As águas residuárias provenientes da indústria do charque são conhecidas por apresentarem elevado teor de cloreto de sódio, aliado a grandes concentrações de matéria orgânica proveniente do sangue liberado ao longo do processo industrial. Esse tipo de água residuária apresenta potencial para degradação biológica, contudo, o cloreto de sódio, em concentração elevada, pode inibir a atividade dos microrganismos e, em alguns casos, levar sistemas biológicos à falência. No presente trabalho, foi avaliada a viabilidade de degradação anaeróbia de efluente sintético de Charqueada contendo elevado teor de cloreto de sódio, em reator anaeróbio tipo UASB (Upflow Anaerobic Sludge Blanket), em escala de laboratório. Foram utilizados 4 reatores, alimentados com água residuária sintética com características similares à água residuária de Charqueada. O reator 1 foi utilizado como controle, o reator 2 recebeu NaCl e os demais (3 e 4) foram operados na presença de NaCl acrescidos de: betaína e potássio com cálcio, respectivamente. Os compostos citados são conhecidos como antagonizantes, por possuirem capacidade de minimizar o efeito inibitório do sódio sobre o processo de digestão anaeróbia. Os reatores foram inoculados com lodo de reator UASB e submetidos à concentração de 5000 mg/L de matéria orgânica, como DQO. A carga orgânica aplicada foi de 5 Kg/m3.d e os reatores não suportaram tal carga. Reiniciou-se a operação com aumento progressivo da DQO de 500 a 2000 mg/L resultando em cargas orgânicas de 0,5 a 2,0 Kg/m3.d, respectivamente. Após estabilização dos reatores, iniciou-se a fase de introdução de cloreto de sódio (1.500 a 13.500 mg/L) e antagonizantes com aumento progressivo a cada fase. Na presença ou ausência de antagonizantes, os reatores 2, 3 e 4 não tiveram o desempenho alterado até a concentração de NaCl de 6000 mg/L. Na presença de 9000 mg/L de NaCl, a betaína se mostrou pouco efetiva como soluto compatível no reator 3 e os antagonizantes do reator 4, potássio e cálcio, apresentaram efeitos estimulatórios. As morfologias encontradas ao longo do experimento foram cocos, víbrios, bacilos, sarcinas, além de morfologias semelhantes a Methanosarcina sp. e Methanosaeta sp. O aumento da concentração de cloreto de sódio provocou a redição da população de Arqueas. / Wastewaters from the charque industry (salted and dried meat) are known for their high levels of sodium chloride, allied to high organic concentrations originary from blood which is disposed along the industrial process. That kind of wastewater presents good potential for biological degradation (relationship DBO/DQO aproximately 0,5). However, the high concentration of sodium chloride can inhibit microorganisms activity and in some cases, take the biological systems to failure. The present work studied the viability of anaerobic degradation of synthetic charqueada wastewater, containing high salinity, in reactor UASB, at laboratory scale. Four reactors were used. Reactor 1 was used as control, reactor 2 received just NaCl and the two others (3 and 4) were operated in the presence of NaCl in increased with betaine and potassium with calcium, respectively. The mentioned compounds are known to be antagonists, for they possess capacity to minimize the inhibitory effect of sodium in the anaerobic digestion process. The reactors were inoculated with UASB sludge and the operation began with concentration from 5000 mg/L of organic matter, expressed as DQO. The organic load applied from 5,0 Kg/m3.d and the reactors did not support such load. The operation was restarted with progressive increase of 500 to 2000 mg/L of DQO, resulting in organic load from 0,5 to 2,0 Kg/m3.d, respectively. After reactors stabilization, the phase of introduction of NaCl (1500 to 13500 mg/L) and antagonists was started, with the progressive increase of sodium chloride and antagonists. In presence or absence of antagonists, reactors 2, 3 and 4 did not present performance alterations until the concentration of NaCl of 6000 mg/L was reached. When NaCl concentrations were from 9000 to 13500 mg/L, betaine was shown to have small effects in the reactor 3 and the antagonists of reactor 4, potassium and calcium, presented stimulant effects. The morphologies found along the experiment were: coconuts, víbrios, bacilli, sarcinas, besides morphologies similar to the Methanosarcina sp. and Methanosaeta sp. When concentrations of sodium chloride were increased the population of Arqueas were reduced.

Os efluentes com elevadas concentrações de lipídios, apesar de demonstrarem elevado potencial para produção de metano podem causar inibição da atividade do consórcio anaeróbio, impossibilitando a produção de biogás. O presente trabalho teve como objetivo o estudo de um reator anaeróbio compartimentado (ABR) com cinco compartimentos tratando efluente simulado de laticínio. Para tanto, a biomassa, foi adaptada por 51 dias e em seguida submetida a tempos de detenção de hidráulica (TDH) de 72h, 24h e 12h, tendo o monitoramento se estendido por 340 dias. Enquanto operando com TDH de 24, o sistema foi submetido a choques hidráulicos e de carga orgânica através da diminuição do TDH para 12 h. O reator absorveu bem a carga, e em 36 horas recuperou os valores prévios de eficiência. Quando operou permanentemente com 12 horas, todavia, problemas de flotação de biomassa e obstruções foram constantes. As eficiências de remoção de matéria orgânica alcançadas para cada condição aplicada foram de 92 ± 3,0; 91 ± 1,8 e 90 ± 2,4% para os TDH de 72h, 24h e 12h, respectivamente. Esses valores foram estatisticamente semelhantes (Anova e L-Fisher). A percentagem de metano no biogás aumentou com a redução de TDH, tendo sido 41 ± 23, 53 ± 27 e 62 ± 12% quando os TDH foram 72, 24 e 12 horas, respectivamente. A produção média de alcalinidade foi de 320±25 mg.CaCO3.L-1, sendo esta observada desde o início da operação. A relação média entre alcalinidade intermediária e parcial foi de 0.1±0.06 em amostras coletadas na saída do sistema. Embora apresentando problemas de obstrução com o menor TDH, o ABR foi eficiente, robusto e confiável ao tratar efluente de laticínios, tendo produzido um efluente líquido de alta qualidade e biogás rico em metano. / Effluents with high concentrations of lipids, although demonstrating high potential for methane production represents potential inhibition of the anaerobic consortium activity, depleting the production of biogas. This project was carried out monitoring an anaerobic hybrid baffled reactor with five compartments (ABR) treating simulated dairy wastewater. The biomass, was adapted for 51 days and then subjected to hydraulic retention times (HDT) of 72h, 24h and 12h, resulting in a monitoring period of 340 days. While operating with 24h of HRT, the system was subjected to three organic and hydraulic shock loads, when its HDT was decreased to 12 h. The reactor absorbed the shock within 36 hours, achieving similar efficiencies to the previous condition. Nevertheless, although presenting high organic matter efficiencies, when permanently operating with HDT of 12 h, clogging problems due to biomass flotation were constant. The organic matter removal efficiencies for each operational condition were 92 ± 3%, 91 ± 1.8%, 90 ± 2,4%. Those values were statistically similar. The methane percentage in the biogas increased with the HDT reduction, being 41 ± 23, 53 ± 27, and 62 ± 12% when the HDT were 72, 24 and 12 hours. Alkalinity production was observed since the beginning of operation. The mean relation between intermediate and partial alkalinity was 0.1±0.006 in samples collected in the system output. Although presenting clogging problems with the smallest HDT, the ABR was efficient, robust and reliable when treating dairy effluents, producing a high quality liquid effluent and a methane rich biogas.

Foi estudada a degradação do pentaclorofenol (PCP) em reator aneróbio horizontal de leito fixo (RAHLF) de volume de 2000 mL. O reator foi inoculado com microrganismos oriundos de reatores aneróbios não previamente adaptados a PCP. Atividade microbiana foi monitorada através de técnicas clássicas na presença do organoclorado na faixa de 2,0 a 13 mg/L de PCP. O reator apresentou eficiência de 97% na remoção de DQO e completo desaparecimento do composto de PCP em todas as concentrações testadas. A fração orgânica foi consumida totalmente na primeira terça parte do reator de acordo com os valores determinados de ácidos voláteis, DQD e PCP. Não foi verificada inibição da atividade de culturas microbianas. Os exames microscópicos, fluorescência e varredura, permitiram verificar o predomínio de microrganismos pertencentes ao Domínio Archea. As técnicas moleculares PCR, ARDRA e hibridação slot-blot confirmaram o predomínio do Domínio Archaea e possibilitaram a verificação de alterações na diversidade das populações após adição de 2 mg PCP/L. Conclui-se que o reator sem prévia adaptação do inóculo foi eficiente para o tratamento do PCP, e os microrganismos relacionados às Archaea metanogênicas acetocláticas podem estar envolvidas na degradação deste composto. / The degradation of pentachlorophenol (PCP) was studied in a 2000 mL. Horizontal Anaerobic Immobilized System (HAIS). The reactor was inoculated with microorganisms obtained from an anaerobic reactor without previous adaptation to the PCP. The microbial activity was evaluated by using classic techniques in order to . monitor its behavior during the HAIS fed with a range of PCP between 2.0 to 13 mg/L. The reactor presented 97% of efficiency in the removal of COD and complete decrease of PCP in alI concentrations tested. The total consumption of organic fraction took place mainly in the first third part of the reactor according the values of volatile fatty acids, COD and PCP obtained. Microbial inhibition was not verified in during HAIS operation. Microscopic examinations allowed certifying the Archaea Domain predominance according the morphologies observed. The molecular techniques polimerase chain reaction (PCR), ARDRA and slot-blot hibridation confirmed the predominance of Archaea Domain and alIowed verifying some changes in the population\'s diversity under additions of 2mg PCP/L. The efficiency of PCP decreased in the anaerobic reactor was related to the presence of Archaea Domain, especially the acetoclastic methanogens, whose where probably involved with the organochlorine compound degradation.

Foi estudada a degradação do pentaclorofenol (PCP) em reator aneróbio horizontal de leito fixo (RAHLF) de volume de 2000 mL. O reator foi inoculado com microrganismos oriundos de reatores aneróbios não previamente adaptados a PCP. Atividade microbiana foi monitorada através de técnicas clássicas na presença do organoclorado na faixa de 2,0 a 13 mg/L de PCP. O reator apresentou eficiência de 97% na remoção de DQO e completo desaparecimento do composto de PCP em todas as concentrações testadas. A fração orgânica foi consumida totalmente na primeira terça parte do reator de acordo com os valores determinados de ácidos voláteis, DQD e PCP. Não foi verificada inibição da atividade de culturas microbianas. Os exames microscópicos, fluorescência e varredura, permitiram verificar o predomínio de microrganismos pertencentes ao Domínio Archea. As técnicas moleculares PCR, ARDRA e hibridação slot-blot confirmaram o predomínio do Domínio Archaea e possibilitaram a verificação de alterações na diversidade das populações após adição de 2 mg PCP/L. Conclui-se que o reator sem prévia adaptação do inóculo foi eficiente para o tratamento do PCP, e os microrganismos relacionados às Archaea metanogênicas acetocláticas podem estar envolvidas na degradação deste composto. / The degradation of pentachlorophenol (PCP) was studied in a 2000 mL. Horizontal Anaerobic Immobilized System (HAIS). The reactor was inoculated with microorganisms obtained from an anaerobic reactor without previous adaptation to the PCP. The microbial activity was evaluated by using classic techniques in order to . monitor its behavior during the HAIS fed with a range of PCP between 2.0 to 13 mg/L. The reactor presented 97% of efficiency in the removal of COD and complete decrease of PCP in alI concentrations tested. The total consumption of organic fraction took place mainly in the first third part of the reactor according the values of volatile fatty acids, COD and PCP obtained. Microbial inhibition was not verified in during HAIS operation. Microscopic examinations allowed certifying the Archaea Domain predominance according the morphologies observed. The molecular techniques polimerase chain reaction (PCR), ARDRA and slot-blot hibridation confirmed the predominance of Archaea Domain and alIowed verifying some changes in the population\'s diversity under additions of 2mg PCP/L. The efficiency of PCP decreased in the anaerobic reactor was related to the presence of Archaea Domain, especially the acetoclastic methanogens, whose where probably involved with the organochlorine compound degradation.

I formally joined Prof. M. R. N. Murthy’s laboratory at the Molecular Biophysics Unit, Indian institute of Science, on 1st August 2001. During that time, the interest in the laboratory was mainly focused on structural studies on a number of capsid mutants of two plant viruses, sesbania mosaic virus and physalis mottle virus, to gain an insight into the virus structure and its assembly. Besides these two projects, there were a few other collaborative projects running in the lab at that time such as NIa protease from pepper vein banding virus and diaminopropionate ammonia lyase from Escherichia coli with Prof. H. S. Savithri, triosephosphate isomerase from Plasmodium falciparum with Prof. P. Balaram and Prof. H. Balaram and a DNA binding protein (TP2) with Prof. M. R. S. Rao. During my first semester, along with my course work, I was assigned to make an attempt to purify and crystallize recombinant NIa protease and TP2 protein. I started with NIa protease which could be purified using one step Ni-NTA affinity column chromatography. Although the expression and protein yield were reasonably good, protein precipitated with in a couple of hours after purification. Attempts were made to prevent the precipitation of the purified enzyme and towards this end we were successful to some extent. However, during crystallization trials most of the crystallization drops precipitated completely even at low protein oncentration. TP2 protein was purified using three-step chromatographic techniques by one of the project assistant in Prof. M. R. S. Rao’s laboratory. Because of low expression level and three step purification protocol, protein yield was not good enough for complete crystallization screening. Hits obtained from our initial screening could not be confirmed because of low protein yield as well as batch to batch variation. My attempts to crystallize these two proteins remained unsuccessful but in due course I had learnt a great deal about the tips and tricks of expression, purification and mainly crystallization. To overcome the problems faced with these two proteins, we decided to make some changes in the gene construct and try different expression systems. By this time (beginning of 2002), I had finished my first semester and a major part of the course work, so we decided to start a new project focusing on some of the unknown enzymes from a metabolic pathway. Dr. Parthasarathy, who had finished his Ph. D. from the lab, helped me in literature work and in finding targets for structural studies. Finally, we decided to target enzymes involved in the propionate etabolism. The pathways for propionate metabolism in Escherichia coli as well as Salmonella typhimurium were just established and there were no structural information available for most of the enzymes involved in these pathways. Since, propionate metabolic pathways were well described in the case of Salmonella typhimurium, we decided to use this as the model organism. We first started with the enzymes present in the propionate catabolic pathway “2-methylcitrate pathway”, which converts propionate into pyruvate and succinate. 2-methylcitrate pathway resembles the well-studied glyoxylate and TCA cycle. Most of the enzymes involved in 2-methylcitrate pathway were not characterized biochemically as well as structurally. First, we cloned all the four enzymes PrpB, PrpC, PrpD and PrpE present in the prpBCDE operon along with PrpR, a transcription factor, with the help of Dr. P.S. Satheshkumar from Prof. H. S. Savithri’s laboratory. Since these five proteins were cloned with either N- or C-terminal hexa-histidine tag, they could be purified easily using one-step Ni-NTA affinity column chromatography. PrpB, PrpC and PrpD had good expression levels but with PrpE and PrpR, more than 50% of the expressed protein went into insoluble fraction, probably due to the presence of membrane spanning domains in these two enzymes. Around this time, crystallization report for the PrpD from Salmonella was published by Ivan Rayment’s group, so after that we focused only on the remaining four proteins leaving out PrpD. Our initial attempts to crystallize these proteins became successful in case of PrpB, 2-methylisocitrate lyase. We collected a complete diffraction data to a resolution of 2.5 Å which was later on extended to a resolution of 2.1 Å using another crystal. Repeated crystallization trials with PrpC also gave small protein crystals but they were not easy to reproduce and size and diffraction quality always remained a problem. Using one good crystal obtained for PrpC, data to a resolution of 3.5 Å could be collected. Unfortunately, during data collection due to failure of the cryo-system, a complete dataset could not be collected. Further attempts to crystallize this protein made by Nandashree, one of my colleagues in the lab at that time, was also without much success. Attempts to purify and crystallize PrpE and PrpR were made by me as well as one of my colleagues, Anupama. In this case, besides crystallization, low expression and precipitation of the protein after purification were major problems. Our attempt to phase the PrpB data using the closest search model (phosphoenolpyruvate mutase) by molecular replacement technique was unsuccessful,probably because of low sequence identity between them (24%). Further attempts were made to obtain heavy atom derivatives of PrpB crystal. We could obtain a mercury derivative using PCMBS. However, an electron density map based on this single derivative was not nterpretable. Around this time, the structure of 2-methylisocitrate lyase (PrpB) from E. coli was published by Grimm et. al. The structure of Salmonella PrpB could easily be determined using the E. coli PrpB enzyme as the starting model. We also solved the structure of PrpB in complex with pyruvate and Mg2+. Our attempts to crystallize PrpB with other ligands were not successful. Using the structures of PrpB and its complex with pyruvate and Mg2+, we carried out comparative studies with the well-studied structural and functional homologue, isocitrate lyase. These studies provided the plausible rationale for different substrate specificities of these two enzymes. Due to unavailability of PrpB substrate commercially and the extensive biochemical and mutational studies carried out by two different groups made us turn our attention to other enzymes in this metabolic pathway. Since our repeated attempts to obtain good diffraction quality crystals of PrpC, PrpE and PrpR continued to be unsuccessful, we decided to target other enzymes involved in propionate metabolism. We looked into the literature for the metabolic pathways by which propionate is synthesized in the Salmonella typhimurium and finally decided to target enzymes present in the metabolic pathway which converts L-threonine to propionate. Formation of propionate from L-threonine is the most direct route in many organisms. During February 2003, we initiated these studies with the last enzyme of this pathway, propionate kinase (TdcD), and within a couple of months we could obtain a well-diffracting crystal in complex with ADP and with a non-hydrolysable ATP analog, AMPPNP. TdcD structure was solved by molecular replacement using acetate kinase as a search model. Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin, the superfamily of phosphotransferases. Examination of the active site pocket in propionate kinase revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. One of the datasets of TdcD obtained in the presence of ATP showed extra continuous density beyond the γ-phosphate. Careful examination of this extra electron density finally allowed us to build diadenosine tetraphosphate (Ap4A) into the active site pocket, which fitted the density very well. Since the data was collected at a synchrotron source to a resolution of 1.98 Å, we could identify the ligand in the active site pocket solely on the basis of difference Fourier map. Later on, co-crystallization trials of TdcD with commercially available Ap4A confirmed its binding to the enzyme. These studies suggested the presence of a novel Ap4A synthetic activity in TdcD, which is further being examined by biochemical experiments using mass-spectrometry as well as thin-layer chromatography experiments. By the end of 2004, we shifted our focus to the first enzyme involved in the anaerobic degradation of L-threonine to propionate, a biodegradative threonine deaminase (TdcB). Sagar Chittori, who had joined the lab as an integrated Ph. D student, helped me in cloning this enzyme. My attempt to crystallize this protein became finally successful and datasets in three different crystal forms were collected. Dataset for TdcB in complex with CMP was collected during a synchrotron trip to SPring8, Japan by my colleague P. Gayathri and Prof. Murthy. TdcB structure was solved by molecular replacement using the N-terminal domain of biosynthetic threonine deaminase as a search model. Structure of TdcB in the native form and in complex with CMP helped us to understand several unanswered questions related to ligand mediated oligomerization and enzyme activation observed in this enzyme. The structural studies carried out on these three enzymes have provided structural as well as functional insights into the catalytic process and revealed many unique features of these metabolic enzymes. All these have been possible mainly due to proper guidance and encouragement from Prof. Murthy and Prof. Savithri. Prof. Murthy’s teaching as well as discussions during the course of investigation has helped me in a great deal to learn and understand crystallography. Collaboration with Prof. Savithri kept me close to biochemistry and molecular biology, the background with which I entered the world of structural biology. The freedom to choose the project and carry forward some of my own ideas has given me enough confidence to enjoy doing research in future.

Os efluentes com elevadas concentrações de lipídios, apesar de demonstrarem elevado potencial para produção de metano podem causar inibição da atividade do consórcio anaeróbio, impossibilitando a produção de biogás. O presente trabalho teve como objetivo o estudo de um reator anaeróbio compartimentado (ABR) com cinco compartimentos tratando efluente simulado de laticínio. Para tanto, a biomassa, foi adaptada por 51 dias e em seguida submetida a tempos de detenção de hidráulica (TDH) de 72h, 24h e 12h, tendo o monitoramento se estendido por 340 dias. Enquanto operando com TDH de 24, o sistema foi submetido a choques hidráulicos e de carga orgânica através da diminuição do TDH para 12 h. O reator absorveu bem a carga, e em 36 horas recuperou os valores prévios de eficiência. Quando operou permanentemente com 12 horas, todavia, problemas de flotação de biomassa e obstruções foram constantes. As eficiências de remoção de matéria orgânica alcançadas para cada condição aplicada foram de 92 ± 3,0; 91 ± 1,8 e 90 ± 2,4% para os TDH de 72h, 24h e 12h, respectivamente. Esses valores foram estatisticamente semelhantes (Anova e L-Fisher). A percentagem de metano no biogás aumentou com a redução de TDH, tendo sido 41 ± 23, 53 ± 27 e 62 ± 12% quando os TDH foram 72, 24 e 12 horas, respectivamente. A produção média de alcalinidade foi de 320±25 mg.CaCO3.L-1, sendo esta observada desde o início da operação. A relação média entre alcalinidade intermediária e parcial foi de 0.1±0.06 em amostras coletadas na saída do sistema. Embora apresentando problemas de obstrução com o menor TDH, o ABR foi eficiente, robusto e confiável ao tratar efluente de laticínios, tendo produzido um efluente líquido de alta qualidade e biogás rico em metano. / Effluents with high concentrations of lipids, although demonstrating high potential for methane production represents potential inhibition of the anaerobic consortium activity, depleting the production of biogas. This project was carried out monitoring an anaerobic hybrid baffled reactor with five compartments (ABR) treating simulated dairy wastewater. The biomass, was adapted for 51 days and then subjected to hydraulic retention times (HDT) of 72h, 24h and 12h, resulting in a monitoring period of 340 days. While operating with 24h of HRT, the system was subjected to three organic and hydraulic shock loads, when its HDT was decreased to 12 h. The reactor absorbed the shock within 36 hours, achieving similar efficiencies to the previous condition. Nevertheless, although presenting high organic matter efficiencies, when permanently operating with HDT of 12 h, clogging problems due to biomass flotation were constant. The organic matter removal efficiencies for each operational condition were 92 ± 3%, 91 ± 1.8%, 90 ± 2,4%. Those values were statistically similar. The methane percentage in the biogas increased with the HDT reduction, being 41 ± 23, 53 ± 27, and 62 ± 12% when the HDT were 72, 24 and 12 hours. Alkalinity production was observed since the beginning of operation. The mean relation between intermediate and partial alkalinity was 0.1±0.006 in samples collected in the system output. Although presenting clogging problems with the smallest HDT, the ABR was efficient, robust and reliable when treating dairy effluents, producing a high quality liquid effluent and a methane rich biogas.

Benzene is chemically stable as it has no substituents which can be biochemically attacked and a well-known toxic contaminant whose anaerobic degradation pathway is still not fully resolved. As only a very few anaerobic benzene-mineralizing pure cultures have been described yet, research was usually done with enrichment cultures dominated by specific organisms capable of benzene degradation under different electron acceptor conditions. Remarkable progress has been made in recent years with regard to the initial mechanism of benzene transformation especially on the putative genes that are involved in anaerobic carboxylation of benzene and the benzoyl-CoA central pathway. Many phylotypes described to be primary benzene degraders in anaerobic enrichment cultures at various electron acceptor conditions belong to the Peptococcaceae. Here, the thesis focused on characterizing the structure and function of anaerobic benzene-mineralizing microbial communities enriched from two hydrocarbon-contaminated sites: hydrocarbon-contaminated sediment from Ogoni in Niger Delta of Nigeria and a benzene-contaminated aquifer in Zeitz (Germany). The Niger Delta is one of the world’s most damaged ecosystem mainly due to hydrocarbon exploration accidents. The natural attenuation potential of Niger Delta subsurface sediment for anaerobic hydrocarbon degradation was investigated using benzene as a model compound under iron-reducing, sulfate-reducing, and methanogenic conditions. Benzene was slowly mineralized under iron-reducing conditions using Fe(III) chelated with nitrilotriacetic acid, or poorly crystalline Fe(III) oxyhydroxides as electron acceptors, analyzed by measurement of 13CO2 produced from added 13C-labelled benzene. The highest mineralization rates were observed in microcosms amended with Fe(III) oxyhydroxides while microcosms amended with Fe(III) nitrilotriacetic acid produced methane. Abundant phylotypes were affiliated to Betaproteobacteriales, Ignavibacteriales, Desulfuromonadales, and Methanosarcinales of the genera Methanosarcina and Methanothrix, illustrating that the enriched benzene mineralizing communities were diverse and may contain more than a single benzene degrader. The study underpins the importance of microbial ecosystem services in contaminant degradation as a sustainable environmental means of mitigating harmful chemicals. Benzene degradation pathways in a benzene-mineralizing, nitrate-reducing enrichment culture from Zeitz was investigated. Benzene mineralization was dependent on the presence of nitrate and correlated to enrichment of a Peptococcaceae phylotype only distantly related to known anaerobic benzene degraders of this family. Its relative abundance decreased after benzene mineralization had terminated, while other abundant taxa - Ignavibacteriaceae, Rhodanobacteraceae and Brocadiaceae - slightly increased. Generally, the microbial community remained diverse despite amendment of benzene as single organic carbon source, suggesting complex trophic interactions between different functional groups. A subunit of the putative anaerobic benzene carboxylase (AbcA) previously detected in Peptococcaceae was identified by metaproteomic analysis suggesting that benzene was activated by carboxylation. Detection of proteins involved in anaerobic ammonium oxidation (Anammox) indicates that benzene mineralization was accompanied by Anammox, facilitated by nitrite accumulation and the presence of ammonium in the growth medium. The results suggest that benzene was activated by carboxylation and further assimilated by a novel Peptococcaceae phylotype and confirm the hypothesis that Peptococcaceae are important anaerobic benzene degraders. Only a few benzene mineralizing anaerobes have been isolated to date. In an attempt using classical isolation techniques to isolate benzene-mineralizing pure cultures from a benzene-mineralizing nitrate-reducing microbial community, two consortia were gained under nitrate-reducing conditions spiked separately with acetate and benzene as sole sources of carbon and energy with media containing ammonium or without ammonium. Both consortia – Bz4 (with ammonium) and Bz7 (without ammonium) - mineralized 13C-labelled acetate under anoxic conditions at 3.3 and 2.7 µM day-1, respectively, revealed by analysis of evolved 13CO2. However, only Bz4 mineralized 13C-labelled benzene (0.298 µM benzene mineralized day-1) generated up to 960.2 ± 0.3 ‰ ẟ13C-CO2 during 184 days while producing only slight amounts of nitrite (4.60 ± 0.004 µM). By 16S rRNA gene amplicon sequencing was determined that the isolated cultures were not pure cultures but still contained several different phylotypes. The gained Bz4 consortium that mineralized benzene under anoxic conditions can be further purified and explored for their metabolic potentials.:Acknowledgments ………………………………………………………................. ii Table of Contents …………………………………………………………………… iii Dissertation Summary ……………………………………………………………… vi Dissertation Zusammenfassung …………………………………………………… viii List of Tables ………………………………………………………………………… x List of Figures ……………………………………………………………………….. xi List of Appendices ………………………………………………………………….. xiii Abbreviations .………………………………………………………….................... xv Chapter 1: Introduction and Research Objectives ……………………………… 1 1.1 Introduction ……………………………………………………………… 2 1.2 Aims and Objectives ………………………………………………….... 4 Chapter 2: Anaerobic Benzene Degradation by Microbial Communities and Pure Cultures …… 6 2.1 Anaerobic benzene degradation – a brief introduction ...…………… 7 2.2 Anaerobic benzene degradation under different electron acceptor conditions … 9 2.2.1 Benzene degradation under methanogenic conditions ……… 9 2.2.2 Benzene degradation under sulfate-reducing conditions …… 14 2.2.3 Benzene degradation under nitrate-reducing conditions …… 20 2.2.4 Benzene degradation under iron-reducing conditions ……… 25 2.3 Anaerobic benzene degradation by pure cultures ………………… 26 2.4 Anaerobic benzene activation mechanisms and associated genes……………… 28 2.4.1 Hydroxylation of benzene …………………………………….… 30 2.4.2 Methylation of benzene ………………………………..………… 34 2.4.3 Carboxylation of benzene ……………………………....………. 34 2.5 Benzoyl-CoA central metabolic pathways ………………………… 37 2.6 Syntrophic interactions in benzene-degrading communities ……… 42 2.7 Prospects for the future ……..……………………………………………… 43 Chapter 3: Anaerobic Benzene Mineralization by Natural Microbial Communities from Niger Delta …………………………………………………………………........... 44 3.1 Introduction …………………………………………………………..... 45 3.2 Materials and Methods ……………………………………………….. 46 3.2.1 Chemicals ………………………………………………………... 46 3.2.2 Site description and sampling procedure ……………………… 47 3.2.3 Setup of enrichment cultures …………………………………… 47 3.2.4 Chemical and microscopic analysis …………………………… 48 3.2.5 Microbial community analysis …………………………………… 49 3.3 Results and Discussion …………………………………………………. 50 3.3.1 Mineralization of benzene at different electron-acceptor conditions …………... 50 3.3.2 Microbial community structure at different electron-acceptor conditions ……... 53 3.4 Conclusion ………………………………………….…………………… 61 Chapter 4: Structure and Functional Capacity of a Benzene-mineralizing, and Nitrate-reducing Microbial Community ……………………………………………......... 62 4.1 Introduction …………………………………………………………..... 63 4.2 Materials and methods ……………………………………………..... 64 4.2.1 Chemicals ………………………………………………………... 64 4.2.2 Microcosm setup and sampling ………………………………… 64 4.2.3 Chemical and physiochemical analyses ……………………… 66 4.2.4 Amplicon and metagenome sequencing ……………………… 67 4.2.5 Protein mass spectrometry ……………………………………. 67 4.2.6 Metaproteome analysis ………………………………………… 68 4.2.7 Cloning and sequencing of putative nitric oxide dismutase (nod) genes ………. 68 4.2.8 Data availability …………………………………………………… 69 4.3 Results …………………………………………………………………………. 70 4.3.1 Benzene mineralization under nitrate-reducing conditions …… 70 4.3.2 Changes in microbial diversity during benzene mineralization . 71 4.3.3 Metaproteome composition ……………………………………… 74 4.3.4 Presence of putative nitric oxide dismutase genes (nod) ……. 76 4.4 Discussion ……………………………………………………………... 76 4.4.1 Putative pathways for nitrate reduction coupled with benzene mineralization … 76 4.4.2 Elucidation of the benzene activation step ……………………… 78 4.4.3 Benzoyl-CoA central pathway ……………………………………. 79 4.4.4 Peptococcacea as putative primary benzene degraders ……… 80 4.4.5 Metabolic function of Anammox bacteria in the community …… 81 Chapter 5: Consortia Dominated by Gammaproteobacteria Isolated from a Denitrifying Benzene-degrading Enrichment Culture and their Capacity to Mineralize Benzene...................... 83 5.1 Introduction ……………………………………………………………… 84 5.2 Materials and methods ………………………………………………… 85 5.2.1 Chemicals ………………………………………………………… 85 5.2.2 Isolation procedure …………………………………………………… 85 5.2.3 Mineralization and nitrite analyses ……………………..……… 86 5.2.4 Genomic DNA extraction and 16S rRNA gene sequencing … 87 5.3 Results …………………………………………………………………. 87 5.4 Discussions …………………………………………………………… 91 Chapter 6: General Conclusions and Outlook …..……………………………… 95 6.1 Conclusions and novelty of the research …………………………… 96 6.2 Ignavibacteriales as benzene degrading consortia under iron-reducing conditions 96 6.3 Insights into benzene activation via carboxylation by Peptococcaceae … 97 6.4 Unraveling growth of Anammox bacteria during benzene mineralization … 98 6.5 Study significance ……………………………………………………… 99 6.6 General outlook ………………………………………………………… 100 References ………………………………………………………………………… 101 Appendices ………………………………………………………………………… 120 Contributions of other Authors …………………………………………………… 160

Nesse trabalho foi estudado a degradação anaeróbia do alquilbenzeno linear sulfonado (LAS), um surfactante amplamente utilizado na fabricação de detergentes e presente em esgoto doméstico e águas residuárias industriais. Para isso foi utilizado reator anaeróbio de leito fluidificado em escala de bancada (1,2 L) preenchido com material suporte para imobilização da biomassa. Quatro diferentes suportes foram testados previamente em reatores de leito fluidificado em menor escala (350 ml): carvão ativado (R1), argila expandida (R2), pérolas de vidro (R3) e areia (R4). Todos os reatores foram inoculados com lodo proveniente de reator UASB utilizado no tratamento de dejetos de suinocultura e alimentados com substrato sintético acrescido de LAS. Os reatores foram mantidos a 30°C e operados com tempo de detenção hidráulica (TDH) de 18 horas. Foi possível constatar que os quatro reatores foram aptos na remoção de matéria orgânica (acima de 84%) e LAS (acima de 81%), respectivamente para concentração inicial média de 550 mg/L e 16,5 mg/L. No entanto, carvão ativado e argila expandida sofreram processo de fragmentação durante a operação do reator. Assim, areia foi o material escolhido para preencher o reator em escala de bancada devido aos bons resultados de remoção do LAS (99%), menor custo e facilidade de aquisição, quando comparada a pérola de vidro. Após 270 dias de operação, com concentrações crescentes de LAS e média de 32,3 mg/L, constatou-se que o reator em escala de bancada removeu 93% de LAS. Todos os suportes adsorveram pouco LAS (máximo de 0,43 mgLAS/gargila) não interferindo significativamente no processo de remoção biológica. Exames microscópicos realizados durante a operação dos reatores mostraram que estiveram presentes microrganismos com morfologias semelhantes a espiroquetas, bacilos, bactérias filamentosas e cocos, entre outros. A biomassa presente no final da operação do reator em escala de bancada e nos reatores menores preenchidos com pérolas de vidro (R3) e areia (R4) foi submetida à técnica de clonagem e sequenciamento do gene 16S RNAr para o Domínio Bacteria. Observou-se que os reatores apresentaram clones relacionados a diversos Filos como Bacteroidetes, Proteobacteria, Verrucomicrobia e Firmicutes, entre outros. / This work presents the anaerobic degradation of linear alkylbenzene sulphonate (LAS), a surfactant widely used in the production of detergents and present in domestic and industrial wastewaters. It was used an anaerobic fluidized bed reactor in bench scale (1,2L) filled with support material for biomass immobilization. Four different supports were previously tested in small scale fluidized bed reactors (350 ml): activated charcoal (R1), expanded clay (R2), glass beads (R3) and sand (R4). All reactors were inoculated with sludge from a UASB reactor treating swine wastewater and were fed with a synthetic substrate supplemented with LAS. The reactors were kept at 30ºC and operated with a hydraulic retention time (HRT) of 18 h. It was possible to note that the four tested reactors were able to remove organic matter (higher than 84%) and LAS (higher than 81%), respectively, to initial mean value of 550 mg/L and 16.5 mg/L. However, activated charcoal and expanded clay both produced shearing during reactor operation. Thus, sand was the chosen material to fill the bench scale reacto because of good results of LAS removal (99%) and smaller cost and affordability compared to glass beads. After 270 days of operation, with crescent LAS concentrations and average of 32,3 mg/L, it was found that the bench scale reactor was able to remove LAS in 93%. All supports adsorb a few LAS (maximun of 0.43 mgLAS/gclay). This value does not interfere in biologic removal process. Microscopic tests done during the reactor´s operation presented microorganisms with morphologies similar to spirochetes, bacillus, filamentous, cocci and others. 16S rRNA gene sequencing and phylogenetic analysis of samples from the bench scale reactor and smaller reactor filled with glass beads (R3) and sand (R4) revealed that these reactors gave rise to broad microbial diversity, with microorganisms belonging to the phyla Bacteroidetes, Proteobacteria, Verrucomicrobia and Firmicutes, and others.

Nesse trabalho foi estudado a degradação anaeróbia do alquilbenzeno linear sulfonado (LAS), um surfactante amplamente utilizado na fabricação de detergentes e presente em esgoto doméstico e águas residuárias industriais. Para isso foi utilizado reator anaeróbio de leito fluidificado em escala de bancada (1,2 L) preenchido com material suporte para imobilização da biomassa. Quatro diferentes suportes foram testados previamente em reatores de leito fluidificado em menor escala (350 ml): carvão ativado (R1), argila expandida (R2), pérolas de vidro (R3) e areia (R4). Todos os reatores foram inoculados com lodo proveniente de reator UASB utilizado no tratamento de dejetos de suinocultura e alimentados com substrato sintético acrescido de LAS. Os reatores foram mantidos a 30°C e operados com tempo de detenção hidráulica (TDH) de 18 horas. Foi possível constatar que os quatro reatores foram aptos na remoção de matéria orgânica (acima de 84%) e LAS (acima de 81%), respectivamente para concentração inicial média de 550 mg/L e 16,5 mg/L. No entanto, carvão ativado e argila expandida sofreram processo de fragmentação durante a operação do reator. Assim, areia foi o material escolhido para preencher o reator em escala de bancada devido aos bons resultados de remoção do LAS (99%), menor custo e facilidade de aquisição, quando comparada a pérola de vidro. Após 270 dias de operação, com concentrações crescentes de LAS e média de 32,3 mg/L, constatou-se que o reator em escala de bancada removeu 93% de LAS. Todos os suportes adsorveram pouco LAS (máximo de 0,43 mgLAS/gargila) não interferindo significativamente no processo de remoção biológica. Exames microscópicos realizados durante a operação dos reatores mostraram que estiveram presentes microrganismos com morfologias semelhantes a espiroquetas, bacilos, bactérias filamentosas e cocos, entre outros. A biomassa presente no final da operação do reator em escala de bancada e nos reatores menores preenchidos com pérolas de vidro (R3) e areia (R4) foi submetida à técnica de clonagem e sequenciamento do gene 16S RNAr para o Domínio Bacteria. Observou-se que os reatores apresentaram clones relacionados a diversos Filos como Bacteroidetes, Proteobacteria, Verrucomicrobia e Firmicutes, entre outros. / This work presents the anaerobic degradation of linear alkylbenzene sulphonate (LAS), a surfactant widely used in the production of detergents and present in domestic and industrial wastewaters. It was used an anaerobic fluidized bed reactor in bench scale (1,2L) filled with support material for biomass immobilization. Four different supports were previously tested in small scale fluidized bed reactors (350 ml): activated charcoal (R1), expanded clay (R2), glass beads (R3) and sand (R4). All reactors were inoculated with sludge from a UASB reactor treating swine wastewater and were fed with a synthetic substrate supplemented with LAS. The reactors were kept at 30ºC and operated with a hydraulic retention time (HRT) of 18 h. It was possible to note that the four tested reactors were able to remove organic matter (higher than 84%) and LAS (higher than 81%), respectively, to initial mean value of 550 mg/L and 16.5 mg/L. However, activated charcoal and expanded clay both produced shearing during reactor operation. Thus, sand was the chosen material to fill the bench scale reacto because of good results of LAS removal (99%) and smaller cost and affordability compared to glass beads. After 270 days of operation, with crescent LAS concentrations and average of 32,3 mg/L, it was found that the bench scale reactor was able to remove LAS in 93%. All supports adsorb a few LAS (maximun of 0.43 mgLAS/gclay). This value does not interfere in biologic removal process. Microscopic tests done during the reactor´s operation presented microorganisms with morphologies similar to spirochetes, bacillus, filamentous, cocci and others. 16S rRNA gene sequencing and phylogenetic analysis of samples from the bench scale reactor and smaller reactor filled with glass beads (R3) and sand (R4) revealed that these reactors gave rise to broad microbial diversity, with microorganisms belonging to the phyla Bacteroidetes, Proteobacteria, Verrucomicrobia and Firmicutes, and others.

O presente trabalho teve por objetivo avaliar a eficiência de remoção do surfactante aniônico alquilbenzeno linear sulfonado (LAS) em reator anaeróbio de leito granular expandido - EGSB (1,5 litros) com recirculação e alimentação com meio mineral. Além de caracterizar filogeneticamente a diversidade de bactérias na presença do surfactante. O sistema foi operado em condição mesofílica em 4 etapas: (I), (II) e (IV) com TDH de 32 horas, e (III) com TDH de 26 horas. Em todas as etapas a DQO foi em média de 609 \'+ OU -\' 137 mg/L e 14 \'+ OU -\' 1,71 mg/L de LAS afluente. As maiores remoções de LAS foram verificada nas etapas II e IV, com valores de 73,6 \'+ OU -\' 5,6% e 63,6 \'+ OU -\' 6,17%, respectivamente de. Na etapa III essa remoção foi de 47,8 \'+ OU -\' 6,2%. Por meio do balanço de massa constatou-se que 56,6% do total de LAS adicionado foram removidos compreendendo 48,4% por biodegradação e 8,2% por adsorção. A remoção de matéria orgânica não foi afetada com a adição do LAS e nem pela exposição prolongada a esse surfactante. Entretanto, a estrutura do grânulo foi comprometida quando da adição do surfactante, observado pelo aumento da concentração de sólidos totais efluente de 0,049 g/L na etapa I (sem LAS), 0,128 g/L na etapa II, 0,064 g/L na etapa III e 0,038 g/L na etapa IV, quando da adição de 14 \'+ OU -\' 1,71 mg LAS/L. Além disso, foi notada diminuição do diâmetro médio dos grânulos no decorrer da operação do reator de 0,36 cm nas etapas I e III para 0,34 cm na etapa IV. Por meio da técnica de tubos múltiplos (NMP) foi constatado aumento das bactérias anaeróbias totais e diminuição das arqueias metanogênicas, em função do tempo de operação do reator. As bactérias redutoras de ferro representaram 8% da biomassa anaeróbia na etapa IV. Por meio do seqüenciamento da região 16S do RNAr para o domínio Bacteria da biomassa da extremidade superior do reator e da biomassa do leito, foi verificado semelhança com os seguintes filos Proteobacteria, Firmicutes e Synergistetes. Notou-se diferença significativa entre as bibliotecas de clones para essas duas amostras. / This study aimed to evaluate the efficiency of removal of linear alkylbenzene sulfonate (LAS) in expanded bed reactor (1.5 liters) using granular sludge (EGSB) with recirculation and feed with mineral medium modified. The system was operated at mesophilic condition in four stages: (I) (II) and (IV) with HRT of 32 hours, and (III) with HRT of 26 hours. At all stages the COD averaged 609 \'+ OR -\' 137 mg/L and 14 \'+ OR -\' 1.71 mg/L LAS influent. The higher removals of LAS were found in stages II and IV, respectively, 73.6 \'+ OR -\' 5.6% and 63.6 \'+ OR -\' 6.17%. In stage III this removal was 47.8 \'+ OR -\' 6.2%. Through mass balance was found that 56.6% of total LAS added were removed by biodegradation comprising 48.4% and 8.2% by adsorption. The organic matter removal was not affected by the addition of LAS and not by prolonged exposure to this surfactant. However, the granule structure was compromised after the addition of surfactant, the observed increase in effluent total solids concentration of 0.049 g/L in stage I (no LAS), 0.128 g/L in stage II, 0.064 g/L in stage III and 0.038 g/L in stage IV when adding 14 \'+ OR -\' 1.71 mg/L. Furthermore, it was noticed significant decrease in mean diameter of the granules during the operation of the reactor of 0.36 cm in stages I and III to 0.34 cm in stage IV. Through the multiple tube method (MPN) was found to increase the total anaerobic bacteria and methanogenic archaea decreased depending on the time of reactor operation. Iron-reducing bacteria accounted for 8% of anaerobic bacteria total in step IV. By sequencing the 16S rRNA for the domain Bacteria biomass from the upper end of the reactor and the biomass of the bed, was found similar to the following phyla Proteobacteria, Firmicutes and Synergistetes. Significant difference was noted between the clone libraries for these two samples.