Theses and Dissertations at Montana State University (MSU)

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Biocorrosion of copper by Oleidesulfovibrio alaskensis G20 biofilms in static and dynamic environments
(Montana State University - Bozeman, College of Engineering, 2024) Keskin, Yagmur; Chairperson, Graduate Committee: Brent M. Peyton; Matthew Fields (co-chair); This is a manuscript style paper that includes co-authored chapters.
This study presents a detailed examination of the intricate relationships between Oleidesulfovibrio alaskensis G20 and copper (101), emphasizing three interconnected perspectives: the kinetics of copper toxicity in three distinct media, the impact of surface finishing on microbiologically influenced corrosion (MIC), and the interaction of G20 biofilms and copper in CDC biofilm reactors. Initially, the study concentrates on the kinetic effects of copper toxicity on the growth of G20. The research meticulously quantifies the detrimental impact of different copper (II) concentrations (6, 12, 16, and 24 micron) on bacterial growth kinetics in three media: LS4D balanced (BAL), electron acceptor-limited (EAL), and electron donor-limited (EDL). Using a non-competitive inhibition model, I50 (concentrations of copper causing 50% inhibition of bacterial growth) values were calculated to be 13.1, 13.87, and 11.31 micron for LS4D BAL, EAL, and EDL media, respectively. The second part of the study shifts its focus to the effect of surface finishing on MIC of copper 101 by G20. The biofilm and corrosion pit depths were measured through a series of sophisticated analyses employing 3D optical profilometry, Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray (EDX), and X-ray Diffraction Analysis (XRD). The research investigates how different levels of surface roughness, applied through metallographic grinding and polishing, influence corrosion. The findings demonstrate a clear pattern of both uniform and pitting corrosion across all surface finishes. Notably, a statistically significant decrease in corrosion rates was observed when the surface roughness of copper was altered from approximately 13?m to about 0.06?m. Finally, the study explores the interaction between G20 biofilms and copper (101) into CDC reactors to understand biofilm development on copper surfaces and its subsequent impact on copper corrosion in a dynamic environment over periods of 7, 9, and 14 days. The results showed robust biofilm formation through hexose and protein analyses and SEM images displaying progressive increases in SRB cell accumulation over time. Localized pit depths were measured and compared to static conditions, and pits showed only a 20% increase in a dynamic environment. These findings offer an improved understanding of the complex interactions between G20 and MIC of copper.
The interaction of heavy metals with the mammalian gut microbiome
(Montana State University - Bozeman, College of Agriculture, 2022) Coe, Genevieve Lea; Chairperson, Graduate Committee: Seth Walk; This is a manuscript style paper that includes co-authored chapters.
Heavy metals are for the most part, naturally occurring elements found in the environment. Some are essential, meaning they are involved in critical biochemical pathways, in all branches of life. Other heavy metals are non-essential and disrupt metabolic functions in most organisms rendering them toxic. The following research explored the interactions of the mammalian gut microbiome with an essential heavy metal, iron, and a non-essential heavy metal, methylmercury, acquired through the diet. The overarching goal was to define and characterize the influence of gut microbial interactions with heavy metals on host health. Novel experimental designs using murine models were designed to examine 1) the consequences of low iron challenge on the murine gut microbiome and whether host iron availability was affected and 2) the potential influence of the gut microbiome in methylmercury elimination rate and demethylation in conventional, germ-free, gnotobiotic, and humanized mice. Culturing in vitro and toxicity assays, 16S sequencing, deep metagenomic sequencing of human stool, bioinformatic analysis, transcriptional analysis of iron biomarkers, quantification of iron and mercury by ICP-MS and HPLC-ICP-MS methods were performed as well as the use of mouse models to examine iron and methylmercury interactions with the gut microbiome in vivo. Our results from this project indicate that the gut microbiome is significantly affected by loss of iron from the diet, and does not fully recover post-iron repletion, while the host is relatively unaffected by low-iron challenge to the gut microbiome. Methylmercury elimination and demethylation is significantly faster and higher, respectively, in mice with a gut microbiome, providing novel evidence in support of a role for the gut microbiome in methylmercury demethylation and elimination. However, exact mechanisms of microbial interactions with methylmercury in the gut have yet to be elucidated. Our data also suggests the possibility of host-mediated mechanisms of methylmercury demethylation, by yet unknown mechanisms that warrant further exploration.
Identification and cultivation of methyllycaconitine degraders from wild ruminants to protect against larkspur poisoning in range cattle
(Montana State University - Bozeman, College of Agriculture, 2021) Grace, Savannah Gray; Chairperson, Graduate Committee: Carl Yeoman and Craig Carr (co-chair); Joanna-Lynn C. Borgogna, Craig A. Carr, Lance B. McNew, Brian Bothner and Carl J. Yeoman were co-authors of the article, 'Degradation of toxic alklaoids in Delphinium occidentale species occurs within the gastrointestinal tract of Montana's wild ruminants' submitted to the journal 'American Society of Animal Science' which is contained within this thesis.; Joanna-Lynn C. Borgogna, Mostafa Elshahed, Lance B. McNew, Brian Bothner, Craig A. Carr and Carl J. Yeoman were co-authors of the article, 'Degradation of the toxic alkaloid, methyllycaconitine by wild ruminant species is predominantly mediated by rumen fungi' submitted to the journal 'American Society of Animal Science' which is contained within this thesis.
Tall larkspur (Delphinium spp.) in the western United States present a serious toxicity danger to rangeland cattle. Consumption of Methyllycaconitine (MLA), the toxic alkaloid in larkspur plants, can cause annual losses of 5-15% of range cattle in grazing pastures with sufficient larkspur. With the wide distribution and abundance of larkspur, wild ruminants in Montana likely encounter tall larkspur while foraging; however, no evidence suggests they are negatively affected by MLA's toxic effects. Therefore, we evaluated: i) whether alkaloids in Delphinium spp., and MLA specifically degraded within ruminal specimens collected from Montana's wild ruminant species over 48 h using in vitro incubations; ii) whether observed degradative activities were abiotic, or mediated by either the fungal or non-fungal (mostly bacterial) residents of the ruminal microbiota in wild ruminant specimens; and iii) if representative microbial isolates individually possessed the ability to degrade MLA within in vitro incubations. Rumen samples were collected from wild ruminant species during the 2019 and 2020 hunting seasons using legal methods by volunteer hunters. In all assays, total alkaloid was measured spectrophotometrically, and MLA by High-Performance Liquid Chromatography Mass Spectrometry (HPLC) from initial and final incubations. Our results demonstrated that, with the exception of white-tailed deer, all wild ruminant species exhibited variable degradative abilities in both total alkaloid (P< 0.001) and MLA (P< 0.001) assays and that such degradation was predominantly mediated by ruminal fungi. Additionally, screening of 15 fungal isolates, representing 10 known genera and 2 isolates of unknown taxonomic identity each obtained from herbivorous hosts, determined all were capable of degrading MLA to some extent. Fungal isolates obtained from wild ruminants exhibited greater degradative activity, with Aestipasuomyces R5 isolated from wild sheep degrading 71% of MLA (P<0.001). Overall, our results indicate that degradation of both total alkaloid and MLA-specifically occurs within the gastrointestinal tract of Montana's wild ruminants and that it is largely influenced by fungal activity. Additionally, fungal strains isolated from wild ruminants are capable of degrading MLA and have the potential to be further used as a direct fed microbial to rangeland cattle as an optimal way to mitigate larkspur toxicosis.
Omics approaches identify molecular mechanisms of arsenic-microbial interactions
(Montana State University - Bozeman, College of Letters & Science, 2019) Rawle, Rachel Anna; Chairperson, Graduate Committee: Timothy R. McDermott and Brian Bothner (co-chair); Yoon-Suk Kang, Brian Bothner, Gejiao Wang and Timothy R. McDermott were co-authors of the article, 'Transcriptomics analysis defines global cellular response of Agrobacterium tumefaciens 5A to arsenite exposure regulated through the histidine kinases phor and aios' in the journal 'Environmental microbiology' which is contained within this dissertation.; Monika Tokmina-Lukaszewska, Zunji Shi, Brian Tripet, Fang Dang, Timothy R. McDermott, Valerie Copie, Gejiao Wang and Brian Bothner were co-authors of the article, 'Metabolic responses to arsenite exposure regulated through histidine kinases phor and aios in Agrobacterium tumefaciens 5A' submitted to the journal 'Environmental microbiology' which is contained within this dissertation.
Arsenic is a class I carcinogen and causes various cancers and diseases. Its toxicity, prevalence, and potential for human exposure has classified arsenic as the number one environmental toxin according to the Environmental Protection Agency. Contamination of groundwater and soil leads to over 200 million human exposures above the health limit. In every environment where arsenic and microbes coexist, microbes are the principal drivers of arsenic speciation, which is directly related to bioavailability, toxicity, and bioaccumulation. These speciation events drive arsenic behavior in the soil, water, and as recent data suggests, human-associated microbiomes. This dissertation details arsenic-microbial interactions through an omics platform, utilizing transcriptomics, metabolomics, and proteomics profiling as a way to globally assess the impacts of arsenic exposure. This work followed two main aims: (1) characterize cell metabolism during arsenic exposure in soil bacterium Agrobacterium tumefaciens 5A, a model organism for arsenite oxidation, and (2) assess the impacts of specific arsenic-processing bacteria within the gut microbiome of mammals. The results of this work provide a foundational understanding for how arsenic speciation events are regulated and how they affect nutrient cycling in environmental systems, which is necessary for bioremediation and health initiatives.
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.
Selecting filter media for phosphorus removal at the Ennis National Fish Hatchery three-stage subsurface flow treatment wetland
(Montana State University - Bozeman, College of Engineering, 2017) Wallis, Jack Enoch; Chairperson, Graduate Committee: Otto Stein; Otto R. Stein, Christopher R. Allen and Ellen G. Lauchnor were co-authors of the article, 'Selecting wetland media for phosphorus removal' submitted to the journal 'Water science and technology' which is contained within this thesis.
In western Montana, phosphorus is one of the most common contaminants found in rivers and streams, threatening the health of aquatic ecosystems. In response to growing water quality concerns and new regulatory requirements, a three-stage treatment wetland was recently constructed at the Ennis National Fish Hatchery to treat wastewater generated by raceway cleaning operations. Currently only the first two stages of this system are complete and over the first two months of operation have removed over 98% of influent chemical oxygen demand, 99% of total suspended solids, 59% of total nitrogen, and 95% of total phosphorus. However, the effluent phosphorus concentration is expected to increase as organic matter accumulating in the wetland mineralizes and the phosphorus adsorption capacity of the wetland media is saturated. To maintain long-term phosphorus removal, the treatment wetland was designed with a filter unit to be filled with media capable of adsorbing large quantities of phosphorus. The purpose of this research is to choose the optimal media for this filter unit, comparing three manufactured materials (lightweight aggregate, juniper biochar, and lodgepole biochar) and four natural materials (limestone, dolomite, shale, and gravel). Batch adsorption experiments were conducted with coarse media in deionized water, coarse media in Blaine Spring Creek water, and fine media in deionized water. The difference between these batch experiments showed that water chemistry and particle size significantly affect phosphorus adsorption for a given material. Based on their high performance in batch experiments, lightweight aggregate and lodgepole biochar were tested in continuous flow columns, along with gravel to provide a baseline performance comparison. Gravel and lightweight aggregate removed more phosphorus in continuous flow columns than in batch experiments, likely due to ongoing precipitation with calcium ions in the influent. Lightweight aggregate was the top performing media in all experiments, and is recommended for use in the filter units at the Ennis National Fish Hatchery treatment wetland. Based on its phosphorus removal capacity in column experiments (1200 mg P kg -1 lightweight aggregate), the filter beds will be saturated in 14 months if the current effluent phosphorus concentration of 2.3 mg L -1 is maintained.
Introducing the ArsR regulated arsenic stimulon
(Montana State University - Bozeman, College of Agriculture, 2017) Saley, Tara Carolyne; Chairperson, Graduate Committee: Timothy McDermott
The United States EPA ranks arsenic as the number one environmental toxin. Since microorganisms are significant drivers of arsenic toxicity and mobility in nature, it is important to understand how microbes detect and react to arsenic. The microbial arsenic resistance operon (ars) is critical for sensing arsenic in the environment and controlling the cellular response to this toxin. The ars operon is minimally comprised of arsRBC, which codes for an ArsR transcriptional repressor, arsenite effluxer, and an arsenate reductase, respectively, with the operon negatively regulated by the transcriptional repressor, ArsR. Our model organism Agrobacterium tumefaciens 5A carries two ars operons, with each containing two arsR genes. We conducted an RNASeq study to examine the regulatory roles of the encoded four ArsR regulatory proteins as a function of +/- arsenite. We report that the regulatory influence of the ArsR proteins extends well beyond the ars operon, with both activation and repression effects. In addition to the expected arsenic resistance response, many cellular functions were impacted, including: phosphate acquisition/metabolism, sugar transport, chemotaxis, copper tolerance, and iron homeostasis. Each of the ArsR proteins uniquely influenced different sets of genes and an arsR regulatory hierarchy was observed, wherein ArsR1 is auto regulatory and negatively regulates arsR4, ArsR4 activates arsR2, and ArsR2 negatively regulates arsR3. ArsR3 is the least active with respect to number of genes regulated. To summarize, this study provides a more complete understanding of how microbial gene expression and biogeochemical cycling may be influenced by arsenic in the environment.
Exposure and risk to non-target receptors for agricultural spray drift of formulation types and adjuvants
(Montana State University - Bozeman, College of Agriculture, 2017) Preftakes, Collin James; Chairperson, Graduate Committee: Robert K. D. Peterson; Jerome J. Schleier III, David K. Weaver, Greg Kruger, Ryan Henry and Robert K. D. Peterson were co-authors of the article, 'Effect of insecticide formulation and adjuvant combination on agricultural spray drift' which is contained within this thesis.; Jerome J. Schleier III, David K. Weaver and Robert K. D. Peterson were co-authors of the article, 'Non-target insect risk assessment of drift reduction insecticide formulations and spray adjuvants' which is contained within this thesis.
Agricultural applications of crop protection products can have negative economic and ecological consequences when spray drift occurs. Among the ways to reduce spray drift is to manipulate the physical properties of the spray solution with formulated products and tank additives, but further research is required to better understand their effects. By measuring spray drift under field conditions and using the results to estimate ecological risk, we characterize the effect on drift for two formulation types and two adjuvants. A field study was conducted where off-target ground deposition and droplet size were measured to indicate spray drift for different combinations of the formulations and adjuvants, accounting for environmental conditions. Each treatment combination was also sprayed in a wind tunnel set up to precisely measure droplet spectra so that these could be related to deposition in the field. Finally, an efficacy experiment was conducted to test for tradeoffs between drift reduction and pest control. Results from the field study suggest that as much as 32% drift reduction was achieved by selecting between the tested formulation types, and as much as 62% by incorporating certain spray adjuvants, but this effect depended on the formulation/adjuvant combination. Treatments with smaller droplet sizes had greater drift, and there was no tradeoff between drift reduction and pest control. We assessed ecological risk for terrestrial insects in non-target habitats using a novel approach to estimate insecticide residue on plant surfaces based on ground deposition data from the field study. Exposure concentrations were combined with a cumulative distribution of species sensitivities to statistically represent the risk of toxicity for the active ingredient that was used and the levels of drift that were observed. We found that substantial reductions in the percentage of insect species affected in an off-target area could be achieved by certain formulation types and adjuvants. This work could be useful for developing a classification scheme for formulated products and tank additives based on their potential for reducing spray drift and ecological risk to non-target receptors.
Toxicity, exposure, and risk of insecticides used for mosquito management on the alfalfa leafcutting bee, Megachile rotundata
(Montana State University - Bozeman, College of Agriculture, 2017) Piccolomini, Alyssa Margaret; Chairperson, Graduate Committee: Robert K. D. Peterson; Shavonn R. Whiten, Michelle L. Flenniken, Kevin M. O'Neill and Robert K. D. Peterson were co-authors of the article, 'Acute toxicity of permethrin, deltamethrin, and etofenprox to the alfalfa leafcutting bee, Megachile rotundata (Hymenoptera: Megachilidae)' which is contained within this thesis.; Michelle L. Flenniken, Kevin M. O'Neill and Robert K. D. Peterson were co-authors of the article, 'Leaf residue toxicity and risk of mosquito insecticides to the bees, Megachile rotundata and Apis mellifera' which is contained within this thesis.; Michelle L. Flenniken, Kevin M. O'Neill, Ruth P. O'Neill, Casey M. Delphia and Robert K. D. Peterson were co-authors of the article, 'The effects of an ultra-low-volume application of etofenprox for mosquito management on Megachile rotundata (Hymenoptera: Megachilidae) larvae and adults in an agricultural setting' which is contained within this thesis.
The alfalfa leafcutting bee, Megachile rotundata F. (Hymenoptera: Megachilidae), is one of the most managed solitary bees and is an important pollinator of many crops, especially alfalfa, Medicago sativa L. However, little is known about its response to insecticides, specifically pyrethroids, which are frequently used to manage populations of adult mosquitoes that inhabit the same areas. Current regulatory requirements for insecticide toxicity to non-target insects focus on one pollinator, the honey bee, Apis mellifera L., but this species does not represent all insect pollinator species in terms of response to insecticides. Therefore, we characterized the toxicity and risk of three pyrethroid insecticides (permethrin, deltamethrin, and etofenprox) on adult M. rotundata in both laboratory and field settings. The median lethal dose, LD 50, was estimated for adult M. rotundata females when exposed to each pyrethroid to serve as a baseline toxicity test to determine the susceptibility of M. rotundata to these insecticides. The range of concentrations for permethrin and etofenprox ranged from 0.0075-0.076 microgram/bee and the range for deltamethrin was 0.0014-0.0075 microgram/bee. The estimated LD 50 results for permethrin, etofenprox, and deltamethrin were 0.057, 0.051, and 0.0016 microgram/bee, respectively. After obtaining the LD 50 values, we compared female respiration rates after dosing of each LD 50 endpoint. In a field study, we applied a formulated version of each active ingredient at the maximum labeled rate of 0.017 kg/ha over an alfalfa field via ultra-low-volume (ULV) applicator and observed mortality of both adult A. mellifera and M. rotundata for 48-hr after exposure. In both species, there was no significant difference in mortality between control and treated groups for any of the formulations. In another field study, a formulated version of etofenprox was applied in an alfalfa field at the half-maximum labeled rate of 0.003 kg/ha and directly targeted to M. rotundata nests. There was no significant difference in mortality between control and treated groups. We also did not observe a significant difference in the number of adults reared between treated vs. control shelters. Results from the field studies suggest that the risk of mortality from these insecticides applied via ULV applicators may be relatively low.
The effect of oxygen supplementation on the toxicity of ammonia (NHâ‚ƒ) in rainbow trout Oncorhynchus mykiss (Richardson)
(Montana State University - Bozeman, College of Letters & Science, 1992) Hanna, Todd David

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