Theses and Dissertations at Montana State University (MSU)

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    Understanding physiological adaptations, metabolic potential and ecology in a novel photoautotrophic alga for biofuel production
    (Montana State University - Bozeman, College of Letters & Science, 2019) Corredor Arias, Luisa Fernanda; Chairperson, Graduate Committee: Matthew Fields; Elliot B. Barnhart, Al Parker, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Impact of temperature, nitrate concentration, PH and bicarbonate addition on biomass and lipid accumulation of a sporulating green alga' which is contained within this dissertation.; Thiru Ramaraj, Huyen Bui, Mensur Dlakic, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Genomic insights into a sporulating, non-motile, oligotrophic green microalga (PW95)' which is contained within this dissertation.; Huyen Bui, Thiru Ramaraj, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Transcriptomic profiling of Chlamydomonas-like PW95 cultivated in coal bed methane production water with the native microbial community' which is contained within this dissertation.; Anna J. Zelaya, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Associations between sympatric bacterial groups and a novel green alga cultivated in coal bed methane production water' which is contained within this dissertation.
    Commercial implementation of microalgal biomass as bio-oil/chemical feedstocks has been difficult to achieve, and challenges include water/nutrient sources, CO 2 delivery, and community dynamics of mixed cultures. We employed an integrated approach to the study of microalgal production systems to advance towards sustainable implementation of industrial microalgal biofuel production using a native alga (Chlamydomonas-like alga, PW95) isolated from Coal Bed Methane (CBM) production water. Our approach was based on the evaluation of PW95 physiological responses to combinations of growth constraints, the determination of its genomic and functional potential, phylogenetic relations and the implementation of an ecosystem view to algal biomass production. PW95 growth and lipid accumulation (biofuel potential) were ascertained in standardized media and CBM water through the evaluation of mixed effects of temperatures, nitrate levels, pH, and bicarbonate to elucidate interactions between multiple environmental variables and nutritional levels. The biofuel potential of PW95 ranges between 20-32% depending on culture conditions and our results suggest an important interaction between low nitrate levels, high temperature, and elevated pH for trade-offs between biomass and lipid production in the alga. Whole genome sequence was employed to predict biological and metabolic capacity in PW95, and the expression of these capabilities during growth in CBM water with the native microbial consortia was evaluated using RNA sequencing. genome determination and assembly resulted in a draft genome size of 92 Mbp with 14,000 genes predicted and 402 pathways mapped in the KEGG database. The gene complement of PW95 provided a glance into life in an oligotrophic environment (CBM water) and evidence of essential metabolic pathways for cell growth, survival and maintenance, also relevant for cultivation and value-added products generation. Microbial composition and shifts during growth were identified, as well as the algal phycosome. During growth in CBM water, PW95 appeared to be supported by a native microbial consortium and differential expression analysis showed basic metabolic functions and adaptive physiological responses. Our findings build on previous knowledge for improved algal culturing for biomass and industry-valued products while exploring the biology of an organism with relevant impact in energy and water resource management.
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    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.
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    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.
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    Bacteriophage in host associated microbial communities examined with continuous culture systems
    (Montana State University - Bozeman, College of Letters & Science, 2018) Dills, Michael Stefan; Co-Chairs, Graduate Committee: Mark J. Young and Seth Walk
    Mechanistic understanding of the role of extracellular and parasitic elements in host ecosystems is currently lacking. Extensive surveys have catalogued a large diversity of bacteriophage which associate differentially with definable host states. This work is an attempt to aid in the development of a coherent model for complex symbiosis within mammalian host ecosystems by investigating the role of bacteriophage in microbial community structure. It details an investigation of continuous culture systems as a platform to study bacteriophage within polymicrobial communities of the human GI tract. It then describes an experiment testing an extracellular community's ability to modulate bacterial community structure.
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