The microbial origin of methane produced from sedimentary basins is a subject of great interest, with implications for the global cycling of carbon as well as natural gas exploration. Despite the growing body of research in sedimentary basin methanogenesis, few studies have sought to understand the subsurface microbial communities that produce methane, the metabolic pathways involved in the decomposition of ancient organic matter, or the components of ancient organic matter that are consumed. This research examined shale-derived dissolved organic matter (DOM) as a potential substrate to support a subsurface methanogenic community in a known microbial shale gas reserve, the Antrim Shale in the Michigan Basin, USA. Experiments were conducted that enriched fermentative and sulfate-reducing microbial communities from Antrim Shale formation waters. Additionally, 1H NMR spectroscopy was used to characterize shale-derived DOM solutions before and after they were used as growth media for fermentative and sulfate-reducing microbial communities, and to characterize the DOM of the Antrim Shale formation waters. The results of the enrichment studies demonstrate that both fermentative and sulfate-reducing microbial communities from the Antrim Shale are capable of growth using shale-derived DOM as their only source of organic carbon; further, the production of methane in a fermentative enrichment demonstrates that methanogenesis can be supported by shale-derived DOM alone. The 1H NMR characterization studies of the shale-derived DOM solutions before and after growth revealed subtle but detectable differences in DOM compositions, indicating the production and consumption of DOM components by the fermentative and sulfate-reducing microbial communities. Characterization analyses of Antrim Shale formation waters suggest that salinity and microbiological activity may influence the liberation of aliphatic and aromatic compounds from shale. The DOM characterization studies also suggest that carboxylic acids may be consumed by methanogenic communities in the Antrim Shale, and aromatic compounds may be produced by the enriched microbial communities and the communities present in the Antrim Shale.

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The archaeal branch of life represents some of the oldest life forms on Earth. Archaea are believed to have diverged from Prokaryotes roughly 3.5 billion years ago and it’s theorized that biological methane production started around this time as well. This would make methanogenesis one of the oldest metabolisms on our planet. Methanogenesis is a process that, so far, is known to be unique to archaea. Since their evolution, methanogens have had massive impacts on Earth’s climate and biology. Methane is an important part of the global carbon cycle, but is also a major greenhouse gas, making it a vital area of research. The methanogen studied in this thesis is referred to as the wild type (WT) and was isolated from an active serpentinization system at Chimaera seep in Çıralı, Antalya Gulf, Turkey. The Chimaera seep is a geological formation analogous to mid-ocean ridges, but exposed and above land. The research in this thesis focuses on the description of the WT, which is believed to be a new species. Describing the WT consisted of the characterization of extremes as well as optimal growth conditions. The WT was found to grow at initial pH levels of 9.0 and 9.5. It grew from 15 °C to 45 °C, but not at 47 °C and not at 12 °C, and had an optimum at 39 °C. The WT had measurable growth up to 40 g/L NaCl, and had its optimum at 0 g/L. The WT grew best with H2/CO2 substrate, but also grew well on formate.

This dissertation research aims to show that there are deterministic microbial distribution patterns based on quantifiable environmental thresholds by determining and rationalizing the relative abundances of hyperthermophilic methanogens, autotrophic iron(III) oxide reducers, and heterotrophic sulfur reducers within deep-sea hydrothermal vents. Organisms of these metabolisms are predicted to be relatively more abundant in different regions depending on environmental conditions such as reduction potential, organic carbon, and hydrogen availability. The relative abundances of these metabolic groups within samples from the Endeavour Segment and Axial Volcano in the northeastern Pacific Ocean were determined. Iron(III) oxide reducers were detected in nearly all samples while methanogens were generally not present or present in concentrations lower than those of the iron(III) reducers. To determine growth constraints and the effect of hydrogen concentration on hyperthermophilic methanogen growth kinetics, Methanocaldococcus jannaschii and two new Methanocaldococcus field isolates were grown at varying hydrogen concentrations. The hydrogen-dependent growth kinetics for all three strains were statistically indistinguishable, exhibiting longer doubling times and lower maximum cell concentrations with decreasing hydrogen concentrations until growth ceased below 17-23 μM. This minimum hydrogen concentration for hyperthermophilic methanogenesis was correlated with field microbiology and fluid geochemistry data from the Endeavour Segment and Axial Volcano. Anomalously high methane concentrations and thermophilic methanogens were only observed in fluid samples where hydrogen concentrations were above this predicted threshold. Aside from anomalous sites, methanogens are predicted to be hydrogen limited, and may rely on hydrogen produced by heterotrophs as suggested by in situ sampling and co-culture experiments. Models and kinetic experiments suggest that iron(III) oxide reducers are not hydrogen limited under the same conditions. A Methanocaldococcus strain that we isolated from Axial Volcano and used in our hydrogen threshold experiments was bioenergetically modeled over its range of growth temperatures, pH, NaCl concentrations, and NH4Cl concentrations. Its methane production rates and growth energies were largely constant but increased at superoptimal temperatures and when nitrogen was limiting. The results of this research demonstrate that the rates of and constraints on metabolic processes can be used to predict the distribution and biogeochemical impact of hyperthermophiles in deep-sea hydrothermal vent systems.

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Anaerobic digestion (AD) is an essential process in wastewater treatment to stabilize waste organic solids and produce biogas. This research is comprised of two projects in the discipline of anaerobic digestion. First, the effect of high sulfate concentration on anaerobic digestion of wastewater sludge was investigated. Secondly, the performance of acetoclastic methanogens Methanosaeta spp. and Methanosarcina spp. were investigated under intensified AD operation conditions (i.e., elevated acetate concentrations, vigorous mixing, etc.). In the sulfate experiments, the cumulative biogas and methane production decreased linearly with increasing initial sulfate doses (0 – 3,300 mg S L-1) and the correlation between the sulfate dose and methane production was verified with theoretical predictions, indicating complete reduction of sulfate to sulfide in AD. The examined sulfate concentrations resulted in no clear negative effects on the COD (chemical oxygen demand) removal or VSS (volatile suspended solids) destruction of the wastewater sludge, indicating that previous findings on sulfide toxicity might have been attributed to potential COD overestimation of digested sludge with high levels of sulfide. To avoid potential misinterpretation of AD performance on sulfide toxicity effects, we proposed a new method for COD correction for digested sludge. In the second project focused on acetoclastic methanogens, vigorous mixing conditions substantially decreased Methanosarcina spp. growth and methane production, and the decreased methanogenesis was more pronounced at higher acetate concentrations. Methanosarcina spp. prefer to grow in clusters and the vigorous mixing can disrupt cluster formation; as a result, reduced chances for cluster formation limited the growth of Methanosarcina spp.. While Methanosarcina spp. growth and methane production increased with the increasing acetate concentration, Methanosaeta spp. growth was unaffected by the examined vigorous mixing and soluble substrate conditions with negligible relative growth. Thus, rapid enrichment of Methanosarcina spp. is critical for successful operation intensified of AD processes under high organic loading conditions. / Thesis / Master of Applied Science (MASc)

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Master of Science / Geology / Matthew Kirk / The Cherokee basin in southeastern Kansas is a declining coalbed methane (CBM) field where little is known about how the CBM formed, the extent to which it continues to form, and what factors influence its formation. An understanding of methanogenic processes and geochemistry could lead to potential enhancement of methane formation in the basin. The objectives of this project are to (1) determine the pathway of methane formation and (2) determine whether geochemistry has influenced gas formation. In order to reach the objectives, we analyzed formation water geochemistry, production history, and gas composition and isotopes. Post Rock Energy Corporation gave us access to 16 wells for sampling purposes. We collected gas samples in Isotubes® for compositional and isotopic analyses at a commercial laboratory. We analyzed major ion chemistry from formation water using standard methods. Co-produced water samples we collected are Na-Cl type with total dissolved solids content ranging from 35,367 to 91,565 mg/L. TDS tended to be highest in samples collected from wells with greater total depth. The pH and temperature of sampled water averaged 7.0 and 19°C with an alkalinity ranging from 3.33 to 8.59. Gas dryness and δ¹³C CH[subscript 4] range from 196 to 4531 and -69.95 to -56.5, respectively, which indicate that methane is being produced biologically. Comparing the δ¹³C CH[subscript 4] to the δD CH[subscript 4], which ranges from -228.2 to -217.2, suggest that the primary pathway of methanogenesis is H[subscript 2]/CO[subscript 2] reduction. We calculated Δ (the difference between δ values) in order to correlate isotope data to produced water chemistry. Samples ΔD and Δ¹³C values range from -189.1 to -168.7 and 61.52 to 69.99. Calculated ΔD[subscript CH4-H2O] and Δ¹³C[subscript CO2-CH4] values approach the range for the acetate/methyl pathways as Clˉ concentration increases, potentially indicating a slight shift in methanogenic pathway in deeper, more saline portions of the basin. The culturing results revealed that living methanogens are still able to utilize H[subscript 2], acetate, and methanol present in co-produced formation water from all tested wells.

Lipídeos contidos em resíduos de laticínios além de representarem uma perda industrial importante, interferem negativamente nos sistemas de tratamento de efluentes inibindo a atividade microbiana do consórcio (Mendes, 2005). O objetivo do presente trabalho foi estudar a degradação anaeróbia de gorduras do leite hidrolisadas com duas enzimas: lipase comercial de Candida rugosa (éster-inespecífica), e preparado enzimático de Geotrichum candidum (lipase éster-específica). Determinaram-se condições ótimas de hidrólise de gorduras do leite no tocante a tempo e temperatura de processo. Após esta etapa, determinou-se a produção metanogênica advinda da estabilização de esterco bovino combinado com gorduras de laticínios hidrolisadas e in natura. As condições ótimas de ação de lipase comercial de C. rugosa em gorduras do leite foram obtidas com temperatura de 40ºC e pH de 6,5. As condições ótimas de ação de preparado enzimático de G. candidum foram obtidas com temperatura de 40ºC e pH de 7,5. Os tempos de processo hidrolítico para a produção máxima de ácidos graxos foram de 8 horas e 16 horas quando se utilizaram preparado enzimático de G. candidum e solução de lipase comercial de C. rugosa respectivamente. As velocidades de processo, bem como os valores de biodegradabilidade anaeróbia, produção metanogênica específica e coeficiente de conversão (Yp/s) indicaram ser muito mais eficiente a utilização lipase de C. rugosa na hidrólise das gorduras do leite, quando o objetivo do processo é a produção de biogás. Além disso, o preparado enzimático de G. candidum ocasionou inibição da atividade metanogênica acetoclástica. Quando se realizou um estudo variando-se a concentração de gordura no tratamento enzimático, obteve-se a maior produção de ácido oléico quando se utilizaram 42 gramas de gordura por grama de enzima. Por outro lado, o melhor fator de conversão entre produto e substrato foi verificado quando a relação entre a massa de gordura e a massa de enzima foi igual a 6g/g. / Lipids in dairy waste as well as representing an important industrial loss, interfere negatively in wastewater treatment systems by inhibiting microbial activity of the consortium. The aim of this work was to study the anaerobic degradation of hydrolyzed milk fats using two enzymes: lipase from Candida rugosa, which is an ester unspecific enzyme; and other specific lipase obtained from Geotrichum candidum. Optimal conditions for hydrolysis of milk fat regarding pH, time and temperature were determined. After that, the methanogenic production from the stabilization of cattle manure combined with fats (hydrolyzed and in nature) was evaluated. The optimum conditions for the action of commercial C. rugosa lipase were obtained at 40ºC and pH 6.5. The optimum conditions for the action of G. candidum preparation were obtained at 40ºC and pH 7.5. Maximum product concentrations were obtained within 8 hours and 16 hours when preparation of G. candidum and C. rugosa lipase were used, respectively. The initial methanogenic production rate, the values of anaerobic biodegradation, the specific methanogenic production and the methane yield as well, showed that the use of C. rugosa lipase in the hydrolysis of fats is more efficient when the goal of the process is the production of biogas. The use of the enzymatic preparation of G. candidum did not cause any benefits, and in addition, caused bigger inhibition of acetoclastic methanogenic activity. When the initial concentration of substrate was varied for the enzymatic treatment, it was possible to verify higher oleic acid accumulated production when 42 grams of fat where used per gram of enzyme. On the other hand, the higher conversion factor between product and substrate was obtained when the relation between the mass of fat and the mass of enzyme was 6g/g.