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.

Modeling uranium transport in acidic contaminated groundwater with base addition.

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 NO(3)(-), SO(4)(2-), 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.

Dynamics of microbial community composition and function during in situ bioremediation of a uranium-contaminated aquifer.

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.

Kinetic analysis and modeling of oleate and ethanol stimulated uranium (VI) bio-reduction in contaminated sediments under sulfate reduction conditions.

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.

Significant association between sulfate-reducing bacteria and uranium-reducing microbial communities as revealed by a combined massively parallel sequencing-indicator species approach.

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.

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.

Responses of microbial community functional structures to pilot-scale uranium in situ bioremediation.

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.

Prediction of uranium and technetium sorption during titration of contaminated acidic groundwater.

This study investigates uranium and technetium sorption onto aluminum and iron hydroxides during titration of acidic groundwater. The contaminated groundwater exhibits oxic conditions with high concentrations of NO(3)(-), SO(4)(2-), U, Tc, and various metal cations. More than 90% of U and Tc was removed from the aqueous phase as Al and Fe precipitated above pH 5.5, but was partially resolublized at higher pH values. An equilibrium hydrolysis and precipitation reaction model adequately described variations in aqueous concentrations of metal cations. An anion exchange reaction model was incorporated to simulate sulfate, U and Tc sorption onto variably charged (pH-dependent) Al and Fe hydroxides. Modeling results indicate that competitive sorption/desorption on mixed mineral phases needs to be considered to adequately predict U and Tc mobility. The model could be useful for future studies of the speciation of U, Tc and co-existing ions during pre- and post-groundwater treatment practices.

Uranium transformations in static microcosms.

Elucidation of complex biogeochemical processes and their effects on speciation of U in the subsurface is critical for developing remediation strategies with an understanding of stability. We have developed static microcosms that are similar to bioreduction process studies in situ under laminar flow conditions or in sediment pores. Uranium L(3)-edge X-ray absorption near-edge spectroscopy analysis with depth in the microcosms indicated that transformation of U(VI) to U(IV) occurred by at least two distinct processes. Extended X-ray absorption fine structure (EXAFS) analysis indicated that initial U(VI) species associated with C- and P-containing ligands were transformed to U(IV) in the form of uraninite and U associated with Fe-bound ligands. Microbial community analysis identified putative Fe(III) and sulfate reducers at two different depths in the microcosms. The slow reduction of U(VI) to U(IV) may contribute the stability of U(IV) within microcosms at 11 months after a decrease in bioreducing conditions due to limited electron donors.

Sequestering uranium and technetium through co-precipitation with aluminum in a contaminated acidic environment.

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.

GeoChip-based analysis of functional microbial communities during the reoxidation of a bioreduced uranium-contaminated aquifer.

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 biostimulation approach may potentially offer an effective means for the bioremediation of U(VI)-contaminated sites.

Potential negative consequences of adding phosphorus-based fertilizers to immobilize lead in soil.

A study of the potential negative consequences of adding phosphate (P)-based fertilizers as amendments to immobilize lead (Pb) in contaminated soils was conducted. Lead-contaminated firing range soils also contained elevated concentrations of antimony (Sb), a common Pb hardening agent, and some arsenic (As) of unknown (possibly background) origin. After amending the soils with triple superphosphate, a relatively soluble P source, column leaching experiments revealed elevated concentrations of Sb, As, and Pb in the leachate, reflecting an initial spike in soluble Pb and a particularly dramatic increase in Sb and As mobility. Minimal As, Sb, and Pb leaching was observed during column tests performed on non-amended control soils. In vitro extractions tests were performed to assess changes in Pb, As, and Sb bioaccessibility on P amendment. Lead bioaccessibility was systematically lowered with increasing P dosage, but there was much less of an effect on As and Sb bioaccessibility than on mobility. Our results indicate that although P amendments may aid in lowering the bioaccessibility of soil-bound Pb, it may also produce an initial increase in Pb mobility and a significant release of Sb and As from the soil, dramatically increasing their mobility and to a lesser extent their bioavailability.

Microbial communities in contaminated sediments, associated with bioremediation of uranium to submicromolar levels.

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 (<30 microg/liter or 0.126 microM). Sediment microbial communities from the treatment zone were compared with those from a control well without biostimulation. Most-probable-number estimations indicated that microorganisms implicated in bioremediation accumulated in the sediments of the treatment zone but were either absent or in very low numbers in an untreated control area. Organisms belonging to genera known to include U(VI) reducers were detected, including 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.

Influence of bicarbonate, sulfate, and electron donors on biological reduction of uranium and microbial community composition.

A microcosm study was performed to investigate the effect of ethanol and acetate on uranium(VI) biological reduction and microbial community changes under various geochemical conditions. Each microcosm contained an uranium-contaminated sediment (up to 2.8 g U/kg) suspended in buffer with bicarbonate at concentrations of either 1 or 40 mM and sulfate at either 1.1 or 3.2 mM. Ethanol or acetate was used as an electron donor. Results indicate that ethanol yielded in significantly higher U(VI) reduction rates than acetate. A low bicarbonate concentration (1 mM) was favored for U(VI) bioreduction to occur in sediments, but high concentrations of bicarbonate (40 mM) and sulfate (3.2 mM) decreased the reduction rates of U(VI). Microbial communities were dominated by species from the Geothrix genus and Proteobacteria phylum in all microcosms. However, species in the Geobacteraceae family capable of reducing U(VI) were significantly enriched by ethanol and acetate in low-bicarbonate buffer. Ethanol increased the population of unclassified Desulfuromonales, while acetate increased the population of Desulfovibrio. Additionally, species in the Geobacteraceae family were not enriched in high-bicarbonate buffer, but the Geothrix and the unclassified Betaproteobacteria species were enriched. This study concludes that ethanol could be a better electron donor than acetate for reducing U(VI) under given experimental conditions, and electron donor and groundwater geochemistry alter microbial communities responsible for U(VI) reduction.

In situ bioreduction of uranium (VI) to submicromolar levels and reoxidation by dissolved oxygen.

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 from < 0.13 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.

Modeling in-situ uranium(VI) bioreduction by sulfate-reducing bacteria.

We present a travel-time based reactive transport model to simulate an in-situ bioremediation experiment for demonstrating enhanced bioreduction of uranium(VI). The model considers aquatic equilibrium chemistry of uranium and other groundwater constituents, uranium sorption and precipitation, and the microbial reduction of nitrate, sulfate and U(VI). Kinetic sorption/desorption of U(VI) is characterized by mass transfer between stagnant micro-pores and mobile flow zones. The model describes the succession of terminal electron accepting processes and the growth and decay of sulfate-reducing bacteria, concurrent with the enzymatic reduction of aqueous U(VI) species. The effective U(VI) reduction rate and sorption site distributions are determined by fitting the model simulation to an in-situ experiment at Oak Ridge, TN. Results show that (1) the presence of nitrate inhibits U(VI) reduction at the site; (2) the fitted effective rate of in-situ U(VI) reduction is much smaller than the values reported for laboratory experiments; (3) U(VI) sorption/desorption, which affects U(VI) bioavailability at the site, is strongly controlled by kinetics; (4) both pH and bicarbonate concentration significantly influence the sorption/desorption of U(VI), which therefore cannot be characterized by empirical isotherms; and (5) calcium-uranyl-carbonate complexes significantly influence the model performance of U(VI) reduction.

Pilot-scale in situ bioremediation of uranium in a highly contaminated aquifer. 1. Conditioning of a treatment zone.

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.

Pilot-scale in situ bioremedation of uranium in a highly contaminated aquifer. 2. Reduction of u(VI) and geochemical control of u(VI) bioavailability.

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.

A nested-cell approach for in situ remediation.

We characterize the hydraulics of an extraction-injection well pair in arbitrarily oriented regional flow by the recirculation ratio, area, and average residence time in the recirculation zone. Erratic regional flow conditions may compromise the performance of the reactor between a single well pair. We propose an alternative four-well system: two downgradient extraction and two upgradient injection wells creating an inner cell nested within an outer cell. The outer cell protects the inner cell from the influence of regional flow. Compared to a two-well system, the proposed four-well system has several advantages: (1) the recirculation ratio within the nested inner cell is less sensitive to the regional flow direction; (2) a transitional recirculation zone between the inner and outer cells can capture flow leakage from the inner cell, minimizing the release of untreated contaminants; and (3) the size of the recirculation zone and residence times can be better controlled within the inner cell by changing the pumping rates. The system is applied at the Field Research Center in Oak Ridge, Tennessee, where experiments on microbial in situ reduction of uranium (VI) are under way.

Mass-transfer limitations for nitrate removal in a uranium-contaminated aquifer.

A field test on in situ subsurface bioremediation of uranium(VI) is underway at the Y-12 National Security Complex in the Oak Ridge Reservation, Oak Ridge, TN. Nitrate has a high concentration at the site, which prevents U(VI) reduction, and thus must be removed. An acidic-flush strategy for nitrate removal was proposed to create a treatment zone with low levels of accessible nitrate. The subsurface at the site contains highly interconnected fractures surrounded by matrix blocks of low permeability and high porosity and is therefore subject to preferential flow and matrix diffusion. To identify the heterogeneous mass transfer properties, we performed a novel forced-gradient tracer test, which involved the addition of bromide, the displacement of nitrate, and the rebound of nitrate after completion of pumping. The simplest conceptualization consistent with the data is that the pore-space consists of a single mobile domain, as well as a fast and a slowly reacting immobile domain. The slowly reacting immobile domain (shale matrix) constitutes over 80% of the pore volume and acts as a long-term reservoir of nitrate. According to simulations, the nitrate stored in the slowly interacting immobile domain in the fast flow layer, at depths of about 12.2-13.7 m, will be reduced by an order of magnitude over a period of about a year. By contrast, the mobile domain rapidly responds to flushing, and a low average nitrate concentration can be maintained if the nitrate is removed as soon as it enters the mobile domain. A field-scale experiment in which the aquifer was flushed with acidic solution confirmed our understanding of the system. For the ongoing experiments on microbial U(VI) reduction, nitrate concentrations must be low in the mobile domain to ensure U(VI) reducing conditions. We therefore conclude that the nitrate leaching out of the immobile pore space must continuously be removed by in situ denitrification to maintain favorable conditions.