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.

<p>Rising atmospheric CO2 concentration, currently about 390 ppm, causes climate change and is expected to reach 500 ppm or higher this century due to human activities. Soils are the largest terrestrial pool of carbon, and changes in soil carbon storage due to plant and microbial activities could affect atmospheric CO2 levels. This dissertation studies soil carbon and microbial responses to an experimental preindustrial-to-future CO2 gradient (250-515 ppm) in a grassland ecosystem. Two contrasting soil types are studied in the gradient, providing insight on how natural ecosystem variation modifies CO2 effects.</p><p>Although total soil organic carbon (SOC) did not change with CO2 treatment after four growing seasons, fast-cycling SOC pools did respond to CO2, particularly in the black clay soil. Microbial biomass increased 18% and microbial activity increased 30% across the CO2 gradient in the black clay, but neither factor changed with CO2 in the sandy loam. Similarly a one-year laboratory soil incubation showed that a fast-cycling SOC pool increased 75% across the CO2 gradient in the black clay. Size fractionation of SOC showed that coarse POM-C, the youngest and most labile fraction, increased four-fold across the CO2 gradient in the black clay, while it increased 50% across the gradient in the sandy loam. CO2 enrichment in this grassland increased the fast-cycling soil organic carbon pool as in other elevated CO2 studies, but only in the black clay soil.</p><p>CO2 also induced changes in microbial community composition, and we explored the functional consequences in a microcosm experiment. Soil collected in the third growing season of CO2 treatment was used to inoculate Indiangrass seedlings grown in the lab. The elevated CO2 soil inoculum had higher microbial biomass C/N (C/N = 21) than the subambient CO2 soil inoculum (C/N = 16), suggesting a difference in community composition. Mean plant height in elevated CO2 soil inoculum (475 ppm) was 57% greater than in subambient CO2 soil inoculum (300 ppm), but the difference was not statistically significant. Similarly, total leaf N from plants in elevated CO2 soil was 28% greater on average than in subambient CO2 soil, but not significantly different. CO2-induced microbial effects on plant growth were either negligible or occurred at finer microbial taxonomic levels, making them difficult to resolve at the whole-community level.</p><p>Soil fungi decompose soil organic matter, and studying fungal community responses to CO2 could improve our understanding of soil carbon responses. We studied fungal communities in the CO2 gradient using Sanger sequencing and pyrosequencing of rDNA. As in our soil C study, fungal community responses to CO2 were mostly linear, and occurred mostly in the black clay soil. Fungal species richness increased linearly with CO2 treatment in the black clay. The relative abundance of Chytridiomycota (chytrids) increased linearly with CO2 in the black clay, while the relative abundance of Glomeromycota (arbuscular mycorrhizal fungi) increased linearly with CO2 in the sandy loam. Increased labile C availability at elevated CO2 and/or decreased inorganic N may explain the increase in fungal species richness and Chytridiomycota abundance in the black clay, while increased P limitation may explain the stimulation of Glomeromycota at elevated CO2 in the sandy loam. Across both soils, fungal species richness increased linearly with soil respiration, an index of decomposition rate (p = 0.01, R2 = 0.46). Adding fungal species may have improved decomposition efficiency, but it is also possible that species richness and decomposition increased due to another factor such as C quantity. Soil type strongly structured both fungal community and arbuscular mycorrhizal fungal community composition.</p><p>Together, these studies suggest that soil C and fungal community responses to CO2 were mostly linear, and were most apparent in the black clay soil. Soil type strongly influenced fungal community composition as well as which phyla responded to CO2. Therefore, soil type could be a useful addition to predictions of soil carbon and microbial responses to future CO2 levels.</p> / Dissertation

Les relations entre le microbiote intestinal et la santé humaine ont été suggérées par les études métagénomiques. Microbial culturomics utilise de nombreuses conditions de culture avec une méthode d'identification rapide par MALDI-TOF, ou par séquençage de l'ARN 16S pour les colonies non identifiées. L'étude pionnière a permis d'identifier 340 bactéries différentes, dont 31 nouvelles espèces bactériennes et 174 espèces bactériennes décrite pour la première de l'intestin humain. Le séquençage du génome de chaque nouvelle espèce a permis de décrire le plus grand génome d'une bactérie isolée chez l'homme (Microvirga massiliensis, 9,3 Mo) et de générer environ 10 000 gènes précédemment inconnus (ORFans) facilitant les futures études métagénomiques. Le pyroséquençage sur les mêmes échantillons a révélé que seulement 51 espèces étaient détectées par les 2 techniques. Culturomics a démontré sa supériorité par rapport au pyroséquençage lors de l'étude d'une selle d'une patiente traitée pour une tuberculose ultra-résistante. Le pyroséquençage de 2 échantillons de selles de patients traités par antibiotiques a révélé 45 à 80% de séquences assignées au phylum des Verrucomicrobia. L'étude par culture de ces mêmes échantillons n'a pas permis d'isoler cette espèce.Grâce à l'étude de 14 échantillons de selles différents par culturomics, nous avons cultivé 520 espèces bactériennes différentes, dont 57 nouvelles espèces bactériennes et 260 espèces décrites pour la première fois à partir du microbiote digestif. Après cette phase de description, les études suivantes pourront tenter d'établir un lien entre les nouvelles espèces bactériennes et le statut clinique des patients étudiés. / Relationships between gut microbiota and human health have been already suggested thanks to metagenomics studies. Microbial culturomics is based on the use of a large number of culture conditions with a rapid identification method by MALDI-TOF or by 16SrRNA amplification and sequencing for the unidentified colonies. The seminal study allowed to identify 340 different bacteria including 31 new bacterial species, 174 bacterial species first described from the human gut. The genome sequencing of each new species allowed to describe the largest genome for a human bacteria (Microvirga massiliensis; 9,3 Mb) and to generate approximately 10,000 previously unknown genes (ORFans) facilitating the future metagenomics studies. In parallel, pyrosequencing performed on the 3 same samples revealed a dramatic low overlapping between the 2 methods with only 51 species detected. In addition, culturomics has demonstrated its superiority than pyrosequencing of a stool from a patient treated for a XDR-tuberculosis. Conversely, the pyrosequencing performed on 2 stool samples of patients treated by antibiotics revealed from 45 to 80% of sequences assigned to Verrucomicrobia although the culturomics study of these same samples did not allowed to culture this species.Currently, thanks to the study of 14 different stool samples by culturomics, we have cultured 520 different bacterial species including 57 new bacterial species and 260 species first described from human gut. After this comprehensive description phase, the following studies will attempt to make a link between new bacterial species and clinical status of the patients studied.

Depuis les années 90, les études portant sur les différentes zones HNLC ont permis d'étudier les effets biologiques et biogéochimiques qu'entrainaient les enrichissements artificiels ou naturels en fer. Il est maintenant bien documenté que l'enrichissement en fer induit des blooms phytoplanctoniques et notamment des blooms de diatomées. En dehors des diatomées, très peu d'informations sont disponibles concernant les autres groupes de protistes et en particulier les protistes hétérotrophes consommateur du phytoplancton. Ce travail a été effectué dans un contexte de fertilisation naturelle en fer, dans la région des îles de Kerguélen (Océan Australe) pendant la campagne KEOPS 2 (Kerguelen Ocean and Plateau compared Study 2) lors de l'initiation du bloom phytoplanctonique et s'est focalisé en particulier sur les protistes hétérotrophes. Des approches moléculaires (tag-pyroséquençages 454) et morphologiques (microscopie) ont été utilisées afin de caractériser la structure des communautés de protistes dans la zone de référence HNLC et dans les différents blooms phytoplanctoniques. l'approche moléculaire a permis (i) de caractériser les communautés de protistes présentes (ii) de mettre en évidence des différences notables entre les structures de protistes dans la région HNLC et la région naturellement enrichie en fer, mais également entre les différents blooms. Les observations microscopiques ont révélé des tendances similaires entre les différentes régions mais aussi des liens significatifs entre les communautés microzooplanctoniques et leurs proies phytoplanctoniques. Les observations microscopiques ont également fournis des valeurs de biomasses des différents compatiments qui ont permis d'estimer le potentiel du microzooplancton en tant que consommateur du phytoplancton ou en tant que source nutritive pour le mésozooplancton. Ce travail représente la première étude caractérisant la communauté des protistes planctoniques dans son ensemble dans un contexte de fertilisation naturelle en fer. / Since the 90s, studies on different HNLC areas allowed to investigated the biological and biogeochemical effects due to artificial or natural iron-enrichment. It is now well documented that iron enrichment induced phytoplankton blooms and more specifically diatom blooms. With the exception of diatoms, very few information is available concerning other protists groups e. g. heterotrophic protists which are consumers of phytoplankton.This work was performed is a natural iron-fertilization context in the Kerguelen Island area (Southern Ocean) during the KEOPS 2 (Kerguelen Ocean and Plateau compared Study 2) cruise at the beginning of the phytoplankton bloom and focused specifically on heterotrophic protists. Molecular (tag-pyrosequencing 454) and morphological (microscopy) approaches were used to characterize the structure of protist communities in the HNLC reference area and in the phytoplankton blooms. The molecular approach allowed (i) to provide a complete picture of the protist communities (ii) to evidence significant differences in protists structures between HNLC and the naturally iron-fertilized area, but also between the different blooms. Microscopic observation revealed similar trends between regions but also significant links between microzooplanctonic communities and their phytoplankton preys. Microscopic observations also provided biomass values from different compartments allowing an estimation of the potential of microzooplankton as phytoplankton consumer or as a nutrient source for mesozooplankton. Above all, this work represents the first study characterizing the global planktonic protists community in the context of natural iron fertilization.

Alquilbenzeno linear sulfonado (LAS) é um surfactante presente em água residuária de lavanderia. Em virtude da complexidade de sua degradação, o presente estudo envolveu a análise de alguns fatores, destacando-se: diversidade de co-substratos; tempo de detenção hidráulico (TDH); e concentração de co-substratos. Avaliou-se a degradação de LAS com diferentes co-substratos (metanol, etanol e extrato de levedura) em reator UASB, em TDH de 24 h e 14±2 mg/L de LAS. A influência de TDH e concentração de co-substratos foram analisadas em sete reatores UASB, com 12±3 mg/L de LAS; TDH de 6, 35 e 80 h, e diferentes concentrações de co-substratos (etanol, metanol e extrato de levedura), expressada pela carga orgânica específica (COE), entre 0,03 e 0,18 gDQO/gSTV.d. Ao final, avaliou-se a degradação de LAS em água residuária de lavanderia diluída, nessa mesma configuração de reator com TDH de 35 h e 10±5 mg/L de LAS. Em todos os ensaios foi utilizado inóculo granulado proveniente de reator UASB empregado no tratamento de água residuária de abatedouro de aves, mantendo-se intacta a forma granulada. No ensaio variando co-substratos, observou-se maior remoção de LAS (50%) na presença de co-substrato complexo (extrato de levedura) que na presença de metanol e etanol (29-41%). Diferença pouco significativa entre as comunidades do domínio Archaea e Bacteria (cerca de 60 e 40%, respectivamente) foi observada na presença de diferentes co-substratos, mediante análise de hibridação fluorescente in situ (fluorescent in situ hybridization FISH). Verificou-se maior influência da concentração de co-substratos na degradação de LAS, seguida pelo TDH. Aplicando a menor COE (0,03 gDQO/gSTV.d), obteve-se alta degradação de LAS (76%), enquanto nos reatores variando TDH foram observadas eficiências de 18% (6 h), 37-53% (35 h) e 55% (80 h). Nos reatores variando TDH e concentração de co-substratos, observou-se significativa remoção de LAS no separador de fases (20-53%; na manta de lodo observou-se 13-43%), relacionada à baixa concentração de co-substratos e condição anaeróbia facultativa nessa região. Por meio da técnica de PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) nas amostras do ensaio variando TDH e concentração de co-substratos, verificou-se maior coeficiente de similaridade na manta de lodo (Archaea: 70-90%; Bacteria: 69-83%), devido à estrutura de grânulo do inóculo utilizado. Verificou-se alta degradação de LAS (82%) no reator com água de lavanderia, atribuída à diversidade de co-substratos (12 ácidos orgânicos voláteis detectados) e à concentração baixa desses co-substratos (COE: 0,03 gDQO/gSTV.d). Mediante análise de pirosequenciamento da região do RNAr 16S de amostras do ensaio com água residuária de lavanderia foram encontrados 147 gêneros, dos quais 32 foram relacionados com a degradação de LAS (gêneros capazes de degradar compostos aromáticos, dessulfonação, e -oxidação). Observou-se significativa abundância relativa (>1%) dos seguintes gêneros relacionados com a degradação de LAS: Comamonas, Dechloromonas, Desulfovibrio, Gemmatimonas, Holophoga, Parvibaculum, Pseudomonas, Rhodanobacter, Sporomusa, Synergistes e Zoogloea. No separador de fases do reator com água de lavanderia, a alta remoção de LAS (90%) e a abundância relativa dos gêneros aeróbios (23%) e anaeróbios (6%) relacionados com a degradação de LAS corroboraram a relação entre remoção de LAS e condição anaeróbia facultativa / Linear alkylbenzene sulfonate (LAS) is a surfactant present in laundry wastewater. Due to the complexity of its degradation, the present study involved the analysis of some features, highlighting: co-substrates diversity; hydraulic retention time (HRT); and co-substrates concentration. The LAS degradation with different co-substrates (methanol, ethanol and yeast extract) was evaluated in UASB reactor, at HRT of 24 h and LAS 14±2 mg/L. The influence of HRT and concentration of co-substrates was analyzed in seven UASB reactors, with LAS 12±3 mg/L; the HRT was 6, 35 and 80 h, and different concentration of co-substrates (methanol, ethanol and yeast extract), as specific organic load rate (SOLR) between 0.03 and 0.18 gCOD/gTVS.d. At the end, the LAS degradation was performed in UASB reactor fed with diluted laundry wastewater, at HRT of 35 h and LAS 10±5 mg/L. In all assays was used a granular inoculum from a UASB reactor employed in treatment of wastewater from poultry slaughterhouse, maintaining the granular form. In the assay varying the co-substrates, it was observed greater LAS removal (50%) in the presence of complex co-substrate (yeast extract) than in the presence of methanol and ethanol (removal: 29-41%). Insignificant difference between the communities from Archaea and Bacteria domain (about 60 and 40%, respectively) was observed in the presence of different co-substrates, according to the fluorescent in situ hybridization (FISH) analysis. It was verified greater influence of cosubstrates concentration than the HRT in the LAS degradation. At the lowest SOLR (0.03 gCOD/gTVS.d), high LAS degradation (76%) was obtained while in the reactors varying the HRT were observed efficiencies of 18% (6 h), 37-53% (35 h) and 55% (80 h). In the reactors varying the HRT and concentration of co-substrates, a significant LAS removal rate (20-53%; in the sludge blanket the rate was 13-43%) was observed in the phase separator, related to the low concentration of co-substrates and the anaerobic facultative condition in this region. By the PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) technique of samples from the assay varying the HRT and concentration of co-substrates, it was verified great similarity coefficient in the sludge blanket (Archaea: 70-90%; Bacteria: 69- 83%) due to the granule structure of the inoculum used. High LAS degradation (82%) was verified in the reactor with laundry wastewater, which was attributed to the diversity of cosubstrates (12 organic volatile acids detected) and the low concentration of co-substrates (SOLR: 0.03 gCOD/gTVS.d). By pyrosequencing analysis of 16S RNAr genes in the samples from assay with laundry wastewater, it was found 147 genus, which 32 were related to the LAS degradation (genus able to degrade aromatic compounds, desulfonation, and - oxidation). A significant relative abundance (>1%) was observed in the following genus related to the degradation of LAS: Comamonas, Dechloromonas, Desulfovibrio, Gemmatimonas, Holophoga, Parvibaculum, Pseudomonas, Rhodanobacter, Sporomusa, Synergistes and Zoogloea. In the phases separator of the reactor with laundry wastewater, the high LAS removal (90%) and the relative abundance of genus aerobic (23%) and anaerobic (6%) related to the degradation of LAS corroborated the relation between LAS removal and the anaerobic facultative condition

Os queijos artesanais, elaborados com leite cru, são apreciados por suas características sensoriais peculiares. Com objetivo de assegurar a inocuidade destes produtos, a legislação brasileira estabeleceu padrões microbiológicos sanitários para estes alimentos. Microorganismos probióticos são conhecidos por sua capacidade de modular favoravelmente a microbiota do hospedeiro por meio da produção de substâncias antimicrobianas, em especial, os ácidos orgânicos e bacteriocinas. Assim, o presente estudo buscou avaliar a utilização de micro-organismos probióticos como alternativa para melhorar a qualidade microbiológica de queijos artesanais usando o Queijo Artesanal da Serra da Mantiqueira como modelo. Para tanto, foi incorporada à microbiota do leite uma preparação composta de 16 cepas de micro-organismos probióticos, obtendo-se uma concentração final de 3x106 UFC/ml de leite. A estratégia adotada consistiu em comparar os queijos inoculados com micro-organismos probióticos em relação aos queijos não inoculados (controle), durante o período de maturação de 45 dias, em três estações do ano (inverno, verão e outono). As amostras foram coletadas nos dias 1, 15, 30 e 45 para a determinação dos parâmetros: umidade, pH, concentrações de carboidratos, ácidos orgânicos e análises microbiológicas. Avaliou-se também a composição da microbiota dos queijos pela técnica do pirossequenciamento dos genes do rRNA 16S. Os resultados permitiram verificar que os gêneros predominantes na microbiota dos queijos foram Streptococcus, Lactobacillus e Lactococcus , em diferentes níveis nas três estações do ano. Foi possível observar que a incorporação das culturas probióticas resultou em diferentes efeitos nas estações avaliadas no que se refere às concentrações de carboidratos e ácido cítrico, bem como de ácidos orgânicos ao final dos 45 dias de maturação, possivelmente devido à variabilidade da microbiota verificada nas diferentes estações. No tocante às características microbiológicas, observou-se que, durante o período de maturação, 95% dos queijos confeccionados nas diferentes condições apresentaram padrões de qualidade em conformidade com o estabelecido na legislação, no que se refere às concentrações de coliformes termotolerantes, estafilococos coagulase positivos, Salmonella sp e Listeria sp independentemente da incorporação das culturas probióticas. Entretanto, a adição das culturas probióticas reduziu a carga microbiana de gêneros da família Enterobacter iaceae , em especial Enterobacter , Citrobacter , Klebsiella , Kluyvera e Obesumbacterium durante processo de maturação. Especificamente, o emprego das culturas probióticas resultou em redução do percentual total de Enterobacter iaceae na microbiota dos queijos de 0,41% para 0,17% no verão, e de 12,42% para 6,71% no outono. Neste contexto, a adição de culturas probióticas pode constituir alternativa viável para melhoria da qualidade microbiológica de queijos elaborados com leite cru por meio do incremento do potencial antagônico da microbiota sobre espécies de micro-organismos potencialmente patogênicos, toxigênicos e deteriorantes, além de agregar valor ao produto final. / Artisanal cheeses, made with raw milk, are appreciated for their sensorial profiles, which differentiates them from industrialized cheeses. In order to ensure safety to consumers, Brazilian legislation established microbiological standards for these products. Probiotics are well known for their ability to favorably modulate the host\'s microbiota by producing antimicrobial compounds, mainly organic acids and bacteriocins. Therefore, this study aimed to evaluate the use of probiotic microorganisms as an alternative to improve the microbiological quality of artisanal cheeses through inhibition of contaminating microorganisms, using Mantiqueira Highlands Artisanal Cheese as a model. To do so, a preparation consisting of 16 probiotic strains was added to the milk, at a final concentration of 3x106 CFU/ml. Additionally, the effects of this practice on pH, carbohydrates and organic acids concentrations over a 45 day ripening period were evaluated. Analyses of cheese bacterial community composition were also undertaken by means of 16S rRNA gene pyrosequencing. The strategy consisted of comparing cheeses made with probiotic cultures with a control group, to which no probiotic culture was added. Over a 45 day ripening period, in three seasons (winter, summer and autumn), samples were collected at days 1, 15, 30 and 45 in order to conduct pH, moisture, carbohydrate, organic acids, microbiological and pyrosequencing analyses. The results showed that the addition of probiotic cultures resulted in distinct effects in the different seasons regarding carbohydrate and organic acids concentrations, possibly due to variability in bacterial community composition. The results also demonstrated that 95% of cheeses were deemed acceptable according to Brazilian law regarding coliforms, positive coagulase staphylococci, Salmonella sp. and Listeria sp, regardless of adding probiotics. However, the incorporation of probiotic microorganisms resulted in reduced populations of genera belonging to Enterobacteriaceae family, especially Enterobacter, Citrobacter, Klebsiella, Kluyvera and Obesumbacterium, throughout the whole ripening process. Specifically, the addition of probiotic cultures reduced the total percentage of Entercobacteriaceae from 0,41% to 0,17% in the summer, and from 12,42% to 6,71% in the fall. In this context, the use of probiotic microorganisms may represent a viable alternative for improving microbiological quality of raw milk cheeses by means of incrementing the antagonistic potential of cheese microbiota towards potentially pathogenic and toxigenic microorganisms.

O guaraná, Paullinia cupana var. sorbilis, espécie nativa do Brasil é uma cultura de relevância para o país, principalmente nos estados da Bahia e Amazonas. O fruto contém substâncias estimulantes, como a cafeína, que conferem ao guaraná um grande potencial exploratório, visto que esta é uma das substâncias estimulantes mais consumidas no mundo. Apesar de sua importância, a cultura do guaraná é pouco estudada, portanto pouco se sabe sobre sua genética e as interações microbianas existentes, tanto em relação aos microrganismos endofíticos e epifíticos, quanto em relação aos patógenos. De tal forma que a cultura do guaraná na região Amazônica vem sendo afetada por condições fitossanitárias desfavoráveis, como a presença do fungo do gênero Colletotrichum, causador da antracnose, doença considerada uma ameaça à produção comercial do guaraná. Assim, visando compreender a dinâmica envolvida na interação planta - microrganismos e o possível controle da antracnose, o presente trabalho acessou a comunidade bacteriana associada às folhas com e sem sintomas de antracnose, de forma independente e dependente de cultivo; bem como, investigou o potencial biotecnológico e de biocontrole dos isolados bacterianos. A comunidade bacteriana acessada de forma dependente e independente de cultivo apresentaram similaridades, como maior valor de riqueza e menor diversidade bacteriana em folhas assintomáticas, quando comparadas com folhas sintomáticas, e com relação ao filo Proteobacteria, mais abundantemente acessado nas duas comunidades. Comparações realizadas com isolados bacterianos acessados epi e endofiticamente de folhas com e sem sintoma da antracnose apontaram que o aparecimento da doença antracnose na folha pareceu ser o fator que mais influenciou na estrutura da comunidade bacteriana. Com relação às diferenças entre tecidos sintomáticos e assintomáticos, foi possível identificar alguns gêneros abundantes mais frequentemente acessados em plantas sintomáticas, sendo que os gêneros Pseudomonas, Stenotrophomonas e Pantoea foram acessados em grande frequência tanto de forma dependente como independente de cultivo. Porém, de forma independente de cultivo foram acessados outros táxons abundantes que diferiram significativamente entre as plantas, sendo que folhas sintomáticas apresentaram Acinetobacter, Acidobacter_GP1 e Sphingobacteria, e em folhas assintomáticas foram acessados os táxons Methylobacterium, Beijerinckia, Bacilli e um grupo de Rhizobiales não classificados. Além disto, os microrganismos endofíticos e epifíticos cultivados foram testados com o fitopatógeno Colletotrichum sp. em ensaios de antagonismos, sendo que isolados do gênero Bacillus, Stenotrophomonas, Pantoea, Erwinia, Entereobacter, Pseudomonas, entre outros, apresentaram inibição do crescimento do fungo fitopatogênico. As bactérias cultivadas ainda foram testadas quanto ao potencial biotecnológico de produção de enzimas hidrolíticas e sideróforo. Muitos isolados apresentaram produção de protease e sideróforo. Foi possível também correlacionar a produção das enzimas amilase, lipase e poligalacturanase com os isolados provenientes de tecidos sintomáticos. / Guarana, Paullinia cupana var. sorbilis, is a native culture of Brazil that has relevance to the country, mainly in the states of Bahia and Amazonas. The fruit contains stimulants substances, such as caffeine, that gives a high exploration potential to the culture, since this is one of the most consumed stimulant substance in the world. Despite its importance, the culture of guarana hasnt gotten much of attention, and little is known about its genetics and microbial interactions with both epiphytic and endophytic microorganisms, and in relation to pathogens. So, the guarana culture in the Amazon region has been affected by bad phytosanitary conditions, such as the presence of the fungi Colletotrichum, which causes anthracnose disease thats considered a threat to guarana commercial production. Thus, to understand the dynamics involved in the interaction plant-microorganism and the possible control of anthracnose, this study accessed the bacterial community associated to leaves with and without anthracnose symptoms, combining culture-dependent and -independent methodologies and also investigated the biotechnology potential and biocontrol of culture-dependent bacteria. The bacterial community accessed by culture-dependent and -independent manner presented similarities, both presented higher bacterial richness but lower bacterial diversity in asymptomatic leaves when compared with symptomatic leaves, and both communities presented the Proteobacteria phylum as the most abundant one. Comparisons between the endophytic and epiphytic bacterial isolates associated with leaves with and without anthracnose symptoms showed that the onset of anthracnose disease on leaf seemed to be the most important factor that modified the bacterial community structure. Regarding the differences between symptomatic and asymptomatic tissue it was possible to identify some genera most frequently accessed in symptomatic plants such as Pseudomonas, Stenotrophomonas and Pantoea accessed in both culture-dependent and independent methodologies. However, the cultureindependent approach of bacterial accessed others abundants taxa that differed significantly between plants. Symptomatic leaves showed Acinetobacter, Acidobacter_GP1 and Sphingobacteria and asymptomatic leaves presented taxa Methylobacterium, Beijerinckia, group Bacilli and a group of unclassified Rhizobiales. In addition, endophytic and epiphytic isolates microorganisms were tested with the pathogen Colletotrichum sp. in antagonism assays, and isolates from the genus Bacillus, Stenotrophomonas, Pantoea, Erwinia, Entereobacter, Pseudomonas, and others, showed inhibition of growth of the fungus. The bacterial isolates were also tested for production of hydrolytic enzymes and siderophore. Many isolates showed protease and siderophore production. It was possible to correlate the production of amylase, lipase and poligalacturanase with isolates from symptomatic tissue.

A correta adoção de medidas de manejo de resistência de insetos-pragas é motivo de preocupação constante, inclusive para as novas tecnologias disponíveis, como as plantas geneticamente modificadas para a expressão de toxinas da bactéria Bacillus thuringiensis (Bt). Portanto, para manter a eficiência das plantas-Bt comercialmente disponíveis, é preconizada a manutenção de áreas de refúgio (livres de plantas Bt) para evitar a rápida seleção de insetos resistentes. No caso de insetos polífagos, a determinação das áreas de refúgio pode levar em consideração a contribuição que fontes não-comerciais e/ou não-transgênicas têm na produção de adultos colonizadores que não sofreram seleção para o evento de transgenia de interesse. A identificação da fonte alimentar pode determinar a procedência de insetos e tornar mais eficiente e segura a implementação de zonas de refúgio e a determinação dos riscos de desenvolvimento de resistência pelo inseto-praga alvo. O objetivo deste trabalho foi o de avaliar o potencial de dois marcadores biológicos, a assinatura isotópica e a microbiota intestinal, para a identificação da fonte de alimento do imaturo pela análise de adultos de Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae), como subsídio para o monitoramento da origem de insetos migrantes em áreas de cultivo Bt para a adoção de estratégias adequadas de manejo de resistência. A análise isotópica de adultos foi realizada utilizando-se insetos criados em 12 plantas hospedeiras distintas, seis de metabolismo C3 e seis de metabolismo C4. Parte dos adultos provenientes dessa criação foram liofilizados, macerados e submetidos à análise isotópica para a determinação dos níveis de ?13C e ?15N. Amostras das plantas utilizadas na alimentação desses insetos também foram submetidas ao mesmo processo de análise. A outra parte dos adultos recémemergidos de S. frugiperda foi utilizada para a determinação da microbiota intestinal pela análise de região do gene do 16S rRNA após sequenciamento em plataforma 454. Os resultados da análise isotópica de carbono para as plantas utilizadas como fonte alimentar apresentaram médias entre -31,37? e -25,07? para plantas C3 e entre -13,03? e -12,26? para plantas C4. Adultos de S. frugiperda oriundos de lagartas criadas em plantas do grupo C3 apresentaram média de ?13C entre - 30,36? e -23,72?, enquanto aqueles do grupo C4 apresentaram média entre - 18,25? e -13,28?. O sequenciamento da microbiota via metagenômica produziu 126,970 sequências com cerca de 421 pb. O alimento influenciou substancialmente a diversidade da microbiota intestinal associada ao intestino de adultos, independentemente do metabolismo fotossintético da planta hospedeira. Análises comparativas de diversidade em que a abundância relativa dos diferentes componentes foi considerada (Unifrac ponderado) permitiu a distinção de praticamente todas as microbiotas. Foram identificadas várias UTOs exclusivamente associadas à microbiota intestinal de adultos provenientes de cada fonte de alimento, mas a maioria apresentou abundância relativa extremamente baixa. Apenas as UTOs 1199 e 2255 foram exclusivamente associados ao milho com abundância relativa superior a 2,5%. / The correct insect resistant management has been a topic of constant concern, even for the newer technologies available, such as genetically modified crops expressing Bacillus thuringiensis (Bt) toxins. The use of refuge areas with non-Bt crops to avoid the fast selection for resistant insects is proposed for maintaining the efficiency of Bt-crops. The implementation of refuge areas for polyphagous insects can consider non-commercial and non-Bt crops as sources of susceptible insects. The identification of the food source used during immature development can make the implementation of refuge areas safer and more efficient and allow for better estimates of risk assessment for insect resistance development. The objective of this study is to determine the potential use of, the isotopic signature and gut microbiota of adults as biological markers to allow for the identification of the food source during the larval stage of Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae). Adults were obtained from larval rearing on 12 food sources, six host-plants with a C3 photosynthetic metabolism and six host-plants with a C4 metabolism. Part of the adults obtained and the food source used during their immature development were lyophilized and macerated, and subjected to ?13C e ?15N isotopic analysis. The remaining adults of S. frugiperda were used to determine the composition of the gut microbiota by metagenomic analysis of the V1-V3 region of the 16S rRNA gene using a 454 sequencing platform. The carbon isotopic signatures obtained for the hostplants used as food source were between -31.37? and -25.07? for C3 plants, and - 13.03? and -12.26? for C4 plants. Adults of S. frugiperda obtained from larval rearing on C3 plants had a carbon isotopic signature between -30.36? and -23.72?, while those from C4 host-plants has between -18.25? and -13.28?. The metagenomic sequencing yielded 126,970 reads with an average of 421bp. The larval food source substantially influenced the diversity of the adult gut microbiota regardless of the plant\'s photosynthesis metabolism. Comparative analysis among gut microbiota in which the relative abundance was taken into account (weight- Unifrac) allowed the discrimination of the majority of the communities. Several OTUs were identified as exclusive to the adult gut microbiota from different food sources, but most of these OTUs were minor components of the community. OTUs 1199 and 2255, both exclusively associated with corn, were the only ones to represent at least 2.5% of their community.

Os queijos artesanais, elaborados com leite cru, são apreciados por suas características sensoriais peculiares. Com objetivo de assegurar a inocuidade destes produtos, a legislação brasileira estabeleceu padrões microbiológicos sanitários para estes alimentos. Microorganismos probióticos são conhecidos por sua capacidade de modular favoravelmente a microbiota do hospedeiro por meio da produção de substâncias antimicrobianas, em especial, os ácidos orgânicos e bacteriocinas. Assim, o presente estudo buscou avaliar a utilização de micro-organismos probióticos como alternativa para melhorar a qualidade microbiológica de queijos artesanais usando o Queijo Artesanal da Serra da Mantiqueira como modelo. Para tanto, foi incorporada à microbiota do leite uma preparação composta de 16 cepas de micro-organismos probióticos, obtendo-se uma concentração final de 3x106 UFC/ml de leite. A estratégia adotada consistiu em comparar os queijos inoculados com micro-organismos probióticos em relação aos queijos não inoculados (controle), durante o período de maturação de 45 dias, em três estações do ano (inverno, verão e outono). As amostras foram coletadas nos dias 1, 15, 30 e 45 para a determinação dos parâmetros: umidade, pH, concentrações de carboidratos, ácidos orgânicos e análises microbiológicas. Avaliou-se também a composição da microbiota dos queijos pela técnica do pirossequenciamento dos genes do rRNA 16S. Os resultados permitiram verificar que os gêneros predominantes na microbiota dos queijos foram Streptococcus, Lactobacillus e Lactococcus , em diferentes níveis nas três estações do ano. Foi possível observar que a incorporação das culturas probióticas resultou em diferentes efeitos nas estações avaliadas no que se refere às concentrações de carboidratos e ácido cítrico, bem como de ácidos orgânicos ao final dos 45 dias de maturação, possivelmente devido à variabilidade da microbiota verificada nas diferentes estações. No tocante às características microbiológicas, observou-se que, durante o período de maturação, 95% dos queijos confeccionados nas diferentes condições apresentaram padrões de qualidade em conformidade com o estabelecido na legislação, no que se refere às concentrações de coliformes termotolerantes, estafilococos coagulase positivos, Salmonella sp e Listeria sp independentemente da incorporação das culturas probióticas. Entretanto, a adição das culturas probióticas reduziu a carga microbiana de gêneros da família Enterobacter iaceae , em especial Enterobacter , Citrobacter , Klebsiella , Kluyvera e Obesumbacterium durante processo de maturação. Especificamente, o emprego das culturas probióticas resultou em redução do percentual total de Enterobacter iaceae na microbiota dos queijos de 0,41% para 0,17% no verão, e de 12,42% para 6,71% no outono. Neste contexto, a adição de culturas probióticas pode constituir alternativa viável para melhoria da qualidade microbiológica de queijos elaborados com leite cru por meio do incremento do potencial antagônico da microbiota sobre espécies de micro-organismos potencialmente patogênicos, toxigênicos e deteriorantes, além de agregar valor ao produto final. / Artisanal cheeses, made with raw milk, are appreciated for their sensorial profiles, which differentiates them from industrialized cheeses. In order to ensure safety to consumers, Brazilian legislation established microbiological standards for these products. Probiotics are well known for their ability to favorably modulate the host\'s microbiota by producing antimicrobial compounds, mainly organic acids and bacteriocins. Therefore, this study aimed to evaluate the use of probiotic microorganisms as an alternative to improve the microbiological quality of artisanal cheeses through inhibition of contaminating microorganisms, using Mantiqueira Highlands Artisanal Cheese as a model. To do so, a preparation consisting of 16 probiotic strains was added to the milk, at a final concentration of 3x106 CFU/ml. Additionally, the effects of this practice on pH, carbohydrates and organic acids concentrations over a 45 day ripening period were evaluated. Analyses of cheese bacterial community composition were also undertaken by means of 16S rRNA gene pyrosequencing. The strategy consisted of comparing cheeses made with probiotic cultures with a control group, to which no probiotic culture was added. Over a 45 day ripening period, in three seasons (winter, summer and autumn), samples were collected at days 1, 15, 30 and 45 in order to conduct pH, moisture, carbohydrate, organic acids, microbiological and pyrosequencing analyses. The results showed that the addition of probiotic cultures resulted in distinct effects in the different seasons regarding carbohydrate and organic acids concentrations, possibly due to variability in bacterial community composition. The results also demonstrated that 95% of cheeses were deemed acceptable according to Brazilian law regarding coliforms, positive coagulase staphylococci, Salmonella sp. and Listeria sp, regardless of adding probiotics. However, the incorporation of probiotic microorganisms resulted in reduced populations of genera belonging to Enterobacteriaceae family, especially Enterobacter, Citrobacter, Klebsiella, Kluyvera and Obesumbacterium, throughout the whole ripening process. Specifically, the addition of probiotic cultures reduced the total percentage of Entercobacteriaceae from 0,41% to 0,17% in the summer, and from 12,42% to 6,71% in the fall. In this context, the use of probiotic microorganisms may represent a viable alternative for improving microbiological quality of raw milk cheeses by means of incrementing the antagonistic potential of cheese microbiota towards potentially pathogenic and toxigenic microorganisms.

O manguezal é um ecossistema costeiro, localizado em regiões de interface entre os ambientes terrestre e marinho, de ocorrência exclusiva em zonas tropicais e subtropicais. Esta localização confere ao sedimento deste ambiente características únicas, como alta salinidade e baixa disponibilidade de oxigênio, associado a grande riqueza em matéria orgânica. O resultado destas condições é a ocorrência de uma restrita diversidade de plantas em tais ambientes, associada a uma grande diversidade de animais, que usam o manguezal para sua proteção e reprodução. O presente estudo mostrou como as comunidades de arquéias (amoA e 16S DNAr) e bactérias (nifH e 16S DNAr) estão estruturadas em sedimentos de manguezais sob distintos estados de preservação. Os perfis de DGGE indicaram alterações na composição das comunidades alvo, ligando sua estruturação com a contaminação do ambiente, enquanto que as quantificações de tais genes por meio de PCR quantitativo em tempo real (qPCR) indicaram alterações apenas na comunidade de arquéias oxidadoras de amônio. A filogenia destes grupos revelou a presença de grupos comumente encontrados em solos e água, alem de grupos particulares, possivelmente relacionado ao processo de especiação no ambiente de manguezal. De maneira geral, os resultados indicam que as comunidades de arquéias e bactérias são responsivas ao estado de contaminação dos manguezais, o que pode gerar um processamento diferencial do nitrogênio nestes sedimentos / The mangrove is a coastal ecosystem, located in the interface regions between the land and sea environments, with exclusive occurrence in tropical and subtropical areas. Such location confers to the mangrove sediments unique characteristics, like as high salinity and low availability of oxygen, associated with the high abundance of organic matter. The result of these conditions is the occurrence of a restrict plant diversity, associated with a great diversity of animals, who use the mangroves for its protection and reproduction. The present study has shown how the communities of archaea (16S DNAr and amoA) and bacteria (16S DNAr and nifH) are structured in mangrove soils under distinct states of preservation. The DGGE patterns indicate alterations in the composition of targeted communities, linking its structure with the environmental contamination, while the quantifications of targeted genes by real time PCR (qPCR) has indicated alterations only in the community of ammonium oxidizers archaea. The phylogeny of these groups revealed the presence of groups commonly found in soils and water samples, besides the occurrence of particular groups, possibly resulted from a speciation process in the mangrove environment. In general, results indicated that archaeal and bacterial communities are responsive to the mangroves contamination, and its alteration can also lead to differential nitrogen processing in these soils