Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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Item In silico approaches to study mass and energy flows in microbial consortia: A syntrophic case study(2009) Taffs, Reed L.; Aston, John E.; Brileya, Kristen A.; Jay, Zackary J.; Klatt, Christian G.; McGlynn, Shawn E.; Inskeep, William P.; Ward, David M.; Carlson, Ross P.Background: Three methods were developed for the application of stoichiometry-based network analysis approaches including elementary mode analysis to the study of mass and energy flows in microbial communities. Each has distinct advantages and disadvantages suitable for analyzing systems with different degrees of complexity and a priori knowledge. These approaches were tested and compared using data from the thermophilic, phototrophic mat communities from Octopus and Mushroom Springs in Yellowstone National Park (USA). The models were based on three distinct microbial guilds: oxygenic phototrophs, filamentous anoxygenic phototrophs, and sulfate-reducing bacteria. Two phases, day and night, were modeled to account for differences in the sources of mass and energy and the routes available for their exchange.ResultsThe in silico models were used to explore fundamental questions in ecology including the prediction of and explanation for measured relative abundances of primary producers in the mat, theoretical tradeoffs between overall productivity and the generation of toxic by-products, and the relative robustness of various guild interactions.Conclusion: The three modeling approaches represent a flexible toolbox for creating cellular metabolic networks to study microbial communities on scales ranging from cells to ecosystems. A comparison of the three methods highlights considerations for selecting the one most appropriate for a given microbial system. For instance, communities represented only by metagenomic data can be modeled using the pooled method which analyzes a community's total metabolic potential without attempting to partition enzymes to different organisms. Systems with extensive a priori information on microbial guilds can be represented using the compartmentalized technique, employing distinct control volumes to separate guild-appropriate enzymes and metabolites. If the complexity of a compartmentalized network creates an unacceptable computational burden, the nested analysis approach permits greater scalability at the cost of more user intervention through multiple rounds of pathway analysis. The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1752-0509/3/114Item Inhibition of microbial arsenate reduction by phosphate(2012-03) Slaughter, D. C.; Macur, Richard E.; Inskeep, William P.The ratio of arsenite (AsIII) to arsenate (AsV) in soils and natural waters is often controlled by the activity of As-transforming microorganisms. Phosphate is a chemical analog to AsV and, consequently, may competitively inhibit microbial uptake and enzymatic binding of AsV, thus preventing its reduction to the more toxic, mobile, and bioavailable form – AsIII. Five As-transforming bacteria isolated either from As-treated soil columns or from As-impacted soils were used to evaluate the effects of phosphate on AsV reduction and AsIII oxidation. Cultures were initially spiked with various P:As ratios, incubated for approximately 48 h, and analyzed periodically for AsV and AsIII concentration. Arsenate reduction was inhibited at high P:As ratios and completely suppressed at elevated levels of phosphate (500 and 1000μM; P inhibition constant (Ki)~20–100 μM). While high P:As ratios effectively shut down microbial AsV reduction, the expression of the arsenate reductase gene (arsC) was not inhibited under these conditions in the AsVreducing isolate, Agrobacterium tumefaciens str. 5B. Further, high phosphate ameliorated AsV-induced cell growth inhibition caused by high (1 mM) As pressure. These results indicate that phosphate may inhibit AsV reduction by impeding AsV uptake by the cell via phosphate transport systems or by competitively binding to the active site of ArsC.Item Microbial community structure and sulfur biogeochemistry in mildly acidic sulfidic geothermal springs in Yellowstone National Park(2013-01) Macur, Richard E.; Jay, Zackary J.; Taylor, William P.; Kozubal, Mark A.; Kocar, Benjamin D.; Inskeep, William P.Geothermal and hydrothermal waters often contain high concentrations of dissolved sulfide, which reacts with oxygen (abiotically or biotically) to yield elemental sulfur and other sulfur species that may support microbial metabolism. The primary goal of this study was to elucidate predominant biogeochemical processes important in sulfur biogeochemistry by identifying predominant sulfur species and describing microbial community structure within high-temperature, hypoxic, sulfur sediments ranging in pH from 4.2 to 6.1. Detailed analysis of aqueous species and solid phases present in hypoxic sulfur sediments revealed unique habitats containing high concentrations of dissolved sulfide, thiosulfate, and arsenite, as well as rhombohedral and spherical elemental sulfur and/or sulfide phases such as orpiment, stibnite, and pyrite, as well as alunite and quartz. Results from 16S rRNA gene sequencing show that these sediments are dominated by Crenarchaeota of the orders Desulfurococcales and Thermoproteales. Numerous cultivated representatives of these lineages, as well as the Thermoproteales strain (WP30) isolated in this study, require complex sources of carbon and respire elemental sulfur. We describe a new archaeal isolate (strain WP30) belonging to the order Thermoproteales (phylum Crenarchaeota, 98% identity to Pyrobaculum/Thermoproteus spp. 16S rRNA genes), which was obtained from sulfur sediments using in situ geochemical composition to design cultivation medium. This isolate produces sulfide during growth, which further promotes the formation of sulfide phases including orpiment, stibnite, or pyrite, depending on solution conditions. Geochemical, molecular, and physiological data were integrated to suggest primary factors controlling microbial community structure and function in high-temperature sulfur sediments.Item In situ analysis of oxygen consumption and diffusive transport in 1 high-temperature acidic iron-oxide microbial mats(2013-03) Bernstein, Hans C.; Beam, Jacob P.; Kozubal, Mark A.; Carlson, Ross P.; Inskeep, William P.The role of dissolved oxygen as a principal electron acceptor for microbial metabolism was investigated within Fe(III)-oxide microbial mats that form in acidic geothermal springs of Yellowstone National Park (USA). Specific goals of the study were to measure and model dissolved oxygen profiles within high temperature (65–75°C) acidic (pH = 2.7–3.8) Fe(III)-oxide microbial mats, and correlate the abundance of aerobic, iron-oxidizing Metallosphaera yellowstonensis organisms and mRNA gene expression levels to Fe(II)-oxidizing habitats shown to consume oxygen. In situ oxygen microprofiles were obtained perpendicular to the direction of convective flow across the aqueous phase/Fe(III)-oxide microbial mat interface using oxygen microsensors. Dissolved oxygen concentrations dropped from ~ 50–60 µM in the bulkfluid/ mat surface to below detection (< 0.3 µM) at a depth of ~ 700 µm (~ 10% of the total mat depth). Net areal oxygen fluxes into the microbial mats were estimated to range from 1.4–1.6 Â¥ 10-4 µmol cm-2 s-1. Dimensionless parameters were used to model dissolved oxygen profiles and establish that mass transfer rates limit the oxygen consumption. A zone of higher dissolved oxygen at the mat surface promotes Fe(III)-oxide biomineralization, which was supported using molecular analysis of Metallosphaera yellowstonensis 16S rRNA gene copy numbers and mRNA expression of haem Cu oxidases (FoxA) associated with Fe(II)-oxidation.Item Yellowstone Lake Nanoarchaeota(2013-09) Clingenpeel, Scott; Jinjun, K.; Macur, Richard E.; Woyke, Tanja; Lovalvo, D.; Varley, J.; Inskeep, William P.; Nealson, Kenneth H.; McDermott, Timothy R.Considerable Nanoarchaeota novelty and diversity were encountered in Yellowstone Lake, Yellowstone National Park (YNP), where sampling targeted lake floor hydrothermal vent fluids, streamers and sediments associated with these vents, and in planktonic photic zones in three different regions of the lake. Significant homonucleotide repeats(HR) were observed in pyrosequence reads and in near full-length Sanger sequences,averaging 112 HR per 1349 bp clone and could confound diversity estimates derived from pyrosequencing, resulting in false nucleotide insertions or deletions (indels). However, Sanger sequencing of two different sets of PCR clones (110bp,1349bp) demonstrated that at least some of these indels are real. The majority of the Nanoarchaeota PCR amplicons were vent associated; however, curiously, one relatively small Nanoarchaeota OTU (71 pyrosequencing reads) was only found in photic zone water samples obtained from a region of the lake furthest removed from the hydrothermal regions of the lake. Extensive pyrosequencing failed to demonstrate the presence of an Ignicoccus lineage in this lake, suggesting the Nanoarchaeota in this environment are associated with novel Archaea hosts. Defined phylogroups based on nearfull-length PCR clones document the significant Nanoarchaeota 16S rRNA gene diversity in this lake and firmly establish a terrestrial clade distinct from the marine Nanoarcheota as well as from other geographical locations.Item Geomicrobiology of sublacustrine thermal vents in Yellowstone Lake: Geochemical controls on microbial community structure and function(2015-10) Inskeep, William P.; Jay, Zackary J.; Macur, Richard E.; Clingenpeel, Scott; Tenney, A.; Lovalvo, D.; Beam, Jacob P.; Kozubal, Mark A.; Shanks, W. C.; Morgan, L. A.; Kan, Jinjun; Gorby, Yuri A.; Yooseph, Shibu; Nealson, Kenneth H.Yellowstone Lake (Yellowstone National Park, WY, USA) is a large high-altitude (2200 m), fresh-water lake, which straddles an extensive caldera and is the center of significant geothermal activity. The primary goal of this interdisciplinary study was to evaluate the microbial populations inhabiting thermal vent communities in Yellowstone Lake using 16S rRNA gene and random metagenome sequencing, and to determine how geochemical attributes of vent waters influence the distribution of specific microorganisms and their metabolic potential. Thermal vent waters and associated microbial biomass were sampled during two field seasons (2007-2008) using a remotely operated vehicle (ROV). Sublacustrine thermal vent waters (circa 50-90°C) contained elevated concentrations of numerous constituents associated with geothermal activity including dissolved hydrogen, sulfide, methane and carbon dioxide. Microorganisms associated with sulfur-rich filamentous ʺstreamerʺ communities of Inflated Plain and West Thumb (pH range 5-6) were dominated by bacteria from the Aquificales, but also contained thermophilic archaea from the Crenarchaeota and Euryarchaeota. Novel groups of methanogens and members of the Korarchaeota were observed in vents from West Thumb and Elliot's Crater (pH 5-6). Conversely, metagenome sequence from Mary Bay vent sediments did not yield large assemblies, and contained diverse thermophilic and nonthermophilic bacterial relatives. Analysis of functional genes associated with the major vent populations indicated a direct linkage to high concentrations of carbon dioxide, reduced sulfur (sulfide and/or elemental S), hydrogen and methane in the deep thermal ecosystems. Our observations show that sublacustrine thermal vents in Yellowstone Lake support novel thermophilic communities, which contain microorganisms with functional attributes not found to date in terrestrial geothermal systems of YNP.