tem similaridade com outros plasmídeos de cepas de X. fastidiosa norte-americanas isoladas de amoreira e videira. A cepa Pr8x possui além de seu cromossomo principal de 2.666.242 pb um plasmídeo, pXF39, de 39.580 pb, o qual contém a maioria das CDS presentes no pXF51. A cepa Hib4, isolada de hibisco, tem o maior cromossomo (2.813.297 pb) e também o maior plasmídeo (pXF64 com 64.251 pb) já descritos para X. fastidiosa. pXF64 apresenta extensa similaridade com o plasmídeo pBVIE04 de Burkholderia vietnamensis cepa G4, sendo descrito pela primeira vez em cepas de X. fastidiosa. Análises comparativas destes genomas possibilitaram a identificação de alterações que podem ser correlacionadas com os fenótipos exibidos por estas cepas, além da variedade e diversidade de regiões relacionadas a bacteriófagos e de plasmídeos que co-existem nas diferentes cepas deste fitopatógeno. / Xylella fastidiosa is a Gram-negative bacteria, of the Gamma-proteobacterium subgroup, non-flagellated that colonizes the xylem of several cultivated and wild plants, where may cause disease. The bacterium is spread by insects known as sharpshooters. Genomes of X. fastidiosa strains isolated from different hosts have been completely or partially sequenced: 9a5c from citrus; Temecula-1 and GB514 from grapevine; Dixon, M12 and M23 from almond tree; Ann-1 from oleander and EB92-1, isolated from elderberry and used as bio-control for Pierce\'s disease of grapevines. Comparative genomics studies associated with approaches from functional genomics and molecular genetics have allowed a detailed study of mechanisms potentially relevant to the colonization of plants and insects by this pathogen as well as to the development of symptoms associated with specific diseases in their respective host plants. Except for 9a5c, all other known genomes are from strains isolated in North America. Here we describe the pyrosequencing of the genomes of strain J1a12, which displays non-virulent phenotype in citrus and of Pr8x and Hib4 strains isolated, respectively, from plum and hibiscus. J1a12 has a main chromosome of 2,788,789 bp and two plasmids, pXF51 and pXF27, respectively of 51,180 bp and 27,268 bp. pXF51 has been described also in the citrus strain 9a5c and pXF27 has similarity with other plasmids found in North American strains isolated from mulberry tree and grapevine. The strain Pr8x has a main chromosome of 2,666,242 bp and one plasmid, pXF39, of 39,580 bp which present similarities with pXF51. Hib4, the strain isolated from hibiscus, has the largest chromosome (2,813,297 bp) and the largest plasmid (pXF64 with 64,251 bp) described for X. fastidiosa. pXF64 shows extensive similarity with the plasmid pBVIE04 of Burkholderia vietnamensis G4 strain and is described for the first time in X. fastidiosa. Comparative analyzes of these genomes have identified several differences that may be correlated with the phenotypes displayed by these strains, in addition to the variety and diversity of regions related to bacteriophages and plasmids that co-exist in different strains of this pathogen.

Thema dieser Arbeit war die klinische sowie molekulargenetische Analyse einer Familie mit der Verdachtsdiagnose autosomal dominante lamelläre Ichthyose (ADLI). Mit direkter Sequenzierung des Loricrin-Gens (LOR) wurde die Mutation 730insG identifiziert und die Diagnose Loricrin-Keratoderma gestellt. Durch Analyse weiterer Patienten wurde gezeigt, dass ADLI keine Loricrin-Keratoderma darstellt. Nach eingehender klinischer Untersuchung und Literaturanalyse konnten für die hier beschriebene Entität folgende Merkmale definiert werden: Als Hauptmerkmale eine honigwabenförmige Palmoplantarkeratose sowie eine leichte Ichthyose, als Nebenmerkmale Pseudoainhums, Autoamputationen, Kollodiumbaby, prominente Fingerknöchel sowie Hyperkeratosen an Knien und Ellenbögen. Die genetisch als Loricrin-Keratoderma charakterisierte Verhornungsstörung in der beschriebenen Familie sollte nunmehr klinisch als honigwabenförmige Palmoplantarkeratose mit Ichthyose bezeichnet werden. Zur Standarddiagnostik von Loricrin-Keratoderma wurde die direkte Sequenzierung von LOR auf DNA-Basis etabliert. Die Mutation 730insG resultiert in einer neuen argininreichen Domäne und einer Verlängerung des Proteins um 22 Aminosäuren. Eine Expressionsanalyse mittels Pyrosequenzierung zeigte eine gleichwertige Expression des mutierten und des Wildtyp-Allels. Dies unterstützt die „gain-of-function“-Theorie für das veränderte Loricrin und stützt die Aussage des für Loricrin-Keratoderma existierenden transgenen Mausmodells. / The main focus of this thesis was the clinical and genetic analysis of a family referred to us with the diagnosis of autosomal dominant lamellar ichthyosis (ADLI). Through direct sequencing of the loricrin gene (LOR) the mutation 730insG was identified and the family was diagnosed as having loricrin keratoderma. By sequencing further patients it was shown that ADLI is not a loricrin keratoderma. Based on refined clinical examination and analysis of the literature the following criteria could be defined for the entity seen: Compulsory features are honeycomb-like palmoplantar keratoderma and ichthyosis, optional features are pseudoainhums, autoamputations, collodion baby, prominent knuckle pads as well as hyperkeratotic lesions on knees and elbows. Therefore the disorder of keratinisation of the family described here genetically characterised as loricrin keratoderma should be clinically termed “honeycomb-like palmoplantar keradoderma with ichthyosis”. To molecularly diagnose loricrin keratoderma direct sequencing of LOR with DNA samples was established. The mutation 730insG results in a new arginine rich domain and an elongation of the protein by 22 residues. Expression analysis showed an equal expression of mutant and wild-type allele. This underlined the “gain-of-function” theory of the modified loricrin and supported the findings in the transgenic mouse model for loricrin keratoderma.

A Mata Atlântica é reconhecida como área de prioridade de conservação na América do Sul, devido ao grande número de espécies endêmicas e constantes ameças à sua biodiversidade em decorrência da substituição da vegetação natural. Embora várias informações sobre a diversidade vegetal e animal estejam disponíveis, pouco se sabe sobre a diversidade de microorganismos existentes no solo desse bioma. A diversidade de fungos do solo foi avaliada em três unidades de conservação da Mata Atlântica do estado de São Paulo: Parque Estadual de Carlos Botelho (PECB), Estação Ecológica de Assis (EEA) e Parque Estadual da Ilha do Cardoso (PEIC). Ao todo, foram analisadas 90 amostras de solo, coletadas em épocas de alta e baixa pluviosidade, e sob a copa de três espécies arbóreas: Cabralea canjerana, Guapira opposita e Maytenus robusta. Foram utilizados métodos independentes (PCR-DGGE e pirosequenciamento) e um método dependente do cultivo (lavagem de solo e filtração de partículas) para a análise da diversidade e estrutura das comunidades de fungos do solo. Os resultados obtidos foram analisados conjuntamente com os dados de atributos químicos do solo e frações da matéria orgânica do solo a fim de verificar suas possíveis relações com as comunidades de fungos. Os resultados obtidos sugerem uma grande diversidade de fungos no solo da Mata Atlântica. Através do método de cultivo, um total de 142 espécies de fungos foi identificado nas três áreas, sendo que a estrutura das comunidades de fungos não foi influenciada pelas espécies arbóreas, mas sim pelas áreas e épocas de amostragem. As comunidades de fungos cultiváveis do PECB e do PEIC foram mais similares entre si do que em relação às comunidades de fungos do solo da EEA, assim como os valores dos atributos químicos do solo dessas áreas foram mais semelhantes entre si. Pelo método de PCR-DGGE, as estruturas das comunidades de fungos das três áreas sofreram influência das espécies arbóreas sob a copas das quais as amostras foram coletadas. Somente a estrutura das comunidades de fungos no solo do PEIC não sofreu influência da época de amostragem. Usando pirosequenciamento, foram obtidas 39.152 sequências da região ITS de fungos, as quais foram agrupadas em 1.800 Unidades Taxonômicas Operacionais (UTOs). A diversidade de fungos na EEA e no PEIC variou em função das espécies arbóreas e épocas de coleta. A análise de NMS de cada área amostrada indicou que as comunidades de fungos do solo são muito homogêneas. O filo Ascomycota foi mais frequentemente detectado, tanto usando metodologia dependente quanto independente de cultivo. De maneira geral, as comunidades de fungos cultiváveis apresentaram maior relação com atributos químicos do solo, áreas e épocas de coleta, e as comunidades de fungos acessadas por PCRDGGE mostraram maior relação com as épocas de coleta e espécies arbóreas sob as quais as amostras de solo foram coletadas. A análise dos metadados usando rede neural revelou uma dependência da diversidade de fungos em relação às concentrações de ácidos húmicos, ácidos fúlvicos e humina no solo, além do pH e concentração de matéria orgânica total. / Brazilian Atlantic Forest is recognized as a top priority for conservation in South America because of its large number of innate species and the threats for the biodiversity due to vegetation changes. Although a plethora of data on plant and animal diversity in the Atlantic Forest is available, the diversity of soil microorganisms in this biome is still mostly unknown. Therefore, soil fungi diversity was evaluated in three Atlantic Forest conservation areas of São Paulo state: Estação Ecológica de Assis (EEA), Parque Estadual de Carlos Botelho (PECB) and Parque Estadual da Ilha do Cardoso (PEIC). A total of 90 soil samples were analyzed, collected in two different seasons (high and low precipitation seasons), and under the canopy projection of three tree species: Cabralea canjerana, Guapira opposita and Maytenus robusta. Two independent cultivation methods (PCR-DGGE and pyrosequencing) and one dependent method (soil washing and particles filtration method) were used for the analysis of soil fungi diversity and community structure. The results of these methods were analyzed together with the data on soil chemical attributes and organic matter fractions in the same samples, in order to verify the possible relations with soil fungi communities. The results suggest a great diversity of soil fungi in the Atlantic Forest. Through the cultivation method, a total of 142 fungi species were identified for the three areas. The structure of fungi communities was not affected by the tree species, but were affected by the sampling areas and seasons. Cultivable fungi community in PECB and PEIC were more similar to each other than in EEAs soil fungi community. In addition, the values of the chemical properties of these areas were more similar to each other. The structure of soil fungi community accessed by PCR-DGGE showed that the three areas were influenced by the tree species under the canopy where the soil samples were collected. Only the structure of PEICs fungi communities was not influenced by the season. By means of pyrosequencing, 39,152 sequences were retained from the ITS rDNA region, which were clustered in 1,800 Operational Taxonomic Unities (OTUs). Fungal diversity in EEA and PECB areas was influenced by the tree species and seasons. NMS analysis of each sampled area showed that soil fungi communities are very homogenous. Ascomycota was the most frequent phylum detected by both dependent and independent cultivation methods. Overall, the communities of cultivable fungi were more related to soil chemical attributes, sampling areas and seasons. Fungal communities accessed by PCR-DGGE showed greater relation with the seasons and tree species where the soil samples were collected. Analysis of metadata using neural network revealed fungal diversity dependence of humic acids, fulvic acids and humin concentrations in soil, as well as pH and organic matter concentrations.