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Organic matter which is insoluble in common solvents and non-oxidising acids often comprises the quantitatively most important fraction of organic matter in sediments. This operationally defined material is usually simply termed 'insoluble organic matter' (IOM) or 'kerogen' when it is isolated from ancient sediments. Indeed, kerogen is regarded as the most abundant form of carbon on the planet. The molecular character of this generic material has not been fully elucidated, principally because of its insolubility which limits instrumental methods of analysis to those applicable to solid substrates. This thesis describes the quantitative isolation of IOM from lacustrine and marine sediments and two species of methanogenic bacteria using a sequential isolation procedure. A range of synthetic IOMs (melanoidins) was also prepared. The dissolution of IOM and melanoidins obtained in this manner was then attempted using the acidic ionic liquid l-ethyl-3- methylimidazolium chloride-aluminium (III) chloride. Two synthetic dendrimers containing similar functional groups to those observed in sedimentary IOM were used to try and assess the mode of action of the ionic liquid. Ionic liquid treatment of the DCM soluble dendrimers resulted in the formation of 7 - 62 % of material that was no longer soluble in DCM, whilst the soluble components had been substantially altered. The ionic liquid was found to non-quantitatively promote ether cleavage, protonation and rearrangement reactions. IOM was isolated from lacustrine Rostherne Mere, UK, sediments (7 - 3 0 % dry weight), Kimmeridge Clay, Dorset (11 - 12 %) and methanogenic bacteria (Methanococcus jannaschii, 3 %; Methanobacterium thermoaiitotrophicum, 0.1 %) using a time-consuming isolation procedure involving over forty separate chemical manipulations. Monitoring of the sequential isolation of IOM and characterisation of the final isolates was carried out using solid-state NMR, IR, elemental analysis, pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), scanning electron microscopy, and the newer surface sensitive technique of time of flight-secondary ion mass spectrometry (ToF-SIMS). Less than 1 % of sedimentary IOM and 5 % of Kimmeridge Clay IOM was soluble in DCM following ionic liquid treatment, whilst alkyl chains were lost from the insoluble portion which also increased in aromaticity. The poor yield recovered following ionic liquid treatment of M. jannaschii IOM (5 %) was attributed to loss of volatile material during hydrolysis. Following ionic liquid treatment 93 - 96 % of the melanoidins remained insoluble in DCM although their character had been altered, becoming more condensed. This ionic liquid dissolution procedure has not provided the substantial progress in elucidating the molecular character of IOM promised by earlier reports.

This feasibility study was performed at Virginia Polytechnic Institute and State University (Blacksburg, VA) in collaboration with BioSystems Technology, Inc. (Blacksburg, VA). In this study, degradability of PCBs (Aroclor 1242) from an aged surface soil was evaluated using serum bottle microcosms containing aceticlastic methanogenic consortium, enriched from a municipal anaerobic digester. Two different experiments, "intermediate feed" and "starve and feed" were conducted by manipulating the methanogenic consortium with different amounts of acetate feeding, during 30 days of incubation. Disappearance of Aroclor 1242 in the microcosms was quantified using gas chromatography (GC). Significant differences in Aroclor 1242 removal between inoculated and uninoculated (control) microcosms were observed suggesting that the methanogenic consortium was responsible for Aroclor 1242 disappearance. However, GC-mass spectrometry (GC-MS) results could not confirm that disappearance of Aroclor 1242 was due to anaerobic dehalogenation. From another experiment, it was confirmed that removal of Aroclor 1242 was not due to evaporation losses during sample extraction. Toxicity of an aged Aroclor 1242 contaminated surface soil was evaluated on an aceticlastic methanogenic consortium, enriched from a municipal anaerobic digester. Microcosms were set up using different amounts of soil and inoculum. Total gas production in the microcosms was monitored during 30 days of incubation, using a glass syringe. Total methane production in the microcosms was quantitated using GC. Toxicity of the soil on the methanogenic inoculum was determined based on the decreased rate of methane production in the microcosms relative to non- soil containing controls. Compared to the control, there was reduction in total methane production in soil containing microcosms. Between 3-27% reduction in total methane production was noticed in microcosms containing different amounts of soil and consortium. Reduction in methane production seemed to increase with increasing amount of soil. Whether this decrease in methane production was due to toxicity of Aroclor 1242 on the methanogenic consortium or due possibly to the toxicity of trapped oxygen in the soil could not be determined. The rate of gas production in the soil microcosm was linear. / Master of Science

Uranium contamination of groundwater from mining and milling operations is an environmental concern. Reductive precipitation of soluble and mobile hexavalent uranium (U(VI)) contamination to insoluble and immobile tetravalent uranium (U(IV)) constitutes the most promising remediation approach for uranium in groundwater. Previous research has shown that many microorganisms are able to catalyze this reaction in the presence of suitable electron-donors. The purpose of this work is to explore lowcost, effective alternatives for biologically catalyzed reductive precipitation of U(VI). Methanogenic granular sludge from anaerobic reactors treating industrial wastewaters was tested for its ability to support U(VI)-reduction. Due to their high microbial diversity, methanogenic granules displayed intrinsic activity towards U(VI)-reduction. Endogenous substrates from the slow decomposition of sludge biomass provided electron-equivalents to support efficient U(VI)-reduction without external electrondonors. Continuous columns with methanogenic granules also demonstrated sustained reduction for one year at high uranium loading rates. One column fed with ethanol, only enabled a short-term enhancement in the uranium removal efficiency, and no enhancement over the long term compared to the endogenous column. Nitrate, a common co-contaminant of uranium, remobilized previously deposited biogenic U(IV). U(VI) also caused inhibition to denitrification. An enrichment culture (EC) was developed from a zero-valent iron (Fe⁰)/sand packed-bed bioreactor. During 28 months, the EC enhanced U(VI)-reduction rates by Fe⁰ compared with abiotic Fe⁰ controls. Additional experiments indicated that the EC prevented the passivation of Fe⁰ surfaces through the use of cathodic H₂ for the reduction of Fe(III) in passivating corrosion mineral phases (e.g. magnetite) to Fe²⁺. This contributed to the formation of secondary minerals more enriched with Fe(II), which are known to be chemically reactive with U(VI). To determine the toxicity of U(VI) to different populations present in uranium contaminated sites, including methanogens, denitrifiers and uranium-reducers, experiments were carried out with anaerobic mixed cultures at increasing U(VI) concentrations. Significant inhibition to the presence of U(VI) was observed for methanogens and denitrifiers. On the other hand uranium-reducing microorganisms were tolerant to high U(VI) concentrations. The results of this dissertation indicate that direct microbial reduction of U(VI) and microbially enhanced reduction of U(VI) by Fe⁰ are promising approaches for uranium bioremediation.

A elevada concentração de cloro das bifenilas policloradas provoca alta toxicidade do composto, o qual dificulta sua biodegradação. A contaminação de PCB no Brasil foi confirmada em estudo realizado na Bahia de Santos-São Vicente (São Paulo), o qual revelou a necessidade de um plano de ação para o controle e remoção de PCB no Brasil. Pretendeu-se assim, na realização da presente pesquisa, verificar quatro hipóteses: (1) A técnica de Microextração em fase sólida é uma metodologia eficaz para avaliação de bifenilas policloradas de amostras de reatores; (2) A condição fermentativa-metanogênica abriga comunidade resistente ao PCB, e removê-lo; (3) A condição desnitrificante abriga comunidade resistente ao PCB, e removê-lo e (4) A remoção de PCB, bem como, a composição microbiana é distinta em cada condição metabólica. Para tanto, reatores em batelada foram montados separadamente com biomassa anaeróbia proveniente de reator UASB usado no tratamento de água residuária de avicultura e biomassa de sistemas de lodos ativados de tratamento de esgoto sanitário. Os reatores operados em condição mesófila foram alimentados com meio sintético, co-substratos, sendo etanol (457 mg.L-1) e formiato de sódio (680 mg.L-1) para os reatores anaeróbios, e somente etanol (598 mg.L-1) para os reatores anóxicos, além de PCB padrão Sigma (congêneres PCBs 10, 28, 52, 153, 138 e 180) em diferentes concentrações, dependendo do objetivo do ensaio. A aplicação do método de extração por SPME com análise em cromatógrafo gasoso com detector por captura de elétrons foi adequada para a determinação dos seis congêneres de PCB. Obteve-se ampla faixa de linearidade, seletividade frente aos vários interferentes, além da robustez do método, utilidade e confiabilidade na identificação e quantificação específica dos seis congêneres de PCB. A Hipótese 1 foi aceita; ou seja, por meio da aplicação da metodologia SPME foi possível quantificar os PCB nos reatores em batelada. Apesar de ter sido comprovada a inibição metanogênica na presença de PCB, com IC50 de 0,03 mg.L<sup-1 (concentração na qual 50&#37; da atividade metanogênica é inibida), a partir da análise dos reatores metanogênicos no Ensaio de Remoção, foi confirmada a remoção de 0,92 mg.L-1, 0,19 mg.L-1, 0,18 mg.L-1, 0,07 mg.L-1, 0,55 mg.L-1 e 0,47 mg.L-1 para os PCBs 10, 28, 52, 153, 138 e 180, respectivamente, para 1,5 mg.L-1 inicial. Thermotogaceae, Sedimentibacter, Anaerolinaceae e Pseudomonas, foram identificadas nos reatores anaeróbios por meio da plataforma Illumina. Representantes de Thermotogaceae e Sedimentibacter foram identificados em sistemas com elevada taxa de remoção de PCB, e representantes do filo Chloroflexi (grupo no qual representantes da Anaerolineae estão inseridos) foram os primeiros microrganismos desalogenadores de PCB identificados. Assim a Hipótese 2 foi aceita; ou seja, por meio de ensaios em batelada foi comprovada a toxicidade do PCB sobre a comunidade anaeróbia, a alteração da composição microbiana influenciada pela presença de PCB e a remoção nesta condição. Verificou-se ainda que na presença de PCB ocorreu a desnitrificação e comparando-se diferentes relações C/N-NO3-, foi estipulado a relação 6,95 como ideal na presença de PCB. Mesmo sendo confirmada a inibição da comunidade anóxica na presença de PCB com IC50 de 1,0 mg.L-1, verificou-se remoção de 1,02 mg.L-1, 0,85 mg.L-1, 1,31 mg.L-1, 1,02 mg.L-1, 0,03 mg.L-1, e 0,09 mg.L-1, para os PCBs 10, 28, 52, 153, 138 e 180, respectivamente, para 1,5 mg.L-1, inicial. Bactérias semelhantes a Aeromonadaceae, Lutispora, Sedimentibacter e Thermotogaceae foram identificadas nos reatores desnitrificantes. Representantes de Aeromonadaceae e Lutispora estão relacionados com o metabolismo desnitrificante e representantes de Thermotogaceae e Sedimentibacter foram identificados em sistemas com elevada taxa de remoção de PCB. A Hipótese 3 foi também aceita; ou seja, por meio de ensaios em batelada foi calculada a relação C/N-NO3- ideal na presença de PCB e foi comprovada a toxicidade do PCB sobre a comunidade anóxica, a alteração da composição microbiana influenciada pela presença de PCB e a remoção nesta condição. A maior remoção de PCB foi verificada para a condição anaeróbia (entre 45&#37; a 100&#37;), quando comparada com a condição anóxica (entre 10&#37; a 95&#37;). Bactérias semelhantes a Sedimentibacter e pertencentes a família Thermotogaceae foram identificadas nas duas condições nutricionais. Entretanto, mesmo verificando-se elevada abundancia relativa desses grupos nos reatores, evidenciou-se distinção entre as biomassas em cada condição. Assim, a Hipótese 4 foi aceita; ou seja, por meio de ensaios em batelada foi comprovada maior eficiência de remoção sob condição anaeróbia e distinta composição microbiana em cada condição. / The high chlorine concentration in polychlorinated biphenyls improves its toxicity, complicating their biodegradation. A study conducted in Santos-São Vicente Bay (São Paulo) confirmed the PCB contamination in Brazil and reveled the need of an action plan to control e remove the PCB in Brazil. In this sense the aim of this study was to evaluate four hypotheses: (1) The Solid Phase Micro Extraction is an efficient methodology to evaluate biphenyl polychlorinated in reactors; (2) The fermentative-methanogenic microorganisms are resistant to PCB and capable to consume it; (3) The denitrifying microorganisms are resilient to the PCB and capable to remove it; (4) The PCB removal, as well the microbial composition is distinct in each condition. Therefore, batch reactors were operated separately inoculated with anaerobic biomass from UASB reactor treating poultry wastewater and biomass from activated sludge treating sewage wastewater. The reactors were feed with synthetic medium, co-substrates, as ethanol (457 mg.L-1) and sodium formate (680 mg.L-1) for the anaerobic reactors, and ethanol (598 mg.L-1) for the anoxic reactors and different concentrations of six congeners of PCB (PCBs 10, 28, 52, 153, 138 and 180) depending on the aim of the assay. The applicability of SPME technique in gas chromatography with electrons capture detection was attested in the analysis of six PCB congeners. Higher linearity, selectivity, accuracy, reproducibility and robustness was obtained in the PCB quantification analyses. The Hypothesis 1 was accepted; since the PCB congeners in the reactors were quantified by the SPME methodology. Although the PCB causes methanogenic inhibition, with IC50 of 0.03 mg.L-1 (concentration in which 50&#37; of the methanogenic metabolism is inhibited), by the Removal Assay was confirmed the anaerobic removal of 0.92 mg.L-1, 0.19 mg.L-1, 0.18 mg.L-1, 0.07 mg.L-1, 0.55 mg.L-1 and 0.47 mg.L-1 to PCB 10, 28, 52, 153, 138 and 180, respectively, for 1.5 mg.L-1 initial. By means of platform Illumina Thermotogaceae, Sedimentibacter, Anaerolinaceae and Pseudomonas, were identified in the anaerobic reactors. The Thermotogaceaeand Sedimentibacter were related to systems with high PCB removal and the Chloroflexi members (Anaerolineae phylum) were the first PCB-dechlorination microorganisms identified. Therefore, the Hypothesis 2 was accepted since the PCB leads to anaerobic inhibition and in the reactors were verified the PCB removal and changes in the microbial composition. Even with PCB, the denitrification metabolism occurs and evaluating different C/N-NO3- relations, the 6.95 was stipulated the ideal in the presence of PCB. Even though the PCB causes inhibition in the denitrification bacteria, with IC50 of 1.0 mg.L-1, by the Removal Assay in the denitrifying reactors, was confirmed the anoxic removal 1.02 mg.L-1, 0.85 mg.L-1, 1.31 mg.L-1, 1.02 mg.L-1, 0.03 mg.L-1 and 0.09 mg.L-1 to PCB 10, 28, 52, 153, 138 and 180, respectively, for 1.5 mg.L-1 initial. Aeromonadaceae, Lutispora, Sedimentibacter and Thermotogaceae were identified in the denitrifying reactors. Members of Aeromonadaceae e Lutispora were related to the denitrification metabolism and Thermotogaceae e Sedimentibacter were identified in systems with high PCB removal rate. The Hypothesis 3 was accepted since the PCB leads to anoxic inhibition and in the reactors were verified the PCB removal and changes in the microbial composition. The anaerobic reactors presented the higher PCB removal percentage (between 45&#37; and 100&#37;) when compared to the anoxic reactors (between 10&#37; and 95&#37;). Member of Sedimentibacter and Thermotogaceae were identifyied in both conditions. However, even with high relative abundance of these two groups in the reactors, it was shown distinct composition in each biomass. Thus, the Hypothesis 4 was accepted since the PCB removal was more efficient in the anaerobic condition and were verified different changes in the microbial composition in each condition.

A atividade metanogênica específica foi estudada neste trabalho a partir do levantamento de diversas metodologias propostas para a realização desse teste . Procurou-se otimizar o teste de atividade metanogênica tomando-se por base a metodologia adotada por Willem de Zeeuw (1984). No desenvolvimento do trabalho experimentou-se várias condições metodológicas para o teste de atividade, objetivando-se a máxima atividade metanogênica específica do lodo, através de um ensaio que fosse simples e rápido. Para isso, utilizando-se de cinco tipos de lodos anaeróbios, estudou-se a influência das quantidades de biomassa e de substrato, da natureza do substrato, da forma de agitação do meio, das soluções de nutrientes e de metais, da redução do meio com nitrogênio, do volume dos frascos-reatores e do tempo de armazenamento do lodo nos resultados dos testes de atividade. Observou-se que, em função do tipo de lodo e da sua atividade metanogênica, devem ser pesquisados o substrato mais adequado e a relação ótima entre a quantidade de substrato e biomassa que conduzam à atividade metanogênica específica máxima durante o teste. Observou-se também que o ensaio pode ser mais simples e mais rápido, comparativamente àquele baseado na metodologia de de Zeeuw (1984). Como conclusão principal são apresentadas novas condições metodológicas para o teste de atividade metanogênica específica para lodos anaeróbios. / The specific methanogenic activity test was studied with basis on several methodologies proposed for carrying out the referred test. The methodology used by Willem de Zeeuw (1984) was employed to optimize this test. Several methodological processes were used for the activity test in an attempt to reach the maximum specific methanogenic activity of the sludge in a simple and quick process. Five types of anaerobic sludges were used and the following parameters were studied: the influence of the amounts of biomass and substrate, the nature of substrate, the liquor mixing, the nutriente and metal solutions, oxygen removal the reactor capacity, and the sludge storage time in the results of the activity tests. It was observed that the more suitable substrate and the optimum relationship between the quantity of substrate and biomass must be researched, sludge, so that the referred according to the type of relationship leads to a maximum specific methanogenic activity. It was observed that the test can be quicker and simpler than de Zeeuw\'s (1984). The main result reached consists of the presentation of new conditions to carry out the test of specific methanogenic activity for anaerobic sludges.

Domestic wastewater is the most abundant type of wastewater. Direct discharge of untreated domestic wastewater has environmental and public health risks due to the presence of organics, nutrients and pathogens. Application of anaerobic processes for the treatment of domestic sewage, which at present is largely treated by aerobic processes, has drawn considerable attention recently. Anaerobic processes can be applied for the removal of organic matter (methanogenesis) and nitrogen (anaerobic ammonium oxidation (Anammox)).The toxicity of fluoride to methanogenesis was investigated. The results indicate that acetoclastic were more susceptible to fluoride than hydrogenotrophic methanogens. The concentration of fluoride causing 50% inhibition (IC50) to acetoclastic ranged from 18.1 to 155.7 mg L-1 while for hydrogenotrophic methanogens was > 400.0 mg L-1.The feasibility of a combined system consisting of anaerobic up-flow anaerobic sludge blanket (UASB) followed by aerobic activated sludge (AS) reactor for removal of carbonaceous and nitrogenous contaminants from strong synthetic sewage (2.5 g chemical oxygen demand (COD) L-1) was also studied. The average combined removal of total COD, volatile fatty acids (VFA) and protein was higher than 89.0%, 99.0% and 97.0%; respectively. Extensive nitrification (96.0%) was observed when dissolved oxygen (DO) concentration was > 2.0 mg L-1. In contrast, only partial nitrification occurred when the AS received high organic loads and/or the DO level was below 2.0 mg L-1.The inhibitory effect of nitrite and nitrate on methanogenesis was evaluated. Methanogenic activity was inhibited by the presence of NOx- compounds (i.e., nitrite and nitrate). The inhibition imparted by nitrate was not due to the nitrate itself, but rather to its reduced intermediate, nitrite. The toxicity of NOx- to methanogens was found to be reversible after all the NOx- were reduced during denitrification.Moreover, the development of Anammox enrichment cultures was evaluated. Anammox cultures were successfully developed using sludge samples collected from municipal wastewater treatment plants (WWTPs) as inocula but not from methanogenic granular sludges. Return activated sludge (RAS) collected from WWTP operating for biological nitrogen removal had the highest intrinsic level of Anammox activity. RAS Anammox culture was developed rapidly within 40 days with a doubling time of 6.8 days.

The deep-sea hypersaline lakes in the Red Sea are among the most challenging, extreme, and unusual environments on the planet Earth. Despite their harshness to life, they are inhabited by diverse and novel members of prokaryotes. Methanogenesis was proposed as one of the main metabolic pathways that drive microbial colonization in similar habitats. However, not much is known about the identities of the methane-producing microbes in the Red Sea, let alone the way in which they could adapt to such poly extreme environments. Combining a range of microbial community assessment, cultivation and omics (genomics, transcriptomics, and single amplified genomics) approaches, this dissertation seeks to fill these gaps in our knowledge by studying archaeal composition, particularly methanogens, their genomic capacities and transcriptomic characteristics in order to elucidate their diversity, function, and adaptation to the deep-sea brines of the Red Sea. Although typical methanogens are not abundant in the samples collected from brine pool habitats of the Red Sea, the pilot cultivation experiment has revealed novel halophilic methanogenic species of the domain Archaea. Their physiological traits as well as their genomic and transcriptomic features unveil an interesting genetic and functional adaptive capacity that allows them to thrive in the unique deep-sea hypersaline environments in the Red Sea.

In theoretic parts diploma work is described mechanism anaerobic of the process, factors which him work and description qualitative determination biogas. Objective practical parts was quantification and qualities produced biogas. Led I'm investigation intent on investigation choice type phytomass and their fitness for production and quality biogas. On fermentative sweetness I'm watched change pH, Cox, decrease organic masses along anaerobic digestion. Further I'm establish entitlements content lignin and CHSK in single figures phytomass. On device OxiTop( control AN12 I'm measured maximum yield and speed development methane and biogas on hundreddweight substrates