Browsing by Author "Macur, Richard E."

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Characterization of methyl tert-butyl ether-degrading bacteria from a gasoline-contaminated aquifer
(2002-01) Kern, Eric Anthony; Veeh, Richard Harold; Macur, Richard E.; Cunningham, Alfred B.
Molecular microbial community analysis was combined with traditional cultivation strategies to investigate the presence of methyl tert-butyl ether (MTBE)-degrading bacteria in a gasoline-contaminated aquifer (Ronan, MT). A bacterial consortium, RS24, which is capable of complete mineralization of MTBE as a sole carbon and energy source was enriched from soil and aquifer materials taken from the contaminated site. The consortium was capable of degrading MTBE at rates up to 0.66 mg d-,1 with corresponding gross biomass yields of 0.25 ± 0.02 mg dry biomass (mg MTBE)-1. Two MTBE-degrading isolates identified as Pseudomonas Ant9 and Rhodococcus koreensis were obtained from the consortium. However, both isolates required the presence of 2-propanol as a cosubstrate for MTBE degradation. Denaturing gradient gel electrophoresis (DGGE) of Polymerase Chain Reaction (PCR)-amplified 16S rDNA confirmed the presence of both isolates in the initial consortium and indicated their disappearance with transfer and subculturing. MTBE degradation and cell growth by the consortium was stimulated by the presence of spent culture medium, suggesting the production of a growth factor during MTBE degradation. These results indicate the presence of naturally occurring MTBE-degrading bacteria in a contaminated aquifer and suggest the potential for natural attenuation or enhanced aerobic oxidation.
An efficient and scalable extraction and quantification method for algal derived biofuel
(2013-09) Lohman, Egan J.; Gardner, Robert D.; Halverson, L.; Macur, Richard E.; Peyton, Brent M.; Gerlach, Robin
Microalgae are capable of synthesizing a multitude of compounds including biofuel precursors and other high value products such as omega-3-fatty acids. However, accurate analysis of the specific compounds produced by microalgae is important since slight variations in saturation and carbon chain length can affect the quality, and thus the value, of the end product. We present a method that allows for fast and reliable extraction of lipids and similar compounds from a range of algae, followed by their characterization using gas chromatographic analysis with a focus on biodiesel-relevant compounds. This method determines which range of biologically synthesized compounds is likely responsible for each fatty acid methyl ester (FAME) produced; information that is fundamental for identifying preferred microalgae candidates as a biodiesel source. Traditional methods of analyzing these precursor molecules are time intensive and prone to high degrees of variation between species and experimental conditions. Here we detail a new method which uses microwave energy as a reliable, single-step cell disruption technique to extract lipids fromlive cultures of microalgae. After extractable lipid characterization (including lipid type (free fatty acids, mono-, di- or tri-acylglycerides) and carbon chain length determination) by GCâ€“FID, the same lipid extracts are transesterified into FAMEs and directly compared to total biodiesel potential by GCâ€“MS. This approach provides insight into the fraction of total FAMEs derived from extractable lipids compared to FAMEs derived fromthe residual fraction (i.e. membrane bound phospholipids, sterols, etc.). This approach can also indicate which extractable lipid compound, based on chain length and relative abundance, is responsible for each FAME. This method was tested on three species of microalgae: the marine diatom Phaeodactylum tricornutum, the model Chlorophyte Chlamydomonas reinhardtii, and the freshwater green alga Chlorella vulgaris. The method is shown to be robust, highly reproducible, and fast, allowing for multiple samples to be analyzed throughout the time course of culturing, thus providing time-resolved information regarding lipid quantity and quality. Total time from harvesting to obtaining analytical results is less than 2 h.
Geochemistry and Mixing Drive the Spatial Distribution of Free-Living Archaea and Bacteria in Yellowstone Lake
(2016-02) Kan, Jinjun; Clingenpeel, Scott; Dow, Charles L.; McDermott, Timothy R.; Macur, Richard E.; Inskeep, William P.; Nealson, Kenneth H.
Yellowstone Lake, the largest subalpine lake in the United States, harbors great novelty and diversity of Bacteria and Archaea. Size-fractionated water samples (0.1–0.8, 0.8–3.0, and 3.0–20 μm) were collected from surface photic zone, deep mixing zone, and vent fluids at different locations in the lake by using a remotely operated vehicle (ROV). Quantification with real-time PCR indicated that Bacteria dominated free-living microorganisms with Bacteria/Archaea ratios ranging from 4037:1 (surface water) to 25:1 (vent water). Microbial population structures (both Bacteria and Archaea) were assessed using 454-FLX sequencing with a total of 662,302 pyrosequencing reads for V1 and V2 regions of 16S rRNA genes. Non-metric multidimensional scaling (NMDS) analyses indicated that strong spatial distribution patterns existed from surface to deep vents for free-living Archaea and Bacteria in the lake. Along with pH, major vent-associated geochemical constituents including CH4, CO2, H2, DIC (dissolved inorganic carbon), DOC (dissolved organic carbon), SO42-, O2 and metals were likely the major drivers for microbial population structures, however, mixing events occurring in the lake also impacted the distribution patterns. Distinct Bacteria and Archaea were present among size fractions, and bigger size fractions included particle-associated microbes (> 3 μm) and contained higher predicted operational taxonomic unit richness and microbial diversities (genus level) than free-living ones (<0.8 μm). Our study represents the first attempt at addressing the spatial distribution of Bacteria and Archaea in Yellowstone Lake, and our results highlight the variable contribution of Archaea and Bacteria to the hydrogeochemical-relevant metabolism of hydrogen, carbon, nitrogen, and sulfur.
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 50-90Â°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.
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 â€“ 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 1000Î¼M; P inhibition constant (Ki)~20â€“100 Î¼M). 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.
Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function
(2010-03) Inskeep, William P.; Rusch, Douglas B.; Jay, Zackary J.; Herrgard, Markus J.; Kozubal, Mark A.; Richardson, Toby H.; Macur, Richard E.; Hamamura, Natsuko; Jennings, Ryan deM.; Fouke, Bruce W.; Reysenbach, Anna-Louise; Roberto, Frank; Young, Mark J.; Schwartz, Ariel; Boyd, Eric S.; Badger, Jonathan H.; Mathur, Eric J.; Ortmann, Alice C.; Bateson, Mary M.; Geesey, Gill G.; Frazier, Marvin
The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14–15,000 Sanger reads per site) was obtained for five high-temperature (>65°C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O2) control microbial community structure and function in YNP geothermal springs.
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.
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.
Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum
(2012-10) Gardner, Robert D.; Cooksey, Keith E.; Mus, Florence; Macur, Richard E.; Moll, Karen M.; Eustance, E. O.; Carlson, Ross P.; Gerlach, Robin; Fields, Matthew W.; Peyton, Brent M.
There is potential for algal-derived biofuel to help alleviate part of the worldâ€™s dependency on petroleum based fuels. However, research must still be done on strain selection, induction of triacylglycerol (TAG) accumulation, and fundamental algal metabolic studies, along with large-scale culturing techniques, harvesting, and biofuel/biomass processing. Here, we have advanced the knowledge on Scenedesmus sp. strain WC-1 by monitoring growth, pH, and TAG accumulation on a 14:10 lightâ€“dark cycle with atmospheric air or 5% CO2 in air (v/v) aeration. Under ambient aeration, there was a loss of pH-induced TAG accumulation, presumably due to TAG consumption during the lower culture pH observed during dark hours (pH 9.4). Under 5% CO2 aeration, the growth rate nearly doubled from 0.78 to 1.53 dâˆ’1, but the pH was circumneutral (pH 6.9) and TAG accumulation was minimal. Experiments were also performed with 5% CO2 during the exponential growth phase, which was then switched to aeration with atmospheric air when nitrate was close to depletion. These tests were run with and without the addition of 50 mM sodium bicarbonate. Cultures without added bicarbonate showed decreased growth rates with the aeration change, but there was no immediate TAG accumulation. The cultures with bicarbonate added immediately ceased cellular replication and rapid TAG accumulation was observed, as monitored by Nile Red fluorescence which has previously been correlated by gas chromatography to cellular TAG levels. Sodium bicarbonate addition (25 mM final concentration) was also tested with the marine diatom Phaeodactylum tricornutum strain Pt-1 and this organism also accumulated TAG.
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.