Identification and characterization of high methane-emitting abandoned oil and gas wells.

Recent measurements of methane emissions from abandoned oil/gas wells show that these wells can be a substantial source of methane to the atmosphere, particularly from a small proportion of high-emitting wells. However, identifying high emitters remains a challenge. We couple 163 well measurements of methane flow rates; ethane, propane, and n-butane concentrations; isotopes of methane; and noble gas concentrations from 88 wells in Pennsylvania with synthesized data from historical documents, field investigations, and state databases. Using our databases, we (i) improve estimates of the number of abandoned wells in Pennsylvania; (ii) characterize key attributes that accompany high emitters, including depth, type, plugging status, and coal area designation; and (iii) estimate attribute-specific and overall methane emissions from abandoned wells. High emitters are best predicted as unplugged gas wells and plugged/vented gas wells in coal areas and appear to be unrelated to the presence of underground natural gas storage areas or unconventional oil/gas production. Repeat measurements over 2 years show that flow rates of high emitters are sustained through time. Our attribute-based methane emission data and our comprehensive estimate of 470,000-750,000 abandoned wells in Pennsylvania result in estimated state-wide emissions of 0.04-0.07 Mt (1012 g) CH4 per year. This estimate represents 5-8% of annual anthropogenic methane emissions in Pennsylvania. Our methodology combining new field measurements with data mining of previously unavailable well attributes and numbers of wells can be used to improve methane emission estimates and prioritize cost-effective mitigation strategies for Pennsylvania and beyond.

Reoxidation of Chromium(III) Products Formed under Different Biogeochemical Regimes.

Hexavalent chromium, Cr(VI), is a widespread and toxic groundwater contaminant. Reductive immobilization to Cr(III) is a treatment option, but its success depends on the long-term potential for reduced chromium precipitates to remain immobilized under oxidizing conditions. In this unique long-term study, aquifer sediments subjected to reductive Cr(VI) immobilization under different biogeochemical regimes were tested for their susceptibility to reoxidation. After reductive treatment for 1 year, sediments were exposed to oxygenated conditions for another 2 years in flow-through, laboratory columns. Under oxidizing conditions, immobilized chromium reduced under predominantly denitrifying conditions was mobilized at low concentrations (≪1 µM Cr(VI); ∼ 3% of Cr(III) deposited) that declined over time. A conceptual model of a limited pool of more soluble Cr(III), and a larger pool of relatively insoluble Cr(III), is proposed. In contrast, almost no chromium was mobilized from columns reduced under predominantly fermentative conditions, and where reducing conditions persisted for several months after introduction of oxidizing conditions, presumably due to the presence of a reservoir of reduced species generated during reductive treatment. The results from this 3-year study demonstrate that biogeochemical conditions present during reductive treatment, and the potential for buildup of reducing species, will impact the long-term sustainability of the remediation effort.

Water Table Dynamics and Biogeochemical Cycling in a Shallow, Variably-Saturated Floodplain.

Three-dimensional variably saturated flow and multicomponent biogeochemical reactive transport modeling, based on published and newly generated data, is used to better understand the interplay of hydrology, geochemistry, and biology controlling the cycling of carbon, nitrogen, oxygen, iron, sulfur, and uranium in a shallow floodplain. In this system, aerobic respiration generally maintains anoxic groundwater below an oxic vadose zone until seasonal snowmelt-driven water table peaking transports dissolved oxygen (DO) and nitrate from the vadose zone into the alluvial aquifer. The response to this perturbation is localized due to distinct physico-biogeochemical environments and relatively long time scales for transport through the floodplain aquifer and vadose zone. Naturally reduced zones (NRZs) containing sediments higher in organic matter, iron sulfides, and non-crystalline U(IV) rapidly consume DO and nitrate to maintain anoxic conditions, yielding Fe(II) from FeS oxidative dissolution, nitrite from denitrification, and U(VI) from nitrite-promoted U(IV) oxidation. Redox cycling is a key factor for sustaining the observed aquifer behaviors despite continuous oxygen influx and the annual hydrologically induced oxidation event. Depth-dependent activity of fermenters, aerobes, nitrate reducers, sulfate reducers, and chemolithoautotrophs (e.g., oxidizing Fe(II), S compounds, and ammonium) is linked to the presence of DO, which has higher concentrations near the water table.

Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi.

The acetyl-CoA "Wood-Ljungdahl" pathway couples the folate-mediated one-carbon (C1) metabolism to either CO2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood-Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH3-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH3-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood-Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities.

Isotopic insights into methane production, oxidation, and emissions in Arctic polygon tundra.

Arctic wetlands are currently net sources of atmospheric CH4 . Due to their complex biogeochemical controls and high spatial and temporal variability, current net CH4 emissions and gross CH4 processes have been difficult to quantify, and their predicted responses to climate change remain uncertain. We investigated CH4 production, oxidation, and surface emissions in Arctic polygon tundra, across a wet-to-dry permafrost degradation gradient from low-centered (intact) to flat- and high-centered (degraded) polygons. From 3 microtopographic positions (polygon centers, rims, and troughs) along the permafrost degradation gradient, we measured surface CH4 and CO2 fluxes, concentrations and stable isotope compositions of CH4 and DIC at three depths in the soil, and soil moisture and temperature. More degraded sites had lower CH4 emissions, a different primary methanogenic pathway, and greater CH4 oxidation than did intact permafrost sites, to a greater degree than soil moisture or temperature could explain. Surface CH4 flux decreased from 64 nmol m(-2)  s(-1) in intact polygons to 7 nmol m(-2)  s(-1) in degraded polygons, and stable isotope signatures of CH4 and DIC showed that acetate cleavage dominated CH4 production in low-centered polygons, while CO2 reduction was the primary pathway in degraded polygons. We see evidence that differences in water flow and vegetation between intact and degraded polygons contributed to these observations. In contrast to many previous studies, these findings document a mechanism whereby permafrost degradation can lead to local decreases in tundra CH4 emissions.

Reactive Transport Model of Sulfur Cycling as Impacted by Perchlorate and Nitrate Treatments.

Microbial souring in oil reservoirs produces toxic, corrosive hydrogen sulfide through microbial sulfate reduction, often accompanying (sea)water flooding during secondary oil recovery. With data from column experiments as constraints, we developed the first reactive-transport model of a new candidate inhibitor, perchlorate, and compared it with the commonly used inhibitor, nitrate. Our model provided a good fit to the data, which suggest that perchlorate is more effective than nitrate on a per mole of inhibitor basis. Critically, we used our model to gain insight into the underlying competing mechanisms controlling the action of each inhibitor. This analysis suggested that competition by heterotrophic perchlorate reducers and direct inhibition by nitrite produced from heterotrophic nitrate reduction were the most important mechanisms for the perchlorate and nitrate treatments, respectively, in the modeled column experiments. This work demonstrates modeling to be a powerful tool for increasing and testing our understanding of reservoir-souring generation, prevention, and remediation processes, allowing us to incorporate insights derived from laboratory experiments into a framework that can potentially be used to assess risk and design optimal treatment schemes.

Effects of varying growth conditions on stable carbon isotope fractionation of trichloroethene (TCE) by tceA-containing Dehalococcoides mccartyi strains.

To quantify in situ bioremediation using compound specific isotope analysis (CSIA), isotope fractionation data obtained from the field is interpreted according to laboratory-derived enrichment factors. Although previous studies that have quantified dynamic isotopic shifts during the reductive dechlorination of trichloroethene (TCE) indicate that fractionation factors can be highly variable from culture-to-culture and site-to-site, the effects of growth condition on the isotope fractionation during reductive dechlorination have not been previously examined. Here, carbon isotope fractionation by Dehalococcoides mccartyi 195 (Dhc195) maintained under a variety of growth conditions was examined. Enrichment factors quantified when Dhc195 was subjected to four suboptimal growth conditions, including decreased temperature (-13.3 ± 0.9‰), trace vitamin B12 availability (-12.7 ± 1.0‰), limited fixed nitrogen (-14.4 ± 0.8‰), and elevated vinyl chloride exposure (-12.5 ± 0.4‰), indicate that the fractionation is similar across a range of tested conditions. The TCE enrichment factors for two syntrophic cocultures, Dhc195 with Desulfovibrio vulgaris Hildenborough (-13.0 ± 2.0‰) and Dhc195 with Syntrophomonas wolfei (-10.4 ± 1.2‰ and -13.3 ± 1.0‰), were also similar to a control experiment. In order to test the stability of enrichment factors in microbial communities, the isotope fractionation was quantified for Dhc-containing groundwater communities before and after two-year enrichment periods under different growth conditions. Although these enrichment factors (-8.9 ± 0.4‰, -6.8 ± 0.8‰, -8.7 ± 1.3‰, -9.4 ± 0.7‰, and -7.2 ± 0.3‰) were predominantly outside the range of values quantified for the isolate and cocultures, all tested enrichment conditions within the communities produced nearly similar fractionations. Enrichment factors were not significantly affected by changes in any of the tested growth conditions for the pure cultures, cocultures or the mixed communities, indicating that despite a variety of temperature, nutrient, and cofactor-limiting conditions, stable carbon isotope fractionations remain consistent for given Dehalococcoides cultures.

Succession of hydrocarbon-degrading bacteria in the aftermath of the deepwater horizon oil spill in the gulf of Mexico.

The Deepwater Horizon oil spill produced large subsurface plumes of dispersed oil and gas in the Gulf of Mexico that stimulated growth of psychrophilic, hydrocarbon degrading bacteria. We tracked succession of plume bacteria before, during and after the 83-day spill to determine the microbial response and biodegradation potential throughout the incident. Dominant bacteria shifted substantially over time and were dependent on relative quantities of different hydrocarbon fractions. Unmitigated flow from the wellhead early in the spill resulted in the highest proportions of n-alkanes and cycloalkanes at depth and corresponded with dominance by Oceanospirillaceae and Pseudomonas. Once partial capture of oil and gas began 43 days into the spill, petroleum hydrocarbons decreased, the fraction of aromatic hydrocarbons increased, and Colwellia, Cycloclasticus, and Pseudoalteromonas increased in dominance. Enrichment of Methylomonas coincided with positive shifts in the δ(13)C values of methane in the plume and indicated significant methane oxidation occurred earlier than previously reported. Anomalous oxygen depressions persisted at plume depths for over six weeks after well shut-in and were likely caused by common marine heterotrophs associated with degradation of high-molecular-weight organic matter, including Methylophaga. Multiple hydrocarbon-degrading bacteria operated simultaneously throughout the spill, but their relative importance was controlled by changes in hydrocarbon supply.

Deep-sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill.

The Deepwater Horizon oil spill resulted in a massive influx of hydrocarbons into the Gulf of Mexico (the Gulf). To better understand the fate of the oil, we enriched and isolated indigenous hydrocarbon-degrading bacteria from deep, uncontaminated waters from the Gulf with oil (Macondo MC252) and dispersant used during the spill (COREXIT 9500). During 20 days of incubation at 5°C, CO(2) evolution, hydrocarbon concentrations and the microbial community composition were determined. Approximately 60% to 25% of the dissolved oil with or without COREXIT, respectively, was degraded, in addition to some hydrocarbons in the COREXIT. FeCl(2) addition initially increased respiration rates, but not the total amount of hydrocarbons degraded. 16S rRNA gene sequencing revealed a succession in the microbial community over time, with an increase in abundance of Colwellia and Oceanospirillales during the incubations. Flocs formed during incubations with oil and/or COREXIT in the absence of FeCl(2) . Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy revealed that the flocs were comprised of oil, carbohydrates and biomass. Colwellia were the dominant bacteria in the flocs. Colwellia sp. strain RC25 was isolated from one of the enrichments and confirmed to rapidly degrade high amounts (approximately 75%) of the MC252 oil at 5°C. Together these data highlight several features that provide Colwellia with the capacity to degrade oil in cold, deep marine habitats, including aggregation together with oil droplets into flocs and hydrocarbon degradation ability.

Phylogenetic microarray analysis of a microbial community performing reductive dechlorination at a TCE-contaminated site.

A high-density phylogenetic microarray (PhyloChip) was applied to track bacterial and archaeal populations through different phases of remediation at Ft. Lewis, WA, a trichloroethene (TCE)-contaminated groundwater site. Biostimulation with whey, and bioaugmentation with a Dehalococcoides-containing enrichment culture were strategies implemented to enhance dechlorination. As a measure of species richness, over 1300 operational taxonomic units (OTUs) were detected in DNA from groundwater samples extracted during different stages of treatment and in the bioaugmentation culture. In order to determine active members within the community, 16S rRNA from samples were analyzed by microarray and ∼600 OTUs identified. A cDNA clone library of the expressed 16S rRNA corroborated the observed diversity and activity of some of the phyla. Principle component analysis of the treatment plot samples revealed that the microbial populations were constantly changing during the course of the study. Dynamic analysis of the archaeal population showed significant increases in methanogens at the later stages of treatment that correlated with increases in methane concentrations of over 2 orders of magnitude. Overall, the PhyloChip analyses in this study have provided insights into the microbial ecology and population dynamics at the TCE-contaminated field site useful for understanding the in situ reductive dechlorination processes.

Timing the onset of sulfate reduction over multiple subsurface acetate amendments by measurement and modeling of sulfur isotope fractionation.

Stable isotope fractionations of sulfur are reported for three consecutive years of acetate-enabled uranium bioremediation at the US Department of Energy's Rifle Integrated Field Research Challenge (IFRC) site. The data show a previously undocumented decrease in the time between acetate addition and the onset of sulfate reducing conditions over subsequent amendments, from 20 days in the 2007 experiment to 4 days in the 2009 experiment. Increased sulfide concentrations were observed at the same time as δ(34)S of sulfate enrichment in the first year, but in subsequent years elevated sulfide was detected up to 15 days after increased δ(34)S of sulfate. A biogeochemical reactive transport model is developed which explicitly incorporates the stable isotopes of sulfur to simulate fractionation during the 2007 and 2008 amendments. A model based on an initially low, uniformly distributed population of sulfate reducing bacteria that grow and become spatially variable with time reproduces measured trends in solute concentration and δ(34)S, capturing the change in onset of sulfate reduction in subsequent years. Our results demonstrate a previously unrecognized hysteretic effect in the spatial distribution of biomass growth during stimulated subsurface bioremediation.

Isotopic tracking of Hanford 300 area derived uranium in the Columbia River.

Our objectives in this study are to quantify the discharge rate of uranium (U) to the Columbia River from the Hanford Site's 300 Area and to follow that U downriver to constrain its fate. Uranium from the Hanford Site has variable isotopic composition due to nuclear industrial processes carried out at the site. This characteristic makes it possible to use high-precision isotopic measurements of U in environmental samples to identify even trace levels of contaminant U, determine its sources, and estimate discharge rates. Our data on river water samples indicate that as much as 3.2 kg/day can enter the Columbia River from the 300 Area, which is only a small fraction of the total load of dissolved natural background U carried by the Columbia River. This very low level of Hanford-derived U can be discerned, despite dilution to <1% of natural background U, 400 km downstream from the Hanford Site. These results indicate that isotopic methods can allow the amounts of U from the 300 Area of the Hanford Site entering the Columbia River to be measured accurately to ascertain whether they are an environmental concern or insignificant relative to natural uranium background in the Columbia River.

Use of carbon stable isotopes to monitor biostimulation and electron donor fate in chromium-contaminated groundwater.

Hexavalent chromium Cr(VI) is a common inorganic contaminant in industrial areas and represents a serious threat to human health due its toxicity. Here we report experimental results from a field-scale investigation of Cr(VI) bio-immobilization at Hanford 100H reservation, a U.S Department of Energy facility (Washington State, USA). Microbial Cr(VI) reduction was stimulated via injection of a13C-labeled sodium lactate solution into the high-permeability aquifer consisting of gravel and coarse sand sediments. Concentrations and carbon isotope ratios of metabolites, including dissolved inorganic carbon and total organic carbon, and compound-specific analysis of acetate and propionate, together with phospholipid fatty acids (biomass) have been analyzed to help provide an understanding of the predominant redox processes accompanying Cr(VI) reduction. Results of our study indicate that the injection of an electron donor caused a sharp decrease of Cr(VI) concentration from ∼32 to ∼10 nM. Cr(VI) reduction was associated with a decrease in the concentration of carboxylic acids, such as lactate (∼6 mM to undetectable), propionate (∼9 mM to undetectable), and acetate (∼6 mM to undetectable), as well as dissolved inorganic carbon (30-10 mM C). Carbon isotope data indicate carbon transfers from the original substrate to organic byproducts and mineralized carbon. Concentrations of metabolites and stable isotope data as well as carbon isotope mass balance calculations were used to monitor biologically mediated reduction of Cr(VI).

Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle.

Beneficial microbial associations enhance the fitness of most living organisms, and wood-feeding insects offer some of the most striking examples of this. Odontotaenius disjunctus is a wood-feeding beetle that possesses a digestive tract with four main compartments, each of which contains well-differentiated microbial populations, suggesting that anatomical properties and separation of these compartments may enhance energy extraction from woody biomass. Here, using integrated chemical analyses, we demonstrate that lignocellulose deconstruction and fermentation occur sequentially across compartments, and that selection for microbial groups and their metabolic pathways is facilitated by gut anatomical features. Metaproteogenomics showed that higher oxygen concentration in the midgut drives lignocellulose depolymerization, while a thicker gut wall in the anterior hindgut reduces oxygen diffusion and favours hydrogen accumulation, facilitating fermentation, homoacetogenesis and nitrogen fixation. We demonstrate that depolymerization continues in the posterior hindgut, and that the beetle excretes an energy- and nutrient-rich product on which its offspring subsist and develop. Our results show that the establishment of beneficial microbial partners within a host requires both the acquisition of the microorganisms and the formation of specific habitats within the host to promote key microbial metabolic functions. Together, gut anatomical properties and microbial functional assembly enable lignocellulose deconstruction and colony subsistence on an extremely nutrient-poor diet.

Attenuating Sulfidogenesis in a Soured Continuous Flow Column System With Perchlorate Treatment.

Hydrogen sulfide production by sulfate reducing bacteria (SRB) is the primary cause of oil reservoir souring. Amending environments with chlorate or perchlorate [collectively denoted (per)chlorate] represents an emerging technology to prevent the onset of souring. Recent studies with perchlorate reducing bacteria (PRB) monocultures demonstrated that they have the innate capability to enzymatically oxidize sulfide, thus PRB may offer an effective means of reversing souring. (Per)chlorate may be effective by (i) direct toxicity to SRB; (ii) competitive exclusion of SRB by PRB; or (iii) reversal of souring through re-oxidation of sulfide by PRB. To determine if (per)chlorate could sweeten a soured column system and assign a quantitative value to each of the mechanisms we treated columns flooded with San Francisco bay water with temporally decreasing amounts (50, 25, and 12.5 mM) of (per)chlorate. Geochemistry and the microbial community structure were monitored and a reactive transport model was developed, Results were compared to columns treated with nitrate or untreated. Souring was reversed by all treatments at 50 mM but nitrate-treated columns began to re-sour when treatment concentrations decreased (25 mM). Re-souring was only observed in (per)chlorate-treated columns when concentrations were decreased to 12.5 mM and the extent of re-souring was less than the control columns. Microbial community analyses indicated treatment-specific community shifts. Nitrate treatment resulted in a distinct community enriched in genera known to perform sulfur cycling metabolisms and genera capable of nitrate reduction. (Per)chlorate treatment enriched for (per)chlorate reducing bacteria. (Per)chlorate treatments only enriched for sulfate reducing organisms when treatment levels were decreased. A reactive transport model of perchlorate treatment was developed and a baseline case simulation demonstrated that the model provided a good fit to the effluent geochemical data. Subsequent simulations teased out the relative role that each of the three perchlorate inhibition mechanisms played during different phases of the experiment. These results indicate that perchlorate addition is an effective strategy for both souring prevention and souring reversal. It provides insight into which organisms are involved, and illuminates the interactive effects of the inhibition mechanisms, further highlighting the versatility of perchlorate as a sweetening agent.

Mechanism of H2S Oxidation by the Dissimilatory Perchlorate-Reducing Microorganism Azospira suillum PS.

The genetic and biochemical basis of perchlorate-dependent H2S oxidation (PSOX) was investigated in the dissimilatory perchlorate-reducing microorganism (DPRM) Azospira suillum PS (PS). Previously, it was shown that all known DPRMs innately oxidize H2S, producing elemental sulfur (So). Although the process involving PSOX is thermodynamically favorable (ΔG°' = -206 kJ â‹… mol-1 H2S), the underlying biochemical and genetic mechanisms are currently unknown. Interestingly, H2S is preferentially utilized over physiological electron donors such as lactate or acetate although no growth benefit is obtained from the metabolism. Here, we determined that PSOX is due to a combination of enzymatic and abiotic interactions involving reactive intermediates of perchlorate respiration. Using various approaches, including barcode analysis by sequencing (Bar-seq), transcriptome sequencing (RNA-seq), and proteomics, along with targeted mutagenesis and biochemical characterization, we identified all facets of PSOX in PS. In support of our proposed model, deletion of identified upregulated PS genes traditionally known to be involved in sulfur redox cycling (e.g., Sox, sulfide:quinone reductase [SQR]) showed no defect in PSOX activity. Proteomic analysis revealed differential abundances of a variety of stress response metal efflux pumps and divalent heavy-metal transporter proteins, suggesting a general toxicity response. Furthermore, in vitro biochemical studies demonstrated direct PSOX mediated by purified perchlorate reductase (PcrAB) in the absence of other electron transfer proteins. The results of these studies support a model in which H2S oxidation is mediated by electron transport chain short-circuiting in the periplasmic space where the PcrAB directly oxidizes H2S to So The biogenically formed reactive intermediates (ClO2- and O2) subsequently react with additional H2S, producing polysulfide and So as end products.IMPORTANCE Inorganic sulfur compounds are widespread in nature, and microorganisms are central to their transformation, thereby playing a key role in the global sulfur cycle. Sulfur oxidation is mediated by a broad phylogenetic diversity of microorganisms, including anoxygenic phototrophs and either aerobic or anaerobic chemotrophs coupled to oxygen or nitrate respiration, respectively. Recently, perchlorate-respiring microorganisms were demonstrated to be innately capable of sulfur oxidation regardless of their phylogenetic affiliation. As recognition of the prevalence of these organisms intensifies, their role in global geochemical cycles is being queried. This is further highlighted by the recently recognized environmental pervasiveness of perchlorate not only across Earth but also throughout our solar system. The inferred importance of this metabolism not only is that it is a novel and previously unrecognized component of the global sulfur redox cycle but also is because of the recently demonstrated applicability of perchlorate respiration in the control of biogenic sulfide production in engineered environments such as oil reservoirs and wastewater treatment facilities, where excess H2S represents a significant environmental, process, and health risk, with associated costs approximating $90 billion annually.

Microbial community responses to organophosphate substrate additions in contaminated subsurface sediments.

BACKGROUND: Radionuclide- and heavy metal-contaminated subsurface sediments remain a legacy of Cold War nuclear weapons research and recent nuclear power plant failures. Within such contaminated sediments, remediation activities are necessary to mitigate groundwater contamination. A promising approach makes use of extant microbial communities capable of hydrolyzing organophosphate substrates to promote mineralization of soluble contaminants within deep subsurface environments. METHODOLOGY/PRINCIPAL FINDINGS: Uranium-contaminated sediments from the U.S. Department of Energy Oak Ridge Field Research Center (ORFRC) Area 2 site were used in slurry experiments to identify microbial communities involved in hydrolysis of 10 mM organophosphate amendments [i.e., glycerol-2-phosphate (G2P) or glycerol-3-phosphate (G3P)] in synthetic groundwater at pH 5.5 and pH 6.8. Following 36 day (G2P) and 20 day (G3P) amended treatments, maximum phosphate (PO4(3-)) concentrations of 4.8 mM and 8.9 mM were measured, respectively. Use of the PhyloChip 16S rRNA microarray identified 2,120 archaeal and bacterial taxa representing 46 phyla, 66 classes, 110 orders, and 186 families among all treatments. Measures of archaeal and bacterial richness were lowest under G2P (pH 5.5) treatments and greatest with G3P (pH 6.8) treatments. Members of the phyla Crenarchaeota, Euryarchaeota, Bacteroidetes, and Proteobacteria demonstrated the greatest enrichment in response to organophosphate amendments and the OTUs that increased in relative abundance by 2-fold or greater accounted for 9%-50% and 3%-17% of total detected Archaea and Bacteria, respectively. CONCLUSIONS/SIGNIFICANCE: This work provided a characterization of the distinct ORFRC subsurface microbial communities that contributed to increased concentrations of extracellular phosphate via hydrolysis of organophosphate substrate amendments. Within subsurface environments that are not ideal for reductive precipitation of uranium, strategies that harness microbial phosphate metabolism to promote uranium phosphate precipitation could offer an alternative approach for in situ sequestration.

Isotopic insights into microbial sulfur cycling in oil reservoirs.

Microbial sulfate reduction in oil reservoirs (biosouring) is often associated with secondary oil production where seawater containing high sulfate concentrations (~28 mM) is injected into a reservoir to maintain pressure and displace oil. The sulfide generated from biosouring can cause corrosion of infrastructure, health exposure risks, and higher production costs. Isotope monitoring is a promising approach for understanding microbial sulfur cycling in reservoirs, enabling early detection of biosouring, and understanding the impact of souring. Microbial sulfate reduction is known to result in large shifts in the sulfur and oxygen isotope compositions of the residual sulfate, which can be distinguished from other processes that may be occurring in oil reservoirs, such as precipitation of sulfate and sulfide minerals. Key to the success of this method is using the appropriate isotopic fractionation factors for the conditions and processes being monitored. For a set of batch incubation experiments using a mixed microbial culture with crude oil as the electron donor, we measured a sulfur fractionation factor for sulfate reduction of -30‰. We have incorporated this result into a simplified 1D reservoir reactive transport model to highlight how isotopes can help discriminate between biotic and abiotic processes affecting sulfate and sulfide concentrations. Modeling results suggest that monitoring sulfate isotopes can provide an early indication of souring for reservoirs with reactive iron minerals that can remove the produced sulfide, especially when sulfate reduction occurs in the mixing zone between formation waters (FW) containing elevated concentrations of volatile fatty acids (VFAs) and injection water (IW) containing elevated sulfate. In addition, we examine the role of reservoir thermal, geochemical, hydrological, operational and microbiological conditions in determining microbial souring dynamics and hence the anticipated isotopic signatures.

Control of sulfidogenesis through bio-oxidation of H2S coupled to (per)chlorate reduction.

We investigated H2S attenuation by dissimilatory perchlorate-reducing bacteria (DPRB). All DPRB tested oxidized H2S coupled to (per)chlorate reduction without sustaining growth. H2S was preferentially utilized over organic electron donors resulting in an enriched (34S)-elemental sulfur product. Electron microscopy revealed elemental sulfur production in the cytoplasm and on the cell surface of the DPRB Azospira suillum. Based on our results, we propose a novel hybrid enzymatic-abiotic mechanism for H2S oxidation similar to that recently proposed for nitrate-dependent Fe(II) oxidation. The results of this study have implications for the control of biosouring and biocorrosion in a range of industrial environments.

Inhibition of microbial sulfate reduction in a flow-through column system by (per)chlorate treatment.

Microbial sulfate reduction is a primary cause of oil reservoir souring. Here we show that amendment with chlorate or perchlorate [collectively (per)chlorate] potentially resolves this issue. Triplicate packed columns inoculated with marine sediment were flushed with coastal water amended with yeast extract and one of nitrate, chlorate, or perchlorate. Results showed that although sulfide production was dramatically reduced by all treatments, effluent sulfide was observed in the nitrate (10 mM) treatment after an initial inhibition period. In contrast, no effluent sulfide was observed with (per)chlorate (10 mM). Microbial community analyses indicated temporal community shifts and phylogenetic clustering by treatment. Nitrate addition stimulated Xanthomonadaceae and Rhizobiaceae growth, supporting their role in nitrate metabolism. (Per)chlorate showed distinct effects on microbial community structure compared with nitrate and resulted in a general suppression of the community relative to the untreated control combined with a significant decrease in sulfate reducing species abundance indicating specific toxicity. Furthermore, chlorate stimulated Pseudomonadaceae and Pseudoalteromonadaceae, members of which are known chlorate respirers, suggesting that chlorate may also control sulfidogenesis by biocompetitive exclusion of sulfate-reduction. Perchlorate addition stimulated Desulfobulbaceae and Desulfomonadaceae, which contain sulfide oxidizing and elemental sulfur-reducing species respectively, suggesting that effluent sulfide concentrations may be controlled through sulfur redox cycling in addition to toxicity and biocompetitive exclusion. Sulfur isotope analyses further support sulfur cycling in the columns, even when sulfide is not detected. This study indicates that (per)chlorate show great promise as inhibitors of sulfidogenesis in natural communities and provides insight into which organisms and respiratory processes are involved.