Theses and Dissertations at Montana State University (MSU)
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Item The stoichiometry of nutrient and energy transfer: from organelles to organisms(Montana State University - Bozeman, College of Engineering, 2016) Hunt, Kristopher Allen; Chairperson, Graduate Committee: Ross Carlson; James P. Folsom, Reed L. Taffs and Ross P. Carlson were co-authors of the article, 'Complete enumeration of elementary flux modes through scalable, demand-based subnetwork definition' in the journal 'Bioinformatics' which is contained within this thesis.; Ashley E. Beck was an author and Hans C. Bernstein and Ross P. Carlson were co-authors of the article, 'Interpreting and designing microbial communities for bioprocess applications, from components to interactions to emergent properties' in the journal 'Biotechnology for biofuel production and optimization' which is contained within this thesis.; Ryan deM. Jennings, William P. Inskeep and Ross P. Carlson were co-authors of the article, 'Stoichiometric modeling of assimilatory and dissimilatory biomass utilization in a microbial community' in the journal 'Environmental microbiology' which is contained within this thesis.; Ryan deM. Jennings, William P. Inskeep and Ross P. Carlson were co-authors of the article, 'Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community' submitted to the journal 'The International Society for Microbial Ecology journal' which is contained within this thesis.; Natasha D. Mallette, Brent M. Peyton and Ross P. Carlson were co-authors of the article, 'Theoretical and practical limitations of hydrocarbon production for a cellulolytic, endophytic filamentous fungus' submitted to the journal 'Metabolic engineering' which is contained within this thesis.All life requires the acquisition and transformation of nutrients and energy, driving processes from cellular nutrient flow to planetary biogeochemical cycling. However, the organisms and communities responsible for these processes are often uncultivable and too complex to observe directly and understand. Stoichiometric modeling, a systems biology approach, analyzes the reactions in an organism and incorporates data from multiple sources to extract biologically meaningful parameters, such as theoretical limits of conversion and yields of a metabolism. These limits and yields quantify relationships between organisms to establish governing principles, from resource requirements to community productivity as a function of population composition. The presented work expanded the stoichiometric modeling algorithm and identified fundamental principles that govern nutrient and energy transfer associated with heterotrophy, community composition, and intracellular compartmentalization. A scalable routine capable of analyzing complex metabolic networks by dividing them into tractable subnetworks was demonstrated for a eukaryotic diatom. The metabolic model contained approximately two billion routes through the network and established an international benchmark for elementary flux mode analysis. Additionally, a heterotrophic archaeon was examined for the resource requirements while consuming 29 different forms of biomass derived dissolved organic carbon. These resource requirements and limitations establish a basis to analyze heterotrophy with regard to the limiting nutrient in a variety of systems. The resulting resource requirements of heterotrophy were incorporated into a community where an iron oxidizing autotroph was hypothesized to be the primary source of carbon and energy. Analysis of the community model and in situ measurements of iron and oxygen utilization indicated additional electron donors were required to account for the observed acquisition of nutrients in some communities. Finally, limits and resource requirements for fungal production of hydrocarbons were identified as a function of carbon and energy partitioning using simulated genetic modifications, providing context regarding endophytic production of bioactive molecules for host resistance as well as endophyte capacity as a petroleum producing alternative.Item Spatial and temporal patterns of antimicrobial action against Staphylococcus Epidermidis biofilms(Montana State University - Bozeman, College of Engineering, 2008) Davison, William Marshall; Chairperson, Graduate Committee: Philip S. Stewart; Joseph D. Seymour (co-chair)This study investigated the spatio-temporal patterns of antimicrobial action against Staphylococcus epidermidis planktonic and biofilm bacteria. Bacteria were stained with a fluorogenic esterase substrate, Calcein-AM, which allowed for the visualization of cells that possessed intact cell membranes. Four different antimicrobial agents were tested for their effect upon cell viability as associated with membrane integrity. The four biocides were Barquat®, glutaraldehyde, chlorine, and nisin. Planktonic bacteria were analyzed with flow cytometry, observing fluorescence loss during 1 h antimicrobial treatment. Treatment with Barquat resulted in initial fluorescence loss, which increased during the treatment period to levels which were present prior to the introduction of biocide, along with a decrease in cell density. Treatments with glutaraldehyde and chlorine resulted in increased average fluorescence intensity for the cell population, accompanied by decreased cell density for chlorine and increased cell density for glutaraldehyde. Nisin treatment resulted in a decrease in CAM fluorescence with an increase in cell density. Viable cell plate counts showed average log reductions in CFU/mL of 3.61, 3.83, 4.12, 4.26, and 4.67 for Barquat, glutaraldehyde, high and low concentrations of chlorine, and nisin treatments, respectively. There was no apparent correlation between plate counts and flow cytometry data. Biofilm bacteria were analyzed with time-lapse confocal scanning laser microscopy, observing fluorescence loss during biocide treatment. Biofilms treated with Barquat lost an average of 91.5% of their initial fluorescence, and clusters decreased in areal coverage by 9%. Fluorescence loss during Barquat treatment suggested the presence of a tolerant subpopulation of bacteria in the interior regions of the biofilm. Glutaraldehyde treatment reduced the average fluorescence by 16%, and cluster area did not change. There was CTC staining after glutaraldehyde treatment only. The high and low concentrations of chlorine treatment showed averages of 100% and 79% reductions in CAM staining, with liquefaction of biomass causing erosion events which reduced areal coverage by 90% and 43%, respectively. Nisin treatment reduced CAM staining by an average of 100%, while shrinking the cluster area by 8%. Corner biofilms showed qualitative differences during treatment than isolated clusters. Mathematically-predicted biocide diffusion times were much faster than experimentally observed fluorescence loss in biofilms.