Theses and Dissertations at Montana State University (MSU)

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Intersection of SARS-CoV-2 and CRISPR-CAS defense systems
(Montana State University - Bozeman, College of Agriculture, 2021) Wiegand, Tanner Roy; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.
Viral predators exploit cellular resources in all domains of life. To defend against these genetic invaders, bacteria and archaea have evolved adaptive immune systems comprised of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins. In this dissertation, I investigate the biological mechanisms and biotechnological applications of CRISPR-Cas systems. The sequences that interspace the eponymous repeats of CRISPR loci are derived from mobile genetic elements, including bacteriophages (i.e., viruses that infect bacteria). When the locus is transcribed into CRISPR-RNA, these spacer sequences guide nucleases to RNA or DNA molecules with complementary sequences, resulting in degradation of the target nucleic acid. While recent work has illuminated many details of CRISPR-RNA-guided surveillance and target interference, the process of new sequence adaptation remains more mysterious. Initially, the goal of this research was to understand how new spacer sequences are acquired and integrated at CRISPR loci. High throughput sequencing of spacers acquired in in vivo adaptation assays revealed that some spacer sequences are reproducibly acquired in the I-F CRISPR system of Pseudomonas aeruginosa, and that the I-F CRISPR-guided surveillance complex enhances the efficiency of new spacer acquisition. We then used bioinformatic and in vitro acquisition assays to show that adaptation in many systems is dependent on the presence and phasing of sequence motifs in the transcriptional leaders of CRISPR loci. Collectively, these results expand our understanding of how CRISPR-Cas systems adapt to new threats. Following the emergence of SARS-CoV-2, and the ensuing international COVID-19 pandemic, my research goals pivoted to developing methods to track the spread of this coronavirus and to understanding how it was evolving. Long read genomic sequencing was used to determine the likely evolutionary origin of SARS-CoV-2 samples isolated from wastewater and human patients. This work led to the identification of isolates with large genomic deletions and shows that while these mutations cause a replication defect in the virus, similar mutations have appeared multiple times, independently in the evolution of SARS-CoV-2. Finally, we show that type III CRISPR-Cas systems can be repurposed for molecular detection of SARS-CoV-2 and investigate how these new diagnostic platforms can be improved.
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.
Resistance temperature devices fabricated using micro electromechanical systems technology designed and characterized for low-temperature applications
(Montana State University - Bozeman, College of Engineering, 2020) Thomae, Madelyn Ruth; Chairperson, Graduate Committee: Stephan Warnat
Research shows that microorganisms play a major role in climate change, but there is a lack of adequate understanding of microbial involvement in climate change and further research is needed for greater understanding. Temperature monitoring lends an insight into the current climatic shifts in Arctic and Antarctic regions. Currently, satellite monitoring is used to track temperature changes in those regions. To further the understanding of the role microorganisms play in the rising temperatures in those regions, in-situ temperature monitoring is needed. Commercially available temperature probes are high in cost and not well-suited for the harsh environment of Arctic and Antarctic regions. Utilizing micro electromechanical systems technology provides a solution for robust low-cost, low-power sensors that can be designed specifically to operate in harsh environments. Gold resistance temperature devices were designed and fabricated using micro electromechanical systems technology with a high spatial resolution capable of detecting microorganisms in subzero applications. The fabricated temperature sensors were calibrated for subzero use and freezing experiments were done to detect any changes due to impurities in the sample. The gold resistance temperature devices were able to withstand prolonged exposure to the harsh experimental environment and provide an accurate spatial temperature gradient throughout the media. The gold resistance temperature devices had negligible effects due to the self-heating phenomenon common in resistance temperature devices. Additionally, the sensors were able to detect variations in the freezing curve of the media with the inclusion of the bacterial isolate Flavobacterium sp. ANT 11 (accession number GU592435) in DI water samples. Future research should focus on (1) furthering the understanding of the microbial interactions in the cooling curves of different medias and (2) integrating electrical impedance spectroscopy sensors to provide knowledge of what impurities are in the sample that could be affecting the freezing curve of the media.
Soil legacy effects alter plant volatile emissions in response to diversified cropping systems
(Montana State University - Bozeman, College of Agriculture, 2020) Malone, Shealyn Chelsea; Chairperson, Graduate Committee: David K. Weaver and Amy Trowbridge (co-chair); David K. Weaver, Fabian Menalled, Tim Seipel, Justin B. Runyon, Lila Hamburg, Megan L. Hofland and Amy M. Trowbridge were co-authors of the article, 'Cropping systems alter crop volatile cues important for insect pests through soil legacy effects' which is contained within this thesis.; David K. Weaver, Tim F. Seipel, Fabian D. Menalled, Megan L. Hofland, Justin B. Runyon and Amy M. Trowbridge were co-authors of the article, 'Soil microbes alter herbivore-induced volatile emissions in response to cereal cropping systems' submitted to the journal 'Plant and soil' which is contained within this thesis.
Soil microbes can influence the emissions of plant volatile organic compounds (VOCs) that serve as host-location cues for insects and their natural enemies. The influence of the soil microbial community on the plasticity of plant VOC synthesis and emissions is particularly important in agricultural settings where crop rotations and management practices cause significant shifts in the soil microbiome. Studies have shown agricultural soils to influence plant-insect interactions through changes in foliar chemistry, but their potential to alter VOC emissions is unknown. To determine the effect of diversified agricultural practices on crop VOC emissions through microbe-mediated soil legacy effects, I measured VOCs from wheat (Triticum aestivum L.) in a series of field and greenhouse experiments. In Chapter II, I determined the effect of the soil microbiome on VOCs in the greenhouse by first measuring VOCs from wheat plants grown in sterilized soil or soil with added inoculum from an agricultural field. Next, to determine the effect of diversified agricultural practices on VOC phenotypes, I measured VOCs from wheat plants in the field in rotation with either fallow or a mixture of cover crops that was terminated by grazing cows. Finally, in Chapter III, I explored the interactive effect of herbivory and the soil microbiome on VOC emissions in a full factorial experiment in which wheat grown in soil inoculum from wheat-fallow or wheat-cover crop rotation that was subjected to larval feeding by the wheat stem sawfly (WSS; Cephus cinctus Norton), a major pest of wheat. Across all studies I found that soils associated with a higher microbial diversity--cover crop soils and inoculated soils--tended to emit more total VOCs and blends that would likely increase pest resistance to the WSS through 1) shifts in key bioactive compounds and 2) enhanced herbivore-induced VOC emissions. Results also suggest that soil microbes may be more likely to alter plant VOCs when plants experience abiotic or biotic stressors. Together, these results suggest that agricultural practices may indirectly influence plant resistance through microbe-altered VOCs, and these effects are more likely to occur when plants experience additional stressors, such as herbivory or drought.
Microbially induced calcium carbonate precipitation: meso-scale optimization and micro-scale characterization
(Montana State University - Bozeman, College of Engineering, 2020) Zambare, Neerja Milind; Chairperson, Graduate Committee: Robin Gerlach and Ellen G. Lauchnor (co-chair); Ellen Lauchnor and Robin Gerlach were co-authors of the article, 'Controlling the distribution of microbially precipitated calcium carbonate in radial flow environments' in the journal 'Environmental science and technology' which is contained within this dissertation.; Robin Gerlach and Ellen Lauchnor were co-authors of the article, 'Spatio-temporal dynamics of strontium partitioning with microbially induced calcium carbonate precipitation in porous media flow cells' submitted to the journal 'Environmental science & technology' which is contained within this dissertation.; Robin Gerlach and Ellen Lauchnor were co-authors of the article, 'Co-precipitation of strontium and barium' submitted to the journal 'Environmental science & technology' which is contained within this dissertation.; Nada Naser, Robin Gerlach and Connie Chang were co-authors of the article, 'Visualizing microbially induced mineral precipitation from single cells using drop-based microfluidics' submitted to the journal 'Nature methods' which is contained within this dissertation.
Microorganisms have the potential to impact processes on a scale orders of magnitude larger than their size. For example, microbe-mineral interactions at the micro-scale can drive macro-scale processes such as rock formation and weathering. Many bioremediation technologies derive inspiration from microbial mineralization processes. Microbially induced calcium carbonate precipitation (MICP) can produce calcium carbonate (CaCO 3) precipitates which can be utilized as a biological cement to strengthen porous media by reducing fluid permeability in subsurface fractures or as an immobilization matrix to remove metal contaminants dissolved in groundwater. To make MICP a feasible and successful bioremediation technology in the world outside the lab, it is necessary to bridge the gap between the meso-scale research studies and macro-scale applications. This thesis focuses on such meso-scale studies but also contributes to bridging the gap in the other direction, i.e., meso-scale to micro-scale to gain a fundamental understanding of the cellular level processes behind MICP. The research presented here investigates two applications of MICP with a focus on controlling precipitate distribution and process efficiency in target environments. Subsurface precipitate distribution and metal partitioning during MICP were studied in novel reactive transport systems that mimic application-environment conditions. A radial flow reactor was used to study the spatial distribution of precipitates in conditions similar to subsurface injection well environments. The distribution and degree of metal partitioning during MICP was investigated in batch reactors and porous media flow cells to study kinetics and reactive transport effects on kinetics. In the radial flow environment, more precipitates formed away from the center injection zone. Results showed that longer reactant residence times and an equimolar ratio of calcium to urea were able to maximize precipitation efficiency. Metal partitioning could be maximized at low reactant flow rates and low metal concentrations. The novel flow cell set up used revealed a spatial decoupling between ureolysis and precipitation. A micro-scale investigation of the fundamental MICP process itself is presented wherein microbe-mineral interactions are observed at the cell level. A semi-correlative approach to investigating individual precipitates in microdroplets is presented, using a multitude of microscopy and microanalysis techniques. The presented studies capture MICP across a range of scales.
Impacts of crop rotations and nitrogen fertilizer on soil biological factors in semi-arid Montana
(Montana State University - Bozeman, College of Agriculture, 2021) Fouts, Willa Constance; Chairperson, Graduate Committee: Catherine A. Zabinski
Evaluating the effects of cropping and fertilizing techniques is key to informing agricultural best practices. We must continue monitoring how we manipulate soils in order to preserve and cultivate high-quality soil ecosystems that can support us in the face of climate change and widespread soil loss and deterioration. We assessed the effects of common agricultural practices in Montana by measuring biological indicators of soil quality in the 18th year of a field plot experiment with 100% and 50% the recommended rate of synthetic nitrogen (N) fertilizer and crop rotations incorporating wheat, fallow, and legumes. The biological indicators measured were four soil extracellular enzymes, potentially mineralizable N (PMN), and microbial biomass. We sampled once in spring 2020 and subsampled in the fall. We also tested whether enzymes and PMN were correlated to aboveground plant residue, which was represented by the sum of the dried plant mass from past two years left on the plots after harvest. Plant residue was positively correlated with the C, N, and S-cycling enzymes and to PMN. The positive correlation between PMN and residue reflects that increased biomass inputs could increase easily mineralizable N. Soil with the high N-rate had a slightly higher geometric mean enzyme activity. This could be from the resulting increase in plant residue. The high N-rate treatment slightly decreased soil PMN but was not affected by crop rotation treatments. Fallow systems had lower enzyme function overall, indicating a lessened fertility and decomposition rate compared to continuously cropped treatments, which keep the soil covered with a crop for more months out of the year. The positive correlations of plant residue, along with the general lower performance of the fallow systems, especially the tilled fallow rotation, support that aboveground biomass inputs are a driver in soil ecosystem function. Continuous no-till crop rotations have increased aboveground plant organic matter, which could increase nutrient cycling and decomposition, and thereby soil biological quality and fertility.
Investigating arsenic-microbiome interactions in the gut using murine models
(Montana State University - Bozeman, College of Letters & Science, 2019) Coryell, Michael Philip; Chairperson, Graduate Committee: Seth Walk; B. A. Roggenbeck and Seth T. Walk were co-authors of the article, 'The human gut microbiome's influence on arsenic toxicity' submitted to the journal 'Current pharmacology reports' which is contained within this thesis.; M. McAlpine, N.V. Pinkham, T.R. McDermott and Seth T. Walk were co-authors of the article, 'The gut microbiome is required for full protection against acute arsenic toxicity in mouse models' in the journal 'Nature communications' which is contained within this thesis.; M. Yoshinaga, T.R. McDermott and Seth T. Walk were co-authors of the article, 'Speciation of excreted arsenicals from germ free and conventional AS3MT knockout mice exposed to inorganic arsenate' which is contained within this thesis.
Drinking water contamination with arsenic is a wide-spread public health concern, potentially affecting over 140 million people across at least 40 different countries. Current understanding of biological and behavioral factors influencing clinical outcomes is insufficient to explain the variation observed in arsenic-related disease prevalence and severity. The intestinal microbiome in humans is a dynamic and active ecosystem with demonstrated potential to mediate arsenic metabolism in vitro and distinct variability between individuals. This dissertation investigates arsenic-microbiome interactions, with a focus on determining how microbiome activity influences host-response and toxicity from arsenic exposures. Chapter 2 overviews common exposure routes, important metabolic pathways, and current evidence of arsenic-microbiome interactions in humans or experimental animal models. Chapter 3, the initial approach was to experimentally perturb the microbiome of common laboratory mice during arsenic exposure, measuring arsenic excretion in the stool and accumulation in host tissues. Arsenic sensitive gene-knockout mice were used to determine the microbiome's influence on subacute arsenic-induced mortality. Disrupting microbiome function--first by antibiotic treatment, then by deriving mice germ free--dramatically reduced survival times during severe arsenic exposures. Transplantation of human fecal communities into germ free mice effectively complemented the loss of function from microbiome disruption in these mice. Chapter 4 examines microbiome's impact on arsenic metabolism in germ free and conventional mice from this same arsenic-sensitive genetic background. These mice are deficient for the primary metabolic pathway involved in arsenic detoxification in both humans and mice, facilitating a more complete experimental isolation of microbiome and host metabolisms. This study provides evidence of microbiome-dependent changes in the elimination routes and metabolic transformation of ingested arsenic and provides a new experimental model for studying arsenic metabolism in the gut.
A study of bio-mineralization for the application of reducing leakage potential of geologically stored CO 2
(Montana State University - Bozeman, College of Engineering, 2019) Daily, Ryanne Leigh; Chairperson, Graduate Committee: Adrienne Phillips
A primary concern of carbon capture and storage (CCS) is leakage of the stored carbon dioxide (CO 2) from the subsurface back to the surface. To ensure long term storage of the CO 2, mitigation strategies are being developed to seal high permeability regions, such as fractures present in the caprock or the near wellbore environment. Ureolysis induced calcium carbonate precipitation (UICP) is a widely investigated technology utilizing the enzymatically driven process of ureolysis to alter the properties of porous media. The advantage of this technology over traditional fracture sealing methods, such as well cement, is the use of low-viscosity aqueous fluids enabling access to smaller fractures. However, CCS reservoirs provide a problematic environment for microbial activity due to the acidity of dissolved CO 2, high pressures, and elevated temperatures. A flow-through pressurized reactor experiment and batch high-pressure ureolysis rate experiments were conducted to investigate the application of UICP technology to mitigate CO 2 migration. First, UICP was induced in two composite rock cores in an environment simulating a CCS reservoir, using a high-pressure axial flow reactor, with an initial and final exposure of the rock cores to a carbonated brine. As a result of UICP, the apparent permeability of the rock cores were reduced by 5-orders of magnitude. The CO 2 challenge increased apparent permeability by 4-orders of magnitude, likely due to a preferential flow path created through the calcium carbonate (CaCO 3) seal, which was found with X-ray microcomputed tomography (micro-CT) imaging. The porosity of the composite rock cores was assessed throughout the experiment with two non-invasive technologies, micro-CT and nuclear magnetic resonance (NMR), both reported a significant decrease in porosity due to UICP and a slight increase after the CO 2 exposure. Second, ureolysis kinetics were assessed in the presence of a pressurized carbonated brine at pressures between 0 and 4 MPa. The kinetic studies were performed in a high-pressure batch reactor connected to high-pressure pH and conductivity probes. Samples could not be taken from the batch reactor without losing pressure; thus, conductivity was used as a surrogate measurement for urea concentration. It was found that, for the pressures tested, JBM urease was capable of hydrolyzing urea in the presence of a pressurized carbonated brine. It was also hypothesized that the rate observed at each experimental pressure may have been dependent on the buffered pH of the system. The combination of these studies suggests that, if the challenge of dissolution could be overcome, bio-mineralization may be used to enhance CCS by reducing the permeability of CO 2 leakage pathways.
Microbial ecology of mosquitos and ticks
(Montana State University - Bozeman, College of Letters & Science, 2017) Pinkham, Nicholas Vernon; Chairperson, Graduate Committee: Seth Walk
Host evolutionary history has been shown to select for distinct host associated microbial communities over large evolutionary time scales. The microbiomes of disease vector have been shown to alter the capacity of their host to vector pathogens. Much remains to be understood about how the microbiome of mosquitos and ticks assemble in situ. We conducted a large-scale investigation of microbiome composition between mosquito species as well as a second investigation of microbiomes of brown dog ticks collected in Iquitos, Peru. Intraspecific and interspecific bacterial community diversity was compared across 26 species of mosquitoes collected in Montana. Previous studies of lab reared mosquitoes report greater variation in microbial communities between species than within. Using 16S rRNA sequencing we observed a large amount of intraspecific variation in microbiomes, as well as different species hosting very similar microbiomes. The tick microbiome was found to be dominated by a few select community members that were seen at an extremely high abundance and resembled intracellular tick-borne pathogens. It is common for ticks to host endosymbionts that closely to human pathogens but are not pathogenic themselves. Negative interactions were seen between the most abundant organism observed in the ticks.
Microbial interactions and the role of environmental stress in natural and synthetic consortia
(Montana State University - Bozeman, College of Letters & Science, 2018) Beck, Ashley Esther; Chairperson, Graduate Committee: Ross Carlson; Kristopher A. Hunt, Hans C. Bernstein and Ross P. Carlson were co-authors of the chapter, 'Interpreting and designing microbial communities for bioprocess applications, from components to interactions to emergent poperties' in the book 'Biotechnology for biofuel production and optimization' which is contained within this thesis.; Kristopher A. Hunt and Ross P. Carlson were co-authors of the article, 'Measuring cellular biomass composition for computational biology applications' submitted to the journal 'Processes, methods in computational biology special issue' which is contained within this thesis.; Hans C. Bernstein, and Ross P. Carlson were co-authors of the article, 'Stoichiometric network analysis of cyanobacterial acclimation to photosynthesis-associated stresses identifies heterotrophic niches' in the journal 'Processes, microbial community modeling: prediction of microbial interactions and community dynamics special issue' which is contained within this thesis.; Kathryn Pintar, Diana Schepens, Ashley Schrammeck, Tim Johnson, Alissa Bleem, Hans C. Bernstein, Tomas Gedeon, Jeffrey J. Heys and Ross P. Carlson were co-authors of the article, 'Escherichia coli co-metabolizes glucose and lactate for enhanced growth' submitted to the journal 'Applied and Environmental Microbiology' which is contained within this thesis.; Ross P. Carlson was a co-author of the article, 'Synthetic consortia engineered for push and pull dynamics show conditional optimality over metabolic generalist' which is contained within this thesis.
Microbial communities are critical underpinnings of most natural processes, e.g. biogeochemical cycling, and can also be harnessed and engineered for a variety of industrial applications. Despite the abundance of detailed physiological characterization of many individual microorganisms, as well as large data sets describing microbial community composition, the area of interspecies interactions requires further research to truly appreciate and harness the potential of microbial capabilities. Using a combination of in silico metabolic modeling and in vitro laboratory approaches linked to guiding ecological theories, this dissertation investigates metabolite exchange as a mechanism of interspecies interactions and focuses on the role of environmental stress in mediating interactions. A stoichiometric metabolic network model was constructed for the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 and was analyzed with elementary flux mode analysis to predict metabolic acclimations to light and oxygen, two common environmental stressors in photoautotrophic habitats. High stress levels were predicted to activate organic byproduct secretion pathways, which opens a niche to support growth of heterotrophic partners. To further investigate metabolite exchange in the laboratory, synthetic consortia were designed through genetic engineering and pairing of Escherichia coli strains to form metabolically partitioned organic acid cross-feeding systems. These controlled systems were used to investigate the impact of division of labor as well as the effect of byproduct detoxification. Kinetic data from these systems were also applied to interpret ecological theories regarding microbial community structure. Altogether, these studies demonstrate an integrated approach to studying microbial community interactions by combining in silico metabolic modeling and in vitro laboratory experiments with ecological theory as a basis for interpretation. This dissertation provides insight into rationale for microbial community structure and highlights the role of environmental stress, particularly byproduct inhibition, in driving microbial consortia interactions.

Theses and Dissertations at Montana State University (MSU)

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