Methanogens are autotrophic Archaea that produce methane as a product of their anaerobic metabolism. They are the largest producers of global methane, contributing over 60% of the total methane budget each year. Methane is an extremely potent greenhouse gas, with emissions providing the second-largest contribution to historical global temperature increases after carbon dioxide. Methanogens have become extremely important industrially as because they are used in the production of biofuels, as well as in treating industrial waste for industrial processes. This report will focus on those successful genetic methods and modifications that have been developed for methanogens and how they have started to contribute to understanding methanogen biochemistry. / text

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Methanogenesis is the biological production of methane and is utilized by methanogenic archaea (methanogens) to generate energy. This process is responsible for 70% of total atmospheric methane, a potent greenhouse gas and an important energy source (natural gas). In the future, reversing methanogenesis in an engineered methanogenic strain could be realized to efficiently convert natural gas into liquid fuels. Methyl coenzyme M reductase (Mcr) catalyzes the final reaction of methanogenesis in methanogens and the first reaction in the anaerobic oxidation of methane (AOM) carried out by the anaerobic methanotrophs (ANME). Cofactor F₄₃₀, a unique nickel-containing tetrapyrrole, serves as the prosthetic group and catalytic component of Mcr. Recently, multiple F₄₃₀ variants have been discovered in several methanogenic species, including Methanococcus maripaludis, Methanosarcina acetivorans, and Methanocaldococcus jannaschii. A novel variant reported here has an exact mass of 1008.3478, a similar absorption spectrum as unmodified F₄₃₀, and associates with purified Mcr from M. acetivorans. Based on the exact mass, this molecule is likely modified with a mercaptopropamide moiety. In some conditions, this modified F₄₃₀ comprises 30-50% of the total F₄₃₀ pool. We also report upon our work to identify the sulfur insertion enzyme required to produce methylthio-F₄₃₀ that functions with Mcr in ANME-1. We hypothesized that the insertion of the methylthio moiety is likely catalyzed by a methylthiotransferase (MTTase) homolog present in ANME. However, purified ANME MTTase does not appear to catalyze this reaction, and instead catalyzes the methylthiolation of N6-threonylcarbamoyladenosine (t6A) in tRNA. / Master of Science in Life Sciences / Methanogens are a unique but diverse group of microorganisms that produce methane to generate their energetic needs. The byproduct of their metabolism is methane gas, most of which escapes into the atmosphere. Methanogens produce 70% of Earth's atmospheric methane, which is a gas that has contributed to 20% of global warming since the start of the industrial era. However, methane, which makes up the majority of natural gas, is also an important source of energy, and natural gas generates 40% of the United States' electricity. An issue with natural gas is, as a gas, it readily leaks out in the extraction and transport process. A solution to this is to convert the gas into liquids, which do not display these negatives. It is possible, through a better understanding of how methanogens work, we could produce a methanogen strain that can efficiently convert methane into liquid fuels. The last methane-generating step in methanogenic metabolism uses a protein known as methyl-coenzyme M reductase (Mcr). To do this, Mcr uses a small molecule known as cofactor F₄₃₀. Recently, variants of the standard F₄₃₀ structure have been described, in both methanogens as well as another microbial group known as the anaerobic methanotrophs (ANME). ANME generate their energy through reversing methanogenic metabolism. The work here involves studying why and how methanogens and ANME make F₄₃₀ variants. The hope is this work will reveal either different functionalities of cofactor F₄₃₀ not previously known, or that they influence Mcr catalysis, potentially in the reverse (methane degradation) direction.

The performance and stability of a thermophilic anaerobic digestion system are inherently dependent on the engineered environment within each reactor. While the selection of operational parameters such as mixing, solids retention time, and digestion temperature are often selected on the basis of certain desirable outcomes such as the deactivation of human pathogens, these parameters have been shown to have a broad impact on the overall sludge digestion process. Since the current time-temperature requirements for biosolids pathogen reduction are most easily met at elevated digestion temperatures within the thermophilic range, it is certainly worth examining the effect of specific digestion temperatures on ancillary factors such as operational stability and the aesthetic quality of biosolids. A series of experiments were carried out in which wastewater sludge was digested at a range of temperatures (35°C, 49°C, 51°C, 53°C, 55°C, 57.5°C). Each reactor was operated for a period at steady state in order to make observations of microbial activity, digestion performance, and biosolids aesthetics as affected solely by digestion temperature. Results of this study show that poor operational stability arises in reactors operated at 57.5°C. Elevated concentrations of hydrogen and short-chain fatty acids in the 57.5°C digesters are evidence that the observed temperature-induced digester failures are related to the temperature sensitivity of hydrogenotrophic (CO₂-reducing) methanogens. Reactors operated at other temperatures performed equally well with respect to solids removal and operational stability. In addition, peak volatile organic sulfur compound (VOSC) production from biosolids treated at 51°C and above was greatly reduced in comparison with mesophilic anaerobic digestion and a lower temperature (49°C) thermophilic system. Since the biosolids methanogenic community appeared to be equally capable of degrading VOSC over the range of thermophilic temperatures, the conclusion is that the activity of VOSC producing organisms in digested and dewatered biosolids is greatly reduced when operating temperature in excess of 51°C are used. This study shows that small changes in an operationally defined parameter such as digestion temperature can have a large impact on the performance and stability of a digestion process. Single minded selection of digestion temperature in order to achieve effective pathogen reduction can result in poor digester performance and the production of an aesthetically unacceptable product. Careful selection, however, of an appropriate digestion temperature can not only ensure successful pathogen reduction in compliance with current regulations, but can also improve the performance, stability, and aesthetic quality of digestion systems employing thermophilic anaerobic digestion. / Master of Science

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As societies around the world become increasingly more dependent on fossil based fuels, the need to investigate alternative fuel sources becomes more pressing. Renewable, biomass-based carbon sources obtained from the biosphere can be gasified to produce synthesis gas, which can in turn be fermented to produce fuel-grade ethanol. A byproduct of ethanol production via fermentation is acetic acid. An optimized ethanol fermentation process should produce a wastewater stream containing less than 2 g/L of acetic acid. This is not enough acid to justify recovery of the acid; however it is a high enough concentration that treatment of the stream is required before it can be discharged. The purpose of this research was to convert the acetic acid into biogas, producing a twoold result: removal of the acid from the wastewater stream and the production of methane, which is a valuable source of energy. Microorganisms known as methanogens will consume acetic acid to produce methane and carbon dioxide under anaerobic conditions. The goal of this research was to optimize methane production from the wastewater stream discharged from an ethanol to syngas facility. Sludge containing methanogenic organisms was obtained from the anaerobic digester of a wastewater treatment facility and used as inoculum in batch reactors containing a synthetic acetic acid solution. Variables such as the type and amount of supplied nutrients, acid concentration, pH, cell acclimation, oxygen exposure, headspace gas composition, and agitation rate were examined. The effects of these parameters on the amount of biogas produced and acetic acid degraded were used to evaluate and optimize reactor performance. Additional experimentation further evaluating methanogenesis at low pH was also conducted using a laboratory scale semi-continuous fermentor. Finally, advanced analytical techniques were used to evaluate changes in organism population with respect to changes in reactor operational parameters. The results of this research were used to estimate kinetic parameters, develop different full-scale reactor design models, and estimate the both the cost of wastewater treatment as well as the value of the methane produced.

Microbial methane from subsurface organic-rich units such as coals and shale support approximately 5% of the United States and Canada's energy needs. In the deep subsurface, microbial methane is formed by the metabolism of primarily CO2, H2, and acetate by methanogens. These metabolites are the by-products of multi-step biodegradation of complex organic matter by microbial consortia. This study investigates microbial methane in the Illinois Basin, which is present in organic-rich shallow glacial sediments (surficial), Pennsylvanian coals (up to 600 m depth), and the Upper Devonian New Albany Shale (up to 900 m depth). Findings from the study show that hydrogeochemical conditions are favorable for methanogenesis in each reservoir, with a decrease in groundwater flushing rates corresponding to a decrease in average reservoir depth and an increase in carbon isotopic fractionation. The deeper reservoirs (coals and shale) were paleopasteurized, necessitating re-inoculation by methanogens. The microbes were likely advectively transported from shallow sediments into the coals and shale, where areas of microbial methanogenesis correlate with freshwater recharge. The recharge in the shale was primarily sourced from paleoprecipitation with minor contributions from glacial meltwater during the Pleistocene (4He ages). All areas sampled in the shale were affected by Pleistocene recharge, however groundwater ages in areas of microbial methanogenesis are younger (average 0.33 Ma) than areas with thermogenic methane (average 1.0 Ma). Estimates of in-situ microbial methane production rates for the shale (10-1000 TCF/Ma) are 104-106 times slower than laboratory rates. Only limited biodegradation is observed in the shale. In-situ stimulation of methane production may be most effective if aimed at increasing production of the supporting microbial consortia as well as methanogens. Trace metal concentrations in the shale are below known levels of inhibition or enhancement, with the exception of Fe, suggesting that microbial methanogenesis is not repressed by any of the measured trace metals and may be improved with the addition of Ag, Co, Cr, Ni, and Zn.

Extensive research has been conducted in numerous coalbed methane (CBM) basins; however, the Williston Basin (WB) remains largely unexamined due to the absence of CBM production despite large coal reserves. CBM in WB coalbeds has been reported, but there has been no systematic study on gas origin and distribution, or hydrogeochemical controls on gas generation to date. This study aims to determine differences in chemistry between groundwaters with and without the presence of CH₄ to better understand factors affecting CBM generation. Results reveal that shallow gas accumulations in WB coalbeds are microbial in origin and formed via CO₂ reduction. CBM is associated with Na-HCO₃ type groundwater with SO₄ concentrations<1 mmole/L due to cation exchange and sulfate reduction, respectively. These groundwaters occur in deeper units of the Fort Union Formation, underlying multiple coalbeds, suggesting that CH₄ is present in waters that have reacted extensively with formations containing low-rank (lignite) coals.