Kenneth Lowe

Effects of nitrate on the stability of uranium in a bioreduced region of the subsurface.

The effects of nitrate on the stability of reduced, immobilized uranium were evaluated in field experiments at a U.S. Department of Energy site in Oak Ridge, TN. Nitrate (2.0 mM) was injected into a reduced region of the subsurface containing high levels of previously immobilized U(IV). The nitrate was reduced to nitrite, ammonium, and nitrogen gas; sulfide levels decreased; and Fe(II) levels increased then deceased. Uranium remobilization occurred concomitant with nitrite formation, suggesting nitrate-dependent, iron-accelerated oxidation of U(IV). Bromide tracer results indicated changes in subsurface flowpaths likely due to gas formation and/or precipitate. Desorption-adsorption of uranium by the iron-rich sediment impacted uranium mobilization and sequestration. After rereduction of the subsurface through ethanol additions, background groundwater containing high levels of nitrate was allowed to enter the reduced test zone. Aqueous uranium concentrations increased then decreased. Clone library analyses of sediment samples revealed the presence of denitrifying bacteria that can oxidize elemental sulfur, H(2)S, Fe(II), and U(IV) (e.g., Thiobacillus spp.), and a decrease in relative abundance of bacteria that can reduce Fe(III) and sulfate. XANES analyses of sediment samples confirmed changes in uranium oxidation state. Addition of ethanol restored reduced conditions and triggered a short-term increase in Fe(II) and aqueous uranium, likely due to reductive dissolution of Fe(III) oxides and release of sorbed U(VI). After two months of intermittent ethanol addition, sulfide levels increased, and aqueous uranium concentrations gradually decreased to <0.1 microM.

A Limited Microbial Consortium Is Responsible for Extended Bioreduction of Uranium in a Contaminated Aquifer

ABSTRACT Subsurface amendments of slow-release substrates (e.g., emulsified vegetable oil [EVO]) are thought to be a pragmatic alternative to using short-lived, labile substrates for sustained uranium bioimmobilization within contaminated groundwater systems. Spatial and temporal dynamics of subsurface microbial communities during EVO amendment are unknown and likely differ significantly from those of populations stimulated by soluble substrates, such as ethanol and acetate. In this study, a one-time EVO injection resulted in decreased groundwater U concentrations that remained below initial levels for approximately 4 months. Pyrosequencing and quantitative PCR of 16S rRNA from monitoring well samples revealed a rapid decline in groundwater bacterial community richness and diversity after EVO injection, concurrent with increased 16S rRNA copy levels, indicating the selection of a narrow group of taxa rather than a broad community stimulation. Members of the Firmicutes family Veillonellaceae dominated after injection and most likely catalyzed the initial oil decomposition. Sulfate-reducing bacteria from the genus Desulforegula, known for long-chain fatty acid oxidation to acetate, also dominated after EVO amendment. Acetate and H2 production during EVO degradation appeared to stimulate NO3 −, Fe(III), U(VI), and SO4 2− reduction by members of the Comamonadaceae, Geobacteriaceae, and Desulfobacterales. Methanogenic archaea flourished late to comprise over 25% of the total microbial community. Bacterial diversity rebounded after 9 months, although community compositions remained distinct from the preamendment conditions. These results demonstrated that a one-time EVO amendment served as an effective electron donor source for in situ U(VI) bioreduction and that subsurface EVO degradation and metal reduction were likely mediated by successive identifiable guilds of organisms.

In Situ Bioremediation of Uranium with Emulsified Vegetable Oil as the Electron Donor

A field test with a one-time emulsified vegetable oil (EVO) injection was conducted to assess the capacity of EVO to sustain uranium bioreduction in a high-permeability gravel layer with groundwater concentrations of (mM) U, 0.0055; Ca, 2.98; NO3(-), 0.11; HCO3(-), 5.07; and SO4(2-), 1.23. Comparison of bromide and EVO migration and distribution indicated that a majority of the injected EVO was retained in the subsurface from the injection wells to 50 m downgradient. Nitrate, uranium, and sulfate were sequentially removed from the groundwater within 1-2 weeks, accompanied by an increase in acetate, Mn, Fe, and methane concentrations. Due to the slow release and degradation of EVO with time, reducing conditions were sustained for approximately one year, and daily U discharge to a creek, located approximately 50 m from the injection wells, decreased by 80% within 100 days. Total U discharge was reduced by 50% over the one-year period. Reduction of U(VI) to U(IV) was confirmed by synchrotron analysis of recovered aquifer solids. Oxidants (e.g., dissolved oxygen, nitrate) flowing in from upgradient appeared to reoxidize and remobilize uranium after the EVO was exhausted as evidenced by a transient increase of U concentration above ambient values. Occasional (e.g., annual) EVO injection into a permeable Ca and bicarbonate-containing aquifer can sustain uranium bioreduction/immobilization and decrease U migration/discharge.

Natural Bacterial Communities Serve as Quantitative Geochemical Biosensors

ABSTRACT Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. IMPORTANCE Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts. Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.

Characterization of soils from an industrial complex contaminated with elemental mercury

Historical use of liquid elemental mercury (Hg(0)l) at the Y-12 National Security Complex in Oak Ridge, TN, USA, resulted in large deposits of Hg(0)l in the soils. The fate and distribution of the spilled Hg(0) are not well characterized. In this study we evaluated analytical tools for characterizing the speciation of Hg in the contaminated soils and then used the analytical techniques to examine the speciation of Hg in two soil cores collected at the site. These include x-ray fluorescence (XRF), soil Hg(0) headspace analysis, and total Hg determination by acid digestion coupled with cold vapor atomic absorption (HgT). XRF was not found to be suitable for evaluating Hg concentrations in heterogeneous soils containing low concentration of Hg or Hg(0) because Hg concentrations determined using this method were lower than those determined by HgT analysis and the XRF detection limit is 20 mg/kg. Hg(0)g headspace analysis coupled with HgT measurements yielded good results for examining the presence of Hg(0)l in soils and the speciation of Hg. The two soil cores are highly heterogeneous in both the depth and extent of Hg contamination, with Hg concentrations ranging from 0.05 to 8400mg/kg. In the first core, Hg(0)l was distributed throughout the 3.2m depth, whereas the second core, from a location 12m away, contained Hg(0)l in a 0.3m zone only. Sequential extractions showed organically associated Hg dominant at depths with low Hg concentration. Soil from the zone of groundwater saturation showed reducing conditions and the Hg is likely present as Hg-sulfide species. At this depth, lateral Hg transport in the groundwater may be a source of Hg detected in the soil at the deeper soil depths. Overall, characterization of soils containing Hg(0)l is difficult because of the heterogeneous distribution of Hg within the soils. This is exacerbated in industrial facilities where fill materials make up much of the soils and historical and continued reworking of the subsurface has remobilized the Hg.

Sources of Mercury to East Fork Poplar Creek Downstream from the Y-12 National Security Complex: Inventories and Export Rates

East Fork Poplar Creek (EFPC) in Oak Ridge, Tennessee, has been heavily contaminated with mercury (also referred to as Hg) since the 1950s as a result of historical activities at the U.S. Department of Energy (DOE) Y-12 National Security Complex (formerly the Oak Ridge Y-12 Plant and hereinafter referred to as Y-12). During the period from 1950 to 1963, spills and leaks of elemental mercury (Hg{sup 0}) contaminated soil, building foundations, and subsurface drainage pathways at the site, while intentional discharges of mercury-laden wastewater added 100 metric tons of mercury directly to the creek (Turner and Southworth 1999). The inventory of mercury estimated to be lost to soil and rock within the facility was 194 metric tons, with another estimated 70 metric tons deposited in floodplain soils along the 25 km length of EFPC (Turner and Southworth 1999). Remedial actions within the facility reduced mercury concentrations in EFPC water at the Y-12 boundary from > 2500 ng/L to about 600 ng/L by 1999 (Southworth et al. 2000). Further actions have reduced average total mercury concentration at that site to {approx}300 ng/L (2009 RER). Additional source control measures planned for future implementation within the facility include sediment/soil removal, storm drain relining, and restriction of rainfall infiltration within mercury-contaminated areas. Recent plans to demolish contaminated buildings within the former mercury-use areas provide an opportunity to reconstruct the storm drain system to prevent the entry of mercury-contaminated water into the flow of EFPC. Such actions have the potential to reduce mercury inputs from the industrial complex by perhaps as much as another 80%. The transformation and bioaccumulation of mercury in the EFPC ecosystem has been a perplexing subject since intensive investigation of the issue began in the mid 1980s. Although EFPC was highly contaminated with mercury (waterborne mercury exceeded background levels by 1000-fold, mercury in sediments by more than 2000-fold) in the 1980s, mercury concentrations in EFPC fish exceeded those in fish from regional reference sites by only a little more than 10-fold. This apparent low bioavailability of mercury in EFPC, coupled with a downstream pattern of mercury in fish in which mercury decreased in proportion to dilution of the upstream source, lead to the assumption that mercury in fish would respond to decreased inputs of dissolved mercury to the streams headwaters. However, during the past two decades when mercury inputs were decreasing, mercury concentrations in fish in Lower EFPC (LEFPC) downstream of Y-12 increased while those in Upper EFPC (UEFPC) decreased. The key assumption of the ongoing cleanup efforts, and concentration goal for waterborne mercury were both called into question by the long-term monitoring data. The large inventory of mercury within the watershed downstream presents a concern that the successful treatment of sources in the headwaters may not be sufficient to reduce mercury bioaccumulation within the system to desired levels. The relative importance of headwater versus floodplain mercury sources in contributing to mercury bioaccumulation in EFPC is unknown. A mercury transport study conducted by the Tennessee Valley Authority (TVA) in 1984 estimated that floodplain sources contributed about 80% of the total annual mercury export from the EFPC system (ORTF 1985). Most of the floodplain inputs were associated with wet weather, high flow events, while much of the headwater flux occurred under baseflow conditions. Thus, day-to-day exposure of biota to waterborne mercury was assumed to be primarily determined by the Y-12 source. The objective of this study was to evaluate the results of recent studies and monitoring within the EFPC drainage with a focus on discerning the magnitude of floodplain mercury sources and how long these sources might continue to contaminate the system after headwater sources are eliminated or greatly reduced.

Temporal Dynamics of In-Field Bioreactor Populations Reflect the Groundwater System and Respond Predictably to Perturbation

Temporal variability complicates testing the influences of environmental variability on microbial community structure and thus function. An in-field bioreactor system was developed to assess oxic versus anoxic manipulations on in situ groundwater communities. Each sample was sequenced (16S SSU rRNA genes, average 10,000 reads), and biogeochemical parameters are monitored by quantifying 53 metals, 12 organic acids, 14 anions, and 3 sugars. Changes in dissolved oxygen (DO), pH, and other variables were similar across bioreactors. Sequencing revealed a complex community that fluctuated in-step with the groundwater community and responded to DO. This also directly influenced the pH, and so the biotic impacts of DO and pH shifts are correlated. A null model demonstrated that bioreactor communities were driven in part not only by experimental conditions but also by stochastic variability and did not accurately capture alterations in diversity during perturbations. We identified two groups of abundant OTUs important to this system; one was abundant in high DO and pH and contained heterotrophs and oxidizers of iron, nitrite, and ammonium, whereas the other was abundant in low DO with the capability to reduce nitrate. In-field bioreactors are a powerful tool for capturing natural microbial community responses to alterations in geochemical factors beyond the bulk phase.

Bacterial community shift and coexisting/coexcluding patterns revealed by network analysis in a uranium-contaminated site after bioreduction followed by reoxidation

ABSTRACT A site in Oak Ridge, TN, USA, has sediments that contain >3% iron oxides and is contaminated with uranium (U). The U(VI) was bioreduced to U(IV) and immobilized in situ through intermittent injections of ethanol. It then was allowed to reoxidize via the invasion of low-pH (3.6 to 4.0), high-nitrate (up to 200 mM) groundwater back into the reduced zone for 1,383 days. To examine the biogeochemical response, high-throughput sequencing and network analysis were applied to characterize bacterial population shifts, as well as cooccurrence and coexclusion patterns among microbial communities. A paired t test indicated no significant changes of α-diversity for the bioactive wells. However, both nonmetric multidimensional scaling and analysis of similarity confirmed a significant distinction in the overall composition of the bacterial communities between the bioreduced and the reoxidized sediments. The top 20 major genera accounted for >70% of the cumulative contribution to the dissimilarity in the bacterial communities before and after the groundwater invasion. Castellaniella had the largest dissimilarity contribution (17.7%). For the bioactive wells, the abundance of the U(VI)-reducing genera Geothrix, Desulfovibrio, Ferribacterium, and Geobacter decreased significantly, whereas the denitrifying Acidovorax abundance increased significantly after groundwater invasion. Additionally, seven genera, i.e., Castellaniella, Ignavibacterium, Simplicispira, Rhizomicrobium, Acidobacteria Gp1, Acidobacteria Gp14, and Acidobacteria Gp23, were significant indicators of bioactive wells in the reoxidation stage. Canonical correspondence analysis indicated that nitrate, manganese, and pH affected mostly the U(VI)-reducing genera and indicator genera. Cooccurrence patterns among microbial taxa suggested the presence of taxa sharing similar ecological niches or mutualism/commensalism/synergism interactions. IMPORTANCE High-throughput sequencing technology in combination with a network analysis approach were used to investigate the stabilization of uranium and the corresponding dynamics of bacterial communities under field conditions with regard to the heterogeneity and complexity of the subsurface over the long term. The study also examined diversity and microbial community composition shift, the common genera, and indicator genera before and after long-term contaminated-groundwater invasion and the relationship between the target functional community structure and environmental factors. Additionally, deciphering cooccurrence and coexclusion patterns among microbial taxa and environmental parameters could help predict potential biotic interactions (cooperation/competition), shared physiologies, or habitat affinities, thus, improving our understanding of ecological niches occupied by certain specific species. These findings offer new insights into compositions of and associations among bacterial communities and serve as a foundation for future bioreduction implementation and monitoring efforts applied to uranium-contaminated sites.

Mercury source zone identification using soil vapor sampling and analysis

Development and demonstration of reliable measurement techniques that can detect and help quantify the nature and extent of elemental mercury (Hg(0)) in the subsurface are needed to reduce uncertainties in the decision-making process and increase the effectiveness of remedial actions. We conducted field tests at the Y-12 National Security Complex in Oak Ridge, Tennessee, USA, to determine if sampling and analysis of Hg(0) vapors in the shallow subsurface (< 0.3 m depth) can be used to as an indicator of the location and extent of Hg(0) releases in the subsurface. We constructed a rigid polyvinyl chloride push probe assembly, which was driven into the ground. Soil gas samples were collected through a sealed inner tube of the assembly and were analyzed immediately in the field with a Lumex and/or Jerome Hg(0) analyzer. Time-series sampling showed that Hg vapor concentrations were fairly stable over time, suggesting that the vapor phase Hg(0) was not being depleted and that sampling results were not sensitive to the soil gas purge volume. Hg(0) vapor data collected at over 200 push probe locations at 3 different release sites correlated very well to areas of known Hg(0) contamination. Vertical profiling of Hg(0) vapor concentrations conducted at two locations provided information on the vertical distribution of Hg(0) contamination in the subsurface. We conclude from our studies that soil gas sampling and analysis can be conducted rapidly and inexpensively at large scales to help identify areas contaminated with Hg(0).

Natural and Accelerated Bioremediation Research (NABIR) Field Research Center (FRC), Oak Ridge Tennessee

The Field Research Center (FRC) in Oak Ridge (Fig. 1), Tennessee supports the U.S. Department of Energys (DOEs) Environmental Remediation Sciences Program (ERSP) goal of understanding the complex physical, chemical, and biological properties of contaminated sites for new solutions to environmental remediation and long-term stewardship. In particular, the FRC provides the opportunity for researchers to conduct studies that promote the understanding of the processes that influence the transport and fate of subsurface contaminants, the effectiveness and long-term consequences of existing remediation options, and the development of improved remediation strategies. It offers a series of contaminated sites around the former S-3 Waste Disposal Ponds and uncontaminated sites in which investigators and students conduct field research or collect samples for laboratory analysis. FRC research also spurs the development of new and improved characterization and monitoring tools. Site specific knowledge gained from research conducted at the FRC also provides the DOE-Oak Ridge Office of Environmental Management (EM) the critical scientific knowledge needed to make cleanup decisions for the S-3 Ponds and other sites on the Oak Ridge Reservation (ORR).