Submitted by ANA LUIZA ROCHA FARIA DUQUE null (analuiza.rduque@gmail.com) on 2016-04-24T19:06:34Z No. of bitstreams: 1 Dissertação Ana Luiza Duque.pdf: 1544679 bytes, checksum: decc5d7d0f47e1235b24c75d5577fd3b (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-26T20:13:00Z (GMT) No. of bitstreams: 1 duque_alrf_me_arafcf.pdf: 1544679 bytes, checksum: decc5d7d0f47e1235b24c75d5577fd3b (MD5) / Made available in DSpace on 2016-04-26T20:13:00Z (GMT). No. of bitstreams: 1 duque_alrf_me_arafcf.pdf: 1544679 bytes, checksum: decc5d7d0f47e1235b24c75d5577fd3b (MD5) Previous issue date: 2016-03-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A microbiota intestinal apresenta impacto direto na saúde do hospedeiro sendo fortemente influenciada pela dieta. O consumo de suco de laranja vem sendo associado à redução do risco de desenvolvimento de doenças crônicas, principalmente devido à presença de compostos bioativos. Os compostos bioativos presentes no suco de laranja, especialmente os polifenóis, também podem estar relacionados com a composição e o metabolismo da microbiota intestinal. O objetivo desse trabalho foi avaliar a influência do suco de laranja fresco e pasteurizado sobre a microbiota intestinal usando o Simulador do Ecossistema Microbiano Humano (SEMH®). O SEMH® foi utilizado para investigar a fermentação do suco de laranja ao longo do cólon e para avaliar as alterações na composição e no metabolismo microbiano. A atividade antioxidante dos sucos e das amostras dos compartimentos do SEMH® também foi avaliada. Foi observado no tratamento com suco de laranja fresco aumento (p≤0,05) das populações de Lactobacillus spp., Enterococcus spp., Bifidobacterium spp. e Clostridium spp. e diminuição (p≤0,05) de enterobactérias, enquanto no tratamento com suco de laranja pasteurizado houve aumento (p≤0,05) da população de Lactobacillus spp. e diminuição (p≤0,05) de enterobactérias. A análise de PCR-DGGE mostrou redução dos valores de riqueza da população de bactérias totais para ambos os sucos. Em relação ao metabolismo microbiano, foi observado aumento (p≤0,05) da produção de ácidos graxos de cadeia curta (AGCC) e diminuição (p≤0,05) do conteúdo de íons amônio no tratamento com os sucos de laranja fresco e pasteurizado. A atividade antioxidante das amostras dos compartimentos do SEMH® no tratamento com os sucos de laranja foi elevada, com ligeira redução em comparação àquela do suco fresco e do suco pasteurizado. A Análise de Componentes Principais (ACP) permitiu diferenciar o tratamento com os sucos dos períodos controle e washout, mostrando que os sucos de laranja fresco e pasteurizado apresentaram impacto sobre a microbiota intestinal. Os sucos mostraram efeito prebiótico e seletivo sobre a microbiota intestinal com aumento de AGCC e bactérias comensais e diminuição de íons amônio, embora com redução dos valores de riqueza da população de bactérias totais. / The gut microbiota has a direct impact on host's health being strongly influenced by diet. Orange juice consumption has been associated with a reduced risk of chronic diseases, largely because of the presence of bioactive compounds. The bioactive compounds present in orange juice, particularly polyphenols, may also be associated with the composition and metabolism of gut microbiota. The aim of this work was to evaluate the influence of fresh orange juice and pasteurized orange juice on gut microbiota using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). SHIME® was used to investigate orange juice fermentation throughout the colon and to assess changes in microbial composition and microbial metabolism. Antioxidant activity of the SHIME® vessels and juice was also evaluated. An increase (p≤0.05) in Lactobacillus spp., Enterococcus spp., Bifidobacterium spp. and Clostridium spp. population was observed in fresh orange juice treatment, as well as a reduction (p≤0.05) in enterobacteria. Regarding pasteurized orange juice treatment, an increase (p≤0.05) in Lactobacillus spp. population and a decrease (p≤0.05) in enterobacteria was observed. The PCR-DGGE analysis showed a reduction in total bacteria population richness values on both juices. According to microbial metabolism, an increasing (p≤0.05) of short-chain fatty acids (SCFA) production and decreasing (p≤0.05) of ammonium was observed for two juices treatments evaluated. The antioxidant activity of the samples from the SHIME® vessels in the orange juice treatments was high, with a slight reduction compared to that of fresh juice and pasteurized juice. Both fresh and pasteurized orange juice influenced on gut microbiota according to Principal Component Analysis (PCA), which enabled to differentiate the orange juice treatments from control and washout periods. Both juices showed a prebiotic and selective effect on gut microbiota which is in agreement with increases in both SCFAs and commensal bacteria, as well as with decreases in ammonium levels, though total bacteria richness values were reduced.

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

A metilamina ('CH IND.3'NH IND.2') é um composto orgânico usado na produção de inseticidas, herbicidas, fungicidas, surfactantes, combustíveis fósseis, explosivos, produtos farmacêuticos, químicos fotográficos, tintas, tecidos, solventes, borrachas e anti-corrosivos. Estudos sobre tratamento de águas residuárias contendo metilamina são escassos e se restringem aos trabalhos envolvendo pesticidas carbamatados. Visando contribuir com os estudos acerca da degradação anaeróbia da metilamina, esta pesquisa estudou a comunidade microbiana e a atividade metanogênica específica em reatores anaeróbios em batelada, inoculados com lodo granular oriundo de reator UASB usado no tratamento de água residuária de abatedouro de aves, sob diferentes condições nutricionais: controle – sem metilamina, 5 mM, 10 mM, 20 mM, 30 mM, 50 mM, 75 mM e 90 mM de metilamina. Os reatores foram incubados sob temperatura de 30°C e agitação de 150 rpm. Desses reatores foram obtidas amostras para a determinação da atividade metanogênica específica (AME), sólidos suspensos voláteis (SVT), nitrogênio amoniacal e exames microscópicos. Ao final do experimento, foram realizados exames da biomassa por meio da técnica do número mais provável (NMP) e análise da diversidade microbiana por PCR/DGGE e seqüenciamento. O aumento da AME foi proporcional ao aumento das concentrações de metilamina, com inibição de produção de metano apenas nos reatores alimentados com 90 mM de metilamina. Os reatores alimentados com 50 mM e 75 mM de metilamina apresentaram os melhores resultados, com valores médios de AME de 0,0804 mmol 'CH IND.4'/g SVT.h e 0,0825 mmol 'CH IND.4'/g SVT.h respectivamente. Nos exames microscópicos foi verificado semelhança de morfologias microbianas em todas as concentrações de metilamina estudadas. Os organismos presentes nos reatores foram Methanosarcina sp., Methanosaeta sp., bacilos, coco-bacilos, filamentos e cocos. Em relação à análise de DGGE, não houve variação significativa nos padrões de bandas, tanto para o domínio Archaea quanto para o domínio Bacteria. Com os resultados da técnica de número mais provável (NMP) observou-se a predominância de arquéias metanogênicas dentre as bactérias anaeróbias totais. / The methylamine ('CH IND.3'NH IND.2') is an organic compound used in the production of insecticides, herbicides, fungicides, surfactants, fossil fuels, explosives, pharmaceuticals, photographic chemicals, paints, textiles, dyes, rubber and anticorrosive chemicals. Some studies about the treatment of wastewater containing methylamine are scarce and limited to works involving carbamate pesticides. This research aimed to study the anaerobic degradation of methylamine, the microbial community and the specific methanogenic activity in anaerobic battled reactors. The reactors were inoculated with granular sludge from a UASB reactor treating poultry wastes. Different nutritional conditions were adopted in the operation of the reactors: control (without methylamine), 5 mM, 10 mM, 20 mM, 30 mM, 50 mM, 75 mM and 90 mM of methylamine. The reactors were incubated under standard conditions: 30ºC and 150 rpm. Samples had been removed from the reactors to determine the specific methanogenic activity, the concentration of volatile suspended solids and ammoniacal nitrogen and the microscopic analysis. At the end of the experiment, the biomass was studied by the most probable number (MPN) technique and by the microbial diversity analysis with PCR and DGGE techniques. The increase of the specific methanogenic activity was proportional to the increase of methylamine concentration. The methane production was inhibited only in the reactor that was fed with 90 mM of methylamine. The reactors that were fed with 50 mM and 75 mM of methylamine showed the best results, with medium values of specific methanogenic activity equal to 0,0804 mmol 'CH IND.4'/g SVT.h and 0,0825 mmol 'CH IND.4'/g SVT.h, respectively. The microscopic analysis showed similarity between the microbial morphologies in all of the reactors. The observed microorganisms were Methanosarcina sp., Methanosaeta sp., rods, cocci and filaments. The DGGE analysis did not show significant variation in the standard profile of the Archaea and Bacteria domains. The results of the MPN technique revealed the predominance of the methanogenic archaea among the total anaerobic bacteria.

As cianobactérias atuam no tratamento de águas residuárias em lagoas de estabilização. Seu estudo torna-se importante tanto pela atuação no referido tratamento como na possível produção de toxinas e seus efeitos nos sistemas aquáticos e saúde da população. Protocolos moleculares para culturas axênicas ou naturais foram testados e adaptados para as amostras analisadas. Este trabalho teve como objetivo avaliar o método molecular de eletroforese em gel de gradiente desnaturante nas espécies de Microcystis (cianobactérias) em um sistema de lagoas de estabilização. Foram utilizadas as técnicas de Reação de Polimerização em Cadeia (PCR) e Eletroforese em Gel de Gradiente Desnaturante (DGGE) do gene RNAr 16S como marcador molecular na análise de cianobactérias no sistema de lagoas de estabilização de São Lourenço da Serra - SP. Este sistema é composto por tratamento primário (caixa de areia), fossa séptica seguida de um sistema australiano (lagoa anaeróbia seguida por uma lagoa facultativa) e na saída do sistema de lagoas um tanque de cloração. Na amostra ambiental realizada na lagoa facultativa obteve-se, através de exame microscópico, o predomínio das espécies Microcystis aeruginosa e Microcystis flos-aquae. Através da técnica molecular de DGGE foi obtido um padrão de aproximadamente 18 bandas com a amostra ambiental. / Cyanobacteria are active in wastewater treatment in stabilization ponds. The study of these microrganisms is of a great importance considering their role in the referred wastewater treatments as well as their potential to produce toxins and their effects in aquatic systems and public health. Molecular protocols for pure or natural cultures were tested and adapted for the collected environmental samples. This work had as objective to evaluate the molecular method of denatuirng gradient gel electrophoresis in the Microcystis species (cyanobacteria) in a system of stabilization ponds. The techniques Polimerase Chain Reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) of the gene 16S rRNA as molecular marker were used in the cyanobacteria analysis in the system of stabilization ponds of São Lourenço da Serra - SP. This system is composed by primary treatment (box of sand), septic tank followed by an Australian system (anaerobic pond proceeded by an facultative pond) and in the exit of the system a clorine tank. In environmental sample accomplished in the facultative pond it was obtained, as microscopic result, the prevalence of the species Microcystis aeruginosa and Microcystis flos-aquae. Through molecular techniques a pattern of approximately 18 bands with these environmental sample was obtained.

Neste trabalho foi realizada a caracterização microbiana, por meio de técnicas de biologia molecular, do lodo granulado de reator anaeróbio de fluxo ascendente e manta de lodo (UASB - Upflow Anaerobic Sludge Blanket Reactor) operado sob condições mesofílicas (30 \'+ OU -\' 2ºC) e sulfetogênicas, com (TDH) de 12 h, na presença de 3,0 \'+ OU -\' 0,7 mg/L de oxigênio dissolvido. Essa caracterização foi realizada no lodo granulado proveniente da manta inferior (P1) e superior (P2) do UASB. O reator UASB de 10,5 L foi alimentado com meio basal Zinder acrescido de solução de vitaminas, metais traços e bicarbonato de sódio (10%). O meio foi preparado diariamente com água de abastecimento público, pH 7-8. Etanol e sulfato de sódio foram utilizados como fonte orgânica e de enxofre, respectivamente. Nestas condições, foram estudadas três diferentes relações de demanda química de oxigênio (DQO)/sulfato (3,0, 1,6 e 2,0). A análise de hibridação in situ fluorescente (FISH) demonstrou que houve predomínio de arquéias metanogênicas, detectadas com a sonda ARC915, em todas as condições operacionais, com médias iguais a 75,9, 77,1 e 85,4% em P1 e 78,6, 73,4 e 83,1% em P2, respectivamente. Methanosaeta sp. foi a arquéia metanogênica acetoclástica predominante confirmada também por seqüenciamento da banda recortada do gel de DGGE (eletroforese em gel de cadeia desnaturante), com similaridade de 96%. As bactérias (sonda EUB338) variaram de 9,6 a 36,2% e de 15,5 a 37,4% em P1 e P2, respectivamente. As bactérias redutoras de sulfato (BRS), detectadas com a sonda SRB385, variaram de 7,9 a 10,8% e de 8,7 a 19,8% em P1 e P2, respectivamente. Outros microrganismos identificados foram Shewanella sp. e Desulfitobacterium hafniense Y51, e a bactéria redutora de sulfato Desulfovibrio vulgaris subsp. vulgaris DP4. A distribuição do tamanho dos grânulos não sofreu mudança significativa no decorrer dos ensaios. Grânulos de 2 a 3 mm, que geralmente representam biomassa ativa no reator, contou com média de 76% do total quantificado; enquanto, grânulos de 1 a 2 mm, os quais sugerem que novos grânulos estavam sendo formados representaram 17% no reator UASB. As concentrações médias de metano e sulfeto no biogás foram iguais a 33 e 1,5 \'mü\'mol/ mL, respectivamente. Os resultados obtidos neste trabalho demonstraram que a presença de oxigênio, na concentração aplicada, não afetou severamente o metabolismo dos microrganismos comumente considerados estritamente anaeróbios. Esse fato foi evidenciado também pelo valor do potencial redox obtido que se manteve aproximadamente constante em -208 mV, mesmo na presença de oxigênio. As eficiências de remoção de matéria orgânica (DQO) e redução de sulfato também alcançaram resultados favoráveis, com médias superiores a 74%. / A microbial characterization of granular sludge from an upflow anaerobic sludge blanket reactor (UASB) was carried out by molecular biology techniques. The reactor with 1,5 L of volume was operated with HRT of 12 h under mesophilic (30 \'+ OR -\' 2ºC) and sulfidogenic conditions, in the presence of 3.0 \'+ OR -\' 0.7 mg \'O IND.2\'/L. The granular sludge samples were withdrawn from the bottom (P1) and upper (P2) parts of the reactor. The synthetic substrate used in the reactor feeding was composed by Zinder basal medium in addition with a solution of vitamins, trace metal and sodium bicarbonate (10%). The Zinder basal medium was prepared daily with tap water with pH value varying from 7 to 8. Concentrations of ethanol and sodium sulfate were applied as organic and sulfur sources, respectively. Three different COD/sulfate ratios (3,0, 1,6 and 2,0) were evaluated in these conditions. The fluorescent in situ hibridization (FISH) analysis demonstrated the predominance of methanogenic archaea, detected by ARC915 specific probe, in all the operational conditions, with mean values of 75.9%, 77.1% and 85.4% in P1 and 78.6%, 73.4% and 83.1% in P2. The sequencing of the excised band of DGGE (denaturing gradient gel electrophoresis) showed similarity of 96% with the acetoclastic archaea Methanosaeta sp. The bacterial community (EUB338 probe) varied from 9.6% to 36.2% in P1 and from 15.5% to 37.4% in P2. Sulfate-reducing bacteria (SRB), detected by SRB385 probe, varied from 7.9% to 10.8% and from 8.7% to 19.8% in P1 and P2, respectively. Other identified microorganisms were Shewanella sp. and Desulfitobacterium hafniense Y51 bacteria, and the sulfate-reducing Desulfovibrio vulgaris subsp. vulgaris DP4. Granule-size distribution did not significantly change during the assays. Granules of size varying from 2 mm to 3 mm, that generally represent the active biomass inside the reactor, accounted for 76% of the total quantified percentage; while, granules of size varying from 1 mm to 2 mm, that suggest the formation of new granules in the reactor, presented mean percentage of 17% of the total. The mean produced concentrations of methane and sulfide in the reactor were equal to 33 \'mü\'mol/mL and 1.5 \'mü\'mol/mL of biogas, respectively. The obtained results indicated that the applied oxygen concentration did not severely affect the metabolism of the strictly anaerobic microorganisms. This fact was evidenced by the obtained result of the oxidation-reduction potential that remained equal to -208 mV, even in the presence of oxygen. The mean removal efficiencies of organic matter (COD) and sulfate also achieved favourable results with values higher than 74%.

The century poison ¡§dioxins¡¨ are hydrophobic compounds that can combine with many organic matters and persist in the environment as well as to accumulate in living organisms. Dioxins caused great risk to the health of living organisms and to the entire ecological environment. We had isolated previously one bacterial species, Pseudomonas mendocina NSYSU, which can use pentachlorophenol (PCP) as its sole carbon source and degrade dioxin compounds. In order to study the feasibility of using this bacterial strain to bioremediate an PCDD/Fs polluted site, four microcosm experiment groups were designed to test the degradation efficiency of this strain: sterile soil group, non-sterile soil group, soya lecithin group and non-sterile soil with soya lecithin group. In addition, we also analyzed the shift of community structure of each microcosm by PCR-DGGE. The results show that the soya lecithin group has the highest efficiency to degrade OCDD/OCDF. After fifty days of reaction, the degradation rates of OCDD/OCDF were 62% and 47% respectively. The microbial diversity analysis indicated that the soya lecithin group presented less abundant from the initial stage, but increasing gradually over time. This might related to the formation of micelles in water phase which contained higher concentration of PCDD/Fs dissolved from the soil particles. Therefore, soya lecithin not only can reduce the toxicity of PCDD/Fs, but also can enhance the bioavailability of the organic pollutants to the microorganisms. In conclusion, monitoring the transition of P. mendocina NSYSU as well as the microbial diversity can provide valuable information during the bioremediation process by applying soya lecithin.

No description available.

The dinoflagellate genus Symbiodinium is a popular research topic in the coral reef molecular biology field. Primarily because these organisms serve as the coral holobiont’s primary source of energy, carrying out photosynthesis, and providing hydrocarbons to the coral host. Previous studies have shown the difficulty of isolating Symbiodinium as well as the inherent problems in trying to quantify the diversity of this genus and to qualify the distinct reactions of different Symbiodinium sp. to changing environmental conditions. The main goals of this study are: (1) to detail the relationship between the genetic classification of the organism and its physiology in regard to photosynthesis with a number of established Symbiodinium cultures; and (2) to isolate Symbiodinium from coral of the central Red Sea. To evaluate the photosynthetic physiology of Symbiodinium, a microsensor was used to measure oxygen concentrations along with a phytoplankton analyzer system that used pulse-amplitude-modulation (Phyto-PAM) to measure fluorescence. In order to identify the particular clade that the isolates belonged to, denaturing gradient gel electrophoresis (PCR-DGGE) was used to identify Symbiodinium based on their internal transcribed spacer 2 (ITS2) region. These techniques helped us to achieve our goals in the following ways: Symbiodinium sp. from a culture collection were classified to the subclade level; species-specific and clade-specific photosynthetic profiles were generated; and a Symbiodinium sp. was isolated from the central Red Sea. This study provided preliminary correlation between the photosynthetic difference and Symbiodinium genetic classification; showed the probable existence of a self-protection system inside the Symbiodinium cells by comparing the difference between the initial oxygen production at the beginning of each light step and the oxygen production after light adaptation; and confirmed the possibility of the isolation of Symbiodinium.

L'ochratoxine A (OTA), principalement produite dans le café par les moisissures A. ochraceus et A. westerdijkiae, suscite une attention particulière pour ses effets néphrotoxiques, immunotoxiques, tératogènes et cancérigènes. La présence d'OTA dans les fèves de café peut être mise en relation avec les conditions de récolte, les conditions de traitement post-récolte et les conditions de stockage et de transport. Dans certains pays producteurs, les dommages causés sur les grains de café par des espèces fongiques sont liés à des teneurs élevées en OTA. La dynamique et la biodiversité des populations microbiennes (bactéries, levures, moisissures) lors des traitements post-récolte du café a été étudiée par une méthode d'analyse moléculaire globale des flores, la PCR-DGGE. Il a été observé une évolution et une diversité des flores microbiennes en fonction du type et des étapes des traitements utilisés, spécifiques des lieux de production. La région génomique ciblée et la proximité phylogénétique sont un obstacle à l'identification des souches ochratoxinogènes par l'approche globale utilisée. De plus, une méthode simple et rapide de différenciation moléculaire d'Aspergillus westerdijkiae et d'Aspergillus ochraceus a été mise au point et couplée avec l'analyse d'image pour permettre la « quantification » d'Aspergillus westerdijkiae. Des phénomènes de compétition/inhibition de la croissance et production d'OTA (supérieurs à 90%) ont été mis en évidence pour des souches d'Aspergillus niger et d'Aspergillus ochraceus faiblement productrices d'OTA vis-à-vis d'Aspergillus westerdijkiae qui est l'une des espèces les plus fortement productrices de la mycotoxine sur café. Les résultats obtenus au cours de ce travail sont importants pour l'amélioration des connaissances sur la dynamique des populations microbiennes au cours des procédés de transformation du café ainsi que pour de possibles applications en prévention et maîtrise de la contamination du café par l'OTA. / Ochratoxin A (OTA) is mainly produced on coffee beans by fungal species Aspergillus ochraceus and Aspergillus westerdijkiae, and is known for its impact on human health through nephrotoxic, immunotox, teratogenic and oncogenic effects.The OTA content in coffee was shown to be closely linked to harvesting conditions, post-harvest processing conditions and especially dry processing, storage and transportation conditions. In some producing countries, damaged caused on beans by fungal communities undoubtedly lead to high OTA contents in coffee. In order to understand the OTA contamination process, the dynamics and biodiversity of microbial populations (bacteria, yeast and moulds) was analyzed during post-harvest treatment by use of a global microbial ecology approach at the molecular level, so-called PCR-DGGE. Specific variations in evolution and diversity of microbial flora were observed as a function of the step and type of treatment, which were specific of the location of production. The genomic region targeted by the global approach and the genetic proximity of ochratoxigenic fungal strains made their study and identification difficult using the the global approach. In addition, a simple and rapid method for the molecular differentiation of A. westerdijkiae and A. ochraceus was established and, coupled with image analysis, allowed the quantification of A. westerdijkiae.Moreover, competition and inhibition effects on growth and OTA production (>90%) could be observed for low OTA producers A. niger and A. ochraceus species towards the high OTA producer A. westerdijkiae species. Results obtained during this study are of importance for understanding microbial population dynamics during coffee transformation processes. Moreover, it provides possible clues for prevention and control of coffee contamination by OTA.

A ampla ocorrência de contaminação de águas por compostos de nitrogênio em concentrações superiores às recomendadas pela legislação tem suscitado interesse no desenvolvimento de tecnologias viáveis de remoção desses compostos. A remoção biológica de nitrogênio apresenta como principais vantagens os custos relativamente reduzidos e a possibilidade de maior eficiência. Compostos reduzidos de enxofre como sulfetos podem ser oxidados a enxofre elementar ou a sulfato por bactérias oxidantes de sulfeto que utilizam nitrato ou nitrito como receptor de elétrons. Esta desnitrificação reduz os requerimentos globais de carbono para a remoção de nutrientes, com menor produção de lodo, proporcionando grande economia. O objetivo desta pesquisa consistiu em contribuir para o conhecimento acerca dos aspectos microbiológicos do processo de desnitrificação com o uso de sulfeto. Foram avaliados os efeitos dos modos de operação dos reatores desnitrificantes sobre a biomassa em cada uma das diferentes configurações e monitorada a colonização microbiana por meio de técnicas de Biologia Molecular como PCR/DGGE, sequenciamento e análises filogenéticas. Os fragmentos do gene RNAr 16S foram relacionados aos gêneros Pseudomonas, Aeromonas, Acidobacteria, Chlroroflexi, Clostridium, Cupriavidus e Ralstonia. Filotipos dos clones para o sistema piloto foram associados a bactérias não cultiváveis e Firmicutes envolvidos na digestão anaeróbia em reatores tratando água residuária, Synergistetes, Deferribacteria e Proteobacteria. Foram identificados micro-organismos presentes nos reatores com reconhecida capacidade para desnitrificação: Pseudomonas, Desulfovibrio desulfuricans e Ralstonia. Amostras de ambos os reatores desnitrificantes apresentaram reações de amplificação positivas com primers específicos para bactérias semelhantes a Thiomicrospira associados a primers universais: 100% das amostras amplificaram com OST1F/1492R e 75% com EUB8F/OSTR1R. Para duas condições de operação do reator em escala de bancada foram identificados micro-organismos semelhantes a Sulfurimonas denitrificans, bactéria autotrófica redutora de nitrato e oxidadora de sulfeto. Chloroflexi também foram encontrados em digestores localizados em plantas de tratamento de águas residuárias recebendo essencialmente efluente doméstico e Propionibacterium foi associada a comunidade microbiana de reatores UASB tratando água residuária industrial. Bactérias em associações sintróficas com participação no ciclo do enxofre e/ou na digestão anaeróbia foram igualmente identificadas: Clostridium sulfidigenes e outros Firmicutes, Synergistetes, clones de bactérias de cultura de enriquecimento e bactérias do gênero Syntrophorhabdus. Os resultados deste trabalho proporcionaram associar a colonização microbiana com o desempenho e as características metabólicas de alguns micro-organismos com reatores desnitrificantes combinados para o tratamento de águas residuárias. / The widespread nitrate contamination in concentrations higher than recommended by legislation has raised interest in technologies for water and wastewater treatment. Biological nitrogen removal is relatively low cost and higher efficiency. Sulfide as electron donor can be oxidized to elemental sulfur or sulfate by sulfide oxidizing bacteria that can use nitrate or nitrite as electron acceptor. This type of denitrification reduces the overall requirements for removal of carbon nutrients and less sludge is produced. The aim of this research was to contribute to microbiological knowledge about denitrification using sulfide. The effects of operation conditions on the denitrifying biomass were monitored through molecular biology techniques such as PCR/DGGE, sequencing and phylogenetic analysis. 16S rRNA gene fragments were related to Pseudomonas, Aeromonas, Acidobacteria, Chlroroflexi, Clostridium, Cupriavidus and Ralstonia. Phylotypes of clones from samples of pilot-scale reactor were associated with non-cultivable bacteria and Firmicutes involved in anaerobic digestion of wastewater, Synergistetes, Deferribacteria and Proteobacteria. Microorganisms with ability to denitrification were identified in both reactors: Pseudomonas, Desulfovibrio desulfuricans and Ralstonia. Samples of denitrifying reactors showed positive amplification with specific primers for Thiomicrospira associated to universal primers: 100% of the samples amplified with OST1F/1492R and 75% EUB8F/OSTR1R. Sulfurimonas denitrificans-like were identified for two operational conditions of the bench scale reactor. Chloroflexi were also found in treatment plants digesters receiving domestic wastewater and Propionibacterium was associated with microbial community of UASB reactors treating industrial wastewater. Syntrophic bacteria participating in the sulfur cycle and/or anaerobic digestion were also identified: Clostridium sulfidigenes and other Firmicutes, Synergistetes, clone enrichment culture bacteria and Syntrophorhabdus. These results provide to associate this microbial colonization and metabolic characteristics of some microorganisms with performance of combined denitrifying reactors for treatment of wastewater.