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In contaminated streams, understanding the role of streambank and streambed source contributions is essential to developing robust remedial solutions. However, identifying relationships can be difficult because of the lack of identifying signatures in source and receptor pools. East Fork Poplar Creek (EFPC) in Oak Ridge, TN, USA received historical industrial releases of mercury that contaminated streambank soils and sediments. Here, we determined relationships between the contaminated streambank soils and sand-sized streambed sediments. Field surveys revealed the spatial trends of the concentrations of inorganic total mercury (Hg) and methyl mercury (MeHg), Hg lability as inferred by sequential extraction, particle size distribution, and total organic carbon. Statistical tests were applied to determine relationships between streambank soil and streambed sediment properties. Concentrations of Hg in streambank soils in the upper reaches averaged 206 mg kg−1 (all as dry weight) (n = 457), and 13 mg kg−1 in lower reaches (n = 321), while sand-sized streambed sediments were approximately 16 mg kg−1 (n = 57). Two areas of much higher Hg and MeHg concentrations in streambank soils were identified and related to localized higher Hg concentrations in the streambed sediments; however, most of the streambank soils have similar Hg concentrations to the streambed sediments. The molar ratio of Hg to organic carbon, correlation between MeHg and Hg, and particle size distributions suggested similarity between the streambank soils and the fine sand-sized fraction (125–250 μm) collected from the streambed sediments. Mercury in the fine sand-sized streambed sediments, however, was more labile than Hg in the streambank soils, suggesting an in-stream environment that altered the geochemistry of sediment-bound Hg. This study revealed major source areas of Hg in streambank soils, identified possible depositional locations in streambed sediments, and highlighted potential differences in the stability of Hg bound to streambank soils and sediments. This work will guide future remedial decision making in EFPC and will aid other researchers in identifying source–sink linkages in contaminated fluvial systems.

This study evaluated uranium sequestration performance in iron-rich (30 g/kg) sediment via bioreduction followed by reoxidation. Field tests (1383 days) at Oak Ridge, Tennessee demonstrated that uranium contents in sediments increased after bioreduced sediments were re-exposed to nitrate and oxygen in contaminated groundwater. Bioreduction of contaminated sediments (1200 mg/kg U) with ethanol in microcosm reduced aqueous U from 0.37 to 0.023 mg/L. Aliquots of the bioreduced sediment were reoxidized with O2, H2O2, and NaNO3, respectively, over 285 days, resulting in aqueous U of 0.024, 1.58 and 14.4 mg/L at pH 6.30, 6.63 and 7.62, respectively. The source- and the three reoxidized sediments showed different desorption and adsorption behaviors of U, but all fit a Freundlich model. The adsorption capacities increased sharply at pH 4.5 to 5.5, plateaued at pH 5.5 to 7.0, then decreased sharply as pH increased from 7.0 to 8.0. The O2-reoxidized sediment retained a lower desorption efficiency at pH over 6.0. The NO3−-reoxidized sediment exhibited higher adsorption capacity at pH 5.5 to 6.0. The pH-dependent adsorption onto Fe(III) oxides and formation of U coated particles and precipitates resulted in U sequestration, and bioreduction followed by reoxidation can enhance the U sequestration in sediment.

Probable maximum precipitation (PMP) is the primary criterion used to design flood protection measures for critical infrastructures such as dams and nuclear power plants. Based on our analy...

A pilot-scale field experiment demonstrated that a one-time amendment of emulsified vegetable oil (EVO) reduced groundwater U(VI) concentrations for 1 year in a fast-flowing aquifer. However, little is known about how EVO amendment stimulates the functional gene composition, structure, and dynamics of groundwater microbial communities toward prolonged U(VI) reduction. In this study, we hypothesized that EVO amendment would shift the functional gene composition and structure of groundwater microbial communities and stimulate key functional genes/groups involved in EVO biodegradation and reduction of electron acceptors in the aquifer. To test these hypotheses, groundwater microbial communities after EVO amendment were analyzed using a comprehensive functional gene microarray. Our results showed that EVO amendment stimulated sequential shifts in the functional composition and structure of groundwater microbial communities. Particularly, the relative abundance of key functional genes/groups involved in EVO biodegradation and the reduction of NO 3 − , Mn(IV), Fe(III), U(VI), and SO 4 2− significantly increased, especially during the active U(VI) reduction period. The relative abundance for some of these key functional genes/groups remained elevated over 9 months. Montel tests suggested that the dynamics in the abundance, composition, and structure of these key functional genes/groups were significantly correlated with groundwater concentrations of acetate, NO 3 − , Mn(II), Fe(II), U(VI), and SO 4 2− . Our results suggest that EVO amendment stimulated dynamic succession of key functional microbial communities. This study improves our understanding of the composition, structure, and function changes needed for groundwater microbial communities to sustain a long-term U(VI) reduction.

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).

© 2017 American Chemical Society. 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.

We amended a shallow fast-flowing uranium (U) contaminated aquifer with emulsified vegetable oil (EVO) and subsequently monitored the biogeochemical responses for over a year. Using a biogeochemical model developed in a companion article (Tang et al., Environ. Sci. Technol. 2013, doi: 10.1021/es304641b) based on microcosm tests, we simulated geochemical and microbial dynamics in the field test during and after the 2-h EVO injection. When the lab-determined parameters were applied in the field-scale simulation, the estimated rate coefficient for EVO hydrolysis in the field was about 1 order of magnitude greater than that in the microcosms. Model results suggested that precipitation of long-chain fatty acids, produced from EVO hydrolysis, with Ca in the aquifer created a secondary long-term electron donor source. The model predicted substantial accumulation of denitrifying and sulfate-reducing bacteria, and U(IV) precipitates. The accumulation was greatest near the injection wells and along the lateral boundaries of the treatment zone where electron donors mixed with electron acceptors in the groundwater. While electron acceptors such as sulfate were generally considered to compete with U(VI) for electrons, this work highlighted their role in providing electron acceptors for microorganisms to degrade complex substrates thereby enhancing U(VI) reduction and immobilization.

© 2016 The Authors. Reoxidation and mobilization of previously reduced and immobilized uranium by dissolved-phase oxidants poses a significant challenge for remediating uranium-contaminated groundwater. Preferential oxidation of reduced sulfur-bearing species, as opposed to reduced uranium-bearing species, has been demonstrated to limit the mobility of uranium at the laboratory scale yet field-scale investigations are lacking. In this study, the mobility of uranium in the presence of nitrate oxidant was investigated in a shallow groundwater system after establishing conditions conducive to uranium reduction and the formation of reduced sulfur-bearing species. A series of three injections of groundwater (200 L) containing U(VI) (5 μM) and amended with ethanol (40 mM) and sulfate (20 mM) were conducted in ten test wells in order to stimulate microbial-mediated reduction of uranium and the formation of reduced sulfur-bearing species. Simultaneous push-pull tests were then conducted in triplicate well clusters to investigate the mobility of U(VI) under three conditions: 1) high nitrate (120 mM), 2) high nitrate (120 mM) with ethanol (30 mM), and 3) low nitrate (2 mM) with ethanol (30 mM). Dilution-adjusted breakthrough curves of ethanol, nitrate, nitrite, sulfate, and U(VI) suggested that nitrate reduction was predominantly coupled to the oxidation of reduced-sulfur bearing species, as opposed to the reoxidation of U(IV), under all three conditions for the duration of the 36-day tests. The amount of sulfate, but not U(VI), recovered during the push-pull tests was substantially more than injected, relative to bromide tracer, under all three conditions and further suggested that reduced sulfur-bearing species were preferentially oxidized under nitrate-reducing conditions. However, some reoxidation of U(IV) was observed under nitrate-reducing conditions and in the absence of detectable nitrate and/or nitrite. This suggested that reduced sulfur-bearing species may not be fully effective at limiting the mobility of uranium in the presence of dissolved and/or solid-phase oxidants. The results of this field study confirmed those of previous laboratory studies which suggested that reoxidation of uranium under nitrate-reducing conditions can be substantially limited by preferential oxidation of reduced sulfur-bearing species.

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 H 2 production during EVO degradation appeared to stimulate NO 3 − , Fe(III), U(VI), and SO 4 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.

This study investigates reactive transport modeling in a column of uranium(VI)-contaminated sediments with base additions in the circulating influent. The groundwater and sediment exhibit oxic conditions with low pH, high concentrations of NO3−, SO42−, U and various metal cations. Preliminary batch experiments indicate that additions of strong base induce rapid immobilization of U for this material. In the column experiment that is the focus of the present study, effluent groundwater was titrated with NaOH solution in an inflow reservoir before reinjection to gradually increase the solution pH in the column. An equilibrium hydrolysis, precipitation and ion exchange reaction model developed through simulation of the preliminary batch titration experiments predicted faster reduction of aqueous Al than observed in the column experiment. The model was therefore modified to consider reaction kinetics for the precipitation and dissolution processes which are the major mechanism for Al immobilization. The combined kinetic and equilibrium reaction model adequately described variations in pH, aqueous concentrations of metal cations (Al, Ca, Mg, Sr, Mn, Ni, Co), sulfate and U(VI). The experimental and modeling results indicate that U(VI) can be effectively sequestered with controlled base addition due to sorption by slowly precipitated Al with pH-dependent surface charge. The model may prove useful to predict field-scale U(VI) sequestration and remediation effectiveness.

A pilot-scale system was established to examine the feasibility of in situ U(VI) immobilization at a highly contaminated aquifer (U.S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter −1 ). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.

Massively parallel sequencing has provided a more affordable and high-throughput method to study microbial communities, although it has mostly been used in an exploratory fashion. We combined pyrosequencing with a strict indicator species statistical analysis to test if bacteria specifically responded to ethanol injection that successfully promoted dissimilatory uranium(VI) reduction in the subsurface of a uranium contamination plume at the Oak Ridge Field Research Center in Tennessee. Remediation was achieved with a hydraulic flow control consisting of an inner loop, where ethanol was injected, and an outer loop for flow-field protection. This strategy reduced uranium concentrations in groundwater to levels below 0.126 μM and created geochemical gradients in electron donors from the inner-loop injection well toward the outer loop and downgradient flow path. Our analysis with 15 sediment samples from the entire test area found significant indicator species that showed a high degree of adaptation to the three different hydrochemical-created conditions. Castellaniella and Rhodanobacter characterized areas with low pH, heavy metals, and low bioactivity, while sulfate-, Fe(III)-, and U(VI)-reducing bacteria ( Desulfovibrio , Anaeromyxobacter , and Desulfosporosinus ) were indicators of areas where U(VI) reduction occurred. The abundance of these bacteria, as well as the Fe(III) and U(VI) reducer Geobacter , correlated with the hydraulic connectivity to the substrate injection site, suggesting that the selected populations were a direct response to electron donor addition by the groundwater flow path. A false-discovery-rate approach was implemented to discard false-positive results by chance, given the large amount of data compared.

Flow-interruption tracer test is an effective approach to identify kinetic mass transfer processes for solute transport in subsurface media. By switching well pumping and resting, one may alter the dominant transport mechanism and generate special concentration patterns for identifying kinetic mass transfer processes. In the present research, we conducted three-phase (i.e., pumping, resting, and pumping) field-scale flow-interruption tracer tests using a conservative tracer bromide in a multiple-well system installed at the US Department of Energy Site, Oak Ridge, TN. A novel modeling approach based on the resting-period measurements was developed to estimate the mass transfer parameters. This approach completely relied on the measured breakthrough curves without requiring detailed aquifer characterization and solving transport equations in nonuniform, transient flow fields. Additional measurements, including hydraulic heads and tracer concentrations in large pumping wells, were taken to justify the assumption that mass transfer processes dominated concentration change during resting periods. The developed approach can be conveniently applied to any linear mass transfer model. Both first-order and multirate mass transfer models were applied to analyze the breakthrough curves at various monitoring wells. The multirate mass transfer model was capable of jointly fitting breakthrough curve behavior, showing the effectiveness and flexibility for incorporating aquifer heterogeneity and scale effects in upscaling effective mass transfer models.

A pilot-scale field test system with an inner loop nested within an outer loop was constructed for in situ U(VI) bioremediation at a US Department of Energy site, Oak Ridge, TN. The outer loop was used for hydrological protection of the inner loop where ethanol was injected for biostimulation of microorganisms for U(VI) reduction/immobilization. After 2 years of biostimulation with ethanol, U(VI) levels were reduced to below drinking water standard (30 microg l(-1)) in the inner loop monitoring wells. To elucidate the microbial community structure and functions under in situ uranium bioremediation conditions, we used a comprehensive functional gene array (GeoChip) to examine the microbial functional gene composition of the sediment samples collected from both inner and outer loop wells. Our study results showed that distinct microbial communities were established in the inner loop wells. Also, higher microbial functional gene number, diversity and abundance were observed in the inner loop wells than the outer loop wells. In addition, metal-reducing bacteria, such as Desulfovibrio, Geobacter, Anaeromyxobacter and Shewanella, and other bacteria, for example, Rhodopseudomonas and Pseudomonas, are highly abundant in the inner loop wells. Finally, the richness and abundance of microbial functional genes were highly correlated with the mean travel time of groundwater from the inner loop injection well, pH and sulfate concentration in groundwater. These results suggest that the indigenous microbial communities can be successfully stimulated for U bioremediation in the groundwater ecosystem, and their structure and performance can be manipulated or optimized by adjusting geochemical and hydrological conditions.

After intervals of groundwater immersion, polyacrylamide hydrogel-encapsulated solid specimens were retrieved, assayed non-destructively for uranium and other elements using x-ray fluorescence spectroscopy, and replaced in groundwater for continued reaction. Desorption dynamics of uranium from contaminated soils and other solids, when moved to uncontaminated groundwater, were fit to a general two-component kinetic retention model with slow-release and fast-release fractions of the total uranium. In a group of Oak Ridge soils with varying ambient uranium contamination (169-1360 mg/kg), the uranium fraction retained under long-term in situ kinetic behavior was strongly correlated (r2 = 0.89) with the residual uranium retained after laboratory sequential extraction of water-soluble and cation-exchangeable fractions of the same soils. To illustrate how potential remedial techniques can be compared to natural attenuation, thermal stabilization of one soil increased the size of its long-term retained fraction from 50 to 88% of the total uranium and increased the in situ retention half-life of the long-term retained fraction from 990 to 40,000 days. Hydrogel encapsulation presents a novel and powerful general method to observe many water-solids interactions in situ for a variety of aqueous media besides groundwater, with a variety of non-destructive analytical methods, and with a variety of solids besides contaminated soil.

A pilot-scale system was established for in situ biostimulation of U(VI) reduction by ethanol addition at the US Department of Energy's (DOE's) Field Research Center (Oak Ridge, TN). After achieving U(VI) reduction, stability of the bioreduced U(IV) was evaluated under conditions of (i) resting (no ethanol injection), (ii) reoxidation by introducing dissolved oxygen (DO), and (iii) reinjection of ethanol. GeoChip, a functional gene array with probes for N, S and C cycling, metal resistance and contaminant degradation genes, was used for monitoring groundwater microbial communities. High diversity of all major functional groups was observed during all experimental phases. The microbial community was extremely responsive to ethanol, showing a substantial change in community structure with increased gene number and diversity after ethanol injections resumed. While gene numbers showed considerable variations, the relative abundance (i.e. percentage of each gene category) of most gene groups changed little. During the reoxidation period, U(VI) increased, suggesting reoxidation of reduced U(IV). However, when introduction of DO was stopped, U(VI) reduction resumed and returned to pre-reoxidation levels. These findings suggest that the community in this system can be stimulated and that the ability to reduce U(VI) can be maintained by the addition of electron donors. This biostimulationmore » approach may potentially offer an effective means for the bioremediation of U(VI)-contaminated sites.« less

This research evaluated a method of controlled base addition for immobilizing uranium (U) and technetium (Tc) through coprecipitation with aluminum (Al) and other metal ions which coexist in a highly contaminated acidic environment. The batch and column experiments indicate that the addition of strong base (NaOH) provided a rapid yet effective means of sequestering U, Tc, and toxic metal ions such as nickel (Ni2+) and cobalt (Co2+) in the sediment and groundwater. Greater than 94% of soluble U (as UO2(2+)) and > 83% of Tc (as TcO4-) can be immobilized at pH above 4.5 by co-precipitation with Al-oxyhydroxides. The presence of sediment minerals appeared to facilitate co-precipitation of these contaminants at lower pH values than those in the absence of sediments. The immobilized U and Tc were found to be stable against dissolution in Ca(NO3)2 solution (up to 50 mM) because of the formation of strong surface complexes between U or Tc and Al-oxyhydroxides. This research concludes that as long as a relatively high pH (> 5) and a low carbonate concentration are maintained, both U and Tc can be effectively immobilized under given site-specific conditions.

Metal and hydrogen ion acidity and extreme nitrate concentrations at Department of Energy legacywaste sites pose challenges for successful in situ U and Tc bioimmobilization. In this study, we investigated a potential in situ biobarrier configuration designed to neutralize pH and remove nitrate and radionuclides from nitric acid-, U-, and Tc-contaminated groundwater for over 21 months. Ethanol additions to groundwater flowing through native sediment and crushed limestone effectively increased pH (from 4.7 to 6.9), promoted removal of 116 mM nitrate, increased sediment biomass, and immobilized 94% of total U. Increased groundwater pH and significant U removal was also observed in a control column that received no added ethanol. Sequential extraction and XANES analyses showed U in this sediment to be solid-associated U(VI), and EXAFS analysis results were consistent with uranyl orthophosphate (UO2)3(PO4)2.4H2O(s), which may control U solubility in this system. Ratios of respiratory ubiquinones to menaquinones and copies of dissimilatory nitrite reductase genes, nirS and nirK, were at least 1 order of magnitude greater in the ethanol-stimulated system compared to the control, indicating that ethanol addition promoted growth of a largely denitrifying microbial community. Sediment 16S rRNA gene clone libraries showed that Betaproteobacteria were dominant (89%) near the source of influent acidic groundwater, whereas members of Gamma- and Alphaproteobacteria and Bacteroidetes increased along the flow path as pH increased and nitrate concentrations decreased, indicating spatial shifts in community composition as a function of pH and nitrate concentrations. Results of this study support the utility of biobarriers for treating acidic radionuclide- and nitrate-contaminated groundwater.

Many geochemical reactions that control aqueous metal concentrations are directly affected by solution pH. However, changes in solution pH are strongly buffered by various aqueous phase and solid phase precipitation/dissolution and adsorption/desorption reactions. The ability to predict acid-base behavior of the soil-solution system is thus critical to predict metal transport under variable pH conditions. This studywas undertaken to develop a practical generic geochemical modeling approach to predict aqueous and solid phase concentrations of metals and anions during conditions of acid or base additions. The method of Spalding and Spalding was utilized to model soil buffer capacity and pH-dependent cation exchange capacity by treating aquifer solids as a polyprotic acid. To simulate the dynamic and pH-dependent anion exchange capacity, the aquifer solids were simultaneously treated as a polyprotic base controlled by mineral precipitation/ dissolution reactions. An equilibrium reaction model that describes aqueous complexation, precipitation, sorption and soil buffering with pH-dependent ion exchange was developed using HydroGeoChem v5.0 (HGC5). Comparison of model results with experimental titration data of pH, Al, Ca, Mg, Sr, Mn, Ni, Co, and SO4(2-) for contaminated sediments indicated close agreement suggesting that the model could potentially be used to predictthe acid-base behavior of the sediment-solution system under variable pH conditions.

In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas , and Pseudomonas . Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase ( gltA ) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.

A travel-time based approach is developed for estimating first-order reaction rate coefficients for transport with nonequilibrium linear mass transfer in heterogeneous media. Tracer transport in the mobile domain is characterized by a travel-time distribution, and mass transfer rates are described by a convolution product of concentrations in the mobile domain and a memory function rather than predefining the mass transfer model. A constant first-order reaction is assumed to occur only in the mobile domain. Analytical solutions in Laplace domain can be derived for both conservative and reactive breakthrough curves (BTCs). Temporal-moment analyses are presented by using the first and second moments of conservative and reactive BTCs and the mass consumption of the reactant for an inverse Gaussian travel-time distribution. In terms of moment matching, there is no need for one to specify the mass transfer model. With the same capacity ratio and the mean retention time, all mass transfer models will lead to the same moment-derived reaction rate coefficients. In addition, the consideration of mass transfer generally yields larger estimations of the reaction rate coefficient than models ignoring mass transfer. Furthermore, the capacity ratio and the mean retention time have opposite influences on the estimation of the reaction rate coefficient: the first-order reaction rate coefficient is positively linearly proportional to the capacity ratio, but negatively linearly proportional to the mean retention time.

The success of sequestration-based remediation strategies will depend on detailed information, including the predominant U species present as sources before biostimulation and the products produced during and after in situ biostimulation. We used X-ray absorption spectroscopy to determine the valence state and chemical speciation of U in sediment samples collected at a variety of depths through the contaminant plume at the Field Research Center at Oak Ridge, TN, before and after approximately 400 days of in situ biostimulation, as well as in duplicate bioreduced sediments after 363 days of resting conditions. The results indicate that U(VI) in subsurface sediments was partially reduced to 10-40% U(IV) during biostimulation. After biostimulation, U was no longer bound to carbon ligands and was adsorbed to Fe/Mn minerals. Reduction of U(VI) to U(IV) continued in sediment samples stored under anaerobic condition at4 degrees C for 12 months, with the fraction of U(IV) in sediments more than doubling and U concentrations in the aqueous phase decreasing from 0.5-0.74 to0.1 microM. A shift of uranyl species from uranyl bound to phosphorus ligands to uranyl bound to carbon ligands and the formation of nanoparticulate uraninite occurred in the sediment samples during storage.

Synthetic resins are shown to be effective in removing uranium from contaminated groundwater. Batch and field column tests showed that strong-base anion-exchange resins were more effective in removing uranium from both near-neutral-pH (6.5)- and high-pH (8)-low-nitrate-containing groundwaters, than metal-chelating resins, which removed more uranium from acidic-pH (5)-high-nitrate-containing groundwater from the Oak Ridge Reservation (ORR) Y-12 S-3 Ponds area in Tennessee, USA. Dowex 1-X8 and Purolite A-520E anion-exchange resins removed more uranium from high-pH (8)-low-nitrate-containing synthetic groundwater in batch tests than metal-chelating resins. The Dowex 21K anion-exchange resin achieved a cumulative loading capacity of 49.8 mg g(-1) before breakthrough in a field column test using near-neutral-pH (6.5)-low-nitrate-containing groundwater. However, in an acidic-pH (5)-high-nitrate-containing groundwater, metal-chelating resins Diphonix and Chelex-100 removed more uranium than anion-exchange resins. In 15 m L of acidic-pH (5)-high-nitrate-containing groundwater spiked with 20 mg L(-1) uranium, the uranium concentrations ranged from 0.95 mg L(-1) at 1-h equilibrium to 0.08 mg L(-1) at 24-h equilibrium for Diphonix and 0.17 mg L(-1) at 1-h equilibrium to 0.03 mg L(-1) at 24-h equilibrium for Chelex-100. Chelex-100 removed more uranium in the first 10 min in the 100mL of acidic-(pH 5)-high-nitrate-containing groundwater ( approximately 5 mg L(-1) uranium); however, after 10 min, Diphonix equaled or out-performed Chelex-100. This study presents an improved understanding of the selectivity and sorption kenetics of a range of ion-exchange resins that remove uranium from both low- and high-nitrate-containing groundwaters with varying pHs.

Chemical analysis and scanning electron microscopy (SEM) microanalysis were carried out on cores of contaminated geological material collected around four closed waste disposal ponds to examine the extent of nitric acid extractable U (U(NA)) association with P, S, and extractable Fe, Al, and Mn oxides within deeply weathered fractured shale. The solid phase in many regimes on the site has been exposed to highly buffered acidic (3.5) groundwater and has been aggressively weathered. Higher correlations occur between U(NA) and total P and S (r2 = 0.76, 0.69, respectively), citrate bicarbonate dithionite extractable Fe (Fed) and Al (Ald) (r2 = 0.87, 0.80, respectively), and acid oxalate extractable or amorphous/poorly crystalline Fe (Feo) (r2 = 0.63) in core material from a field plot known as Area 1 compared to core material from another field plot known as Area 3. In Area 3 core material, linear regression analysis of U(NA) and total P and S, and Fed, Ald and Feo gave r2 values of 0.67, 0.4, 0.06, 0.24, and 0.45, respectively. These results showed similar relationships with SEM-wavelength dispersive spectroscopy (WDS) mapping of this material. It is noteworthy that Area 1 geological material has not been as aggressively weathered as Area 3 material due to its physical location from the waste source. In all of the cores, most of the Fe and Al oxides were crystalline, while most of the Mn oxides were amorphous. The greater adsorption and/or fixation of anion complexes of P-U (uranium phosphate) and S onto Fe and Al oxides from Area 1 cores compared to Area 3 core material is probably due to a higher amount of crystalline Fe and Al oxides compared to amorphous Fe and Al oxides and higher Al substitution in Fe oxides in Area 1. This unique study illustrates the relationships between U(NA), total P and S, and Al, Fe and Mn oxides in fractured shale under field conditions which can be used in planning remediation of this site and other similar sites.

This paper presents a reaction-based water quality transport model in subsurface flow systems. Transport of chemical species with a variety of chemical and physical processes is mathematically described by M partial differential equations (PDEs). Decomposition via Gauss–Jordan column reduction of the reaction network transforms M species reactive transport equations into two sets of equations: a set of thermodynamic equilibrium equations representing N E equilibrium reactions and a set of reactive transport equations of M – N E kinetic-variables involving no equilibrium reactions (a kinetic-variable is a linear combination of species). The elimination of equilibrium reactions from reactive transport equations allows robust and efficient numerical integration. The model solves the PDEs of kinetic-variables rather than individual chemical species, which reduces the number of reactive transport equations and simplifies the reaction terms in the equations. A variety of numerical methods are investigated for solving the coupled transport and reaction equations. Simulation comparisons with exact solutions were performed to verify numerical accuracy and assess the effectiveness of various numerical strategies to deal with different application circumstances. Two validation examples involving simulations of uranium transport in soil columns are presented to evaluate the ability of the model to simulate reactive transport with complex reaction networks involving both kinetic and equilibrium reactions.

Site characterization is an essential initial step in determining the feasibility of remedial alternatives at hazardous waste sites. Physico-chemical and mineralogical characterization of U-contaminated soils in deeply weathered saprolite at Area 2 of the DOE Field Research Center (FRC) site, Oak Ridge, TN, was accomplished to examine the feasibility of bioremediation. Concentrations of U in soil-saprolite (up to 291 mg kg^-1 in oxalate-extractable Uo) were closely related to low pH (ca. 4-5), high effective cation exchange capacity without Ca (64.7-83.2 cmolc kg_1), amorphous Mn content (up to 9910 mg kg_1), and the decreased presence of relative clay mineral contents in the bulk samples (i.e., illite 2.5-12 wt. %, average 32 wt. %). The pH of the fill material ranged from 7.0 to 10.5, whereas the pH of the saprolite ranged from 4.5 to 8. Uranium concentration was highest (about 300 mg kg^-1) at around 6 m below land surface near the saprolite-fill interface. The pH of ground water at Area 2 tended to be between 6 and 7 with U concentrations of about 0.9 to 1.7 mg L^-1. These site specific characteristics of Area 2, which has lower U and nitrate con-tamination levels and more neutral ground water pH comparedmore » with FRC Areas 1 and 3 (ca. 5.5 and _4, respectively), indicate that with appropriate addition of electron donors and nutrients bioremediation of U by metal reducing microorganisms may be possible.« less

High levels of nitrate are present in groundwater migrating from the former waste disposal ponds at the Y-12 National Security Complex in Oak Ridge, TN. A field-scale denitrifying fluidized bed reactor (FBR) was designed, constructed, and operated with ethanol as an electron donor for the removal of nitrate. After inoculation, biofilms developed on the granular activated carbon particles. Changes in the bacterial community of the FBR were evaluated with clone libraries (n = 500 partial sequences) of the small-subunit rRNA gene for samples taken over a 4-month start-up period. Early phases of start-up operation were characterized by a period of selection, followed by low diversity and predominance by Azoarcus-like sequences. Possible explanations were high pH and nutrient limitations. After amelioration of these conditions, diversification increased rapidly, with the appearance of Dechloromonas, Pseudomonas, and Hydrogenophaga sequences. Changes in NO3, SO4, and pH also likely contributed to shifts in community composition. The detection of sulfate-reducing-bacteria-like sequences closely related to Desulfovibrio and Desulfuromonas in the FBR have important implications for downstream applications at the field site.

Although humic substances occur ubiquitously in soil and groundwater, their effect on the biological reduction of uranium(VI) and subsequent reoxidation of U(IV) is poorly understood. This study investigated the role of humics in enhancing the bioreduction of U(VI) in laboratory kinetic studies, in field push-pull tests, and in the presence or absence of metal ions such as Ca2+ and Ni2+, which are known to inhibit the biological reduction of U(VI). Results from laboratory experiments indicate that, under strict anaerobic conditions, the presence of humic materials enhanced the U(VI) reduction rates (up to 10-fold) and alleviated the toxicity effect of Ni2+ on microorganisms. Humic acid was found to be more effective than fulvic acid in enhancing the reduction of U(VI). Such an enhancement effect is attributed to the ability of these humics in facilitating electron-transfer reactions and/or in complexing Ca2+ and Ni2+ ions. Similarly, field push-pull tests demonstrated a substantially increased rate of U(VI) reduction when humic acid was introduced into the site groundwater. However, humics were also found to form complexes with reduced U(IV) and increased the oxidation of U(IV) (when exposed to oxygen) with an oxidation halflife on the order of a few minutes. Both of these processes render uranium soluble and potentially mobile in groundwater, depending on site-specific and dynamic geochemical conditions. Future studies must address the stability and retention of reduced U(IV) under realistic field conditions (e.g., in the presence of dissolved oxygen and low concentrations of complexing organics).

The objectives of this study were to examine (1) the distribution of U and Th in dolomitic gravel fill and shale saprolite, and (2) the removal of uranium from acidic groundwater by dolomitic gravel through precipitation with amorphous basaluminite at the U.S. DOE Oak Ridge Integrated Field Research Challenge (ORIFRC) field site west of the Oak Ridge Y-12 National Security Complex in East Tennessee. Media reactivity and sustainability are a technical concern with the deployment of any subsurface reactive media. Because the gravel was placed in the subsurface and exposed to contaminated groundwater for over 20 years, it provided a unique opportunity to study the solid and water phase geochemical conditions within the media after this length of exposure. This study illustrates that dolomite gravel can remove U from acidic contaminated groundwater with high levels of Al3+, Ca2+, NO3−, and SO42− over the long term. As the groundwater flows through high pH carbonate gravel, U containing amorphous basaluminite precipitates as the pH increases. This is due to an increase in groundwater pH from 3.2 to ∼6.5 as it comes in contact with the gravel. Therefore, carbonate gravel could be considered as a possible treatment medium for removal and sequestration of U and other pH sensitive metals from acidic contaminated groundwater. Thorium concentrations are also high in the carbonate gravel. Thorium generally shows an inverse relationship with U from the surface down into the deeper saprolite. Barite precipitated in the shallow saprolite directly below the dolomitic gravel from barium present in the acidic contaminated groundwater.

Zero-valent iron (Fe0)-based permeable reactive barriertreatment has been generating great interest for passivegroundwater remediation, yet few studies have paid particularattention to the microbial activity and characteristics withinand in the vicinity of the Fe0-barrier matrix. The presentstudy was undertaken to evaluate the microbial population andcommunity composition in the reducing zone of influence byFe0 corrosion in the barrier at the Oak Ridge Y-12 Plantsite. Both phospholipid fatty acids and DNA analyses were usedto determine the total microbial population and microbialfunctional groups, including sulfate-reducing bacteria,denitrifying bacteria, and methanogens, in groundwater andsoil/iron core samples. A diverse microbial community wasidentified in the strongly reducing Fe0 environment despitea relatively high pH condition within the Fe0 barrier (up topH ∼ 10). In comparison with those found in the backgroundsoil/groundwater samples, the enhanced microbial populationranged from ∼ 1 to 3 orders of magnitude and appeared to increase from upgradient of the barrier to downgradient soil. Inaddition, microbial community composition appeared to change overtime, and the bacterial types of microorganismsincreased consistently as the barrier aged. DNA analysisindicated the presence of sulfate-reducing and denitrifyingbacteria in the barrier and its surrounding soil. However, theactivity of methanogens was found to be relatively low,presumably as a result of the competition by sulfate/metal-reducing bacteria and denitrifying bacteria because of the unlimited availability of sulfate and nitrate in the site groundwater. Results of this study provide evidenceof a diverse microbial population within and in the vicinity ofthe iron barrier, although the important roles of microbial activity, either beneficially or detrimentally, on the longevityand enduring efficiency of the Fe0 barriers are yet to be evaluated.

There is a limited amount of information about the effects of mineral precipitates and corrosion on the lifespan and long-term performance of in situ Fe0 reactive barriers. The objectives of this p...

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.

We conducted batch and recirculating column titration tests with contaminated acidic sediments with controlled CO2 in the headspace, and extended the geochemical model by Gu et al. (2003, GCA) to better understand and quantify the reactions governing trace metal fate in the subsurface. The sediment titration curve showed slow pH increase due to strong buffering by Al precipitation and CO2 uptake. Assuming precipitation of basaluminite at low saturation index (SI=-4), and decreasing cation exchange selectivity coefficient (kNa\Al=0.3), the predictions are close to the observed pH and Al; and the model explains 1) the observed Ca, Mg, and Mn concentration decrease by cation exchange with sorbed Al, and 2) the decrease of U by surface complexation with Fe hydroxides at low pH, and precipitation as liebigite (Ca2UO2(CO3)3:10H2O) at pH>5.5. Without further adjustment geochemical parameters, the model describes reasonably well previous sediment and column titration tests without CO2 in the headspace, as well as the new large column test. The apparent inhibition of U and Ni decrease in the large column can be explained by formation of aqueous carbonate complexes and/or competition with carbonate for surface sites. These results indicated that ignoring labile solid phase Al would underestimate base requirement in titrationmore » of acidic aquifers.« less

We conducted microcosm tests and biogeochemical modeling to study U(VI) reduction in contaminated sediments amended with emulsified vegetable oil (EVO). Indigenous microorganisms in the sediments degraded EVO and stimulated Fe(III), U(VI), and sulfate reduction, and methanogenesis. Acetate concentration peaked in 100-120 days in the EVO microcosms versus 10-20 days in the oleate microcosms, suggesting that triglyceride hydrolysis was a rate-limiting step in EVO degradation and subsequent reactions. Acetate persisted 50 days longer in oleate- and EVO- than in ethanol-amended microcosms, indicating that acetate-utilizing methanogenesis was slower in the oleate and EVO than ethanol microcosms. We developed a comprehensive biogeochemical model to couple EVO hydrolysis, production, and oxidation of long-chain fatty acids (LCFA), glycerol, acetate, and hydrogen, reduction of Fe(III), U(VI) and sulfate, and methanogenesis with growth and decay of multiple functional microbial groups. By estimating EVO, LCFA, and glycerol degradation rate coefficients, and introducing a 100 day lag time for acetoclastic methanogenesis for oleate and EVO microcosms, the model approximately matched observed sulfate, U(VI), and acetate concentrations. Our results confirmed that EVO could stimulate U(VI) bioreduction in sediments and the slow EVO hydrolysis and acetate-utilizing methanogens growth could contribute to longer term bioreduction than simple substrates (e.g., ethanol, acetate, etc.) in the subsurface.

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.

Geophysical measurements, and electrical resistivity tomography (ERT) data in particular, are sensitive to properties that are related (directly or indirectly) to hydrological processes. The challenge is in extracting information from geophysical data at a relevant scale that can be used to gain insight about subsurface behavior and to parameterize or validate flow and transport models. Here, we consider the use of ERT data for examining the impact of recharge on subsurface contamination at the S-3 ponds of the Oak Ridge Integrated Field Research Challenge (IFRC) site in Tennessee. A large dataset of time-lapse cross-well and surface ERT data, collected at the site over a period of 12 months, is used to study time variations in resistivity due to changes in total dissolved solids (primarily nitrate). The electrical resistivity distributions recovered from cross-well and surface ERT data agrees well, and both of these datasets can be used to interpret spatiotemporal variations in subsurface nitrate concentrations due to rainfall, although the sensitivity of the electrical resistivity response to dilution varies with nitrate concentration. Using the time-lapse surface ERT data interpreted in terms of nitrate concentrations, we find that the subsurface nitrate concentration at this site varies as a function of spatial position, episodic heavy rainstorms (versus seasonal and annual fluctuations), and antecedent rainfall history. These results suggest that the surface ERT monitoring approach is potentially useful for examining subsurface plume responses to recharge over field-relevant scales.

Shifts in terminal electron-accepting processes during biostimulation of uranium-contaminated sediments were linked to the composition of stimulated microbial populations using DNA-based stable isotope probing. Nitrate reduction preceded U(VI) and Fe(III) reduction in [ 13 C]ethanol-amended microcosms. The predominant, active denitrifying microbial groups were identified as members of the Betaproteobacteria , whereas Actinobacteria dominated under metal-reducing conditions.

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{sub 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,more » 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

Microcosm tests with uranium contaminated sediments were performed to explore the feasibility of using oleate as a slow-release electron donor for U(VI) reduction in comparison to ethanol. Oleate degradation proceeded more slowly than ethanol with acetate produced as an intermediate for both electron donors under a range of initial sulfate concentrations. A kinetic microbial reduction model was developed and implemented to describe and compare the reduction of sulfate and U(VI) with oleate or ethanol. The reaction path model considers detailed oleate/ethanol degradation and the production and consumption of intermediates, acetate and hydrogen. Although significant assumptions are made, the model tracked the major trend of sulfate and U(VI) reduction and describes the successive production and consumption of acetate, concurrent with microbial reduction of aqueous sulfate and U(VI) species. The model results imply that the overall rate of U(VI) bioreduction is influenced by both the degradation rate of organic substrates and consumption rate of intermediate products.

To understand how contaminants affect microbial community diversity, heterogeneity, and functional structure, six groundwater monitoring wells from the Field Research Center of the U.S. Department of Energy Environmental Remediation Science Program (ERSP; Oak Ridge, TN), with a wide range of pH, titrate, and heavy metal contamination were investigated. DNA from the groundwater community was analyzed with a functional gene array containing 2006 probes to detect genes involved in metal resistance, sulfate reduction, organic contaminant degradation, and carbon and nitrogen cycling. Microbial diversity decreased in relation to the contamination levels of the wells. Highly contaminated wells had lower gene diversity but greater signal intensity than the pristine well. The microbial composition was heterogeneous, with 17-70% overlap between differentwells. Metal-resistant and metal-reducing microorganisms were detected in both contaminated and pristine wells, suggesting the potential for successful bioremediation of metal-contaminated groundwaters. In addition, results of Mantel tests and canonical correspondence analysis indicate that nitrate, sulfate, pH, uranium, and technetium have a significant (p < 0.05) effect on microbial community structure. This study provides an overall picture of microbial community structure in contaminated environments with functional gene arrays by showing that diversity and heterogeneity can vary greatly in relation to contamination.

An in situ passive sampling and gas chromatographic protocol was developed for analysis of the major and several minor fixed gases (He, Ne, H2, N2, O2, CO, CH4, CO2, and N2O) in groundwater. Using argon carrier gas, a HayeSep DB porous polymer phase, and sequential thermal conductivity and reductive gas detectors, the protocol achieved sufficient separation and sensitivity to measure the mixing ratio of all these gases in a single 0.5 mL gas sample collected in situ, stored, transported, and injected using a gastight syringe. Within 4 days of immersion in groundwater, the simple passive in situ sampler, whether initially filled with He or air, attained an equivalent and constant mixing ratio for five of the seven detected gases. The abundant mixing ratio of N2O, averaging 2.6%, indicated that significant denitrification is likely ongoing within groundwater contaminated with uranium, acidity, nitrate, and organic carbon from a group of four closed radioactive wastewater seepage ponds at the Oak Ridge Field Research Center. Over 1000 passive gas samples from 12 monitoring wells averaged 56% CO2, 32.4% N2, 2.6% O2, 2.6% N2O, 0.21% CH4, 0.093% H2, and 0.025% CO with an average recovery of 95 +/- 14% of the injected gas volume.

Microbial enumeration, 16S rRNA gene clone libraries, and chemical analysis were used to evaluate the in situ biological reduction and immobilization of uranium(VI) in a long-term experiment (more than 2 years) conducted at a highly uranium-contaminated site (up to 60 mg/liter and 800 mg/kg solids) of the U.S. Department of Energy in Oak Ridge, TN. Bioreduction was achieved by conditioning groundwater above ground and then stimulating growth of denitrifying, Fe(III)-reducing, and sulfate-reducing bacteria in situ through weekly injection of ethanol into the subsurface. After nearly 2 years of intermittent injection of ethanol, aqueous U levels fell below the U.S. Environmental Protection Agency maximum contaminant level for drinking water and groundwater (Desulfovibrio, Geobacter, Anaeromyxobacter, Desulfosporosinus , and Acidovorax spp. The predominant sulfate-reducing bacterial species were Desulfovibrio spp., while the iron reducers were represented by Ferribacterium spp. and Geothrix spp. Diversity-based clustering revealed differences between treated and untreated zones and also within samples of the treated area. Spatial differences in community structure within the treatment zone were likely related to the hydraulic pathway and to electron donor metabolism during biostimulation.

Groundwater within Area 3 of the U.S. Department of Energy (DOE) Environmental Remediation Sciences Program (ERSP) Field Research Center at Oak Ridge, TN (ORFRC) contains up to 135 microM uranium as U(VI). Through a series of experiments at a pilot scale test facility, we explored the lower limits of groundwater U(VI) that can be achieved by in-situ biostimulation and the effects of dissolved oxygen on immobilized uranium. Weekly 2 day additions of ethanol over a 2-year period stimulated growth of denitrifying, Fe(III)-reducing, and sulfate-reducing bacteria, and immobilization of uranium as U(IV), with dissolved uranium concentrations decreasing to low levels. Following sulfite addition to remove dissolved oxygen, aqueous U(VI) concentrations fell below the U.S. Environmental Protection Agengy maximum contaminant limit (MCL) for drinking water (30/microg L(-1) or 0.126 microM). Under anaerobic conditions, these low concentrations were stable, even in the absence of added ethanol. However, when sulfite additions stopped, and dissolved oxygen (4.0-5.5 mg L(-1)) entered the injection well, spatially variable changes in aqueous U(VI) occurred over a 60 day period, with concentrations increasing rapidly from0.13 to 2.0 microM at a multilevel sampling (MLS) well located close to the injection well, but changing little at an MLS well located further away. Resumption of ethanol addition restored reduction of Fe(III), sulfate, and U(VI) within 36 h. After 2 years of ethanol addition, X-ray absorption near-edge structure spectroscopy (XANES) analyses indicated that U(IV) comprised 60-80% of the total uranium in sediment samples. Atthe completion of the project (day 1260), U concentrations in MLS wells were less than 0.1 microM. The microbial community at MLS wells with low U(VI) contained bacteria that are known to reduce uranium, including Desulfovibrio spp. and Geobacter spp., in both sediment and groundwater. The dominant Fe(III)-reducing species were Geothrix spp.

A simple in-situ passive dissolved gas groundwater sampler, comprised of a short length of silicone tubing attached to a gastight or other syringe, was adapted and tested for in-situ collection of equilibrium gas samples. Sampler retrieval after several days of immersion in groundwater allowed the direct injection of the sample onto a gas chromatograph (GC), simplifying field collection and sample handling over the commonly used "bubble stripping" method for H2 analyses. A GC was modified by sequencing a thermal conductivity (TC) detector followed by a reductive gas (RG) detector so that linear calibration of H2 over the range 0.2-200,000 ppmv was attained using a 0.5-mL gas sample; inclusion of the TC detector allowed the simultaneous quantification of other fixed gases (O2, CO2, He, and Ne) to which the RG detector was not responsive. Uptake kinetics for H2 and He indicated that the passive sampler reached equilibrium within 12 h of immersion in water. Field testing of these passive samplers revealed unusually large equilibrium gas-phase H2 concentrations in groundwater, ranging from 0.1 to 13.9%, by volume, in 11 monitoring wells surrounding four former radiological wastewater disposal ponds at the Y-12 plant in Oak Ridge, Tennessee.

In situ microbial reduction of soluble U(VI) to sparingly soluble U(IV) was evaluated at the site of the former S-3 Ponds in Area 3 of the U.S. Department of Energy Natural and Accelerated Bioremediation Research Field Research Center, Oak Ridge, TN. After establishing conditions favorable for bioremediation (Wu, et al. Environ. Sci. Technol. 2006, 40, 3988-3995), intermittent additions of ethanol were initiated within the conditioned inner loop of a nested well recirculation system. These additions initially stimulated denitrification of matrix-entrapped nitrate, but after 2 months, aqueous U levels fell from 5 to approximately 1 microM and sulfate reduction ensued. Continued additions sustained U(VI) reduction over 13 months. X-ray near-edge absorption spectroscopy (XANES) confirmed U(VI) reduction to U(IV) within the inner loop wells, with up to 51%, 35%, and 28% solid-phase U(IV) in sediment samples from the injection well, a monitoring well, and the extraction well, respectively. Microbial analyses confirmed the presence of denitrifying, sulfate-reducing, and iron-reducing bacteria in groundwater and sediments. System pH was generally maintained at less than 6.2 with low bicarbonate level (0.75-1.5 mM) and residual sulfate to suppress methanogenesis and minimize uranium mobilization. The bioavailability of sorbed U(VI) was manipulated by addition of low-level carbonate (5 mM) followed by ethanol (1-1.5 mM). Addition of low levels of carbonate increased the concentration of aqueous U, indicating an increased rate of U desorption due to formation of uranyl carbonate complexes. Upon ethanol addition, aqueous U(VI) levels fell, indicating that the rate of microbial reduction exceeded the rate of desorption. Sulfate levels simultaneously decreased, with a corresponding increase in sulfide. When ethanol addition ended but carbonate addition continued, soluble U levels increased, indicating faster desorption than reduction. When bicarbonate addition stopped, aqueous U levels decreased, indicating adsorption to sediments. Changes in the sequence of carbonate and ethanol addition confirmed that carbonate-controlled desorption increased bioavailability of U(VI) for reduction.

To evaluate the potential for in situ bioremediation of U(VI) to sparingly soluble U(IV), we constructed a pilot test facility at Area 3 of the U.S. Department of Energy Natural and Accelerated Bioremediation Research (NABIR) Field Research Center (FRC) in Oak Ridge, TN. The facility is adjacent to the former S-3 Ponds which received trillions of liters of acidic plating wastes. High levels of uranium are present, with up to 800 mg kg(-1) in the soil and 84-210 microM in the groundwater. Ambient groundwater has a highly buffered pH of approximately 3.4 and high levels of aluminum (12-13 mM), calcium (22-25 mM), and nitrate (80-160 mM). Adjusting the pH of groundwater to approximately 5 within the aquifer would deposit extensive aluminum hydroxide precipitate. Calcium is present in the groundwater at levels that inhibit U(VI) reduction, but its removal by injection of a high pH solution would generate clogging precipitate. Nitrate also inhibits U(VI) reduction and is present at such high concentrations that its removal by in situ denitrification would generate large amounts of N2 gas and biomass. To establish and maintain hydraulic control, we installed a four well recirculation system parallel to geologic strike, with an inner loop nested within an outer loop. For monitoring, we drilled three boreholes perpendicular to strike across the inner loop and installed multilevel sampling tubes within them. A tracer pulse with clean water established travel times and connectivity between wells and enabled the assessment of contaminant release from the soil matrix. Subsequently, a highly conductive region of the subsurface was prepared for biostimulation by removing clogging agents and inhibitors and increasing pH. For 2 months, groundwater was pumped from the hydraulically conductive zone; treated to remove aluminum, calcium, and nitrate, and supplemented with tap water; adjusted to pH 4.3-4.5; then returned to the hydraulically conductive zone. This protocol removed most of the aqueous aluminum and calcium. The pH of the injected treated water was then increased to 6.0-6.3. With additional flushing, the pH of the extracted water gradually increased to 5.5-6.0, and nitrate concentrations fell to 0.5-1.0 mM. These conditions were judged suitable for biostimulation. In a companion paper (Wu et al., Environ. Sci. Technol. 2006, 40, 3978-3987), we describe the effects of ethanol addition on in situ denitrification and U(VI) reduction and immobilization.

Microbial communities were characterized at contaminated sites that had elevated levels of nitrate, nickel, aluminum, and uranium (up to 690 mM, 310 microM, 42 mM, and 30 microM, respectively). The bacterial community structure based upon clonal libraries of the SSU rRNA genes (screened clones = 876) was diverse at the background site, but the three acidic samples had decreased diversity and the majority of clones were closely related to Azoarcus and Pseudomonas species. Arthrobacter and Novosphingobium sequences were recovered from the background samples but not the acidic sites, and similar pseudomonad populations were present at the background and acidic sites albeit at different relative abundances. Heterologous sequence coverage analyses indicated the microbial communities at the contaminated sites were very similar (p = 0.001) but different from the background site. Bacterial isolates (n = 67) classified as beta-or gamma-Proteobacteria, high G+C Gram-positive or low G+C Gram-positive were obtained from the background and one contaminated sample, and some of the isolates had less than 95% sequence identity with previously observed microorganisms. Despite variations in nitrate and heavy metal levels and different proximities to the source ponds, the three acidic samples had similar microbial populations. However, the least contaminated site (lowest nitrate and aluminum) had increased diversity compared to the other acidic samples. The results suggested that the combined contamination has decreased the microbial diversity, and Azoarcus populations were observed at a drastically increased frequency compared to the background site that had a more even distribution of multiple taxa.

A down-well aquifer microbial sampling system was developed using glass wool or Bio-Sep beads as a solid-phase support matrix. Here we describe the use of these devices to monitor the groundwater microbial community dynamics during field bioremediation experiments at the U.S. Department of Energy Natural and Accelerated Bioremediation Research Program's Field Research Center at the Oak Ridge National Laboratory. During the 6-week deployment, microbial biofilms colonized glass wool and bead internal surfaces. Changes in viable biomass, community composition, metabolic status, and respiratory state were reflected in sampler composition, type of donor, and groundwater pH. Biofilms that formed on Bio-Sep beads had 2-13 times greater viable biomass; however, the bead communities were less metabolically active [higher cyclopropane/monoenoic phospholipid fatty acid (PLFA) ratios] and had a lower aerobic respiratory state (lower total respiratory quinone/ PLFA ratio and ubiquinone/menaquinone ratio) than the biofilms formed on glass wool. Anaerobic growth in these systems was characterized by plasmalogen phospholipids and was greater in the wells that received electron donor additions. Partial 16S rDNA sequences indicated that Geobacter and nitrate-reducing organisms were induced by the acetate, ethanol, or glucose additions. DNA and lipid biomarkers were extracted and recovered without the complications that commonly plague sediment samples due to the presence of clay or dissolved organic matter. Although microbial community composition in the groundwater or adjacent sediments may differ from those formed on down-well biofilm samplers, the metabolic activity responses of the biofilms to modifications in groundwater geochemistry record the responses of the microbial community to biostimulation while providing integrative sampling and ease of recovery for biomarker analysis.

The potential to stimulate an indigenous microbial community to reduce a mixture of U(VI) and Tc(VII) in the presence of high (120 mM) initial NO3- co-contamination was evaluated in a shallow unconfined aquifer using a series of single-well, push-pull tests. In the absence of added electron donor, NO3-, Tc(VII), and U(VI) reduction was not detectable. However, in the presence of added ethanol, glucose, or acetate to serve as electron donor, rapid NO3- utilization was observed. The accumulation of NO2-, the absence of detectable NH4+ accumulation, and the production of N2O during in situ acetylene-block experiments suggest that NO3- was being consumed via denitrification. Tc(VII) reduction occurred concurrently with NO3- reduction, but U(VI) reduction was not observed until two or more donor additions resulted in iron-reducing conditions, as detected by the production of Fe(II). Reoxidation/remobilization of U(IV) was also observed in tests conducted with high (approximately 120 mM) but not low (approximately 1 mM) initial NO3- concentrations and not during acetylene-block experiments conducted with high initial NO3-. These results suggest that NO3(-)-dependent microbial U(IV) oxidation may inhibit or reverse U(VI) reduction and decrease the stability of U(IV) in this environment. Changes in viable biomass, community composition, metabolic status, and respiratory state of organisms harvested from down-well microbial samplers deployed during these tests were consistent with the conclusions that electron donor additions resulted in microbial growth, the creation of anaerobic conditions, and an increase in activity of metal-reducing organisms (e.g., Geobacter). The results demonstrate that it is possible to stimulate the simultaneous bioreduction of U(VI) and Tc(VII) mixtures commonly found with NO3- co-contamination at radioactive waste sites.

Publisher Summary This chapter presents a study in which a permeable iron reactive barrier was installed in late November 1997 at the U.S. Department of Energy's Y-12 National Security Complex in Oak Ridge, Tennessee. The overall goal of this study was to determine the effectiveness of the use of zero valent iron (Fe 0 ) to retain or remove uranium and other contaminants such as technetium and nitrate in groundwater. The long-term performance issues were investigated by studying the biogeochemical interactions between Fe 0 and groundwater constituents and the mineralogical and biological characteristics over an extended field operation. Results from nearly 3 years of monitoring indicated that the Fe 0 barrier was performing effectively in removing contaminant radionuclides such as uranium and technetium. In addition, a number of groundwater constituents such as bicarbonates, nitrate, and sulfate were found to react with the Fe 0 . Both nitrate and sulfate were reduced within or in the influence zone of the Fe 0 with a low redox potential. An increased anaerobic microbial population was also observed within and in the vicinity of the Fe 0 barrier, and these microorganisms were at least partially responsible for the reduction of nitrate and sulfate in groundwater. Decreased concentrations of Ca2+ and bicarbonate in groundwater occurred as a result of the formation of minerals such as aragonite (CaCO 3 ) and siderite (FeCO 3 ), which coincided with the Fe 0 corrosion and an increased groundwater pH. A suite of mineral precipitates was identified in the Fe 0 barrier system, including amorphous iron oxyhydroxides, goethite, ferrous carbonates and sulfides, aragonite, and green rusts, which were found to be responsible for the cementation and possibly clogging of Fe 0 filings observed in a number of core samples from the barrier.