Scholarly Work - Center for Biofilm Engineering
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Item Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community(2018-09) Hunt, Kristopher A.; Jennings, Ryan deM.; Inskeep, William P.; Carlson, Ross P.Interactions among microbial community members can lead to emergent properties, such as enhanced productivity, stability, and robustness. Iron-oxide mats in acidic (pH 2–4), high-temperature (> 65 °C) springs of Yellowstone National Park contain relatively simple microbial communities and are well-characterized geochemically. Consequently, these communities are excellent model systems for studying the metabolic activity of individual populations and key microbial interactions. The primary goals of the current study were to integrate data collected in situ with in silico calculations across process-scales encompassing enzymatic activity, cellular metabolism, community interactions, and ecosystem biogeochemistry, as well as to predict and quantify the functional limits of autotroph-heterotroph interactions. Metagenomic and transcriptomic data were used to reconstruct carbon and energy metabolisms of an important autotroph (Metallosphaera yellowstonensis) and heterotroph (Geoarchaeum sp. OSPB) from the studied Fe(III)-oxide mat communities. Standard and hybrid elementary flux mode and flux balance analyses of metabolic models predicted cellular- and community-level metabolic acclimations to simulated environmental stresses, respectively. In situ geochemical analyses, including oxygen depth-profiles, Fe(III)-oxide deposition rates, stable carbon isotopes and mat biomass concentrations, were combined with cellular models to explore autotroph-heterotroph interactions important to community structure-function. Integration of metabolic modeling with in situ measurements, including the relative population abundance of autotrophs to heterotrophs, demonstrated that Fe(III)-oxide mat communities operate at their maximum total community growth rate (i.e. sum of autotroph and heterotroph growth rates), as opposed to net community growth rate (i.e. total community growth rate subtracting autotroph consumed by heterotroph), as predicted from the maximum power principle. Integration of multiscale data with ecological theory provides a basis for predicting autotroph-heterotroph interactions and community-level cellular organization.Item Soil depth and temperature effects on microbial degradation of 2,4-d(1996) Veeh, Richard Harold; Inskeep, William P.; Camper, Anne K.Numerous soil factors and climatic conditions affect the degradation rate of pesticides in soils. A major soil factor influencing herbicide degradation is the composition and abundance of the microbiota, which has been shown to vary considerably with soil depth. Another important variable affecting microbial growth and degradation kinetics is temperature. Soil samples from 0- to 30-, 30- to 60-, and 60- to 120-cm depths of two Montana soils were placed in reaction flasks and treated with 14C-labeled 2,4-D at representative field use rates at temperatures of 10, 17, and 24°C. A carrier gas was used to continuously evacuate evolved 14CO2 into NaOH traps as a measure of 2,4-D degradation. Comparisons of the effects of soil depth and temperature were made by fitting experimental data to both first-order and logistic kinetic models. Degradation rates of 2,4-D decreased significantly with increasing soil depth and were positively correlated with bacterial plate counts. Effects of temperature on degradation rate constants were adequately described using the Arrhenius equation. Degradation rates of 2,4-D and bacterial enumerations were positively correlated with changes in soil organic C as a function of soil depth. These results support the idea that changes in organic C with soil depth could be used as a parameter for estimating changes in degradation rate as a function of soil depth. Efforts to model the transport of 2,4-D in soils should account for variation in degradation rate as a function of soil depth and temperature.Item Use of soil column test apparatus for evaluation of in situ bioremediation potential(1990) Jones, Warren L.; Sturman, Paul J.; Ellison, C.; Camper, Anne K.; Inskeep, William P.; Rogers, Robert D.; Cunningham, Alfred B.Item Impact of ferrihydrite and anthraquinone-2,6-disulfonate on the reductive transformation of 2,4,6-trinitrotoluene by a gram-positive fermenting bacterium(2005-09) Borch, Thomas; Inskeep, William P.; Harwood, Jace A.; Gerlach, RobinBatch studies were conducted to explore differences in the transformation pathways of 2,4,6-trinitrotoluene (TNT) reduction by a Gram-positive fermenting bacterium (Cellulomonas sp. strain ES6) in the presence and absence of ferrihydrite and the electron shuttle anthraquinone-2,6-disulfonate (AQDS). Strain ES6 was capable of TNT and ferrihydrite reduction with increased reduction rates in the presence of AQDS. Hydroxylaminodinitrotoluenes, 2,4-dihydroxylamino-6-nitrotoluene (2,4-DHANT), and tetranitroazoxytoluenes were the major metabolites observed in ferrihydrite- and AQDS-free systems in the presence of pure cell cultures. Ferrihydrite enhanced the production of amino derivatives because of reactions with microbially produced surface-associated Fe(II). The presence of AQDS in the absence of ferrihydrite promoted the fast initial formation of arylhydroxylamines such as 2,4-DHANT. However, unlike in pure cell systems, these arylhydroxylamines were transformed into several unidentified polar products. When both microbially reduced ferrihydrite and AQDS were present simultaneously, the reduction of TNT was more rapid and complete via pathways that would have been difficult to infer solely from single component studies. This study demonstrates the complexity of TNT degradation patterns in model systems where the interactions among bacteria, Fe minerals, and organic matter have a pronounced effect on the degradation pathway of TNT.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 Stoichiometric modelling of assimilatory and dissimilatory biomass utilisation in a microbial community(2016-08) Hunt, Kristopher A.; Jennings, Ryan deM.; Inskeep, William P.; Carlson, Ross P.Assimilatory and dissimilatory utilisation of autotroph biomass by heterotrophs is a fundamental mechanism for the transfer of nutrients and energy across trophic levels. Metagenome data from a tractable, thermoacidophilic microbial community in Yellowstone National Park was used to build an in silico model to study heterotrophic utilisation of autotroph biomass using elementary flux mode analysis and flux balance analysis. Assimilatory and dissimilatory biomass utilisation was investigated using 29 forms of biomass-derived dissolved organic carbon (DOC) including individual monomer pools, individual macromolecular pools and aggregate biomass. The simulations identified ecologically competitive strategies for utilizing DOC under conditions of varying electron donor, electron acceptor or enzyme limitation. The simulated growth environment affected which form of DOC was the most competitive use of nutrients; for instance, oxygen limitation favoured utilisation of less reduced and fermentable DOC while carbon-limited environments favoured more reduced DOC. Additionally, metabolism was studied considering two encompassing metabolic strategies: simultaneous versus sequential use of DOC. Results of this study bound the transfer of nutrients and energy through microbial food webs, providing a quantitative foundation relevant to most microbial ecosystems.Item Terminal oxidase diversity and function in Metallosphaera yellowstonensis: Gene expression and protein modeling suggest mechanisms of Fe(II) oxidation in the sulfolobales(2011-03) Kozubal, Mark A.; Dlakic, Mensur; Macur, Richard E.; Inskeep, William P.Metallosphaera yellowstonensis is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b558/566, several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex.An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores.Item 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.