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.     

Physicochemical heterogeneity controls on uranium bioreduction rates at the field scale

It has been demonstrated in laboratory systems that U(VI) can be reduced to immobile U(IV) by bacteria in natural environments. The ultimate efficacy of bioreduction at the field scale, however, is often challenging to quantify and depends on site characteristics. In this work, uranium bioreduction rates at the field scale are quantified, for the first time, using an integrated approach. The approach combines field data, inverse and forward hydrological and reactive transport modeling, and quantification of reduction rates at different spatial scales. The approach is used to explore the impact of local scale (tens of centimeters) parameters and processes on field scale (tens of meters) system responses to biostimulation treatments and the controls of physicochemical heterogeneity on bioreduction rates. Using the biostimulation experiments at the Department of Energy Old Rifle site, our results show that the spatial distribution of hydraulic conductivity and solid phase mineral (Fe(III)) play a critical role in determining the field-scale bioreduction rates. Due to the dependence on Fe-reducing bacteria, field-scale U(VI) bioreduction rates were found to be largely controlled by the abundance of Fe(III) minerals at the vicinity of the injection wells and by the presence of preferential flow paths connecting injection wells to down gradient Fe(III) abundant areas.     

Uranium and technetium bio-immobilization in intermediate-scale physical models of an in situ bio-barrier

We investigated the long-term effects of ethanol addition on U and Tc mobility in groundwater flowing through intermediate-scale columns packed with uncontaminated sediments. The columns were operated above-ground at a contaminated field site to serve as physical models of an in situ bio-barrierfor U and Tc removal from groundwater. Groundwater containing 4 microM U and 520 pM Tc was pumped through the columns for 20 months. One column received additions of ethanol to stimulate activity of indigenous microorganisms; a second column received no ethanol and served as a control. U(VI) and Tc(VII) removal was sustained for 20 months (approximately 189 pore volumes) in the stimulated column under sulfate- and Fe(III)-reducing conditions. Less apparent microbial activity and only minor removal of U(VI) and Tc(VII) were observed in the control. Sequential sediment extractions and XANES spectra confirmed that U(IV) was present in the stimulated column, although U(IV) was also detected in the control; extremely low concentrations precluded detection of Tc(IV) in any sample. These results provide additional evidence that bio-immobilization may be effective for removing U and Tc from groundwater. However, long-term effectiveness of bio-immobilization may be limited by hydraulic conductivity reductions or depletion of bioavailable Fe(III).     

Uranium reduction in sediments under diffusion-limited transport of organic carbon

Costly disposal of uranium (U) contaminated sediments is motivating research on in situ U(VI) reduction to insoluble U(IV) via directly or indirectly microbially mediated pathways. Delivery of organic carbon (OC) into sediments for stimulating U bioreduction is diffusion-limited in less permeable regions of the subsurface. To study OC-based U reduction in diffusion-limited regions, one slightly acidic and another calcareous sediment were treated with uranyl nitrate, packed into columns, then hydrostatically contacted with tryptic soy broth solutions. Redox potentials, U oxidation state, and microbial communities were well correlated. At average supply rates of 0.9 micromol OC (g sediment)(-1) day(-1), the U reduction zone extended to only about35-45 mm into sediments. The underlying unreduced U(VI) zone persisted over 600 days because the supply of OC was diffusion-limited and metabolized within a short distance. These results also suggestthat low U concentrations in groundwater samples from OC-treated sediments are not necessarily indicative of pervasive U reduction because interior and exterior regions of such sediment blocks can contain primarily U(VI) and U(IV), respectively.     

Natural humics impact uranium bioreduction and oxidation

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).     

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.     

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.     

Oxidative Dissolution of Biogenic Uraninite in Groundwater at Old Rifle, CO

Reductive bioremediation is currently being explored as a possible strategy for uranium-contaminated aquifers such as the Old Rifle site (Colorado). The stability of U(IV) phases under oxidizing conditions is key to the performance of this procedure. An in situ method was developed to study oxidative dissolution of biogenic uraninite (UO?), a desirable U(VI) bioreduction product, in the Old Rifle, CO, aquifer under different variable oxygen conditions. Overall uranium loss rates were 50-100 times slower than laboratory rates. After accounting for molecular diffusion through the sample holders, a reactive transport model using laboratory dissolution rates was able to predict overall uranium loss. The presence of biomass further retarded diffusion and oxidation rates. These results confirm the importance of diffusion in controlling in-aquifer U(IV) oxidation rates. Upon retrieval, uraninite was found to be free of U(VI), indicating dissolution occurred via oxidation and removal of surface atoms. Interaction of groundwater solutes such as Ca2? or silicate with uraninite surfaces also may retard in-aquifer U loss rates. These results indicate that the prolonged stability of U(IV) species in aquifers is strongly influenced by permeability, the presence of bacterial cells and cell exudates, and groundwater geochemistry.     

Extraction of oxidized and reduced forms of uranium from contaminated soils: effects of carbonate concentration and pH

Uranium may present in soil as precipitated, sorbed, complexed, and reduced forms, which impact its mobility and fate in the subsurface soil environment. In this study, a uranium-contaminated soil was extracted with carbonate/ bicarbonate at varying concentrations (0-1 M), pHs, and redox conditions in an attempt to evaluate their effects on the extraction efficiency and selectivity for various forms of uranium in the soil. Results indicate that at least three differentforms of uranium existed in the contaminated soil: uranium(VI) phosphate minerals, reduced U(IV) phases, and U(VI) complexed with soil organic matter. A small fraction of U(VI) could be sorbed onto soil minerals. The mechanism involved in the leaching of U(VI) by carbonates appears to involve three processes which may act concurrently or independently: the dissolution of uranium(VI) phosphate and other mineral phases, the oxidation-complexation of U(IV) under oxic conditions, and the desorption of U(VI)-organic matter complexes at elevated pH conditions. This study suggests that, depending on site-specific geochemical conditions, the presence of small quantities of carbonate/bicarbonate could result in a rapid and greatly increased leaching and the mobilization of U(VI) from the contaminated soil. Even the reduced U(IV) phases (only sparingly soluble in water) are subjected to rapid oxidation and therefore potential leaching into the environment.     

Remediation and recovery of uranium from contaminated subsurface environments with electrodes

Previous studies have demonstrated that Geobacter species can effectively remove uranium from contaminated groundwater by reducing soluble U(VI) to the relatively insoluble U(IV) with organic compounds serving as the electron donor. Studies were conducted to determine whether electrodes might serve as an alternative electron donor for U(VI) reduction by a pure culture of Geobacter sulfurreducens and microorganisms in uranium-contaminated sediments. Electrodes poised at -500 mV (vs a Ag/AgCl reference) rapidly removed U(VI) from solution in the absence of cells. However, when the poise at the electrode was removed, all of the U(VI) returned to solution, demonstrating that the electrode did not reduce U(VI). If G. sulfurreducens was present on the electrode, U(VI) did not return to solution until the electrode was exposed to dissolved oxygen. This suggeststhat G. sulfurreducens on the electrode reduced U(VI) to U(IV) which was stably precipitated until reoxidized in the presence of oxygen. When an electrode was placed in uranium-contaminated subsurface sediments, U(VI) was removed and recovered from groundwater using poised electrodes. Electrodes emplaced in flow-through columns of uranium-contaminated sediments readily removed U(VI) from the groundwater, and 87% of the uranium that had been removed was recovered from the electrode surface after the electrode was pulled from the sediments. These results suggest that microorganisms can use electrons derived from electrodes to reduce U(VI) and that it may be possible to remove and recover uranium from contaminated groundwater with poised electrodes.     

Sorption and binary exchange of nitrate, sulfate, and uranium on an anion-exchange resin

Competitive ion-exchange reactions were studied on a strong-base anion-exchange resin to remove NO3- and uranium from a contaminated groundwater containing high levels of NO3- (approximately 140 mM), SO4(2-) (approximately 10 mM), and U(VI) (approximately 0.2 mM). Results indicate that although SO4(2-) carries divalent negative charges, it showed the least selectivity for sorption by the Purolite A-520E resin, which is functionalized with triethylamine exchange sites. Nitrate was the most strongly sorbed. Sorption selectivity followed the order of NO3- > Cl- > SO4(2-) under the experimental conditions. Nitrate competitively sorbed and displaced previously sorbed SO4(2-) in a column flow-through experiment and resulted in a high elution front of SO4(2-) in the effluent. Although the concentration of uranium in groundwater is orders of magnitude lower than that of NO3- or SO4(2-), it was found to be strongly sorbed by the anion-exchange resin. Because the most stable uranium species in oxic and suboxic environments is the UO2(2+) cation, its strong sorption by anion-exchange resins is hypothesized to be the result of the co-ion effect of NO3- by forming anionic UO2(NO3)3- complexes in the resin matrix. These observations point out a potential alternative remediation strategy that uses strong-base anion-exchange resins to remove uranium from this site-specific groundwater, which has a low pH and a relatively high NO3- concentration.     

Inhibition of a U(VI)- and sulfate-reducing consortia by U(VI)

The stimulation of microbial U(VI) reduction is currently being investigated as a means to reduce uranium's mobility in groundwater, but little is known about the concentration at which U(VI) might inhibit microbial activity, or the effect of U(VI) on bacterial community structure. We investigated these questions with an ethanol-fed U(VI)- and sulfate-reducing enrichment developed from sediment from the site of an ongoing field biostimulation experiment at Area 3 of the Oak Ridge Field Research Center (FRC). Sets of triplicate enrichments were spiked with increasing concentrations of U(VI) (from 49 microm to 9.2 mM). As the U(VI) concentration increased to 224 microM, the culture's production of acetate from ethanol slowed, and at or above 1.6 mM U(VI) little acetate was produced over the time frame of the experiment. An uncoupling inhibition model was applied to the data, and the inhibition coefficient for U(VI), Ku, was found to be approximately 100 microM U(VI), or 24 mg/L, indicating the inhibitory effect is relevant at highly contaminated sites. Microbial community structure at the conclusion of the experiment was analyzed with terminal restriction fragment length polymorphism (T-RFLP) analysis. T-RFs associated with Desulfovibrio-like organisms decreased in relative abundance with increasing U(VI) concentration, whereas Clostridia-like T-RFs increased.     

Speciation of uranium in sediments before and after in situ biostimulation

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 at < 4 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 to < 0.1 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.     

Effects of organic carbon supply rates on uranium mobility in a previously bioreduced contaminated sediment

Bioreduction-based strategies for remediating uranium (U)-contaminated sediments face the challenge of maintaining the reduced status of U for long times. Because groundwater influxes continuously bring in oxidizing terminal electron acceptors (O2, NO3(-)), it is necessary to continue supplying organic carbon (OC) to maintain the reducing environment after U bioreduction is achieved. We tested the influence of OC supply rates on mobility of previously microbial reduced uranium U(IV) in contaminated sediments. We found that high degrees of U mobilization occurred when OC supply rates were high, and when the sediment still contained abundant Fe(III). Although 900 days with low levels of OC supply minimized U mobilization, the sediment redox potential increased with time as did extractable U(VI) fractions. Molecular analyses of total microbial activity demonstrated a positive correlation with OC supply and analyses of Geobacteraceae activity (RT-qPCR of 16S rRNA) indicated continued activity even when the effluent Fe(II) became undetectable. These data support our hypothesis on the mechanisms responsible for remobilization of U under reducing conditions; that microbial respiration caused increased (bi)carbonate concentration and formation of stable uranyl carbonate complexes, thereby shifted U(IV)/U(VI) equilibrium to more reducing potentials. The data also suggested that low OC concentrations could not sustain the reducing condition of the sediment for much longer time. Bioreduced U(IV) is not sustainable in an oxidizing environment for a very long time.     

Uranium biomineralization as a result of bacterial phosphatase activity: insights from bacterial isolates from a contaminated subsurface

Uranium contamination is an environmental concern at the Department of Energy's Field Research Center in Oak Ridge, Tennessee. In this study, we investigated whether phosphate biomineralization, or the aerobic precipitation of U(VI)-phosphate phases facilitated by the enzymatic activities of microorganisms, offers an alternative to the more extensively studied anaerobic U(VI) bioreduction. Three heterotrophic bacteria isolated from FRC soils were studied for their ability to grow and liberate phosphate in the presence of U(VI) and an organophosphate between pH 4.5 and 7.0. The objectives were to determine whether the strains hydrolyzed sufficient phosphate to precipitate uranium, to determine whether low pH might have an effect on U(VI) precipitation, and to identify the uranium solid phase formed during biomineralization. Two bacterial strains hydrolyzed sufficient organophosphate to precipitate 7395% total uranium after 120 h of incubation in simulated groundwater. The highest rates of uranium precipitation and phosphatase activity were observed between pH 5.0 and 7.0. EXAFS spectra identified the uranyl phosphate precipitate as an autunite/meta-autunite group mineral. The results of this study indicate that aerobic heterotrophic bacteria within a uranium-contaminated environment that can hydrolyze organophosphate, especially in low pH conditions, may play an important role in the bioremediation of uranium.     

Bioreduction of uranium: environmental implications of a pentavalent intermediate

The release of uranium and other transuranics into the environment, and their subsequent mobility, are subjects of intense public concern. Uranium dominates the inventory of most medium- and low-level radioactive waste sites and under oxic conditions is highly mobile as U(VI), the soluble uranyl dioxocation (UO2)2+. Specialist anaerobic bacteria are, however, able to reduce U(VI)to insoluble U(IV), offering a strategy for the bioremediation of uranium-contaminated groundwater and a potential mechanism for the biodeposition of uranium ores. Despite the environmental importance of U(VI) bioreduction, there is little information on the mechanism of this transformation. In the course of this study we used X-ray absorption spectroscopy (XAS) to show that the subsurface metal-reducing bacterium Geobacter sulfurreducens reduces U(VI) by a one-electron reduction, forming an unstable (UO2)+ species. The final, insoluble U(IV) product could be formed either through further reduction of U(V) or through its disproportionation. When G. sulfurreducens was challenged with the chemically analogous (NpO2)+, which is stable with respect to disproportionation, it was not reduced, suggesting that it is disproportionation of U(V) which leads to the U(IV) product. This surprising discrimination between U and Np illustrates the need for mechanistic understanding and care in devising in situ bioremediation strategies for complex wastes containing other redox-active actinides, including plutonium.     

Reoxidation of bioreduced uranium under reducing conditions

Nuclear weapons and fuel production have left many soils and sediments contaminated with toxic levels of uranium (U). Although previous short-term experiments on microbially mediated U(VI) reduction have supported the prospect of immobilizing the toxic metal through formation of insoluble U(IV) minerals, our longer-term (17 months) laboratory study showed that microbial reduction of U can be transient, even under sustained reducing conditions. Uranium was reduced during the first 80 days, but later (100-500 days) reoxidized and solubilized, even though a microbial community capable of reducing U(VI) was sustained. Microbial respiration caused increases in (bi)-carbonate concentrations and formation of very stable uranyl carbonate complexes, thereby increasing the thermodynamic favorability of U(IV) oxidation. We propose that kinetic limitations including restricted mass transfer allowed Fe-(III) and possibly Mn(IV) to persist as terminal electron acceptors (TEAs) for U reoxidation. These results show that in-situ U remediation by organic carbon-based reductive precipitation can be problematic in sediments and groundwaters with neutral to alkaline pH, where uranyl carbonates are most stable.     

In-situ evidence for uranium immobilization and remobilization

The in-situ microbial reduction and immobilization of uranium was assessed as a means of preventing the migration of this element in the terrestrial subsurface. Uranium immobilization (putatively identified as reduction) and microbial respiratory activities were evaluated in the presence of exogenous electron donors and acceptors with field push-pull tests using wells installed in an anoxic aquifer contaminated with landfill leachate. Uranium(VI) amended at 1.5 microM was reduced to less than 1 nM in groundwater in less than 8 d during all field experiments. Amendments of 0.5 mM sulfate or 5 mM nitrate slowed U(VI) immobilization and allowed for the recovery of 10% and 54% of the injected element, respectively, as compared to 4% in the unamended treatment. Laboratory incubations confirmed the field tests and showed that the majority of the U(VI) immobilized was due to microbial reduction. In these tests, nitrate treatment (7.5 mM) inhibited U(VI) reduction, and nitrite was transiently produced. Further push-pull tests were performed in which either 1 or 5 mM nitrate was added with 1.0 uM U(VI) to sediments that already contained immobilized uranium. After an initial loss of the amendments, the concentration of soluble U(VI) increased and eventually exceeded the injected concentration, indicating that previously immobilized uranium was remobilized as nitrate was reduced. Laboratory experiments using heat-inactivated sediment slurries suggested that the intermediates of dissimilatory nitrate reduction (denitrification or dissimilatory nitrate reduction to ammonia), nitrite, nitrous oxide, and nitric oxide were all capable of oxidizing and mobilizing U(IV). These findings indicate that in-situ subsurface U(VI) immobilization can be expected to take place under anaerobic conditions, but the permanence of the approach can be impaired by disimilatory nitrate reduction intermediates that can mobilize previously reduced uranium.     

Uranium reoxidation in previously bioreduced sediment by dissolved oxygen and nitrate

Flow-through sediment column experiments examined the reoxidation of microbially reduced uranium with either oxygen or nitrate supplied as the oxidant. The uranium was reduced and immobilized via long-term (70 days) acetate biostimulation resulting in 62-92% removal efficiency of the 20 microM influent uranium concentration. Uranium reduction occurred simultaneously with iron reduction as the dominant electron accepting process. The columns were reoxidized by discontinuing the supply of acetate and either replacing the anaerobic gas used to purge the influent media with a gas mixture containing 20% oxygen (resulting in a dissolved oxygen concentration of 0.27 mM) or adding 1.6 mM nitrate to the influent media. Both oxygen and nitrate resolubilized the majority (88 and 97%, respectively) of the uranium precipitated during bioreduction within 54 days. Although oxygen is more thermodynamically favorable an oxidant than nitrate, nitrate-dependent uranium oxidation occurred significantly faster than oxygen-dependent uranium oxidation at the beginning of our experiment due, in part, to oxygen reacting more strongly with other reduced compounds. Nitrate breakthrough at the effluent of the column occurred within 12 h, which was significantly earlier than when oxygen was detected at the effluent (26 days). Although, over time, the majority of uranium was reoxidized by either oxidant, these results indicate that the type of oxidant and its reactivity with other reduced compounds will influence the fate of reduced uranium during a short-term oxidation event that may occur during a uranium bioremediation scenario.     

Method to attenuate U(VI) mobility in acidic waste plumes using humic acids

Acidic uranium (U) groundwater plumes have resulted from acid-extraction of plutonium during the Cold War and from U mining and milling operations. A sustainable method for in situ immobilization of U under acidic conditions is not yet available. Here, we propose to use humic acids (HAs) for in situ U immobilization in acidic waste plumes. Our laboratory batch experiments show that HA can adsorb onto aquifer sediments rapidly, strongly and practically irreversibly. Adding HA greatly enhanced U adsorption capacity to sediments at pH below 5.0. Our column experiments using historically contaminated sediments from the Savannah River Site under slow flow rates (120 and 12 m/year) show that desorption of U and HA were nondetectable over 100 pore-volumes of leaching with simulated acidic groundwaters. Upon HA-treatment, 99% of the contaminant [U] was immobilized at pH ≤ 4.5, compared to 5% and 58% immobilized in the control columns at pH 3.5 and 4.5, respectively. These results indicate that HA-treatment is a promising in situ remediation method for acidic U waste plumes. As a remediation reagent, HAs are resistant to biodegradation, cost-effective, nontoxic, and easily introducible to the subsurface.