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Item 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.Item Aerobic bacterial methane synthesis in the human gastrointestinal tract(Montana State University - Bozeman, College of Agriculture, 2023) Jackson, Thomas Robert; Chairperson, Graduate Committee: Seth WalkAerobic 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.Item Phycosomal dynamics in xenic cultures of the alkalitolerant green Microalga chlorella sp. SLA-04(Montana State University - Bozeman, College of Agriculture, 2023) Miller, Isaac Robert; Chairperson, Graduate Committee: Matthew Fields; This is a manuscript style paper that includes co-authored chapters.The production of microalgal biomass and biofuel is an important component of the transition away from a petroleum-based economy. Industrial scale microalgal cultures are often xenic, meaning they are comprised of microalgae as well as a phycosome (i.e., microbiome). The microalgal field has begun to appreciate the ubiquity and potential influence of the phycosome, but there remains a critical need for comprehensive research to unravel the intricate metabolic and ecological relationships between microalgae and the respective phycosome that can be comprised of mainly bacteria but also other microorganisms (i.e., archaea, fungi, protists, viruses). Phycosome research is essential for potentially using these interactions to enhance the stability, productivity, and cost-efficiency of industrial microalgal cultivation. Chlorella sp. SLA-04 is an oleaginous, alkalitolerant microalga isolated from the alkaline Soap Lake (Washington, USA). Under alkaline conditions, SLA-04 can be grown to high biomass levels without reliance on the delivery of concentrated CO 2, an improvement in producing competitively priced biomass and biofuel. The high pH, high alkalinity systems are able to capture CO 2 directly from the air in open systems (e.g., raceway ponds) but the open systems can be dynamic in terms of stability and productivity. Despite growing knowledge of the importance of phycosomes in open production systems, little is known about how alterations to cultivation conditions can be used to maintain a xenic system with controllable outputs, especially under high pH, high alkalinity conditions. The work outlined in this dissertation employed long term temporal community studies, open outdoor raceway experiments, diel-cycle-resolved temporal sampling coupled with activity-based probing (bioorthogonal non-canonical amino acid tagging (BONCAT)), and quantitative measures of algal physiology to better understand the relationship between microalgal phenotype and the respective phycosomes. SLA-04 phycosome composition and culture physiology were consistent over time when maintained in xenic cultures under low and high alkalinity. When xenic cultures were used in successive open, outdoor raceway experiments, compositional community changes coincided with seasonal temperature and light shifts, providing evidence that abiotic and biological environmental stresses impact directly and indirectly SLA-04 productivity and phycosome composition. By employing temporally resolved sampling and probing the relationship between diel-cycle-dependent metabolism and the phycosome, we identified active bacterial populations that may play a role in culture productivity. Expanding beyond augmenting SLA-04 productivity, aggregation of xenic cultures was assessed as a quantifiable phenotype, uncovering a relationship between aggregation, taxonomic composition and algal growth conditions (i.e., alkalinity level). All together, these results represent an initial description of the ecology (e.g., composition, succession, activity) of alkaline microalgae cultures and provide methodology and perspective for future phycosome studies.Item Microbiomes and zoonoses: dynamics of the black flying fox (Pteropus alecto) gastrointestinal microbiome(Montana State University - Bozeman, College of Agriculture, 2022) Jones, Devin Nicole; Chairperson, Graduate Committee: Raina K. Plowright; This is a manuscript style paper that includes co-authored chapters.Land-use change is increasingly recognized as a driver of spillover of zoonotic pathogens. Australian black flying foxes (Pteropus alecto) are experiencing extensive loss of habitat which reduces available food, particularly in winter. Hendra virus (HeV, family: Paramyxoviridae) was isolated from horses and humans in 1994 and P. alecto was later identified as the reservoir host. As habitat loss threatens these bat populations, and Hendra virus continues to spill over to horses annually, it is important to understand factors that influence bat health and viral shedding. Because gastrointestinal tract (GIT) microbiomes are important for host health and are understudied in flying foxes, the goal of this research was to understand the natural dynamics of the P. alecto GIT microbiome and its associations with diet, body composition, markers of inflammation, and viral shedding. We sampled Pteropus alecto near Brisbane from 2018-2020. We captured bats returning from foraging and collected rectal swabs to determine the GIT microbiome using 16S rRNA amplicon sequencing. In addition to feces for dietary analysis, we also collected samples to measure health and infection, including blood to measure neutrophil-to-lymphocyte ratios, urine to detect Hendra virus, and bioelectrical impedance analysis to measure body fat. These data enabled us to determine how the P. alecto GIT microbiome varied within individuals over time and in the context of physiological, ecological, and dietary shifts. Lastly, we asked if we could predict health outcomes using the GIT microbiome. We found that P. alecto GIT microbiomes are highly dynamic over time, through different life stages, between foraging strategies, and that the type of diet is associated with GIT microbiome diversity. Bats consuming native foods had lower GIT microbiome diversity compared to those consuming introduced and cultivated foods. Despite associations between body fat and HeV infection, the GIT microbiome was not able to predict these health outcomes. These results suggest that P. alecto GIT microbiomes are highly dynamic and may not contribute significantly to host health. Future research should incorporate more health metrics or other approaches to microbiome profiling to determine if the GIT microbiome could be used as a biomarker of health.Item Diet-induced alterations to postprandial metabolism and the gut microbiota(Montana State University - Bozeman, The Graduate School, 2022) Wilson, Stephanie Michelle-Gandia; Chairperson, Graduate Committee: Mary P. Miles; This is a manuscript style paper that includes co-authored chapters.Obesity is a key component of a cluster of metabolic risk factors for chronic disease which include dyslipidemia, hyperglycemia, and hypertension. Stark changes in diet and lifestyle contribute to growing metabolic disorder prevalence. Many Americans regularly consume foods low in fiber and rich in fat and sugar, which can negatively influence glucose and lipid metabolism over time. A nutritionally poor diet exerts deleterious effects on the gastrointestinal microbial community which has larger host health implications. As a modifiable risk factor, diet can be part of the solution to counter the rise of chronic disease. However, dietary responses as it pertains to glucose and lipid metabolism display high interindividual variability. This interindividual variability with diet can also be observed at the microbial level in our gastrointestinal system. In metabolically at-risk adults, we examined postprandial responses and the efficacy of a long-term lentil dietary intervention in improving postprandial glycemic responses. We further assessed the effect of an antioxidant-rich juice from the Aronia melanocarpa berry and its anti-inflammatory potential against a high fat diet in a gnotobiotic mouse model to evaluate how specific gut microbial communities transferred from metabolically at-risk adults with different inflammatory profiles may impact dietary responses. Postprandial triglyceride responses in adults with overweight and obesity can be partially explained by central adiposity, insulin resistance, and the ability to switch between glucose and fat metabolism to a metabolic stress. A long-term dietary intervention with lentils, a fiber-rich pulse crop, worked in a dose-dependent manner to reduce insulin resistance in adults with increased central adiposity without an increase in gastrointestinal symptoms, a common deterrent to incorporating fiber-foods in the diet. Our gnotobiotic mouse experiments revealed donor- dependent changes in dietary responses. We observed protective effects of Aronia juice in mice, particularly in mice from the low inflammation stool donor. Metabolomic changes in phospholipids such as phosphatidylcholine and sphingomyelin were further detected, with changes respectively unique to Aronia juice and high-fat diet. Taken together, this dissertation provides an improved understanding of our metabolic responses and microbial alterations to the foods we consume, and how these responses influence the progression of metabolic diseases.