Theses and Dissertations at Montana State University (MSU)
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Item The biofilm matrix in sulfate-reducing bacterial biofilms: potential roles for electron mediators and large proteins(Montana State University - Bozeman, College of Letters & Science, 2019) Krantz, Gregory Peter; Chairperson, Graduate Committee: Matthew Fields; Kilean Lucas, Erica L.-Wunderlich, Linh T. Hoang, Recep Avci, Gary Siuzdak and Matthew W. Fields were co-authors of the article, 'Bulk phase resource ratio alters carbon steel corrosion rates and endogenously produced extracellular electron transfer mediators in a sulfate-reducing biofilm' in the journal 'Biofouling' which is contained within this dissertation.; Peter J. Walian, Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Large extracellular proteins sense hydrodynamic force and drive biofilm formation in Desulfovibrio vulgaris' which is contained within this dissertation.; Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Characterization of extracellular biofilm mutants cultivated on 1018 carbon steel in Desulfovibrio vulgaris Hildenborough' which is contained within this dissertation.Sulfate-reducing bacteria grow and form biofilms in soil and benthic environments across much of the Earth's surface. Formation of these prevalent biofilms requires the secretion of an extracellular polymeric substance (EPS) to allow the cells to stick together, as well as adhere to a surface. The specific interactions that occur between EPS components of an SRB biofilm are poorly understood. The data presented in this dissertation suggest the presence of two extracellular mechanisms utilized in these communities. The first mechanism was observed in a study altering the lactate (electron donor) and sulfate (electron acceptor) ratios to create limiting nutrient conditions in Desulfovibrio alaskensis G20 (G20) biofilms. G20 was grown under two conditions: electron donor limited (EDL) and electron acceptor limited (EAL) conditions. When grown on a 1018 carbon steel substrate, the G20 consumes all of the available lactate, and once limited, it turns to the high energy electrons in the Fe 0 for growth. Corrosion rates in the steel increased two fold compared to the EAL condition. Global metabolomic analysis revealed increased lumichrome levels under the EDL condition, which suggested higher flux through the riboflavin/FAD biosynthetic pathway. Previous research showed that synthetically adding riboflavin and FAD increases the corrosion rate of a SRB biofilm on 1018 carbon steel, and paired with these results, suggest G20 produces a flavin-based extracellular electron transfer molecule endogenously, and uses it to harvest high energy electrons from Fe 0 when limited for electron donor. The second mechanism was observed in Desulfovibrio vulgaris Hildenborough (DvH) biofilms grown on glass. Two proteins, DVU1012 and DVU1545 were found to be the most abundant extracellular peptides in a DvH biofilm. Single deletion strains for these proteins grew biofilm similar to the wild type strain, but a double deletion strain had decreased ability to form biofilm, demonstrating that at least one of the peptides must be present in order to form a biofilm. Exposure to increased shear force caused an large increase in wild-type biofilm biomass, yet eliminated the double mutant biofilm. These proteins are required for a DvH biofilm to respond to shear force.Item Metabolic interactions and activity partitioning in a methanogenic, interdomain biofilm(Montana State University - Bozeman, College of Letters & Science, 2019) Camilleri, Laura Beth; Chairperson, Graduate Committee: Matthew Fields; Kristopher A. Hunt, Aurelien Mazurie, Jennifer Kuehl, Alex Michaud, James Connolly, Egan Lohman, Zack Miller, Adam M. Deutschbauer and Matthew W. Fields were co-authors of the article, 'Differential gene expression of a bacterial-archaeal interdomain biofilm producing methane' submitted to the journal 'Biofilms' which is contained within this dissertation.; B.P. Bowen, C.J. Petzold, T.R. Northen and M.W. Fields were co-authors of the article, 'Activity partitioning in an archaeal-bacterial biofilm' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methanococcus maripaludis factor causes slowed growth in Desulfovibrio vulgaris Hildenborough' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Growth effects of sulfopyruvate and sulfoacetate on the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, and the methanogenic archaeon Methanococcus maripaludis S2' submitted to the journal 'Scientific reports' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methane production in Pelosinus fermentans JBW45' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.Biofilms are an ancient survival strategy in which communities of organisms can grow as a cohesive unit, generally attached to a surface and/or at interfaces. Despite the paradigm that 99% of microorganisms grow as a biofilm in the environment, current research methods are largely limited to monoculture planktonic studies. Although more investigations are trying to improve culture complexity by evaluating interactions between two or more populations, experiments are still more readily performed with microorganisms in the planktonic growth mode. The research presented here aims to elucidate the complexity of interactions between two microorganisms from different domains of life that results in enhanced metabolism due to localization of cells in close proximity within an anaerobic biofilm. Desulfovibrio vulgaris Hildenborough (DvH) and Methanococcus maripaludis S2 (Mmp) form a syntrophic mutualism when grown in sulfate-limited media that requires electron flux from DvH to Mmp through what is commonly assumed to be interspecies hydrogen transfer, thereby establishing cross-feeding. The biofilm has been shown to promote a stable and more even carrying capacity for both populations that is likely linked to improved hydrogen transfer (and/or other potential carbon and electron co-metabolites) as compared to planktonic populations. Transcriptomic and proteomic analyses, utilizing RNA-seq and deuterated water respectively, were used to elucidate genes and proteins that contribute to the biofilm growth mode that results in a more efficient metabolism for the syntrophic co-culture (defined by biomass per substrate flux). The results demonstrate the expression of many genes with unknown functions, and others that contribute to cell-cell interactions as well as active proteins in electron processing (e.g., lactate oxidation) in DvH and CO2 reduction (e.g., methanogenesis) in Mmp. A metabolic model of the coculture provided reinforcement for transcriptomic assumptions and aided in the identification of a sulfonate and other amino acids as important syntrophic metabolites. Assessment of biofilm co-culture activity utilizing a new method, Biorthogonal Noncanonical Amino Acid Tagging (BONCAT), showed Mmp was less active in the uptake of a methionine analog as compared to DvH. Alternate assessments confirmed that Mmp was in fact active (based upon methane generation) although translational activity was below the detection limit. Further investigation of the system under sulfate stress showed that the metabolic pairing is more stable than previously thought and could indicate survival strategies that drive the seemingly 'mutualistic' relationship as a forced cooperation. The sulfate stress response coincided with observed lags in DvH growth when grown in Mmp spent medium that was associated with a decoupling of lactate-oxidation and sulfate-reduction. Together the results demonstrate metabolic interactions and activity partitioning within a methanogenic archaeal-bacterial biofilm. The dogma of mutualism being synonymous with equal reciprocity is challenged as it pertains to this model biofilm system. Moreover, this unique bacterial-archaeal biofilm represents interdomain interactions that could represent systems that contributed shared metabolic processes that lead to the development of eukaryotic life.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 Ecophysiology of Methanococcus maripaludis and Desulfovibrio vulgaris : the role of structure in relation to function(Montana State University - Bozeman, College of Letters & Science, 2013) Brileya, Kristen Annis; Chairperson, Graduate Committee: Matthew Fields; Laura B. Camilleri and Matthew W. Fields were co-authors of the article, 'Biofilm growth mode optimizes carrying capacity of interacting populations' submitted to the journal 'The ISME journal' which is contained within this thesis.; James M. Connolly, Carey Downey, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Taxis toward hydrogen gas by Methanococcus maripaludis' submitted to the journal 'Science' which is contained within this thesis.Sulfate-reducing bacteria (SRB) and methanogenic archaea are known to interact in anaerobic environments under a range of conditions, and the nature of the interaction is dependent on the concentration of available carbon and energy sources, electron acceptors and the metabolic potential of the specific genera present. In the absence of sulfate, SRB can participate in a mutualism by product inhibition with hydrogenotrophic methanogens. SRB reduce protons to form hydrogen gas as a methanogenic substrate, and methanogens prevent inhibition of hydrogenase activity in the SRB by consuming the evolved hydrogen, making the interaction beneficial to both parties. This type of interaction can occur in nature when organisms are free-swimming or attached to a surface as biofilm. The impact of structured biofilm on ecosystem function at various scales is becoming increasingly clear, as this growth mode concentrates biomass which can increase the capacity for compound immobilization, affect hydrodynamic flow paths, and leave cells in altered physiological states. In spite of this, few studies have systematically characterized mutualistic interactions within the biofilm state using model organisms. Syntrophic continuous culture of Desulfovibrio vulgaris Hildenborough and Methanococcus maripaludis was monitored from inoculation through steady state for biofilm and planktonic community structure and function in terms of biomass production, lactate oxidation, and methane production. This biofilm was structurally distinct from monoculture biofilms grown under the same conditions and yield of biomass per lactate mass flux or methane produced was much higher when biofilm was present under lactate limitation. The results suggested that biofilm helped optimize carrying capacity of the syntrophic culture. Observations in coculture biofilm of attraction by M. maripaludis to a surface in the presence of a hydrogen-producing biofilm indicated a tactic response in the archaeum. Movement toward favorable conditions, or chemotaxis is a strategy employed across all three domains of life, yet chemotaxis in archaea is still poorly described, and no previous work has demonstrated taxis toward a hydrogen source, like a syntrophic partner, in spite of its role in electron flow in anaerobic communities. Here we present the first direct observation of taxis toward hydrogen, or "hydrogenotaxis" in the archaeum M. maripaludis.Item Microbial conversion of biodiesel by-products to biofuel(Montana State University - Bozeman, College of Letters & Science, 2010) O'Shea, Kelly Frances; Chairperson, Graduate Committee: Matthew FieldsBiodiesel is an alternative liquid transportation fuel derived from biological oils. It is a renewable form of transportation fuel that can be easily integrated into society's current infrastructure. Biodiesel is cleaner burning than petroleum, emitting less carbon pollution and harmful toxins (i.e. sulfur, benzene). One of the major by-products from biodiesel production is crude glycerin. With the increased production of biodiesel, glycerin production will continue to increase. Glycerin was once considered a valuable co-product but now is considered a low-value by-product. In the following study, different co/tricultures of sulfate reducing bacteria (SRB) and methanogens were grown with crude glycerin as a means to convert the waste product into a renewable energy source, methane. The SRBs, Desulfovibrio vietnamensis and Desulfovibrio alcoholovorans 6133, were grown syntrophically, in different co/triculture combinations, with Methanococcus maripaludis, Methanoculleus marisnigri, and Methanosarcina acetivorans. Co/tricultures were investigated for the ability to produce methane via the utilization of pure glycerol, fractionated glycerin, and crude glycerin as carbon and energy sources. In order to gain insight into cellular physiology, glycerol, acetate, free fatty acid, and methane concentrations were measured throughout growth. The co/tricultures grew fastest on pure glycerol and experienced a lag phase in growth on fractionated glycerin and longer lag phases when transferred to crude glycerin. However, methane yields were similar on all three carbon sources. Methane production depended on the carbon source and culture composition. Co/tricultures growing on pure glycerol and fractionated glycerin displayed a decrease of methane production as growth rate increased. The opposite was seen with growth on crude glycerin. With most cultures, the addition of M. acetivorans increased methane concentrations significantly. M. acetivorans displayed the capability of utilizing the by-product, acetate, from SRB oxidation of glycerol and the methanol layer from fractionated and crude glycerin. M. acetivorans appeared to interfere with the coculturing of D. vietnamensis and M. marisnigri based on decreased methane production. Cocultures with M. maripaludis grew poorly and produced little methane when grown on the supernatant of M. acetivorans. This is the first study to characterize the utilization of crude glycerin from biodiesel production by syntrophic cultures of SRB and methanogenic archaea.Item Analysis of microbial biofilm community composition within constructed wetlands(Montana State University - Bozeman, College of Letters & Science, 2010) Faulwetter, Jennifer Lynn; Chairperson, Graduate Committee: Anne Camper; Vincent Gagnon, Carina Sundberg, Florent Chazarenc, Mark D. Burr, Jacques Brisson, Anne K. Camper and Otto R. Stein were co-authors of the article, 'Microbial processes influencing performance of treatment wetlands: a review' in the journal 'Ecological engineering' which is contained within this thesis.; Mark D. Burr, Otto R. Stein and Anne K. Camper were co-authors of the article, 'Characterization of sulfate reducing bacteria in constructed wetlands' in the journal 'Proceedings of the 11th international conference on wetland systems for water pollution control, Indore, India' which is contained within this thesis.; Mark D. Burr, Albert E. Parker, Otto R. Stein and Anne K. Camper were co-authors of the article, 'The effect of plant species and sample location on bacterial biofilm communities associated with constructed wetland microcosms' in the journal 'The international society for microbial ecology journal' which is contained within this thesis.; Mark D. Burr, Albert E. Parker, Otto R. Stein and Anne K. Camper were co-authors of the article, 'The influence of sulfate reducing bacteria and ammonia oxidizing bacteria on nutrient cycling in constructed wetland microcosms' in the journal 'Microbial ecology' which is contained within this thesis.; Mark D. Burr, Alfred B. Cunningham, Frank M. Stewart, Anne K. Camper and Otto R. Stein were co-authors of the article, 'Floating treatment wetlands for domestic wastewater treatment' in the journal 'Water science and technology' which is contained within this thesis.Constructed wetlands (CWs) are ecologically-based water treatment systems that provide cost-effective amelioration of waterborne pollutants. Fundamental understanding of removal mechanisms, especially microbial processes, limits greater usage of constructed wetlands as a wastewater treatment system. The influence of plant species selection, season, and organic load rate on pollutant removal was previously linked to the redox condition of the sub-surface wetland environment. The goal of this research was to determine which of these environmental variables (including spatial location within the CW) influenced the dominant microbial populations and/or the activity of various sub-populations. Once identified, a constructed wetland might be optimized for growth of microorganisms involved in removal of a specific pollutant. To assess environmental factors, microbial population samples were taken in six locations (effluent, 3 root and 2 gravel areas) within replicate unplanted microcosms and wetland microcosms planted with Deschampsia cespitosa or Leymus cinereus during the summer (24°C) and winter (4°C) seasons. Microcosms were fed a synthetic domestic wastewater in 20-day batches for at least 12 months prior to sampling. The most recent techniques in molecular biology including denaturing gradient gel electrophoresis (DGGE) and quantitative PCR were utilized and included treatment with and without propidium monoazide (PMA) to distinguish between "live" and "dead" microbial communities. Primer sets targeted the entire bacterial community (16S rDNA) and two functional groups, nitrifying bacteria (amoA gene) and sulfate reducing bacteria (dsrB gene). Results indicated that overall microbial community structure (16S rDNA) was affected by general location within the microcosm (effluent, root, gravel) as well the plant species present. Specific microbial groups appeared to be affected differently with relative gene quantities of sulfate reducing bacteria and nitrifying bacteria being influenced by a combined effect of plant species and season. For dsrB, D. cespitosa had the lowest relative gene quantities overall. Both genes were more abundant in the summer season, indicating seasonal importance. Location within the microcosms was also important, with anoxic environments (column bottom) being more important for dsrB presence and a diverse population of cultivated sulfate reducers. The roots were an important location for both microbial diversity and activity for all genes investigated.