Les activités anthropiques entraînent une contamination des sédiments de rivière en de nombreux polluants et en particulier en éléments traces métalliques (ETM). Si la majorité des ETM se retrouvent piégés dans les sédiments, ceux-ci peuvent être remobilisés et passer en solution dans certaines conditions physico-chimiques et sous l’action des microorganismes autochtones. Les métaux relargués peuvent alors constituer un danger potentiel pour les organismes vivants dans les sédiments et dans la colonne d'eau. Dans le cas des sols, l’impact de l’activité microbienne autochtone sur la mobilité des ETM a souvent été rapporté. Cependant une telle activité de solubilisation n’a été que rarement étudiée dans le cas des sédiments. Une telle connaissance est pourtant importante pour la prédiction du comportement des métaux contenus dans les sédiments et la gestion de ces derniers, notamment lors de leur stockage suite aux opérations de dragage. Dans ce contexte, l’objectif de cette thèse a été de comprendre et d’évaluer l’importance de certains des processus microbiens et chimiques de mobilité des ETM dans les sédiments en conditions anaérobies. La première phase de notre étude qui a consisté à incuber des sédiments de Seine et de Marne en milieu anaérobie dopé en glucose avait pour objectif d’étudier la corrélation entre le métabolisme microbien et le comportement des métaux en solution et dans les sédiments. Dans ces conditions opératoires, une forte solubilisation du fer et du manganèse (sous forme réduite) associée à une solubilisation de métaux traces (Co, Cu, Ni) a été mise en évidence, ce qui a laissé supposer l’intervention de bactéries ferri-réductrices dans les phénomènes observés. Une activité fermentaire importante a été observée et caractérisée par la production d’acides organiques majoritaires tels que les acides acétique et butyrique. Un tel résultat souligne l’importance des bactéries fermentatrices dans les phénomènes de dissolution observés. La deuxième étape de ce travail a consisté à confirmer l’importance de l’activité ferri-réductrice et à en identifier les acteurs principaux. Les analyses moléculaires menées ont montré que les bactéries ferri-réductrices majoritairement identifiées, appartiennent aux espèces Clostridium butyricum et Paenibacillus polymyxa. L’utilisation d’un modèle géochimique nous a permis de montrer que les voies métaboliques supportant la réduction du fer et la mobilité des métaux étaient les fermentations butyrique et acétique. La troisième étape a consisté à comparer les impacts directs (réduction enzymatique) et indirects (propriétés des acides organiques produits) de Paenibacillus polymyxa et Clostridium butyricum sur la mobilité du fer, du manganèse et des autres métaux. Une telle étude a montré que les acides organiques produits (acétique, lactique, succinique, propionique et butyrique) ont un très faible impact sur la solubilisation aux pH rencontrés dans les sédiments et que la réduction enzymatique microbienne est le principal mécanisme de dissolution des éléments métalliques en milieu anaérobie / Antropic activities lead to the metallic contamination of river sediments. Most of trace metals are sorbed on sediments but a part of them can be released into aquatic environment when environmental conditions are modified. This is often due by the autochthonous microbial activity. Microbial activites and their consequences on the mobility of metals have been widely studied in soils. Metals are released through direct or indirect microbial mechanisms. Such studies in the case of sediments are very seldom. However, it can be usefull to understand the microbial mechanisms of metal release in sediments, and particularly for a good management of dredged sediments. In this environmental framework, the aim of this research work was to understand and to evaluate the role of the microbial and chemical mechanisms in the release of metals from river sediments in anaerobic conditions. Firstly, sediments from the Marne and Seine rivers were incubated in anaerobic conditions. A high solubilisation of iron and manganese occurred associated to the solubilisation of trace metals (Co, Cu, Ni, Pb). Meanwhile, organic acids were produced and the medium was acidified. Thus fermentation was supposed to be the main process of microbial metabolism. Furthermore these observations led us to suppose the presence of iron-reducing bacteria. In a second step, the extent of the iron-reducing activity was studied. The main iron-reducing bacteria identified in the Marne sediments belonged to the species Clostridium butyricum and Paenibacillus polymyxa. The use of a geochemical model revealed that fermentation and reduction of iron(III) were the main metabolic pathways. Finally direct (enzymatic reduction) and indirect (complexation with organic acids, acidification) impacts of iron-reducing bacteria on the release of metals were compared. Acidification and organic acids had a weak impact on metal solubility in the range of studied pH (between 6,5 and 5). Enzymatic reduction is the main mechanisms of metal release in anaerobic conditions. Indeed the metallic concentrations can be 40 times higher in the presence of iron-reducing bacteria

Bifenilas policloradas (PCBs) são compostos de difícil degradação presentes na composição de ascarel, muito utilizado como fluidos dielétricos e isolantes. Neste contexto, a presente pesquisa teve como objetivo avaliar a diversidade de microrganismos em biofilmes de reatores anaeróbios na presença de PCB empregando Métodos de Microbiologia de Anaeróbios Estritos e de Biologia Molecular. Em reator anaeróbio horizontal de leito fixo (RAHLF), alimentado com etanol, formiato, Triton X-100 (0,1%) e ascarel (1 mL/L), operado com tempo de detenção hidráulica (TDH) de 24 horas, foi retirado a comunidade microbiana do biofilme da espuma de poliuretano. Os grupos microbianos encontrados por meio da clonagem e sequenciamento do gene RNAr 16S para o domínio Bacteria foram relacionados aos filos Thermogae, Proteobacteria (Brachymonas petroleovorans, 100% de similaridade e Methylobacillus, 98% de similaridade), Firmicutes (Clostridium, 97% de similaridade, Syntrophomonas, 100% de similaridade e Sporomusa com 100% de similaridade), Synegistetes (Synergistes, 98% de similaridade), Spirochaetes (Leptonema illini, 98% de similaridade), Aminanaerobia, Deferribacteres, Chlorobi, Chloroflexi e Armatimonadetes. Além disso, como nesse biofilme foram identificadas bactérias redutoras de ferro, procedeu-se a sua quantificação por meio da técnica de tubos múltiplos (NMP, Número Mais Provável) obtendo 5,26 x \'10 POT.12\' NMP/g STV de bactérias redutoras de ferro. Ensaio em batelada foi realizado separadamente sob duas condições: (1) metanogênica e (2) ferro redutora. Em ambas as condições foram adicionadas aroclor 1260 (PCB). Os reatores, sob condição metanogênica, foram alimentados com meio de cultivo Angelidaki e substratos orgânicos (formiato e etanol), além de aroclor 1260 (0,2 \'mü\'g/L). Para simular a condição redutora de ferro foi acrescido ao meio de cultura Angelidaki, EDTA férrico (1,86 g/L). A produção de metano, na presença de aroclor 1260 foi de 3,8 x \'10 POT.-4\' mmol \'CH IND.4\'/g STV. A presença de bactérias ferro redutoras foi confirmada indiretamente pela taxa média de redução férrica (90%) nos reatores em batelada, após 60 dias de operação. Por meio de PCR/DGGE, elaborou-se um dendograma das amostras deste ensaio em batelada (metanogênico e redutor de ferro) comparativamente com as do reator RAHLF (biofilme presente na parede do reator e no material suporte). Os reatores em batelada apresentaram similaridade entre si de 79% e 92% para os domínios Bacteria e Archaea, respectivamente. As amostras do reator RAHLF foram 80% (Bacteria) e 96% (Archaea) similares. A existência de bactérias degradadoras de PCB, bem como, bactérias redutoras de ferro no biofilme anaeróbio contribuiu com informações sobre o consórcio microbiano e sua diversidade. / Polychlorinated biphenyls (PCBs) are compounds of difficult degradation, a component of askarel, which were used widely as coolants and lubricants. Hence, this study evaluated the diversity of microorganisms in the presence of PCBs in anaerobic reactors. For such, methods as Strict Anaerobic Microbiology and Molecular Biology were employed. The microbial community of the biofilm, developed in a fixed horizontal bed anaerobic reactor (RAHLF), was studied using the technique of cloning and sequencing of RNAr 16S gene for the Bacteria domain. The reactor had immobilized cells in polyurethane foam with ethanol and formate as a carbon source, Triton X-100 (0.1%) and polychlorinated biphenyls (1 mL/L), and operated with 24 hours HRT. The microbial groups found in this biofilm were related to phyla Thermogae, Proteobacteria (Brachymonas petroleovorans, 100% similarity and Methylobacillus, 98% similarity), Firmicutes (Clostridium, 97% similarity Syntrophomonas, and 100% similarity with Sporomusa 100% similarity), Synegistetes (Synergistes, 98% similarity), Spirochaetes (Leptonema Illini, 98% similarity), Aminanaerobia, Deferribacteres, Chlorobi, Chloroflexi and Armatimonadetes. Furthermore, as bacteria that reduce iron were found, we proceeded the quantification by the multiple tube method (MPN) for this group, obtaining 5.26 x \'10 POT.12\' MPN/g STV of iron-reducing bacteria. The batch reactors evaluated the growth of microorganisms in two condictions: (1) methanogenic e (2) iron reduction, both had the presence of PCBs (Aroclor 1260). The reactor, under methanogenic condition, was fed with synthetic substrate Angelidaki, ethanol and formate, used as carbon source, and aroclor 1260 (0.2 \'mü\'g /L). To simulate the condition of iron reducing, the same synthetic substrate was supplemented with ferric EDTA (1.86 g/L). The production of methane in the presence of aroclor 1260, was 3.8 x \'10 POT.-4\' mmol \'CH IND.4\'/g STV. The presence of iron reducing bacteria, after 60 days, was confirmed indirectly by the average rate of iron ferric reduction (90%). Filogenetics analysis (PCR/DGGE) compared the samples of this batch reactor - methanogenic and reduction of iron ferric -, with the samples of RAHLF - the biofilm in the reactor wall and the support material. The two condictions in batch reactors showed similarity of 79% and 92% respectively for the Bacteria and Archaea domain. Therefore, both samples of RAHLF showed 80% (Bacteria) and 96% (Archaea) of similarity. In other words, more similarity were presented due configuration of the reactor as well as the type of PCB added. As a result, the existence of PCBs degrading bacteria and iron-reducing bacteria in anaerobic biofilm, provided informations about the microbial consortium and its diversity in the presence of PCB.

Bifenilas policloradas (PCBs) são compostos de difícil degradação presentes na composição de ascarel, muito utilizado como fluidos dielétricos e isolantes. Neste contexto, a presente pesquisa teve como objetivo avaliar a diversidade de microrganismos em biofilmes de reatores anaeróbios na presença de PCB empregando Métodos de Microbiologia de Anaeróbios Estritos e de Biologia Molecular. Em reator anaeróbio horizontal de leito fixo (RAHLF), alimentado com etanol, formiato, Triton X-100 (0,1%) e ascarel (1 mL/L), operado com tempo de detenção hidráulica (TDH) de 24 horas, foi retirado a comunidade microbiana do biofilme da espuma de poliuretano. Os grupos microbianos encontrados por meio da clonagem e sequenciamento do gene RNAr 16S para o domínio Bacteria foram relacionados aos filos Thermogae, Proteobacteria (Brachymonas petroleovorans, 100% de similaridade e Methylobacillus, 98% de similaridade), Firmicutes (Clostridium, 97% de similaridade, Syntrophomonas, 100% de similaridade e Sporomusa com 100% de similaridade), Synegistetes (Synergistes, 98% de similaridade), Spirochaetes (Leptonema illini, 98% de similaridade), Aminanaerobia, Deferribacteres, Chlorobi, Chloroflexi e Armatimonadetes. Além disso, como nesse biofilme foram identificadas bactérias redutoras de ferro, procedeu-se a sua quantificação por meio da técnica de tubos múltiplos (NMP, Número Mais Provável) obtendo 5,26 x \'10 POT.12\' NMP/g STV de bactérias redutoras de ferro. Ensaio em batelada foi realizado separadamente sob duas condições: (1) metanogênica e (2) ferro redutora. Em ambas as condições foram adicionadas aroclor 1260 (PCB). Os reatores, sob condição metanogênica, foram alimentados com meio de cultivo Angelidaki e substratos orgânicos (formiato e etanol), além de aroclor 1260 (0,2 \'mü\'g/L). Para simular a condição redutora de ferro foi acrescido ao meio de cultura Angelidaki, EDTA férrico (1,86 g/L). A produção de metano, na presença de aroclor 1260 foi de 3,8 x \'10 POT.-4\' mmol \'CH IND.4\'/g STV. A presença de bactérias ferro redutoras foi confirmada indiretamente pela taxa média de redução férrica (90%) nos reatores em batelada, após 60 dias de operação. Por meio de PCR/DGGE, elaborou-se um dendograma das amostras deste ensaio em batelada (metanogênico e redutor de ferro) comparativamente com as do reator RAHLF (biofilme presente na parede do reator e no material suporte). Os reatores em batelada apresentaram similaridade entre si de 79% e 92% para os domínios Bacteria e Archaea, respectivamente. As amostras do reator RAHLF foram 80% (Bacteria) e 96% (Archaea) similares. A existência de bactérias degradadoras de PCB, bem como, bactérias redutoras de ferro no biofilme anaeróbio contribuiu com informações sobre o consórcio microbiano e sua diversidade. / Polychlorinated biphenyls (PCBs) are compounds of difficult degradation, a component of askarel, which were used widely as coolants and lubricants. Hence, this study evaluated the diversity of microorganisms in the presence of PCBs in anaerobic reactors. For such, methods as Strict Anaerobic Microbiology and Molecular Biology were employed. The microbial community of the biofilm, developed in a fixed horizontal bed anaerobic reactor (RAHLF), was studied using the technique of cloning and sequencing of RNAr 16S gene for the Bacteria domain. The reactor had immobilized cells in polyurethane foam with ethanol and formate as a carbon source, Triton X-100 (0.1%) and polychlorinated biphenyls (1 mL/L), and operated with 24 hours HRT. The microbial groups found in this biofilm were related to phyla Thermogae, Proteobacteria (Brachymonas petroleovorans, 100% similarity and Methylobacillus, 98% similarity), Firmicutes (Clostridium, 97% similarity Syntrophomonas, and 100% similarity with Sporomusa 100% similarity), Synegistetes (Synergistes, 98% similarity), Spirochaetes (Leptonema Illini, 98% similarity), Aminanaerobia, Deferribacteres, Chlorobi, Chloroflexi and Armatimonadetes. Furthermore, as bacteria that reduce iron were found, we proceeded the quantification by the multiple tube method (MPN) for this group, obtaining 5.26 x \'10 POT.12\' MPN/g STV of iron-reducing bacteria. The batch reactors evaluated the growth of microorganisms in two condictions: (1) methanogenic e (2) iron reduction, both had the presence of PCBs (Aroclor 1260). The reactor, under methanogenic condition, was fed with synthetic substrate Angelidaki, ethanol and formate, used as carbon source, and aroclor 1260 (0.2 \'mü\'g /L). To simulate the condition of iron reducing, the same synthetic substrate was supplemented with ferric EDTA (1.86 g/L). The production of methane in the presence of aroclor 1260, was 3.8 x \'10 POT.-4\' mmol \'CH IND.4\'/g STV. The presence of iron reducing bacteria, after 60 days, was confirmed indirectly by the average rate of iron ferric reduction (90%). Filogenetics analysis (PCR/DGGE) compared the samples of this batch reactor - methanogenic and reduction of iron ferric -, with the samples of RAHLF - the biofilm in the reactor wall and the support material. The two condictions in batch reactors showed similarity of 79% and 92% respectively for the Bacteria and Archaea domain. Therefore, both samples of RAHLF showed 80% (Bacteria) and 96% (Archaea) of similarity. In other words, more similarity were presented due configuration of the reactor as well as the type of PCB added. As a result, the existence of PCBs degrading bacteria and iron-reducing bacteria in anaerobic biofilm, provided informations about the microbial consortium and its diversity in the presence of PCB.

Made available in DSpace on 2014-12-17T15:18:11Z (GMT). No. of bitstreams: 1 AmandaLSS.pdf: 2422936 bytes, checksum: d5a0f2bb2f42d87bacd7a1d2c3f5c6bc (MD5) Previous issue date: 2009-03-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Bacteria trom Shewanella and Geobacter ganera are the most studied iron-reducing microorganisms particularly due to their electron transport systems and contribution to some industrial and environmental problems, including steel corrosion, bioenergy and bioremediation of petroleum-impacted sites. The present study was focused in two ways: the first is an in silico comparative ecogenomic study of Shewanella spp. with sequenced genomes, and the second is an experimental metagenomic work to detect iron-reducing Shewanella through PCR-DGGE of a metabolic gene. The in silico study resulted in positive correIation between copy number of 16S rDNA and genome size in Shewanella spp., with clusters of rrn near lhe origin of replication. This way, the genus is inferred as opportunist. There are no compact genomes and their sequences length varied, ranging from 4306142 nt in S. amazonensis SB2B to 5935403 nt in S. woodyi ATCC 51908, without correIation to temperature range characteristic of each specie. Intragenomic 16S rDNA sequences possess little divergence, but reasonable to resuIt in different phyIogenetic trees, depending on the sequence that is chosen to compare. For moIecuIar detection of iron-reducing Shewanella, it is proposed the mtrB gene as new biomarker. because it codes to a fundamental protein at Fe (III)-reduction. The specific primers were designed and evaluated in silico and resulted in a fragment of 360 pb. In the second study, these primers were tested in a genomic sample from S. oneidensis MR-1, amplifying the expected region. After this successfuI resuIt, the primer set was used as a tool to assess the iron-reducing communities of ShewaneIla genus under an environmental stress, i.e. crude oil contamination in mangrove sediment in Rio Grande do Norte State (Brazil). The primers presented high specificity and the reactions performed resulted in one single band of ampIification in the metagenomic samples. The fingerprinting obtained at DGGE reveaIed temporal variation of Shewanella spp. in analyzed samples. The resuIts presented show the detection of a biotechnological important group of microorganisms, the iron-reducing Shewanella spp. using a metabolic gane as target. It is concluded there are eight or more 16S rDNA sequences in Shewanella genus, with little divergence among them that affects the phylogeny; the pair of primers designed to ampIify mtrB sequences is a viable alternative to detect iron-reducing ShewanelIa in metagenomic approaches; such bacteria are present in the mangrove sediment anaIyzed, with temporal variations in the samples. This is the first experimental study that screened the iron-reducing Shewanella genus in a metagenomic experiment of mangrove sediments subjected to oil contamination through a key metabolic gene / Bact?rias dos g?neros Shewanella e Geobacter s?o os microrganismos redutores de ferro mais estudados. Esse interesse ocorre particularmente devido aos seus sistemas de transporte de el?trons e contribui??o em alguns problemas industriais e ambientais, tais como corros?o de oIeodutos, bioenergia e biorremedia??o de locais contaminados com petr?leo. O presente estudo foi tocado em duas partes: a primeira ? um estudo ecogen?mico comparativo de ShewanelIa spp. com genomas seq?enciados, e a segunda ? um trabalho metagen?mico experimental para detectar Shewanella redutoras de ferro atrav?s de PCR-DGGE de um gene metab?lico. O estudo in silico resultou em correla??o positiva entre o n?mero de c?pias 16S rDNA e tamanho do genoma em ShewaneIla spp., com agrupamentos de rrn. pr?ximo ? origem de replica??o. Desta maneira, o g?nero ? inferido como oportunista. N?o existem genomas compactos e o tamanho de suas sequ?ncias variam de 4306142 nt em S. amazonensis SB2B at? 5935403 nt em S. woodyi ATCC 51908, sem correla??o com a faixa de temperatura caracter?stica de cada esp?cie. Sequ?ncias intragen?micas de 16S rDNA possuem pouca diverg?ncia. mas razo?vel para resultar em diferentes ?rvores filogen?ticas. dependendo da sequ?ncia que ? escolhida para compara??o. Para a detec??o moIecuIar de ShewanelIa redutoras de ferro, ? proposto o gene mtrB como um novo biomarcador, por ser codante de uma prote?na fundamental na redu?ao de Fe (III). Os primers espec?ficos foram desenhados e avaliados in silico e resultou em um fragmento de 360 pb. No segundo estudo, esses primers foram testados em . amostra gen?mica de S. oneidensis MR-1, amplificando a regi?o esperada. Depois desse resultado favor?vel, o par de primers foi utilizado como ferramenta para acessar as comunidades redutoras de ferro do g?nero ShewanelIa sob um stress ambiental - contamina??o com ?leo cru em sedimento de mangue, no Estado do Grande do Norte (Brasil). Os primers apresentaram alta especificidade e as rea??es resultaram em banda ?nica de amplifica??o das amostras metagen?micas. O perfil obtido no DGGE revelou varia??o temporal de ShewanelIa spp. nas amostras analisadas. Os resultados apresentados mostram a defec??o de um grupo de microrganismos biotecnologicamente importante. ShewaneIla spp. redutoras de ferro, usando um gene metab?lico como alvo. Concluiu-se que existem oito ou mais sequ?ncias 16S rDNA no g?nero ShewaneIla, com pouca diverg?ncia entre elas que afetam a filogenia; o par de primers desenhados para amplificar sequ?ncias mtrB ? uma alternativa vi?vel para detectar Shewanella redutoras de ferro em abordagens metagen?micas; tais bact?rias est?o presentes no sedimento de mangue analisado, com varia??es temporais nas amostras. Este ? o primeiro estudo experimental que examina Shewanella redutoras de ferro em um experimento metagen?mico de sedimento de mangue submetido a contamina??o por ?leo atrav?s de um gene metab?lico

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 ferro é um importante elemento em reações catalíticas no meio ambiente, pois possui a capacidade de ser reduzido ou oxidado. Duas espécies de ferro solúvel podem estar presentes em amostras ambientais, o Fe (II) e o Fe (III). Métodos analíticos capazes de diferenciar e quantificar estas duas espécies de ferro são muito importantes para a compreensão dos processos metabólicos dos diversos microrganismos, e também para entender a atuação destes microrganismos na remobilização do coagulante utilizado em estações de tratamento de esgotos (ETEs), nas quais é possível utilizar como coagulante o FeCl3. Porém não há trabalhos publicados que recuperam o ferro coagulado utilizando bactérias redutoras de ferro. Os objetivos deste trabalho são: 1) avaliar o método colorimétrico de fenantrolina para quantificação de Fe (II) e os principais interferentes nessas análises; e 2) avaliar o potencial do Fe (II) gerado via metabolismo das bactérias redutoras de ferro como coagulante de matéria orgânica e inorgânica de águas residuárias. Os resultados para o método colorimétrico de fenantrolina são confiáveis somente para leituras de amostras que contenham Fe (II), mas não diferencia e quantifica corretamente espécies de Fe (III) em todos os valores de pH. A separação das diferentes espécies de ferro foi feita utilizando membrana de acetato de celulose com porosidade de 0,2 m e ajustando o valor do pH para valores entre 4 e 5. Para obtenção das concentrações de Fe (II) e Fe (III), é necessário realizar a leitura em amostras filtradas e não filtradas, pois o Fe (II) passa pela membrana e o Fe (III) fica retido. Desta forma, é possível realizar a distinção das espécies de ferro, e em seguida realizar a quantificação com testes colorimétricos, seja em campo ou em laboratório. A diferenciação das espécies de ferro se mostrou importante para quantificar corretamente o Fe (III) e o Fe (II) durante o tratamento de águas residuárias utilizando Fe (III) como coagulante na forma de FeCl3. Na comparação com a recuperação ácida, a biológica se mostrou mais eficiente por não apresentar metais pesados remobilizados na fração líquida, recuperando 58% do ferro quando adicionado o glicerol como fonte de carbono. Durante a remobilização do ferro houve a produção do metano, gás de interesse econômico. A escolha do coagulante e da concentração foi determinada pela remoção da turbidez, sendo o melhor coagulante para água residuária do CRUSP o FeCl3 na concentração de 60 mg/ L de Fe, pois removeu 99% da turbidez, 98% do fosfato, 85% dos carboidratos e 100% de proteínas presentes na água residuária. Aplicando-se o coagulante remobilizado (400 mg/L), foi possível remover 85% da turbidez. O ferro recuperado servirá novamente como coagulante, favorecendo a redução dos custos com o tratamento de água residuária. / Iron is an important element in catalytical action in the environment as it has an ability to be filtered or oxidized. Soluble iron species may be present in environmental samples, Fe (II) and Fe (III). Analytical methods capable of differentiating and quantifying these two iron species are very important for the remobilization of coagulation in sewage treatment plants (ETEs), in which FeCl3 can be used as a coagulant. It is not a job that recovers coagulated iron with iron reducing units. The objectives of this work are: 1) to evaluate the colorimetric method of phenanthroline for quantification of Fe (II) and the main interferents in these analyzes; and 2) to evaluate the potential of Fe (II) through the metabolism of iron-reducing bacteria as a coagulant of organic and inorganic wastewater. The results for the colorimetric method of phenanthroline are only for the readings of samples containing Fe (II), but do not differentiate and quantify the Fe (III) species at all pH values. The separation of the fish fiber species was left to the cellulose acetate test with the porosity of 0.2 m and adjusting the pH value to values between 4 and 5. For the concentration of Fe (II) and Fe (III), it is necessary to read in filtered and unfiltered samples, as Fe (II) passes through the membrane and Fe (III) is retained. In this way, it is possible to perform an analysis of the iron species, and then perform quantification with colorimetric tests, either in the field or in the laboratory. Differentiation of iron species has become important in correctly quantifying Fe (III) and Fe (II) during wastewater treatment using Fe (III) as a coagulant in the form of FeCl3. In comparison with an acid replica, a biological recovery is done through large amounts of remobilized in the liquid fraction, recovering 58% of the iron when the glycerol as carbon source. During the remobilization of the iron there was a production of methane, gas of economic interest. The choice of the coagulant and the capacity was determined by the removal of the turbidity, being the best coagulant for the residual water of the CRUSP the FeCl3 in the concentration of 60 mg/L of Fe, since it removed 99% of the turbidity, 98% of the phosphate, 85% of carbohydrates and 100% of proteins present in the wastewater. Applying the remobilized coagulant (400 mg/L), it was 85% turbidity remover. The recovered iron will again serve as a coagulant, favoring the reduction of costs with the treatment of wastewater.

Land disposal of solid waste is a vital component of any solid waste management system. Design, operation and closure of municipal solid waste (MSW) landfills are required by regulations to control leachate and gases generated during the life, closure,and post-closure of the facility. Clogging of leachate drainage and removal systems in landfills is a common phenomenon and has been acknowledged in several landfills throughout the United States and abroad. This project was conducted in two phases. Phase I was completed in February of 2005 and Phase II was completed in August of 2006. Leachate characteristics data obtained in Phase I was processed and analyzed, along with supplementary data obtained in Phase II on liquid and solid phase testing. Leachate samples from the landfill and lysimeters indicated the presence of iron and sulfate-reducing bacteria. These bacteria are known to facilitate biologically induced precipitate formation.The mechanism by which biologically ind uced precipitate may form begins with oxidizing acetate by iron and sulfate-reducing bacteria, reducing sulfate to sulfide and ferric iron to ferrous, and then forming calcium carbonate, iron sulfate, and possibly dolomite and other minerals.The results show that the clogging mechanism is driven by two major processes: transformation of volatile acids to substrates by iron and sulfate-reducing bacteria causing local pH and total carbonate to increase, which accelerate calcium carbonate precipitation, and thermodynamically favored reactions in supersaturated conditions based on saturation indices of calcium, sulfide, iron, and other species with respect to minerals. For each 1 mg of consumed volatile acids there were 1.7 mg of calcium, 0.28 mg of sulfate, and 0.03 mg of iron removed. Field and lysimeter precipitate samples were analyzed (using X-Ray Diffraction, Scanning Electron microscopy, and Electron Dispersive Spectroscopy) and correlated with geochemical modeling of leachate const ituents. Precipitate analyses showed the presence of calcium carbonate, brushite (calcium phosphate),and dolomite, where as geochemical modeling showed that calcium carbonate, hydroxyapatite (complex of calcium phosphate), dolomite, pyrite, and siderite may be formed from field and lysimeter leachate constituents. The results also showed that submerged and stagnant conditions in the leachate collction systems accelerate the precipitation process.

L’objectif principal de cette étude est d’évaluer le rôle de l’activité de bactéries hydrogénotrophes et ferri-réductrices sur le processus de corrosion anoxique en utilisant des indicateurs géochimiques. Il est considéré que le couple redox H2/Fe(III) est un moteur important pour les activités bactériennes qui peuvent ainsi affecter les vitesses de corrosion par la déstabilisation des couches de passivation (i.e. magnétite, Fe3O4). Les résultats indiquent que la magnétite de synthèse est déstabilisée en présence de bactéries hydrogénotrophes et ferri-réductrices due à la réduction du Fe(III) structural couplée à l’oxydation de l’H2. La quantité de Fe(III) bioréduit est augmentée en présence de concentrations croissantes en H2 dans le système: 4% H2 < 10% H2 < 60% H2. De plus, les résultats indiquent que la réaction de corrosion est différente selon la composition de la solution et la surface de contact de l’échantillon métallique (poudre de fer ou coupon en acier au carbone). Les produits de corrosion solides sont différents pour chaque échantillon étudié: vivianite, sidérite et chukanovite sont les principales phases minérales identifiées dans les expériences avec de la poudre de fer, tandis que vivianite et magnétite sont identifiées en présence de coupons en acier au carbone. Les résultats montrent que la vitesse de corrosion est quasiment deux fois plus importante en présence de bactéries après 5 mois de réaction. Cette étude apporte une nouvelle approche sur la compréhension des phénomènes de biocorrosion, l’identification des mécanismes physico-chimiques et la détermination des paramètres contrôlant la vitesse de corrosion. / The main objective of this study is to evaluate the role of hydrogenotrophic and IRB activities on anoxic corrosion process by using geochemical indicators. It is assumed that the redox couple H2/Fe(III) is an important driver for bacterial activities potentially affecting the corrosion rate by destabilization of passive layers (i.e. magnetite, Fe3O4). Our results indicate that synthetized Fe3O4 is destabilized in the presence of hydrogenotrophic IRB due to structural Fe(III) reduction coupled to H2 oxidation. The extent of Fe(III) bioreduction is notably enhanced with the increase in the H2 concentration in the system: 4% H2 < 10% H2 < 60% H2. Moreover, the results indicate that corrosion extent changes according to the solution composition and the surface of metallic sample (iron powder and carbon steel coupon). The solid corrosion products are different for each sample: vivianite, siderite and chukanovite are the main mineral phases identified in the experiments with iron powder, while vivianite and magnetite are identified with carbon steel coupons. Our results demonstrate that corrosion rate is increased almost two-fold in the presence of bacteria after 5 months of reaction. This study gives new insights regarding the understanding of biocorrosion phenomena, identification of physicochemical mechanisms, and determination of key parameters controlling the corrosion rate.

En France, il est envisagé de stocker en formation géologique profonde les déchets radioactifs vitrifiés à haute activité et vie longue dans un conteneur en acier inoxydable chemisé par un surconteneur en acier non allié. Les principaux produits de corrosion attendus à la surface de ce dernier, i.e. la sidérite (FeIICO3) et la magnétite (FeIIFeIII2O4), jouent un rôle protecteur contre la corrosion en tant que couche passivante. Ce travail de thèse visait à étudier l’influence des groupes métaboliques bactériens réducteurs du fer ferrique (IRB) et des nitrates (NRB) sur les transformations de ces produits de corrosion en anoxie. Des souches modèles de NRB (Klebsiella mobilis) et IRB (Shewanella putrefaciens) ont, dans un premier temps, été incubées en présence de suspension de sidérite ou de magnétite, afin d’exacerber les processus de transformation du fer du fait d’une surface spécifique élevée, puis dans un second temps, en présence de films électrogénérés de ces produits pour se rapprocher des conditions d’un acier non allié corrodé en anoxie. Ces souches bactériennes sont capables de transformer la sidérite et la magnétite par des processus microbiens directs ou indirects et de conduire à la formation de rouille verte carbonatée (FeII4FeIII2(OH)12CO3). Ce composé occupe une place centrale dans le cycle biogéochimique du fer en anoxie en tant que transitoire commun à plusieurs réactions microbiennes mobilisant le fer sous deux états d’oxydation différents FeII et FeIII. L’originalité de ce travail de thèse est donc de montrer que des métabolismes bactériens inaccoutumés tels que les NRB ou les IRB sont susceptibles de jouer un rôle dans les processus de biocorrosion / Radioactive waste is one of the major problems facing the nuclear industry. To circumvent this issue France plans to store vitrified high-level nuclear waste in a stainless steel container, placed into a non-alloy steel overpack, at a depth of 500m in an argillaceous formation. The main iron corrosion products formed at the surface of the non-alloy steel are siderite (FeIICO3) and magnetite (FeIIFeIII2O4). These compounds are formed in the anoxic conditions present in the nuclear waste repository and play a protective role against corrosion as a passive layer. This work aims to investigate the activity of nitrate-reducing bacteria (NRB, Klebsiella mobilis) and iron-reducing bacteria (IRB, Shewanella putrefaciens) during the transformation of siderite and magnetite, especially those involved in anoxic iron biogeochemical cycle. Klebsiella mobilis and Shewanella putrefaciens were first incubated with siderite or magnetite suspensions (high surface specific area) in order to exacerbate the microbial iron transformation, subsequently incubated with a magnetite/siderite film synthesized by anodic polarization at applied current density. The transformation of siderite and magnetite by direct or indirect microbial processes led to the formation of carbonated green rust (FeII4FeIII2(OH)12CO3). As a transient phase shared by several bacterial reactions involving FeII and FeIII, this compound is the cornerstone of the anoxic iron biogeochemical cycle. The novelty of this thesis is the consideration of bacterial metabolisms of NRB and IRB often overlooked in biocorrosion processes

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.