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Aerobic bacterial methane synthesis in the human gastrointestinal tract
(Montana State University - Bozeman, College of Agriculture, 2023) Jackson, Thomas Robert; Chairperson, Graduate Committee: Seth Walk
Aerobic bacterial methane synthesis constitutes a paradigm-shifting novel metabolism recently described in aquatic environments. It challenges the traditional model of methanogenesis as being a strictly anaerobic process carried out by archaeal methanogens. To date, the presence of aerobic bacterial methane synthesis has not been studied within the context of the human gastrointestinal tract. The goal of this work was to investigate the possibility of the presence of such metabolisms in the human gut microbiome. To investigate this, fecal samples from six individuals were first screened for the ability to produce methane under aerobic conditions. Bacteria from two of those fecal samples were isolated and evaluated for their ability to utilize methylamine, a known substrate involved in aerobic bacterial methane synthesis, as a sole nitrogen source. The ability of those isolates to produce methane under aerobic conditions from methylamine was then evaluated. Additionally, a flask-independent culture-based assay was developed in order to screen larger numbers of future isolates for the ability to utilize methylamine as a sole nitrogen source. This work demonstrates the first evidence of aerobic bacterial methane synthesis from members of the human gastrointestinal tract, finding two isolates capable of producing methane under aerobic conditions. Such findings broaden the understanding of methane-generating pathways that may have implications for the development of dysbiosis and atherosclerosis in human hosts.
Evaluation of methanotrophic activity and growth in a methanotrophic-heterotrophic co-culture
(Montana State University - Bozeman, College of Engineering, 2021) Kilic, Ayse Bengisu; Chairperson, Graduate Committee: Ellen G. Lauchnor; Erika J. Espinosa-Ortiz, Brent M. Peyton and Ellen Lauchnor were co-authors of the article, 'Methane-based bioreactor configurations in value-added product development: a review' submitted to the journal 'Journal of bioscience and bioengineering' which is contained within this thesis.; Erika J. Espinosa-Ortiz, Brent M. Peyton and Ellen Lauchnor were co-authors of the article, 'Evaluation of methanotrophic activity and growth in a methanotrophic-heterotrophic co-culture' submitted to the journal 'Engineering in life sciences' which is contained within this thesis.
Methane is a potent greenhouse gas (GHG) and accounts for 20-30% of the GHG emissions globally. In nature, methane is utilized as a sole carbon and energy source by a group of bacteria referred to as methanotrophs. Methanotrophs have been reported to have the ability to form close associations with other microorganisms such as heterotrophic bacteria in the environment. Therefore, understanding methanotrophic activity and growth in a microbial consortium with heterotrophic bacteria is of interest from an environmental and biotechnology perspective. In this study, a methanotroph; Methylocystis sp. NLS7 and a heterotrophic bacterium, Pseudomonas chlororaphis, were co-cultivated in a methane-fed bioreactor with a dialysis membrane device used to separate the species physically. It was hypothesized that the co-culture would exhibit enhanced methanotrophic activity and microbial growth of NLS7 in NLS7- P. chlororaphis co-culture. The methane-oxidation rate and microbial growth rate of NLS7 were evaluated as a functional response variable to the presence of P. chlororaphis. In addition, the effects of NLS7 growth were evaluated on the growth of P. chlororaphis. Our findings indicated that the presence of P. chlororaphis does not have any beneficial effects on Methylocystis sp. NLS7 activity and growth. However, the growth of P. chlororaphis in the co-culture with solely methane as a carbon source indicated that P. chlororaphis is likely gaining carbon and energy from by-products of methane oxidation by Methylocystis sp. NLS7 since P. chlororaphis could not utilize methane as a carbon and energy source. The results of this study give us an important insight into the activity and the growth of methanotrophic consortia in methane-driven ecosystem.
Hydrogen production from mechanically-activated basalt under experimental conditions simulating subglacial environments
(Montana State University - Bozeman, College of Letters & Science, 2019) Mitchell, Kari Rebecca; Chairperson, Graduate Committee: Mark L. Skidmore
Shearing of rocks containing silicate followed by reaction with water has previously been shown to produce hydrogen under experimental conditions relevant to subglacial environments. The abiotic production of hydrogen, carbon dioxide, methane, and other hydrocarbon gases has also been demonstrated in laboratory comminution experiments on rocks from glaciated catchments. Thus, the generation of these biologically useful gases (e.g. hydrogen and methane) beneath glaciers could serve as a source of reductant capable of sustaining microbial ecosystems beneath the ice. Despite the ubiquitous nature of basalt on both Earth and other planetary bodies, production of hydrogen and other gases from basalt through mechanical shearing and reaction with water has not been demonstrated. Basalts were collected from glaciated catchments in Iceland to test whether hydrogen and other gases were produced under laboratory conditions simulating glacial comminution. Rock samples were milled under an inert atmosphere, after which water was added and hydrogen and methane production measured over time. An average of 6.6 nmol hydrogen and 2.6 nmol methane per gram rock were produced after 168 hours from basalt samples tested; additionally, hydrogen peroxide and radicals were produced during grinding. The abiogenic production of hydrogen and methane under these simulated subglacial basaltic environments demonstrated in this study also has implications for supporting subglacial microbial communities during periods of extended glaciation, such as glacial-interglacial cycles in the Pleistocene and during the pervasive low-latitude glaciation of the Cryogenian. This mechanism of hydrogen production also has implications for the potential for life on icy worlds like Mars.
Metabolic interactions and activity partitioning in a methanogenic, interdomain biofilm
(Montana State University - Bozeman, College of Letters & Science, 2019) Camilleri, Laura Beth; Chairperson, Graduate Committee: Matthew Fields; Kristopher A. Hunt, Aurelien Mazurie, Jennifer Kuehl, Alex Michaud, James Connolly, Egan Lohman, Zack Miller, Adam M. Deutschbauer and Matthew W. Fields were co-authors of the article, 'Differential gene expression of a bacterial-archaeal interdomain biofilm producing methane' submitted to the journal 'Biofilms' which is contained within this dissertation.; B.P. Bowen, C.J. Petzold, T.R. Northen and M.W. Fields were co-authors of the article, 'Activity partitioning in an archaeal-bacterial biofilm' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methanococcus maripaludis factor causes slowed growth in Desulfovibrio vulgaris Hildenborough' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Growth effects of sulfopyruvate and sulfoacetate on the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, and the methanogenic archaeon Methanococcus maripaludis S2' submitted to the journal 'Scientific reports' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methane production in Pelosinus fermentans JBW45' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.
Biofilms are an ancient survival strategy in which communities of organisms can grow as a cohesive unit, generally attached to a surface and/or at interfaces. Despite the paradigm that 99% of microorganisms grow as a biofilm in the environment, current research methods are largely limited to monoculture planktonic studies. Although more investigations are trying to improve culture complexity by evaluating interactions between two or more populations, experiments are still more readily performed with microorganisms in the planktonic growth mode. The research presented here aims to elucidate the complexity of interactions between two microorganisms from different domains of life that results in enhanced metabolism due to localization of cells in close proximity within an anaerobic biofilm. Desulfovibrio vulgaris Hildenborough (DvH) and Methanococcus maripaludis S2 (Mmp) form a syntrophic mutualism when grown in sulfate-limited media that requires electron flux from DvH to Mmp through what is commonly assumed to be interspecies hydrogen transfer, thereby establishing cross-feeding. The biofilm has been shown to promote a stable and more even carrying capacity for both populations that is likely linked to improved hydrogen transfer (and/or other potential carbon and electron co-metabolites) as compared to planktonic populations. Transcriptomic and proteomic analyses, utilizing RNA-seq and deuterated water respectively, were used to elucidate genes and proteins that contribute to the biofilm growth mode that results in a more efficient metabolism for the syntrophic co-culture (defined by biomass per substrate flux). The results demonstrate the expression of many genes with unknown functions, and others that contribute to cell-cell interactions as well as active proteins in electron processing (e.g., lactate oxidation) in DvH and CO2 reduction (e.g., methanogenesis) in Mmp. A metabolic model of the coculture provided reinforcement for transcriptomic assumptions and aided in the identification of a sulfonate and other amino acids as important syntrophic metabolites. Assessment of biofilm co-culture activity utilizing a new method, Biorthogonal Noncanonical Amino Acid Tagging (BONCAT), showed Mmp was less active in the uptake of a methionine analog as compared to DvH. Alternate assessments confirmed that Mmp was in fact active (based upon methane generation) although translational activity was below the detection limit. Further investigation of the system under sulfate stress showed that the metabolic pairing is more stable than previously thought and could indicate survival strategies that drive the seemingly 'mutualistic' relationship as a forced cooperation. The sulfate stress response coincided with observed lags in DvH growth when grown in Mmp spent medium that was associated with a decoupling of lactate-oxidation and sulfate-reduction. Together the results demonstrate metabolic interactions and activity partitioning within a methanogenic archaeal-bacterial biofilm. The dogma of mutualism being synonymous with equal reciprocity is challenged as it pertains to this model biofilm system. Moreover, this unique bacterial-archaeal biofilm represents interdomain interactions that could represent systems that contributed shared metabolic processes that lead to the development of eukaryotic life.
The role of adsorbed phase volume on the thermodynamics of supercritical methane adsorption on microporous carbon
(Montana State University - Bozeman, College of Engineering, 2019) Remington, Emily Lynn; Chairperson, Graduate Committee: Sarah L. Codd; Nicholas P. Stadie (co-chair)
Experimental determination of the isosteric heat of adsorption at the fluidsolid interface is an important undertaking in the chemical sciences since this fundamental thermodynamic quantity is closely related to the binding energy of the adsorbate on the adsorbent surface. The usual methods employed to calculate the isosteric heat from measured gas adsorption equilibria, however, are unsuited to the treatment of adsorption under a high-pressure adsorptive fluid (where the difference in molar volume between the two phases becomes small and depends significantly on that of the adsorbed phase). Herein we employ a methodological approach to the thermodynamic analysis of adsorption up to high pressures in the supercritical regime, with a specific focus on methane adsorption on microporous carbonaceous materials at T/T c between 1.25-2.75 and P/P c up to 2. The aim is to achieve a meritorious description of the thermodynamics of the adsorbed phase with as few independent parameters as possible. We compare several simple approaches to estimating the molar volume of the adsorbed phase, and demonstrate that among the several well-known sources of error involved in the isosteric approach, that attributed to molar volume estimations is not itself prohibitive to achieving meritorious results. We contrast the isosteric approach with that of the so-called 'isoexcess' methodology, and thereby shed new insights into the key role of the finite adsorbed phase volume in assessments of adsorption thermodynamics.
Measuring methane emissions from American bison (Bison bison L.) using eddy covariance
(Montana State University - Bozeman, College of Agriculture, 2019) Cook, Adam Anderson; Chairperson, Graduate Committee: Paul C. Stoy
American bison (Bison bison L.) have recovered from the brink of extinction over the past century. Bison offer potential environmental benefits as they re-occupy their native range, but many specific impacts of bison reintroduction are not well understood. Methane emissions are known to be a major climate impact of ruminants, but few measurements for bison exist due to challenges caused by their mobile grazing habits and safety issues associated with direct measurements. Here, we measure the methane and carbon dioxide fluxes from a bison herd on winter range using the eddy covariance technique. Methane emissions were negligible (mean = 0.0024 micromole m -2 s -1, SD = 0.0102 micromole m -2 s -1) before and after bison grazed in the area sampled by the eddy covariance flux footprint with the exception of a single spike possibly attributable to thawing soil or the presence of white-tailed deer (Odocoileus virginianus Z.). Methane fluxes when bison were present in the study area averaged 0.041 micromole m -2 s -1 (SD = 0.046 micromole m -2 s -1), similar to previous measurements over sheep and cattle pastures, but with little diurnal pattern due to a lack of consistent bison movement habits over the course of each day. An eddy covariance flux footprint analysis coupled to bison location estimates from automated camera images calculated methane flux with a median of 56.5 micromole s -1 per animal and a mean of 91.6 micromole s-1 per animal, approximately 50 and 75% of established emission rates for range cattle, respectively. Eddy covariance measurements are a promising way to measure methane and carbon dioxide flux from large ruminants on native range and we recommend comparisons amongst alternate grazing systems to help identify management strategies that are cognizant of climate.
Land-atmosphere exchange of carbon and energy at a tropical peat swamp forest in Sarawak, Malaysia
(Montana State University - Bozeman, College of Agriculture, 2017) Tang Che Ing, Angela; Chairperson, Graduate Committee: Paul C. Stoy; Paul C. Stoy, Kevin K. Musin, Edward B. Aeries, Joseph Wenceslaus, Mariko Shimizu, Ryuichi Hirata and Lulie Melling were co-authors of the article, 'The role of biophysical drivers in controlling the variability of net ecosystem CO 2 exchange in a tropical peat forest in Sarawak, Malaysian Borneo' submitted to the journal 'Global change biology' which is contained within this thesis.; Paul C. Stoy, Kevin K. Musin, Edward B. Aeries, Joseph Wenceslaus, Mariko Shimizu, Ryuichi Hirata and Lulie Melling were co-authors of the article, 'The exchange of water and energy between a tropical peat forest and the atmosphere: seasonal trends and comparison against global tropical rainforests' submitted to the journal 'Geophysical research letters' which is contained within this thesis.; Paul C. Stoy, Kevin K. Musin, Edward B. Aeries, Joseph Wenceslaus, Mariko Shimizu, Ryuichi Hirata and Lulie Melling were co-authors of the article, 'Eddy covariance measurements of methane flux at a tropical peat forest in Sarawak, Malaysian Borneo' submitted to the journal 'Agricultural and forest meteorology' which is contained within this thesis.
Tropical peatlands comprise 11% of the global peat area of ca. 400 Mha and are estimated to store about 89 Gt of carbon (C). However, considerable uncertainties remain about their present role in global C cycle as interannual ecosystem-scale measurements of undisturbed tropical peat forests have not been measured to date. Hence, an eddy covariance tower was instrumented in a tropical peat forest in Sarawak, Malaysia over four years from 2011 to 2014. We found that the forest was a net source of CO 2 to the atmosphere during every year of measurement. The inter-annual variation in net ecosystem CO 2 exchange (NEE) was largely modulated by the variation in gross primary production (GPP), which was jointly controlled by vapor pressure deficit (VPD) and leaf area index (LAI) in addition to photosynthetically active photon flux density (PPFD). Greater reduction of GPP in 2011 and 2012, are likely attributed to the relative low atmospheric transmission due to massive peat fires in Indonesia. Similarly, no analysis to our knowledge has measured whole-ecosystem methane (CH 4) flux from a tropical peat forested wetland to date despite their importance to global CH 4 budget. The two-month average of C-CH 4 flux measurements, on the order of 0.024 g C-CH 4 m -2 d -1, suggests that tropical peat forests are not likely to be disproportionally important to global CH 4 flux. Results demonstrate a limited ability for simple models to capture the variability in the diurnal pattern of CH 4 efflux, but also consistent responses to soil moisture, water table height, and precipitation over daily to weekly time scales. The sensible heat flux (H) and latent heat flux (LE) and their ratio (the Bowen ratio, Bo) at the study ecosystem were relatively invariant compared to other tropical rainforests. The average daily LE across the calendar year tended to be higher at MY-MLM (11 MJ m -2 day -1) than most other tropical rainforest ecosystems in the FLUXNET2015 database. Results demonstrate important differences in the seasonal patterns in water and energy exchange in tropical rainforest ecosystems that need to be captured by models to understand how ongoing changes in tropical rainforest extent impact the global climate system.
Microbially mediated biogeochemical cycles in polar ice covered lakes
(Montana State University - Bozeman, College of Agriculture, 2016) Michaud, Alexander Bryce Olson; Chairperson, Graduate Committee: John C. Priscu; Trista J. Vick-Majors, Mark L. Skidmore and John C. Priscu were co-authors of the article, 'Field testing of a clean, hot water drill used for access to subglacial aquatic environments' submitted to the journal 'Antarctic science' which is contained within this dissertation.; Mark L. Skidmore, Andrew C. Mitchell, Trista J. Vick-Majors, John C. Priscu, Carlo Barbante, Clara Turetta and Will vanGelder were co-authors of the article, 'Solute sources and geochemical processes in subglacial Lake Whillans, west Antarctica' in the journal 'Geology' which is contained within this dissertation.; John E. Dore, Trista J. Vick-Majors, Mark L. Skidmore and John C. Priscu were co-authors of the article, 'Microbial methane transformations beneath the west Antarctic ice sheet' submitted to the journal 'Science' which is contained within this dissertation.; This dissertation contains one article of which Alexander Bryce Olson Michaud is not the main author.
Lakes are important sites for globally-relevant biogeochemical cycles mediated by microorganisms. In the Arctic, seasonally ice covered thermokarst lakes are a large component in Earth's carbon cycle due to their methane emissions from organic carbon degradation. In the Antarctic, over 400 unexplored lakes exist beneath the Antarctic ice sheet with unknown biogeochemical contributions to the Earth system. This dissertation seeks to investigate the biogeochemical role of microorganisms in the lake habitat and how they interact with the seasonal and permanent ice covers of lakes in polar environments. Microbiologically clean hot water drilling was used to access a subglacial lake beneath Antarctica's ice to collect, for the first time, intact sediment and water samples. Laboratory experiments on Arctic and Antarctic, seasonally and perennially, respectively, ice covered lakes were used to investigate the impact of lake ice freezing regimes on microorganisms. My results show that subglacial lake sediments beneath the West Antarctic Ice Sheet contain solute ratios that suggest relict marine sediments were deposited during previous interglacial periods. Microbial activity overprints the marine geochemical signature to produce fluxes of ions into the Subglacial Lake Whillans water column, which ultimately drains to the Southern Ocean. Microbial activity in Subglacial Lake Whillans is partially fueled by biologically-formed methane diffusing from below our deepest collected (~38 cm) subglacial sediment samples. The ice above Subglacial Lake Whillans appears to be an important source of molecular oxygen for microorganisms to drive oxidative physiologies. My experimental evidence shows microorganisms incorporate into lake ice cover to, potentially, avoid increasing stressors from progressive lake ice freezing. Taken together, the results from this dissertation reinforce the hypothesis that subglacial environments beneath the Antarctic ice sheet are habitats for life. Further, the microorganisms in subglacial lakes participate in globally-relevant biogeochemical cycles. Here, I extend the extent of the biosphere and show sediments at the base of ice sheets are an active component of the Earth system.
Development of a singly-resonant optical parametric oscillator for carbon cycle science
(Montana State University - Bozeman, College of Engineering, 2015) Jones, Briana Lynn; Chairperson, Graduate Committee: Kevin S. Repasky
The human impact on the global carbon cycle is affecting the health of the environment by changing the balance between incoming and outgoing radiation as well as altering other geochemical cycles such as the nitrogen and water cycles. Although carbon dioxide makes up most of the greenhouse gas emissions, methane has a much greater impact on climate change due to its warming potential on a per molecule basis. Improved understanding of the spatial distribution of methane is necessary to quantify the anthropogenic impacts and mitigate future damage. A differential absorption lidar (DIAL) is proposed for spatially mapping methane concentrations. The system requires a laser transmitter that can produce over 3 mJ of pulse energy with a repetition rate of 1 kHz and output wavelength of 1.654 micron as well as a narrow linewidth on the order of 3 MHz. Modeling predicts that a system with these specifications can achieve measurement error of less than 2% relative to ambient levels of methane. Laser sources with these specifications are not commercially available, and the goal of this work is to evaluate the potential for a singly-resonant optical parametric oscillator (OPO) for the DIAL laser transmitter. The OPO is based on large-aperture periodically-poled magnesium-oxide-doped lithium niobate as the nonlinear optical material. Results from the OPO indicate that energies on the order of 1 mJ are possible with the experimental setup presented when operating at 20 Hz repetition rate. The OPO produced a linewidth of 10.5 GHz, measured on a system with resolution of 6.6 GHz. Future work includes optimization of the OPO to increase the output energy from the system to 3 mJ by improving mode-matching into the cavity and increasing the energy into the system. Additionally a method to precisely measure the linewidth of the OPO output is necessary as well as a pump laser that operates at 1 kHz to test the performance of the OPO at 1 kHz. The initial results show promise for the use of the OPO as the DIAL laser transmitter and with improvements, the OPO should meet the requirements for the DIAL system.
Methane flux from recently exposed subglacial sediments, Robertson Glacier, Canada
(Montana State University - Bozeman, College of Letters & Science, 2014) Spotts, Terra Marie; Chairperson, Graduate Committee: Mark L. Skidmore
Methane is over 20 times more effective than CO 2 as a greenhouse gas. Thus, its atmospheric concentration and the processes controlling it are important components of the global climate system. Recent research has shown methanogenesis in subglacial sediments. However, the net contribution from subglacial systems to the global methane budget is poorly understood due to a dearth of empirical data. Using measurements via the static chamber method, the flux of methane from recently exposed subglacial sediments at Robertson Glacier, Canadian Rockies was quantified. Methane concentrations were measured from surface gas flux chambers in transects both parallel and perpendicular to the glacier terminus. Over 300 measurements were made during the 2012 melt season (July to September) and used to determine both spatial and temporal variability in the gas fluxes. The chamber farthest from the glacier terminus, approximately 50 m down valley, had an average flux close to zero whereas the chambers nearest the terminus had the highest average fluxes. The average methane efflux from the sediment surface to atmosphere was 0.22 micromoles m -2 d -1. The highest methane efflux during the season, 11.0 micromoles m -2 d -1, was measured in close proximity to the glacier terminus. Shallow sediment cores were collected adjacent to the static chambers and vertical gas concentration profiles were measured from the cores. Within the profiles, methane concentrations were greater than atmospheric concentrations at all depths. Additionally, CO 2, CO and H 2 gas concentrations were analyzed in the cores to evaluate potential microbial metabolic pathways of methane production. Previous studies on methane fluxes from glacial sediments in Greenland and the Swiss Alps used single time point flux measurements during a melt season from multiple locations. This study concludes that such point measurements are unlikely representative for determining a net seasonal flux as they do not consider temporal variability. There was a two order of magnitude difference between the annual source contribution of methane based on the average melt season flux and the highest measured surface flux. This indicates that single sampling periods may significantly over or underestimate the net seasonal flux of methane from recently exposed glacial sediments to the atmosphere.

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