Theses and Dissertations at Montana State University (MSU)

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Thermoregulatory adaptations of the wetsalts tiger beetle, Cicindelidia haemorrhagica, in Yellowstone National Park
(Montana State University - Bozeman, College of Agriculture, 2021) Bowley, John Lawrence; Chairperson, Graduate Committee: Robert K. D. Peterson; This is a manuscript style paper that includes co-authored chapters.
The wetsalts tiger beetle, Cicindelidia haemorrhagica (LeConte) is found throughout Yellowstone National Park (YNP) associated with active thermal hot spring areas with surface temperatures that exceed 50 °C. Previous studies suggest that YNP C. haemorrhagica behaviorally thermoregulate differently than populations from Idaho not associated with hot springs. However, the cause of this behavioral difference is unknown. Therefore, our study had five objectives: 1) compare internal temperatures of YNP and Idaho adult C. haemorrhagica in similar environmental conditions; 2) determine if the cuticular wax influences YNP beetle internal body temperature; 3) measure and analyze venter reflectivity of YNP and Idaho beetles; 4) compare YNP and Idaho beetle abdominal structures using scanning electron microscopy; and 5) analyze thermal video recordings of YNP adults to estimate times spent on different surface temperatures. Internal temperatures of freshly killed adults were recorded using thermocouples inserted into the abdomen and were suspended over a heated water bath. Our results found a mean difference of 1.03 °C between YNP and Idaho beetle internal temperatures, suggesting that the exoskeleton reflects more heat in YNP adults. Our results suggest that the wax does not play a significant role in temperature reduction. The dissected exoskeletal ventral abdominal face experienced a mean difference of 0.58 °C compared to an imitation venter, suggesting the venter was primarily responsible for heat reflection. Spectrophotometric differences revealed greater reflective properties of YNP beetle venters. Scanning electron microscopic images indicated no visible structural differences between YNP and Idaho beetles. Thermal video analysis indicated YNP adults spent an increasing amount of time on surface temperatures greater than 20 °C, but decreasing amounts of time on surfaces temperatures greater than 40 °C. Beetles from only one YNP location were on surfaces exceeding 50 °C. Our results suggest that physical properties of the abdominal venter of YNP beetles are likely responsible for improved heat reflection from bottom-up heating of hot spring areas. Improved heat reflection resulted in lower internal temperatures of YNP beetles relative to Idaho.
Identification of cellulolytic hot spring organisms through bioorthogonal labeling
(Montana State University - Bozeman, College of Letters & Science, 2021) Reichart, Nicholas John; Chairperson, Graduate Committee: Roland Hatzenpichler; This is a manuscript style paper that includes co-authored chapters.
Microbial physiology is the study of the metabolism and function of microorganisms. The recent expansion of genomic diversity has outpaced the description of physiology. To better understand microbial metabolisms and environmental processes, more detailed research is needed for both novel and undescribed microbes. While many new methods are being developed to describe in situ microbial activity, this dissertation implements bioorthogonal non-canonical amino acid tagging as a proxy to track metabolic activity of microbes under close to environment conditions. Using differential analyses on hot spring microbial communities, we were able to show that certain microbial taxa had preferential activity towards specific incubation amendments. Previous activity-based studies had shown that hot springs were a unique environment for discovering cellulolytic microbes that could be used in industrial processing of plant biomass. Herein, we used computational analysis to screen publicly available metagenomic datasets to identify the enzymatic potential of hot springs worldwide. The wide diversity of taxa and biomass degrading enzymes were investigated and hot springs were further highlighted as a system that could be used to find improvement for the industry of plant biomass degradation and processing. To build upon the cellulolytic potential found in hot spring metagenomic datasets, bioorthogonal non-canonical amino acid tagging coupled with fluorescence-activated cell sorting was applied to the biotechnological relevant field of plant biomass degradation to identify microbes involved in the cellulolytic process. Examination of the active microbes revealed difference in the community when supplemented with cellulose. Taken together, the work in this dissertation served to expand and apply the recent development of activity-based studies used to describe environmental microbial populations, with a focus on plant biomass degradation.
Analysis of complex samples by mass spectrometry leads to insights into system dynamics
(Montana State University - Bozeman, College of Letters & Science, 2021) Peach, Jesse Thomas; Chairperson, Graduate Committee: Brian Bothner; James Larson, Sutton Kanta, Eric Boltinghouse, Rebecca Mueller, Ganesh Balasubramanian, Mohammed Refai, Brent Peyton and Brian Bothner were co-authors of the article, 'Optimization of thermal small molecule and protein mass spectrometry analysis' submitted to the journal 'Analytical biochemistry' which is contained within this dissertation.; Rebecca Mueller, Dana Skorupa, Margaux Mesle, Sutton Kanta, Eric Boltinghouse, Bailey Sharon, Valerie Copie, Brian Bothner and Brent Peyton were co-authors of the article, 'Longitudinal meta-analysis of the Five Sisters Hot Springs in Yellowstone National Park reveals a dynamic thermoalkaline environment' submitted to the journal 'Environmental microbiology' which is contained within this dissertation.; Stephanie M. Wilson, Logan D. Gunderson, Lizzi Frothingham, Tan Tran, Seth T. Walk, Carl J. Yeoman, Brian Bothner and Mary P. Miles were co-authors of the article, 'Temporal metabolic response yields a dynamic biosignature of inflammation' submitted to the journal 'iScience' which is contained within this dissertation.
Systems biology offers a holistic approach to biological science. In its most complete form, systems biology requires comprehensive data encompassing all of the parts or molecules across a set of hierarchical networks. To obtain and analyze the comprehensive and large datasets required for systems biology analysis, biologists have taken advantage of new technology and computational tools. Over the last few decades, advances in computational modeling and analysis technology has dramatically increased the efficacy of systems biology and the understanding of the natural world. However, systems biology is still an emerging discipline. The overwhelming scale of potential biological data that has yet to be described, coupled with interpretation and application obstacles, leaves much work to be accomplished. One aspect of systems biology that needs development is the interpretation and analysis of temporal biological data. Temporal data reveals more about biological phenomena than static data as biology is inherently dynamic. This dissertation explores the benefits of temporal profiling of complex samples to make time-resolved conclusions about complicated biological questions. Three research projects are the backbone of this document, with a chapter being devoted to each. Chapter 2 describes the development of a comprehensive method for extraction and mass spectrometry analysis of several different fractions from hot spring sediment. Chapter 3 delves into a multi-omics analysis tracking changes over the course of three years in a thermoalkaline spring system in Yellowstone National Park. It defines how specific extracellular small molecules correlate with microbial fitness. Specifically, how unique nitrogen and sulfur containing molecules in the sediment drive archaeal abundance and diversity. The final chapter introduces the concept of a 'dynamic biosignature', a set of metabolites that have similar responses to known biomarkers, in this case pro-inflammatory cytokines. A cohort of metabolites was identified that provided mechanistic insight into the inflammatory response. Overall, this dissertation provides examples of systems biology analysis and provides evidence that static, single time-point datasets fail to capture that which is the essence of biology - change.
Virus host interactions at the single cell level in hot springs of Yellowstone National Park
(Montana State University - Bozeman, College of Letters & Science, 2019) Munson-McGee, Jacob Hampton; Chairperson, Graduate Committee: Mark J. Young; Jamie C. Snyder and Mark J. Young were co-authors of the article, 'Introduction to archaeal viruses' in the journal 'Genes' which is contained within this dissertation.; Ross Hartman was an author and Mark J. Young were co-authors of the article, 'vFish for the quantification of viral infection in natural environments' submitted to the journal 'Environmental microbiology' which is contained within this dissertation.; Erin K. Field, Mary Bateson, Colleen Rooney, Ramunas Stepanauskas and Mark J. Young were co-authors of the article, 'The identification and characterization of a nanoarchaeota, its cellular host and a nanoarchaeal virus across Yellowstone National Park hot springs' which is contained within this dissertation.; Colleen Rooney and Mark J. Young were co-authors of the article, 'An uncultivated virus infecting a nanoarchaeal parasite in the hot springs of Yellowstone National Park' submitted to the journal 'Virology' which is contained within this dissertation.; Shengyun Peng, Samantha Dewerff, Ramunas Stepanauskas, Rachel J. Whitaker, Joshua Weitz and Mark J. Young were co-authors of the article, 'A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments' in the journal 'The ISME journal' which is contained within this dissertation.
Viruses are the most abundant biological entities on the planet and virus-host interactions are some of the most important factors in shaping microbial community structure and function and global chemical cycling. The high temperature low pH hot spring of Yellowstone National Park contain simplified microbial communities of 8-10 Archaeal species, and comparatively simple viral communities. These idealized communities that contain only viruses and their Archaeal hosts represent a model natural environment for the study of viruses and their hosts. This work presented here builds on previous population level studies of the viral and microbial communities to examine virus-host interactions at the single cell level. The identification of viral infection has long been a scourge of environmental virologist. In order to identify viral infection in natural environments we have adapted Fluorescent in situ hybridization (FISH) techniques to directly identify viral sequences. We further advance this technique to be compatible with flow cytometry analysis for the rapid quantification of viral infection of uncharacterized viruses in natural environments. This technique is used to quantify viral infection of two different viruses, previously only characterized by metagenomic sequencing analysis, in four geographically separate low pH high temperature hot springs of Yellowstone National Park. Finally, we combine viral and cellular metagenomics with cellular transcriptomics and single cell genomics to identify virus host interactions at the single cell level and identify viruses that are replicating in the hot springs. This work suggests that a majority of cells in the hot springs are interacting with viruses and that a majority of the cells are interacting with multiple viruses at any given time. We also identify RNA sequences from a majority of the viral types present in the hot springs suggesting that viral replication is occurring and is an important force in determining the structure and function of the microbial communities in these hot springs. Together these works represent a significant advancement of our understanding of virus host interactions in natural environments as well as new techniques to be used in future studies.
Geomicrobiology of hydrogen in Yellowstone Hot Springs
(Montana State University - Bozeman, College of Letters & Science, 2019) Lindsay, Melody Rose; Chairperson, Graduate Committee: Eric Boyd; Daniel R. Colman, Maximiliano J. Amenabar, Kirsten E. Fristad, Kristopher M. Fecteau, Randall V. Debes, John R. Spear, Everett L. Shock, Tori M. Hoehler and Eric S. Boyd were co-authors of the article, 'Geological source and biological fate of hydrogen in Yellowstone hot springs' which is contained within this dissertation.; Maximiliano J. Amenabar, Kristopher M. Fecteau, R. Vincent Debes II, Maria Clara Fernandes, Kirsten E. Fristad, Huifang Xu, Tori M. Hoehler, Everett L. Shock and Eric S. Boyd were co-authors of the article, 'Subsurface processes influence oxidant availability and chemoautotrophic hydrogen metabolism in Yellowstone hot springs' in the journal 'Geobiology' which is contained within this dissertation.
Hydrogen (H 2) connects the geosphere and biosphere in rock-hosted ecosystems and has likely done so since early in Earth's history. High temperature hydrothermal environments, such as hot springs, can be enriched in H 2 and were likely widespread on early Earth. As such, linking the geological processes that supply H 2 to contemporary hot springs and the distribution of extant thermophilic organisms that can utilize H 2 as a component of their energy metabolism can provide insights into the environment types that supported early H 2 dependent life. Using a series of geochemical proxies, I developed a model to describe variable H 2 concentrations in Yellowstone National Park (YNP) hot springs. The model invokes interaction between water and crustal minerals that generates H 2 that can partition into the vapor phase during decompressional boiling of ascending hydrothermal waters. Fractures and faults in bedrock, combined with topographic features such as high elevation, allow for vapor to migrate and concentrate in certain areas of YNP leading to elevated concentrations of H 2. Metagenomes from chemosynthetic communities in YNP springs sourced with vapor-phase gas are enriched in genes coding for enzymes predicted to be involved in H 2-oxidation. A spring in an area of YNP (Smokejumper, SJ3) sourced with vapor-phase gas, that has the highest concentration of H 2 measured in YNP, and that is enriched in hydrogenase encoding genes was chosen to further examine the biological fate of H 2. SJ3 harbors a hyperdiverse community that is supported by mixing of oxidized meteoric fluids and volcanic gases. Transcripts coding for genes involved in H 2 uptake and CO 2 fixation were detected. The processes that control the availability of oxidants and their effect on the activity and abundance of H 2 dependent organisms was also investigated in two paired hot springs. H 2-oxidizing chemoautotrophs utilized different oxidants in the two springs and this underpinned differences in H2 oxidation activity and their identity. Together, these observations indicate that the subsurface geological processes of decompressional boiling and phase separation influence the distribution, identity, and activity of hydrogenotrophs through their combined effects on the availability of H 2 and oxidants.
Theory-based demarcation of hot spring microbial mat species from large DNA sequence datasets
(Montana State University - Bozeman, College of Agriculture, 2018) Wood, Jason Michael; Chairperson, Graduate Committee: David M. Ward; Eric D. Becraft, Danny Krizanc, Frederick M. Cohan, and David M. Ward were co-authors of the article, 'Ecotype simulation 2: an improved algorithm for efficiently demarcating microbial species from large sequence datasets' submitted to the journal 'BMC bioinformatics' which is contained within this thesis.; Jason M. Wood, Frederick M. Cohan and David M. Ward were co-authors of the article, 'Biogeography of American Northwest Hot Spring A/B'-lineage Synechococcus populations' submitted to the journal 'Frontiers in microbiology' which is contained within this thesis.
The identification of closely related, ecologically distinct populations within microbial communities is paramount to understanding the structure and function of these communities. Microbial systematists have long used differences in DNA sequence relatedness to categorize the observed diversity in a community of microbes without including ecological theory to identify whether or not the identified groups are ecologically distinct. Ecotype Simulation, an evolutionary simulation algorithm based on the Stable Ecotype Model of microbial species and speciation, has been used successfully to study the diversification of thermophilic A/B'-lineage Synechococcus living in the effluent channels of alkaline-siliceous hot springs in Yellowstone National Park. However, Ecotype Simulation is an extremely slow program that is unable to handle the quantity of data produced by modern DNA sequencing technologies. I introduce a new version of this algorithm, called Ecotype Simulation 2, that permits the rapid analyses of microbial diversity from very large DNA sequence datasets. Results from this new version of the Ecotype Simulation algorithm compare favorably with results from the old version, but with analyses performed much more quickly on a much greater quantity of sequences sampled. The new algorithm was used to analyze three datasets. First, the biogeography of thermophilic A/B0-lineage Synechococcus living in hot springs of the American Northwest was analyzed. Results suggested a surprising amount of endemism among springs sampled, as well as implications for adaptations to physical and chemical environmental features not seen before. Second, Ecotype Simulation 2 was used to study the history of change in Synechococcus populations, seasonally (winter to summer) and over a twenty-five year period. Results suggested changes in population abundances and distribution seasonally, but stability in population genetic structure over many years. Finally, Ecotype Simulation 2 was used to study the populations of other predominant phototrophic microbes living along temperature and depth gradients in the same microbial mat community. Results suggested that the algorithm and the Stable Ecotype Model can successfully predict ecological diversity within all predominant mat taxa. Ecotype Simulation 2 provides the means for other microbiologists to base their understanding of the communities they study on evolutionary and ecological principals.
Transport of dissolved and particulate material in biofilm-lined tubes and channels
(Montana State University - Bozeman, College of Letters & Science, 2015) Jackson, Benjamin David; Chairperson, Graduate Committee: Tianyu Zhang; Isaac Klapper (co-chair)
This dissertation develops two models for biofilm-lined channels. The first model seeks to address the rate at which cells move in or out of the flow in a natural hot spring drainage channel. This is done by building a one- and then two-dimensional partial differential equation model of the stream. The model is parameterized using data gathered at Mushroom Spring in Yellowstone National Park in 2011 and 2012. Using this data, we predict erosion and adhesion rates at steady state in upper and lower regions of the stream. The second model describes the utilization of urea by biofilms in an artificial tube flow reactor. The goal of this model is to determine kinetic parameters for ureolytic biofilms. The model is created by deriving two coupled steady state ordinary differential equations, which are parametrized using experimental data. Once the model is fully described, an inverse problem is formulated and solved using a Markov Chain Monte Carlo method. From this model we obtain first order kinetic parameters for a particular strain of E. coli, and discuss results for Michaelis-Menten kinetics. These two model systems are linked by a set of intersecting elements. First, both models concern biofilm-lined channels. Second, in each model these biofilms are found in a streamflow system in which some component transfers from the flow to the biofilm or vice-versa. Third, both systems are represented by low dimensional mathematical models which seek to summarize complex physical behaviors using broad, summarizing parameters. Fourth, in both scenarios the parameters of interest are estimated by combining experimental measurements and mathematical modeling. Finally, error plays an important role in model efficacy. The effects of error are implicit in the first model, but explicitly analyzed in the second.
Inorganic carbon fixation and trophic interactions in high-temperature geothermal springs of Yellowstone National Park, WY, USA
(Montana State University - Bozeman, College of Agriculture, 2015) Jennings, Ryan deMontmollin; Chairperson, Graduate Committee: William P. Inskeep; Laura M. Whitmore, James J. Moran, Helen W. Kreuzer and William P. Inskeep were co-authors of the article, 'Carbon dioxide fixation by metallosphaera yellowstonensis and acidothermophilic iron-oxidizing microbial communities from Yellowstone National Park' in the journal 'The American Society for Microbiology Applied and Environmental Microbiology journal' which is contained within this thesis.; James J. Moran, Zackary J. Jay, Jacob P. Beam, Laura M. Whitmore, Mark A. Kozubal, Helen W. Kreuzer, and William P. Inskeep were co-authors of the article, 'The extent and mechanisms of carbon dioxide fixation across geochemically diverse high-temperature microbial communities' submitted to the journal 'Nature publishing group nature geosciences journal' which is contained within this thesis.; Kristopher A. Hunt, Ross P. Carlson and William P. Inskeep were co-authors of the article, 'Genome-enabled multi-scale analysis of autotroph-heterotroph interactions in a high-temperature microbial community' submitted to the journal 'The International Society for Microbial Ecology journal' which is contained within this thesis.
Numerous chemotrophic microorganisms inhabit high-temperature (> 65 °C) systems of Yellowstone National Park (WY, USA). Prior geochemical and metagenome characterization has identified the primary electron donors and acceptors and phylotypes distributed across a range in pH and geochemical conditions. Although several chemolithoautotrophs are expected to play a direct role in the fixation of inorganic C in these communities, little work has directly identified the importance of this process in situ. Consequently, the primary goal of this thesis was to evaluate the role of CO 2 fixation across numerous types of geothermal habitats and to explore autotroph-heterotroph interactions that may control community composition. Genes encoding enzymes for inorganic C fixation pathways were identified in assembled genome sequence corresponding to the predominant autotrophs (Crenarchaeota and Aquificales) observed in Fe(III)-oxide mats, sulfur sediments, and filamentous streamer communities. Carbon isotope (13 C) mixing models were used to interpret the 13C compositional values of microbial samples as a function of 13C-dissolved inorganic C (DIC) and 13 C-organic C (DOC and/or landscape sources). The relative abundance of autotrophs versus heterotrophs identified in complementary metagenome analysis and respective CO 2-fixation fractionation factors were utilized in site-specific mixing models to calculate minimum contributions of DIC-derived microbial C across 15 different microbial communities. Genome sequence was also used to develop stoichiometric reaction networks for a primary autotroph (Metallosphaera yellowstonensis) and heterotroph ('Geoarchaeota') important in acidic Fe(III)-oxide mats. Possible modes of biomass production were evaluated for different C sources and/or electron donors as a function of oxygen cost. The total oxygen flux was also used to predict the rate of Fe(II)-oxidation, and these values were compared to Fe(III)-oxide deposition rates and oxygen fluxes measured in situ. Stoichiometric modeling and elementary flux mode analysis established an optimum autotroph to heterotroph ratio (2.4:1) for DIC-derived biomass dependent on Fe(II) as the electron donor. Comparison of predicted Fe(II)-oxidation rates with observed Fe(III)-oxide deposition rates and oxygen flux measurements using microelectrodes suggest the importance of other oxygen consuming processes. Results from this thesis demonstrated the importance of inorganic C fixation in numerous geochemically distinct high-temperature microbial habitats, and the potential for DIC-derived biomass to support other hyperthermophilic heterotrophic organisms.
Geobiological interactions of archaeal populations in acidic and alkaline geothermal springs of Yellowstone National Park, WY, USA
(Montana State University - Bozeman, College of Agriculture, 2015) Beam, Jacob Preston; Chairperson, Graduate Committee: William P. Inskeep; Zackary J. Jay, Mark A. Kozubal and William P. Inskeep were co-authors of the article, 'Niche specialization of novel thaumarchaeota to oxic and hypoxic acidic geothermal springs in Yellowstone National Park' in the journal 'The International Society for Microbial Ecology journal' which is contained within this thesis.; Hans C. Bernstein, Zackary J. Jay, Mark A. Kozubal, Ryan deM. Jennings, Susannah G. Tringe and William P. Inskeep were co-authors of the article, 'Assembly and succession of iron oxide microbial mat communities in acidic geothermal springs' submitted to the journal 'Geobiology' which is contained within this thesis.; Zackary J. Jay, Markus C. Schmid, Margaret F. Romine, Douglas B. Rusch, Ryan deM. Jennings, Mark A. Kozubal, Susannah G. Tringe, Michael Wagner and William P. Inskeep were co-authors of the article, 'In situ ecophysiology of an uncultured lineage of aigarchaeota from an oxic hot spring filamentous 'streamer' community' in the journal 'International Society for Microbial Ecology journal' which is contained within this thesis.
Microbial communities in high-temperature acidic and alkaline geothermal springs contain abundant, novel Archaea whose role in biogeochemical cycling and community function in microbial mats is not described. This thesis utilized a complementary suite of analyses that included aqueous and solid phase geochemistry, community genomics, phylogenomics, targeted 16S rRNA gene sequencing, community transcriptomics, and microscopy to elucidate the role of novel archaeal populations in acidic sulfur and iron rich hot springs in Norris Geyser Basin, Yellowstone National Park (YNP), and alkaline microbial 'streamer' communities in Lower Geyser Basin, YNP. Novel members of the archaeal phylum, Thaumarchaeota were identified in oxic iron oxide mats and hypoxic elemental sulfur sediments in acidic geothermal springs. These two different groups of Thaumarchaeota likely utilize organic carbon as electron donors and exhibited metabolic capacities based on the presence and absence of oxygen (e.g., heme copper oxidases). The assembly and succession of iron oxide mats in acidic geothermal springs showed later colonization (> 40 d) of Thaumarchaeota. Early colonizers (< 7 d) of Fe(III)-oxide mats include Hydrogenobaculum spp. (Aquificales) and the iron-oxidizing Metallosphaera yellowstonensis (7 - 14 d), which accrete copious amounts of Fe(III)-oxides. Interaction of Hydrogenobaculum and M. yellowstonensis is important to mat formation and subsequent later colonization of heterotrophic archaea (> 40 d). The succession of these communities follows a repeatable pattern, which exhibits interplay among oxygen flux, hydrodynamics, and microbial growth. The biogeochemical and micromorphological signatures may be important for the interpretation of ancient Fe(III)-oxide geothermal deposits. Interactions between Archaea and Aquificales are also important in oxic, alkaline 'streamer' communities, which contain a novel Aigarchaeota population and Thermocrinis spp. This Aigarchaeota population (Candidatus "Calditenuis aerorheumensis") exhibits a filamentous morphology and was intricately associated with Thermocrinis spp. C. aerorheumensis is an aerobic chemoorganotroph. Oxygen is the predominant electron acceptor of C. aerorheumensis, and mRNA transcripts were elevated for heme copper oxidase complexes. Organic carbon electron donors may come from bacteria in close proximity and/or dissolved organic carbon. Archaeal interactions with Aquificales contribute to higher-order level properties (e.g., biomineralization, metabolite sharing) that are important in the formation of hot spring microbial mats and streamer communities.
Geothermal habitats as sites for year-round transmission of Fasciola hepatica
(Montana State University - Bozeman, College of Agriculture, 1995) Potts, Robert Stanley

Theses and Dissertations at Montana State University (MSU)

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