Theses and Dissertations at Montana State University (MSU)
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Item Evaluation of methanotrophic activity and growth in a methanotrophic-heterotrophic co-culture(Montana State University - Bozeman, College of Engineering, 2021) Kilic, Ayse Bengisu; Chairperson, Graduate Committee: Ellen G. Lauchnor; Erika J. Espinosa-Ortiz, Brent M. Peyton and Ellen Lauchnor were co-authors of the article, 'Methane-based bioreactor configurations in value-added product development: a review' submitted to the journal 'Journal of bioscience and bioengineering' which is contained within this thesis.; Erika J. Espinosa-Ortiz, Brent M. Peyton and Ellen Lauchnor were co-authors of the article, 'Evaluation of methanotrophic activity and growth in a methanotrophic-heterotrophic co-culture' submitted to the journal 'Engineering in life sciences' which is contained within this thesis.Methane is a potent greenhouse gas (GHG) and accounts for 20-30% of the GHG emissions globally. In nature, methane is utilized as a sole carbon and energy source by a group of bacteria referred to as methanotrophs. Methanotrophs have been reported to have the ability to form close associations with other microorganisms such as heterotrophic bacteria in the environment. Therefore, understanding methanotrophic activity and growth in a microbial consortium with heterotrophic bacteria is of interest from an environmental and biotechnology perspective. In this study, a methanotroph; Methylocystis sp. NLS7 and a heterotrophic bacterium, Pseudomonas chlororaphis, were co-cultivated in a methane-fed bioreactor with a dialysis membrane device used to separate the species physically. It was hypothesized that the co-culture would exhibit enhanced methanotrophic activity and microbial growth of NLS7 in NLS7- P. chlororaphis co-culture. The methane-oxidation rate and microbial growth rate of NLS7 were evaluated as a functional response variable to the presence of P. chlororaphis. In addition, the effects of NLS7 growth were evaluated on the growth of P. chlororaphis. Our findings indicated that the presence of P. chlororaphis does not have any beneficial effects on Methylocystis sp. NLS7 activity and growth. However, the growth of P. chlororaphis in the co-culture with solely methane as a carbon source indicated that P. chlororaphis is likely gaining carbon and energy from by-products of methane oxidation by Methylocystis sp. NLS7 since P. chlororaphis could not utilize methane as a carbon and energy source. The results of this study give us an important insight into the activity and the growth of methanotrophic consortia in methane-driven ecosystem.Item 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.Item 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.Item Emergence of cooperative behavior in microbial consortia(Montana State University - Bozeman, College of Letters & Science, 2018) Schepens, Diana Ruth; Chairperson, Graduate Committee: Tomas GedeonCooperative microbial communities and their impact are ubiquitous in nature. The complexities of the cross-feeding interactions within such communities invite the application of mathematical models as a tool which can be used to investigate key influences in the emergence of cooperative behavior and increased productivity of the community. In this work, we develop and investigate a differential equation model of competition within a chemostat between four microbial strains utilizing a substrate to produce two necessary metabolites. The population of our chemostat includes a wild type strain that generalizes in producing both metabolites, two cross-feeding cooperator strains that each specialize in producing one of the two metabolites, and a cheater strain that produces neither metabolite. Using numerical methods we consider three key characteristics of the microorganisms and investigate the impact on the emergence of mutual cross-feeding in the community. First, we investigate the impact that substrate input concentration and the rate and type (active vs. passive) of metabolite transport between cells has on the emergence of cooperation and multi-stabilities resulting from the competition. Second, we investigate the role that resource allocation within metabolic pathways plays in the results of the competition between cells with different metabolite production strategies. Introducing metabolite production cost into the model leads to new outcomes of the competition, including stable coexistence between different strains. Lastly, we examine the effect that an initial population of a non-cooperative cheater strain has on the outcome of competition. Our results show that the emergence of a cross-feeding consortia relies on the availability and efficient use of resources, ease of transport of metabolites between cells, and limited existence of cheaters.Item 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.Item 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 WalkMechanistic 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.Item Systems analysis of engineered and natural microbial consortia(Montana State University - Bozeman, College of Engineering, 2013) Bernstein, Hans Christopher; Chairperson, Graduate Committee: Ross Carlson; Ross P. Carlson was a co-author of the article, 'Microbial consortia engineering for cellular factories: in vitro to in silico systems' in the journal 'Computational and structural biotechnology journal' which is contained within this thesis.; Ross P. Carlson was a co-author of the article, 'Design, construction and characterization methodologies for synthetic microbial consortia' in the book 'Methods in molecular biology, engineering multicellular system' which is contained within this thesis.; Steven D. Paulson and Ross P. Carlson were co-authors of the article, 'Synthetic Escherichia coli consortia engineered for syntrophy demonstrate enhanced biomass productivity' in the journal 'Journal of biotechnology' which is contained within this thesis.; Maureen Kessano, Karen Moll, Terence Smith, Robin Gerlach, Ross P. Carlson, Brent M. Peyton, Robert D. Gardner and Ronald C. Sims were co-authors of the article, 'Direct measurement and characterization of active photosynthesis zones inside biofuel producing and waste-water remediating algal biofilms' submitted to the journal 'Biotechnology and bioengineering' which is contained within this thesis.; Jacob P. Beam, Mark A. Kozubal, Ross P. Carlson and William P. Inskeep were co-authors of the article, 'In situ analysis of oxygen consumption and diffusive transport in high-temperature acidic iron-oxide microbial mats' in the journal 'Environmental Microbiology' which is contained within this thesis.; Alissa Bleem, Steven Davis and Ross P. Carlson were co-authors of the article, 'Chacterization of an artificial photoautotrophic-heterotrophic biofilm consortium composed of synechococcus PCC 7002 and Escherichia coli MG1655' which is contained within this thesis.Microorganisms are ubiquitous and typically exist within complex interacting communities or consortia. Microbial consortia are capable of cooperating in a coordinated fashion to extract mass and free energy from their environment. Chemical and biological engineers have long been keen to harness microbial processes for the development of technologies with applications ranging from energy capture to environmental remediation to human health. The pursuit of novel microbial biotechnologies has given rise to the relatively new discipline of microbial consortia engineering, which differs from and expands upon more traditional monoculture based practices. Many successful examples of applied and/or engineered microbial consortia mimic fundamental ecological strategies observed from nature, highlighting the importance for engineers to study natural biological phenomena. The overarching goal for this dissertation was to observe and quantitatively characterize interactions and physical phenomena occurring within select microbial consortial systems. The technical research presented here explores microbial consortia on three main fronts: (i) metabolically engineered heterotrophic systems, (ii) photoautotrophic-heterotrophic biofilm systems and (iii) naturally occurring thermo-acidophilic microbial mat systems. The metabolically engineered systems were designed to mimic a common ecological strategy involving syntrophic metabolite exchange via primary-productivity coupled with secondary consumption of potentially inhibitory byproducts (i.e., acetic acid). This system exhibited enhanced biomass productivity as compared to monoculture controls. The primary-productivity concept was also explored, in a more traditional sense, by characterizing production, consumption and exchanges of oxygen within photoautotrophic-heterotrophic biofilm systems. Tight spatial coupling of oxygenic-photosynthesis and aerobic-respiration was observed in both biofuel producing and waste-water remediating biofilm communities. The role of oxygen as an important terminal electron acceptor was also investigated in pristine Fe(III)-oxide microbial mats from geothermal springs located in Yellowstone National Park (USA). For these systems, oxygen availability defines ecological niche environments that spatially govern specific community member abundances and activities. Classical chemical engineering reaction and diffusion analysis was used to model concentration dependent oxygen consumption kinetics and establish that these mats are likely mass transfer limited. Both primary-productivity and microbially mediated oxygen reactions are interrelated, cross-cutting themes throughout this dissertation. The research described here is interdisciplinary chemical engineering that utilizes fundamental microbial ecology as a foundational platform.