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Methyl-coenzyme M reductase (MCR) is the key enzyme involved in the production of methane by methanogenic archaea and its consumption by anaerobic methanotrophs (ANME). MCR is a multimeric complex composed of six different subunits arranged in a 2α, 2β, 2γ configuration that requires two molecules of its nickel-containing tetrapyrrole prosthetic group, coenzyme F430. Additionally, the α subunits of MCR house a variety of different post-translational modifications across both methanogens and ANME. In methanogens, MCR is encoded in a conserved mcrBDCGA gene cluster, which encodes accessory proteins McrD and McrC. These are believed to be involved in the assembly and activation of MCR, respectively. However, one or both accessory proteins are often omitted from the operon in other MCR-containing archaea as is the case in ANME. MCR knowledge is mostly limited to methanogens due to difficulties associated with large-scale cultivation of ANME and other MCR-containing archaea. Due to the complexity of MCR, studies on this enzyme are also largely limited to native enzymes. Developing methods for the detailed biochemical characterization ANME MCRs would be highly desirable since these enzymes are proposed to be optimized for methane oxidation and thus have immense potential for bioenergy and greenhouse gas mitigation applications. In addition to containing the necessary machinery for the production of an assembled and active MCR, model methanogens are easier to culture and have established genetic manipulation techniques, making them ideal candidates for the development of heterologous expression systems. Thus, here we sought to generate such a system for the study of various ANME MCRs in the methanogen, Methanococcus maripaludis. We report the successful expression and purification of an ANME-2d MCR, marking a significant step toward the development of a heterologous MCR expression system. Additionally, our attempts to purify various recombinant MCRs revealed the importance of including accessory proteins, particularly McrD, within expression constructs. Therefore, we also sought to functionally characterize McrD, which we show is likely an MCR chaperone that plays a key role in MCR maturation. Taken together, our work has provided key insights into MCR assembly as well as provided a foundation for the eventual development of MCR based biocatalytic systems to be used for methane mitigation strategies and bioenergy platforms. / Doctor of Philosophy / Life is divided into three domains known as Bacteria, Eukarya, and Archaea. Methanogens are anerobic microbes belonging to the domain Archaea, which can be found across a wide variety of oxygen deprived environments. These organisms can turn different carbon-containing compounds into energy and methane gas in a process known as methanogenesis. This results in roughly 90 billion tons of biologically produced methane, making methanogenesis a key point of interest for potential greenhouse gas mitigation. The methane-generating step of methanogenesis is performed by methyl-coenzyme M reductase (MCR), a large enzyme composed of two α subunits, two β subunits, and two γ subunits. Additionally, this enzyme harbors a nickel-containing cofactor which is responsible for catalyzing the difficult methane formation reaction. In addition to the MCR-encoding genes, MCR gene clusters contain two extra genes that encode accessory proteins, named McrC and McrD, which are believed to play an important role in the activation and the assembly of the enzyme, respectively. Relatives of methanogens known as Anerobic Methanotrophs (ANME) are a different type of archaea which consume methane by reversing methanogenesis in a process known as anerobic methane oxidation. Because of their ability to consume methane, there is a large interest in studying MCR from these organisms to potentially use it for methane mitigation strategies and for bioenergy applications to convert methane to more usable liquid fuels. However, due to the high difficulty of growing ANME in a lab setting, studying any biochemical processes from ANME is a difficult task. Luckily, genetic manipulation techniques are available for many methanogens, making them ideal candidates to study MCR from ANME organisms. In this work, we sought to develop a system to express and purify MCR from different methanogens and ANME in a methanogenic host, Methanococcus maripaludis. We also sought to understand the role and importance of accessory protein McrD, especially with respect to developing a proper expression system for MCRs. We were able to successfully express a ANME MCR in M. maripaludis and found that McrD is an important aspect to consider when expressing MCRs in a methanogen, although it is not essential for this protein to exist within the MCR gene cluster. This work sets the stage for the future biotechnological use of MCR for methane mitigation and bioenergy applications.

Methanogens are supposed to be one of the first life forms that emerged and evolved on early Earth inan environment of high depths and pressure utilizing the chemical energy provided by serpentinization.Serpentinization is a geological process that involves the transformation of low-silica ultramafic rocks,which are present in the lower oceanic crust and upper mantle. During this transformation minerals arereacting with water producing H2. Methanogens that can utilize this H2 to reduce the available CO2(hydrogenotrophs) can thrive in an environment like that, taking advantage of the substrate and energyflow that exists and thrive at these ecosystems. Methanobacterium oryzae is a hydrogenotrophicmethanogen that belongs to the order Methanobacteriales and has been isolated from a rice field inPhilippines. Methanobacterium oryzae strain FPi and a strain (wild strain) similar to that, isolated froman ophiolitic outcrop in an active serpentine site in Chimaera, Antalya, Turkey in 2017, have been usedin this study and have been tested for their survivability and adaptation abilities in different iron and O2concentrations. The two strains were cultivated in mediums with 4 different iron and 2 different O2concentrations for a duration ranging from 9 to 21 weeks. Gas chromatography was used to analyzeweekly gas samples for CH4 and CO2 concentrations that have been used as growth indicators. Scanningelectron microscope pictures have been taken to assess cell presence and contamination as well asidentify mineral precipitates. The results indicate the importance of iron for these species showinglimited or no growth when treated with no iron and enhanced growth at higher concentrations of iron.Furthermore, O2 has hindered or inhibited growth in most of the samples, but lower oxygenconcentrations seem to be tolerated by some specimens throughout the experimental time. Furtherresearch for the detection of the mechanisms behind the survivability of the methanogens with no ironand with oxygen in the medium is needed to further reveal the limits of life and provide moreinformation about the organisms that might have been the first ones that inhabited our planet.

Trimethylamine, (CH3)3N, (TMA), odors are often associated with limed and polymer conditioned sludges. This odor has a fishy smell and can be a nuisance to the community surrounding a wastewater treatment plant or land application site. Several factors are thought to determine the amount of TMA generated from limed biosolids. These are, the presence of cationic polymer, the polymer dose, the time between addition of polymer and lime stabilization, shear imparted on the sludge in the dewatering process and dewatered cake solids concentration. All of these were investigated in this study. The results showed that TMA could be generated from sludge that did not contain polymer but the concentrations were low compared to sludge conditioned with cationic polymer. As the polymer dose increased, the TMA increased. Shear also showed to play an important role for TMA production. In addition to higher shear increasing the polymer demand, shear in itself can increase TMA generation. However, the most important factor in generating TMA was the time between conditioning and liming. If this time was minimized, little TMA was produced, even at high polymer doses. Data also suggests that methanogens play an important role in the breakdown of TMA. / Master of Science

Tidal freshwater wetlands (TFW) are at significant risk of loss or alteration due to global climate change, and saltwater intrusion from sea level rise is of particular concern for these habitats due to their proximity to coastal areas. A space-for-time model was used to investigate the effects of saltwater intrusion on soil methanogen communities along naturally occurring salinity gradients on the Waccamaw, James, and Hudson Rivers. Amplification of the methyl coenzyme-M reductase (mcrA) functional gene was used in qPCR, reverse transcription qPCR, and T-RFLP to measure the abundance, activity, and community composition of soil methanogens. Both the abundance and activity of methanogens decreased with increasing salinity, and the both total and active methanogen community composition shifted in response to changes in salinity. This research demonstrates that saltwater intrusion will alter carbon cycling in TFWs, potentially altering their ability to sequester carbon and keep pace with rising sea level.

Neste trabalho investigou-se o desenvolvimento de biofilmes anaeróbios em um sistema de laboratório chamado de \"Modified Robbins Device\" (MRD). O objetivo específico foi o de comparar a organização das células anaeróbias, particularmente daquelas que são comuns em lodos de esgoto, sobre superfícies hidrofílicas (vidro) e hidrofóbicas (polipropileno). A hibridação in situ com sondas fluorescentes complementares ao RNAr 16S específicas para domínio e grupos e a microscopia confocal de varredura a laser foram utilizadas para verificar a composição microbiana dos biofilmes, bem como do inóculo. Foram realizados dois tipos de experimentos, um com culturas puras de metanogênicas e outro com células oriundas de lodo granulado anaeróbio. As culturas puras de metanogênicas, Methanobacterium formicicum (DSM 1535), Methanosaeta concilii (DSM 3671) e Methanosarcina barkeri (DSM 800) foram usadas como inóculo para a formação dos biofilmes no interior do MRD durante 9 dias. Os resultados mostraram que as três espécies colonizaram ambas as superfícies após o segundo e sétimo dia de ensaio. No segundo experimento, o MRD foi inoculado com um consórcio microbiano anaeróbio e a formação do biofilme foi estudada durante 22 dias. As amostras dos biofilmes bem como aquelas retiradas do frasco-reservatório de células apresentaram composição microbiana semelhante, ambas foram dominadas por Archaeae metanogênicas hidrogenotróficas relacionadas com membros da família Methanobacteriaceae, já que foram detectadas com a sonda MB1174. Este grupo contribuiu com cerca de 44 a 90% do total de células coradas com DAPI e foi morfologicamente semelhante à Methanobacterium e Methanobrevibacter. As células detectadas com a sonda específica para membros da ordem Methanomicrobiales (MG1200) representaram cerca de 2 a 18,0% do total de células coradas com DAPI no frasco-reservatório e de 0,1 a 2,0% nas amostras dos biofilmes. Estas células foram ) morfologicamente semelhantes à Methanospirillum, também uma metanogênica hidrogenotrófica. Não foram detectadas células pertencentes à família Methanosarcinaceae, pois a hibridação com a sonda MSMX860 foi negativa. Células que hibridaram com a sonda específica para o Domínio Bacteria (EUB338) representaram cerca de 2 a 18% do total de células coradas com DAPI. Os resultados mostraram que as Archaeae metanogênicas hidrogenotróficas que foram predominantes no inóculo também dominaram os biofilmes que se desenvolveram em ambas as superfícies, vidro e polipropileno. Os dados desse trabalho sugerem que a hidrofobicidade do material suporte não influenciou o desenvolvimento e a composição microbiana dos biofilmes anaeróbios, considerando as condições específicas dos ensaios realizados. / In this study the development of anaerobic biofilms using a laboratory system called modified robbins device (MRO) were investigated. We were especially interested in comparing the organization of anaerobic cells, particularly those that are very common in domestic sewage sludge, in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescence in situ hybridization (FISH) with domain and group speci fie probes that target intracell ular 16S rRNA and confocal laser scanning microscopy (CLSM) were used to investigate the microbial composition of both the inoculum and anaerobic biofilms. Two sets of experiments were carried, one with pure methanogenic organisms and the other with cells from a mesophilic anaerobic granular sludge. The pure methanogenic cultures, Methanobacterium formicicum (OSM 1535); Methanosaeta conci/ii (OSM 3671) and Methanosarcina barkeri (OSM 800) were used to seed the MRD to allow the development of biofilms over 9 days. The results showed that ali the three species were colonizing both surfaces after 2 and 7 days of experimental period. In the second experiment, the biofilm reactor was seeded with a microbial anaerobic consortium and biofilm forrnation was studied during 22 days. Biofilm and culture vessel samples showed nearly the same microbial composition, both were dominated by hydrogenotrophic methanogenic Archaea related to the Methanobacteriaceae as detected by the specific probe (MBI174). This group accounted for 44 to 90% of the OAPI-stained cells and morphologically resembled Methanobacterium and Methanobrevibacter. Cells detected with the Methanomicrobiales specific probe (MG 1200) accounted for 2 to 18.0% of the OAPI-stained cells in the culture vessel and 0.1 to 2.0% in the biofilm samples. These cells were morphologically similar to Methanospiriltum, also a hydrogenotrophic methanogen. No cells were detected by the Methanosarcinaceae specific probe (MSMX860). Cells which hybridized to the Bacteria specific probe (EUB338) accounted for the remaining 3 to 18% of the DAPI-stained cells. The results showed that the hydrogenotrophic methanogenic Archaea cells predominated in the inoculum and the biofilms that developed on both surfaces, glass and polypropylene. Our data suggest that the hydrophobicity of the support material did not influence the development and the microbial composition of anaerobic biofilms, considering specific conditions of the experiments.

Neste trabalho investigou-se o desenvolvimento de biofilmes anaeróbios em um sistema de laboratório chamado de \"Modified Robbins Device\" (MRD). O objetivo específico foi o de comparar a organização das células anaeróbias, particularmente daquelas que são comuns em lodos de esgoto, sobre superfícies hidrofílicas (vidro) e hidrofóbicas (polipropileno). A hibridação in situ com sondas fluorescentes complementares ao RNAr 16S específicas para domínio e grupos e a microscopia confocal de varredura a laser foram utilizadas para verificar a composição microbiana dos biofilmes, bem como do inóculo. Foram realizados dois tipos de experimentos, um com culturas puras de metanogênicas e outro com células oriundas de lodo granulado anaeróbio. As culturas puras de metanogênicas, Methanobacterium formicicum (DSM 1535), Methanosaeta concilii (DSM 3671) e Methanosarcina barkeri (DSM 800) foram usadas como inóculo para a formação dos biofilmes no interior do MRD durante 9 dias. Os resultados mostraram que as três espécies colonizaram ambas as superfícies após o segundo e sétimo dia de ensaio. No segundo experimento, o MRD foi inoculado com um consórcio microbiano anaeróbio e a formação do biofilme foi estudada durante 22 dias. As amostras dos biofilmes bem como aquelas retiradas do frasco-reservatório de células apresentaram composição microbiana semelhante, ambas foram dominadas por Archaeae metanogênicas hidrogenotróficas relacionadas com membros da família Methanobacteriaceae, já que foram detectadas com a sonda MB1174. Este grupo contribuiu com cerca de 44 a 90% do total de células coradas com DAPI e foi morfologicamente semelhante à Methanobacterium e Methanobrevibacter. As células detectadas com a sonda específica para membros da ordem Methanomicrobiales (MG1200) representaram cerca de 2 a 18,0% do total de células coradas com DAPI no frasco-reservatório e de 0,1 a 2,0% nas amostras dos biofilmes. Estas células foram ) morfologicamente semelhantes à Methanospirillum, também uma metanogênica hidrogenotrófica. Não foram detectadas células pertencentes à família Methanosarcinaceae, pois a hibridação com a sonda MSMX860 foi negativa. Células que hibridaram com a sonda específica para o Domínio Bacteria (EUB338) representaram cerca de 2 a 18% do total de células coradas com DAPI. Os resultados mostraram que as Archaeae metanogênicas hidrogenotróficas que foram predominantes no inóculo também dominaram os biofilmes que se desenvolveram em ambas as superfícies, vidro e polipropileno. Os dados desse trabalho sugerem que a hidrofobicidade do material suporte não influenciou o desenvolvimento e a composição microbiana dos biofilmes anaeróbios, considerando as condições específicas dos ensaios realizados. / In this study the development of anaerobic biofilms using a laboratory system called modified robbins device (MRO) were investigated. We were especially interested in comparing the organization of anaerobic cells, particularly those that are very common in domestic sewage sludge, in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescence in situ hybridization (FISH) with domain and group speci fie probes that target intracell ular 16S rRNA and confocal laser scanning microscopy (CLSM) were used to investigate the microbial composition of both the inoculum and anaerobic biofilms. Two sets of experiments were carried, one with pure methanogenic organisms and the other with cells from a mesophilic anaerobic granular sludge. The pure methanogenic cultures, Methanobacterium formicicum (OSM 1535); Methanosaeta conci/ii (OSM 3671) and Methanosarcina barkeri (OSM 800) were used to seed the MRD to allow the development of biofilms over 9 days. The results showed that ali the three species were colonizing both surfaces after 2 and 7 days of experimental period. In the second experiment, the biofilm reactor was seeded with a microbial anaerobic consortium and biofilm forrnation was studied during 22 days. Biofilm and culture vessel samples showed nearly the same microbial composition, both were dominated by hydrogenotrophic methanogenic Archaea related to the Methanobacteriaceae as detected by the specific probe (MBI174). This group accounted for 44 to 90% of the OAPI-stained cells and morphologically resembled Methanobacterium and Methanobrevibacter. Cells detected with the Methanomicrobiales specific probe (MG 1200) accounted for 2 to 18.0% of the OAPI-stained cells in the culture vessel and 0.1 to 2.0% in the biofilm samples. These cells were morphologically similar to Methanospiriltum, also a hydrogenotrophic methanogen. No cells were detected by the Methanosarcinaceae specific probe (MSMX860). Cells which hybridized to the Bacteria specific probe (EUB338) accounted for the remaining 3 to 18% of the DAPI-stained cells. The results showed that the hydrogenotrophic methanogenic Archaea cells predominated in the inoculum and the biofilms that developed on both surfaces, glass and polypropylene. Our data suggest that the hydrophobicity of the support material did not influence the development and the microbial composition of anaerobic biofilms, considering specific conditions of the experiments.

Methanogens, members of the domain Archaea, are unique in their ability to reduce carbon substrates to methane. Coenzyme M (CoM) is required in all methanogenic pathways. The biosynthesis of this coenzyme has been well studied in Class I Methanogens, but in Class II Methanogens, such as Methanosarcina acetivorans, little is known. The first step in the biosynthetic pathway might be catalyzed by cysteate synthase (CS), which converts phosphoserine to cysteate by the addition of sulfite. The 46 kDa enzyme was successfully purified from inclusion bodies and characterized. The identity of the product was confirmed by liquid chromatography-mass spectrometry (LC-MS) results as well as by derivatization of the reaction product coupled with high pressure liquid chromatography (HPLC) analysis. Kinetic analysis showed that the enzyme has a K [subscript m] of 0.43 mM for its substrate, phosphoserine, and a K [subscript m] of 0.05 mM for its required nucleophile, sulfite. Four compounds were found to be inhibitors and IC₅₀ values were determined. The results show that CS carries out a new reaction and narrows the gap in our knowledge of Class II Methanogen CoM biosynthesis. In the second part of this dissertation, five enzymes in a newly discovered but poorly characterized pathway for the degradation of halogenated aromatic compounds in Leptothrix cholodnii SP-6 were examined. The pathway reportedly culminates in the production of 2-chloroacetaldehyde, a well-known alkylating agent. In order to determine if 2-chloroacetaldehyde is produced and how the organism survives in its presence, the pathway intermediates are being identified. To this end, 4-oxalocrotonate tautomerase (4-OT), 4-oxalocrotonate decarboxylase (4-OD), vinylpyruvate hydratase (VPH), pyruvate aldolase (PA) and acetaldehyde dehydrogenase (AAD) were cloned, expressed and characterized. 4-OT was found to process the 5-(chloro)-2-hydroxymuconate, but only when the equilibrium was shifted by the addition of 4-OD and VPH. Steady state kinetic analysis showed that while there is a slight decrease in K [subscript m] for the halogenated substrate when compared to the non-halogenated substrate, indicating a difference in binding. There is also a 30-fold decrease in the turnover number, indicating a preference for the non-halogenated substrate. The identity of the product, 5-(chloro)-2-oxo-4-hydroxypentanoate, was verified by ¹H NMR spectroscopy. A stereochemical analysis was also carried out. / text

Earthworm-microbial interactions with emphasis on the passage effects of Eisenia spp. on microbial community were investigated. The study was focused on earthworm potential to regulate functional microbiota in cattle-impacted soils. Microbial communities were studied through a combination of polar lipid analyses, molecular, and culturing methods.

Les communautés microbiennes benthiques participent activement au recyclage de la matière organique et de fait au fonctionnement biogéochimique des écosystèmes lacustres. Ces communautés comportent de nombreux phyla mais leur diversité fonctionnelle est encore incomplètement connue. Ce travail vise à appréhender les modifications de structure et d'abondance des gènes fonctionnels en lien avec la distribution spatiale verticale (liée au gradient d'oxydoréduction), la variabilité spatiale horizontale (zone côtière vs pélagique) et la dynamique saisonnière liée au brassage de la colonne d'eau (ré-oxygénation des interfaces benthiques), de même que l'identification de la diversité des méthanotrophes et des méthanogènes.Pour cette étude, des carottes sédimentaires ont été prélevées sur un transect zone côtière – zone pélagique, à différentes dates au cours d'un cycle annuel. Chaque carotte sédimentaire a été analysée dans la verticalité entre l'interface eau-sédiment et 20 cm. Les communautés microbiennes participant au cycle du méthane ont été ciblées par 2 gènes de fonction et étudiées en termes de structure, de diversité et d'abondance; par ailleurs, des marqueurs phylogénétiques ont été utilisés pour caractériser les communautés bactériennes et archéennes totales.Les résultats de l'étude spatiale montrent que, si à l'échelle locale (station d'échantillonnage) une relative homogénéité des communautés microbiennes (totales et fonctionnelles) est observée, des variabilités fortes sont détectées d'une part à l'échelle des transects horizontaux en lien avec des changements de conditions environnementales et d'autre part dans la verticalité des sédiments sous l'effet des conditions d'oxydoréduction. La communauté bactérienne étant la plus affectée dans la verticalité, avec des changements de structure entre toutes les strates étudiées. Dans cette même étude, une analyse comparative de la structure des communautés (totales et fonctionelles) a démontré que l'analyse d'échantillons individuels permettait d'obtenir un plus grand nombre d'OTU que l'analyse des mêmes échantillons regroupés en pools.Les résultats de l'étude de la dynamique temporelle des communautés méthanogènes et méthanotrophes révèlent des changements de structure et abondance, principalement à l'interface eau - sédiment en lien avec la dynamique d'oxygénation du lac. Quant à l'analyse de la diversité, elle montre une dominance des Methanomicrobiales (Methanoregula principalement) pour les méthanogènes, mais les Methanosarcinales (Methanosarcina) et les Methanobacteriales (Methanobacterium) ont également été identifiés. Pour les méthanotrophes, la diversité est dominée par Methylobacter en zone profonde et par Methylococcus en zone côtière, les méthanotrophes de Type II (Methylosinus et Methylocystis) ont aussi été identifiés.L'ensemble de ces travaux souligne l'importance de prendre en compte, à la fois la variabilité spatiale (horizontale et verticale) et la variabilité temporelle, des communautés méthanogènes et méthanotrophes lors de l'étude de ces communautés. Les changements quant à leurs structures et leurs abondances sont des paramètres non négligeables pour comprendre les processus impliqués dans le cycle du méthane. / Benthic microbial communities are actively involved in organic matter recycling and fact biogeochemical functioning of lake ecosystems. These communities comprise many phyla but their functional diversity is still incompletely known. This study is focused on the benthic microbial communities involved in the methane cycle in lacsutrine suystems. We aimed understanding the structural and abundance changes of functional genes related to the vertical distribution (redox gradient in sediment), the horizontal variability (coastal vs. pelagic benthic zone) and seasonal dynamics related to mixing of the water column (re-oxygenation of benthic interface). The composition of methanotrophic and methanogenic communities was characterized by sequencing analyses.For this study, sedimentary cores were sampled along a transect from coastal to pelagic zone, at different times during an annual cycle. In addition, each sediment core was analyzed in its verticality from the water-sediment interface to 20 cm depth. Microbial communities involved in the cycle of methane (methanogenesis and methanotrophy) were targeted by 2 functional genes (mcrA and pmoA). Furthermore, phylogenetic markers were used to characterize the total bacterial and archaeal communities. These communities are studied in terms of structure (genotyping), diversity (sequencing) and abundance (qPCR, DNA) of their functional genes.The results of the study showed that, on a spatial scale, a low heterogeneity was detected for a given sampling station in terms of structure of microbial communities (total and functional), however, a high variability was detected both at an horizontal scale along a transect (costal vs. pelagic zone), due to contrasted environmental conditions, and at a vertical scale (upper to deeper layers in the core) under the effect of redox conditions. The bacterial community being the most affected in the verticality, with structural changes among all strata studied. In the same study, a comparative analysis of the structure (for all of the communities), between pooled samples and individual samples, demonstrated that the analysis of individual samples provided a greater number of OTU for the majority of microbial communities.Moreover the study of the temporal dynamic of methanogen and methanotroph communities revealed changes in the structure and abundance, mainly at the water - sediment interface, according to the oxygenation levels that varied through time. The analysis of diversity showed a dominance of Methanomicrobiales (Methanoregula mainly) for methanogens, but Methanosarcinales (Methanosarcina) and Methanobacteriales (Methanobacterium) were also identified. The methanotrophs' community was dominated by Methylobacter on deeper stations and by Methylococcus in coastal station. Type II methanotrophs (Methylosinus and Methylocystis) were also identified.This work highlights the importance of taking into account both the spatial variability (horizontal and vertical) and the temporal variability of methanogen and methanotroph communities. Changes on their structures and abundances are significant parameters for understanding the processes involved in the methane cycle.