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Item Nutrient limitation alters metabolism, CR(VI) response, and biofilm matrix composition in desulfovibrio vulgaris Hildenborough(Montana State University - Bozeman, College of Letters & Science, 2017) Franco, Lauren Christine; Chairperson, Graduate Committee: Matthew Fields; Grant Zane, Sadie Steinbeisser, Judy Wall and Matthew W. Fields were co-authors of the article, 'Cr(VI) reduction and physiological toxicity is impacted by resource ratio in Desulfovibrio vulgaris Hildenborough' submitted to the journal 'Applied microbiology and microbiology' which is contained within this thesis.; Julijana Ivanisevic, Gary Siuzdak and Matthew W. Fields were co-authors of the article, 'Nutrient limitation causes decline in metabolites important for cell cycle progression in bacterial biofilm' submitted to the journal 'Applied microbiology and microbiology' which is contained within this thesis.; Siva Wu, Michael Joo, Joel Mancuso, Jonathan Remis, Amita Gorur, Ambrose Leung, Danielle M. Jorgens, Joaquin Correa, Manfred Auer and Matthew W. Fields were co-authors of the article, 'Extracellular membrane structures in Desulfovibrio vulgaris Hildenborough biofilms' which is contained within this thesis.; Chris Petzold and Matthew W. Fields were co-authors of the article, 'Outer membrane vesicles and associated proteins produced by Desulfovibrio vulgaris Hildenborough biofilms' submitted to the journal 'Applied microbiology and microbiology' which is contained within this thesis.Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that live in anoxic environments and play critical roles in biogechemical cycling, namely linkages between the carbon and sulfur cycles. Desulfovibrio vulgaris Hildenborough (DvH) is a model organism for SRB that has been studied for its ability to reduce toxic heavy metals to insoluble forms and its involvement in microbially induced corrosion in oil pipelines and other industrial settings. The described work investigated how the availability of electron donor/carbon sources and electron acceptors affected Cr(VI) reduction, metabolism, and biofilm growth and composition in DvH. DvH was grown planktonically at 20°C and 30°C in batch mode or as a biofilm under continuous flow at 20°C. In the second chapter of this dissertation, it is established that electron acceptorlimitation (EAL) predisposes cells to Cr(VI) toxicity compared to a balanced electron donor to electron acceptor (BAL) condition and electron donor-limited (EDL) condition. The effect of nutrient limitation on DvH biofilms is investigated, and microscopy revealed unique extracellular membranous structures that have not previously been observed. The extracellular structures were heterogeneously distributed, connected to cells, co-localized with metal precipitates, and more prevalent under EAL compared to BAL condition. Differential staining indicated that the structures were composed of lipid, consistent with the observation that these structures are membrane derived. Metabolomic analysis revealed an up-regulation of fatty acids under the EAL condition, which was confirmed and quantified via GC-MS. Down-regulated metabolites for biofilm grown under the EAL condition included those involved in DNA turnover, N-cycling, and peptidoglycan turnover, indicating that EAL may induce a switch from growth to fatty acid production that may coordinate with alternative electron transfer mechanisms. Outer membrane vesicles (OMVs) were purified from DvH biofilm and proteins detected in OMVs included porins, lipoproteins, hydrogenases, and oxidative stress response proteins. The results presented here show that nutrient limitation and resource ratio affect DvH physiology in both biofilm and planktonic growth modes. The analysis of the DvH biofilm matrix highlights the importance of investigating extracellular capabilities that are unique to the biofilm growth mode and has implications for activities and physiological states in the environment.Item Community analysis of groundwater and surrogate sediment samples during electron donor and electron acceptor injections into a chromium-contaminated site in Hanford, Washington, USA(Montana State University - Bozeman, College of Letters & Science, 2013) Bowen De Leon, Kara Beth; Chairperson, Graduate Committee: Matthew Fields; Bradley D. Ramsay was a co-author and Matthew W. Fields was the corresponding author of the article, 'Quality-score refinement of SSU rRNA gene pyrosequencing differs across gene region for environmental samples' in the journal 'Microbial ecology' which is contained within this thesis.; Boris Faybishenko, Bradley D. Ramsay, Darrell R. Newcomer and Terry C. Hazen were co-authors and Matthew W. Fields was the corresponding author of the article, 'Stimulation for in situ Cr(VI) bioreduction causes convergence of groundwater and sediment-adhered bacterial populations with differing population networks' submitted to the journal 'ISME journal' which is contained within this thesis.; Boris Faybishenko, Bradley D. Ramsay, Darrell R. Newcomer and Terry C. Hazen were co-authors and Matthew W. Fields was the corresponding author of the article, 'Microbial community changes with episodic nitrate during in situ stimulation for Cr(VI) reduction at the Hanford 100-H site' submitted to the journal 'Applied and environment microbiology' which is contained within this thesis.; Robin Gerlach and Brent M. Peyton were co-authors and Matthew W. Fields was the corresponding author of the article, 'Archaeal and bacterial communities in alkaline hot springs in Heart Lake Geyser Basin, Yellowstone National Park' in the journal 'Extremophiles' which is contained within this thesis.; Mary Lynn Young, Laura B. Camilleri, Steven D. Brown, Jeffrey M. Skerker and Adam M. Deutschbauer were co-authors and Matthew W. Fields was the corresponding author of the article, 'Draft genome sequence of Pelosinus fermentans JBW45 isolated during in situ stimulation for Cr(VI) reduction' in the journal 'Journal of bacteriology' which is contained within this thesis.Heavy-metal contamination is a common problem in the industrialized world today. Bioremediation, the use of microorganisms or microbial processes to degrade or reduce contaminants, has been found to be successful for many different contaminants and environments. As part of the World War II Manhattan Project, eight single-pass reactors in the Hanford Nuclear Reservation in Washington were used for plutonium production for ~43 years. Columbia River water, used to cool these reactors, was contaminated with heavy metals and radionuclides and stored in retention basins before discharge or leakage into the ground or river. Hexavalent chromium (Cr(VI)), a carcinogen and mutagen, was used as a corrosion inhibitor in the cooling systems and is a common contaminant at the Hanford Site. In 2008, a polylactate compound was injected into Cr(VI)-contaminated groundwater in the Hanford 100-H area to stimulate the resident microbial community and resulted in Cr(VI) levels below background levels for ~3 months. Temporal and spatial community analyses of the groundwater and surrogate sediment samples via small-subunit rRNA gene pyrosequencing indicated an enrichment of Pseudomonas and fermentative organisms upon injection. The sediment and groundwater communities, while different pre-injection, converged on a similar, fermentative community after injection. Correlation analyses suggested that nitrogen-metabolism and fermentation might be driving community changes. Nitrate is another common contaminant at Hanford and an influx of nitrate, a competing terminal electron acceptor, into the Hanford 100-H site may be detrimental to the bioremediation process. To test this, nitrate was injected into the system following a lactate injection to stimulate the resident community. The nitrate injection resulted in a drastic community shift from metal-reducing organisms to denitrifiers and an increase in Cr(VI) concentrations to above background levels. Long after the nitrate injection, denitrifiers continued to be predominant in the groundwater community; however, the sediment community was comprised of denitrifying and metal-reducing populations. This suggests that the effects of an episodic nitrate event are long lasting, but the sediment community is more resilient than the groundwater community. These community analyses have revealed organisms and metabolic properties of interest during polylactate or nitrate perturbation and can be used to improve future bioremediation strategies.Item Factors influencing the fate of chromium in soils : microbial ecology, physiology and metal transformation studies(Montana State University - Bozeman, College of Letters & Science, 2011) Field, Erin Kirby; Chairperson, Graduate Committee: Robin Gerlach; Seth D'Imperio, Amber R. Miller, Michael R. VanEngelen, Robin Gerlach, Brady D. Lee, William A. Apel, and Brent M. Peyton were co-authors of the article, 'Application of molecular techniques to elucidate the influence of cellulosic waste on the bacterial community structure at a simulated low-level-radioactive-waste site' in the journal 'Applied and Environmental Microbiology' which is contained within this thesis.; John P. Blaskovich, Brent M. Peyton, and Robin Gerlach were co-authors of the article, 'The influence of carbon source on hexavalent chromium toxicity and reduction by an environmental Arthrobacter sp. isolate' in the journal 'Environmental Toxicology and Chemistry' which is contained within this thesis.; Robin Gerlach, Sridhar Viamajala, Laura K. Jennings, Alfred B. Cunningham, Brent M. Peyton, and William A. Apel were co-authors of the article, 'Influence of carbon source, iron minerals, and electron shuttling compounds on hexavalent chromium reduction by Cellulomonas sp. strain ES6' in the journal 'Chemosphere' which is contained within this thesis.Chromium is a common contaminant in soil environments, especially at Department of Energy sites. The fate of chromium in the environment is influenced by a number of factors including the microbial processes and the interactions with soil constituents such as carbon sources, iron minerals and electron shuttling compounds. The overall goals of these studies were to isolate and characterize microorganisms that may play a role in the fate of chromium in soil systems, identify the microbial community associated with a simulated low-level waste site and determine the role carbon source, iron minerals and electron shuttles play on Cr(VI) toxicity and reduction by Arthrobacter spp. and Cellulomonas sp. strain ES6. Three strategies for isolating potential cellulose-degrading isolates (direct isolation on agar plates, liquid enrichments and soil laden continuous flow columns) were implemented through which thirteen isolates were obtained. Clone library, PhyloChip and quantitative PCR analyses of the bacterial community within a simulated low-level waste site at the Idaho National Laboratory suggested that the presence of cellulosic waste influenced the bacterial community structure with soil depth at the site. The influence of potential cellulose degradation products as carbon sources on Cr(VI) toxicity and reduction by Arthrobacter sp. isolate EF01 and A. aurescens TC1 was assessed through bench-scale studies. Results indicated that Cr(VI) toxicity to both Arthrobacter spp. was tied to carbon metabolism. Additional studies with Cellulomonas sp. strain ES6 demonstrated that Cr(VI) reduction by this organism was also influenced by carbon source as well as by the addition of electron shuttles and iron minerals. Overall, the results of this dissertation provide insight into the potential interactions between microorganisms, soil constituents and chromium in situ.