Uranium Redistribution Due to Water Table Fluctuations in Sandy Wetland Mesocosms.

To understand better the fate and stability of immobilized uranium (U) in wetland sediments, and how intermittent dry periods affect U stability, we dosed saturated sandy wetland mesocosms planted with Scirpus acutus with low levels of uranyl acetate for 4 months before imposing a short drying and rewetting period. Concentrations of U in mesocosm effluent increased after drying and rewetting, but the cumulative amount of U released following the dry period constituted less than 1% of the total U immobilized in the soil during the 4 months prior. This low level of remobilization suggests, and XANES analyses confirm, that microbial reduction was not the primary means of U immobilization, as the U immobilized in mesocosms was primarily U(VI) rather than U(IV). Drying followed by rewetting caused a redistribution of U downward in the soil profile and to root surfaces. Although the U on roots before drying was primarily associated with minerals, the U that relocated to the roots during drying and rewetting was bound diffusely. Results show that short periods of drought conditions in a sandy wetland, which expose reduced sediments to air, may impact U distribution without causing large releases of soil-bound U to surface waters.

Uranium immobilization in an iron-rich rhizosphere of a native wetland plant from the Savannah River Site under reducing conditions.

The hypothesis of this study was that iron plaques formed on the roots of wetland plants and their rhizospheres create environmental conditions favorable for iron reducing bacteria that promote the in situ immobilization of uranium. Greenhouse microcosm studies were conducted using native plants (Sparganium americanum) from a wetland located on the Savannah River Site, Aiken, SC. After iron plaques were established during a 73-day period by using an anoxic Fe(II)-rich nutrient solution, a U(VI) amended nutrient solution was added to the system for an additional two months. Compared to plant-free control microcosms, microcosms containing iron plaques successfully stimulated the growth of targeted iron reducing bacteria, Geobacter spp. Their population continuously increased after the introduction of the U(VI) nutrient solution. The reduction of some of the U(VI) to U(IV) by iron reducing bacteria was deduced based on the observations that the aqueous Fe(II) concentrations increased while the U(VI) concentrations decreased. The Fe(II) produced by the iron reducing bacteria was assumed to be reoxidized by the oxygen released from the roots. Advanced spectroscopic analyses revealed that a significant fraction of the U(VI) had been reduced to U(IV) and they were commonly deposited in association with phosphorus on the iron plaque.

Profiling in situ microbial community structure with an amplification microarray.

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO(3)(-)) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO(3), but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications.

Distribution of microbial biomass and potential for anaerobic respiration in Hanford Site 300 Area subsurface sediment.

Subsurface sediments were recovered from a 52-m-deep borehole cored in the 300 Area of the Hanford Site in southeastern Washington State to assess the potential for biogeochemical transformation of radionuclide contaminants. Microbial analyses were made on 17 sediment samples traversing multiple geological units: the oxic coarse-grained Hanford formation (9 to 17.4 m), the oxic fine-grained upper Ringold formation (17.7 to 18.1 m), and the reduced Ringold formation (18.3 to 52 m). Microbial biomass (measured as phospholipid fatty acids) ranged from 7 to 974 pmols per g in discrete samples, with the highest numbers found in the Hanford formation. On average, strata below 17.4 m had 13-fold less biomass than those from shallower strata. The nosZ gene that encodes nitrous oxide reductase (measured by quantitative real-time PCR) had an abundance of 5 to 17 relative to that of total 16S rRNA genes below 18.3 m and <5 above 18.1 m. Most nosZ sequences were affiliated with Ochrobactrum anthropi (97 sequence similarity) or had a nearest neighbor of Achromobacter xylosoxidans (90 similarity). Passive multilevel sampling of groundwater geochemistry demonstrated a redox gradient in the 1.5-m region between the Hanford-Ringold formation contact and the Ringold oxic-anoxic interface. Within this zone, copies of the dsrA gene and Geobacteraceae had the highest relative abundance. The majority of dsrA genes detected near the interface were related to Desulfotomaculum spp. These analyses indicate that the region just below the contact between the Hanford and Ringold formations is a zone of active biogeochemical redox cycling.

Microbial functional gene diversity with a shift of subsurface redox conditions during In Situ uranium reduction.

To better understand the microbial functional diversity changes with subsurface redox conditions during in situ uranium bioremediation, key functional genes were studied with GeoChip, a comprehensive functional gene microarray, in field experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO). The results indicated that functional microbial communities altered with a shift in the dominant metabolic process, as documented by hierarchical cluster and ordination analyses of all detected functional genes. The abundance of dsrAB genes (dissimilatory sulfite reductase genes) and methane generation-related mcr genes (methyl coenzyme M reductase coding genes) increased when redox conditions shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes detected were primarily from Geobacter sp. and decreased with lower subsurface redox conditions. Statistical analysis of environmental parameters and functional genes indicated that acetate, U(VI), and redox potential (E(h)) were the most significant geochemical variables linked to microbial functional gene structures, and changes in microbial functional diversity were strongly related to the dominant terminal electron-accepting process following acetate addition. The study indicates that the microbial functional genes clearly reflect the in situ redox conditions and the dominant microbial processes, which in turn influence uranium bioreduction. Microbial functional genes thus could be very useful for tracking microbial community structure and dynamics during bioremediation.

Uranium reduction and microbial community development in response to stimulation with different electron donors.

Stimulating microbial reduction of soluble U(VI) to less soluble U(IV) shows promise as an in situ bioremediation strategy for uranium contaminated groundwater, but the optimal electron donors for promoting this process have yet to be identified. The purpose of this study was to better understand how the addition of various electron donors to uranium-contaminated subsurface sediments affected U(VI) reduction and the composition of the microbial community. The simple electron donors, acetate or lactate, or the more complex donors, hydrogen-release compound (HRC) or vegetable oil, were added to the sediments incubated in flow-through columns. The composition of the microbial communities was evaluated with quantitative PCR probing specific 16S rRNA genes and functional genes, phospholipid fatty acid analysis, and clone libraries. All the electron donors promoted U(VI) removal, even though the composition of the microbial communities was different with each donor. In general, the overall biomass, rather than the specific bacterial species, was the factor most related to U(VI) removal. Vegetable oil and HRC were more effective in stimulating U(VI) removal than acetate. These results suggest that the addition of more complex organic electron donors could be an excellent option for in situ bioremediation of uranium-contaminated groundwater.

Physiological and taxonomic description of the novel autotrophic, metal oxidizing bacterium, Pseudogulbenkiania sp. strain 2002.

A lithoautotrophic, Fe(II) oxidizing, nitrate-reducing bacterium, strain 2002 (ATCC BAA-1479; =DSM 18807), was isolated as part of a study on nitrate-dependent Fe(II) oxidation in freshwater lake sediments. Here we provide an in-depth phenotypic and phylogenetic description of the isolate. Strain 2002 is a gram-negative, non-spore forming, motile, rod-shaped bacterium which tested positive for oxidase, catalase, and urease. Analysis of the complete 16S rRNA gene sequence placed strain 2002 in a clade within the family Neisseriaceae in the order Nessieriales of the Betaproteobacteria 99.3% similar to Pseudogulbenkiania subflava. Similar to P. sublfava, predominant whole cell fatty acids were identified as 16:17c, 42.4%, and 16:0, 34.1%. Whole cell difference spectra of the Fe(II) reduced minus nitrate oxidized cyctochrome content revealed a possible role of c-type cytochromes in nitrate-dependent Fe(II) oxidation. Strain 2002 was unable to oxidize aqueous or solid-phase Mn(II) with nitrate as the electron acceptor. In addition to lithotrophic growth with Fe(II), strain 2002 could alternatively grow heterotrophically with long-chain fatty acids, simple organic acids, carbohydrates, yeast extract, or casamino acids. Nitrate, nitrite, nitrous oxide, and oxygen also served as terminal electron acceptors with acetate as the electron donor.

Effects of diesel and interactions with copper and other metals in an estuarine sediment microbial community.

Estuarine sediment microcosms were treated with combinations of diesel, copper (at two levels), and a mixture of heavy metals (mercury, cadmium, lead, and chromium; at two levels) mimicking the contaminant loadings found in harbor sediments. The effects on the microbial community were monitored by polar lipid fatty acid analysis. Diesel addition increased microbial biomass, caused shifts in some fatty acid structural groups, and decreased starvation biomarkers. Incorporation of diesel hydrocarbons into lipids was expressed as an increase in the proportion of odd-carbon-number fatty acids. No treatment with the metals mixture (mercury, cadmium, lead, and chromium) alone significantly changed any parameter derived from the polar lipid fatty acids, but the increase in microbial biomass from diesel addition was higher with the metals mixture, possibly because of indirect effects caused by reductions in grazing resulting from metal-induced toxicity to bacteriovorous nematodes. Copper also modified the effects of diesel addition, preventing biomass increase but not diesel degradation, suggesting that some of the energy gained from diesel oxidation was expended combating copper toxicity. In the present study, observations indicate that metals in general, and copper in particular, can modify the response of sedimentary microorganisms to petroleum-hydrocarbon contaminants.

Multiply methyl-branched fatty acids and diacids in the polar lipids of a microaerophilic subsurface microbial community.

A previously unreported series of di- and tri-methylated fatty acids, as well as saturated and monounsaturated diacids were identified in polar lipids isolated from environmental subsurface sediment samples. Mechanisms are proposed for their formation, but their origin and role in cell membranes remains unknown.

Investigating freshwater periphyton community response to uranium with phospholipid fatty acid and denaturing gradient gel electrophoresis analyses.

Periphyton communities can be used as monitors of ecosystem health and as indicators of contamination in lotic systems. Measures of biomass, community structure, and genetic diversity were used to investigate impacts of uranium (U) exposure on periphyton. Laboratory exposures of periphyton in river water amended with 238U were performed for 5 days, followed by 2 days of U depuration in unamended river water. Productivity as measured by biomass was not affected by concentrations up to 100 microg238U L(-1). Phospholipid fatty acid (PLFA) profiles and denaturing gradient gel electrophoresis (DGGE) banding patterns revealed no changes in community or genetic structure related to U exposure. We suggest that the periphyton community as a whole was not significantly impacted by exposures of 238U up to a concentration of 100 microgL(-1). These findings have significance for the assessment and prediction of U impacts on aquatic ecosystems.

Uranium removal from groundwater via in situ biostimulation: Field-scale modeling of transport and biological processes.

During 2002 and 2003, bioremediation experiments in the unconfined aquifer of the Old Rifle UMTRA field site in western Colorado provided evidence for the immobilization of hexavalent uranium in groundwater by iron-reducing Geobacter sp. stimulated by acetate amendment. As the bioavailable Fe(III) terminal electron acceptor was depleted in the zone just downgradient of the acetate injection gallery, sulfate-reducing organisms came to dominate the microbial community. In the present study, we use multicomponent reactive transport modeling to analyze data from the 2002 field experiment to identify the dominant transport and biological processes controlling uranium mobility during biostimulation, and determine field-scale parameters for these modeled processes. The coupled process simulation approach was able to establish a quantitative characterization of the principal flow, transport, and reaction processes based on the 2002 field experiment, that could be applied without modification to describe the 2003 field experiment. Insights gained from this analysis include field-scale estimates of the bioavailable Fe(III) mineral threshold for the onset of sulfate reduction, and rates for the Fe(III), U(VI), and sulfate terminal electron accepting processes.

Uranium fate in wetland mesocosms: Effects of plants at two iron loadings with different pH values.

Small-scale continuous flow wetland mesocosms (∼0.8 L) were used to evaluate how plant roots under different iron loadings affect uranium (U) mobility. When significant concentrations of ferrous iron (Fe) were present at circumneutral pH values, U concentrations in root exposed sediments were an order of magnitude greater than concentrations in root excluded sediments. Micro X-ray absorption near-edge structure (µ-XANES) spectroscopy indicated that U was associated with the plant roots primarily as U(VI) or U(V), with limited evidence of U(IV). Micro X-ray fluorescence (µ-XRF) of plant roots suggested that for high iron loading at circumneutral pH, U was co-located with Fe, perhaps co-precipitated with root Fe plaques, while for low iron loading at a pH of ∼4 the correlation between U and Fe was not significant, consistent with previous observations of U associated with organic matter. Quantitative PCR analyses indicated that the root exposed sediments also contained elevated numbers of Geobacter spp., which are likely associated with enhanced iron cycling, but may also reduce mobile U(VI) to less mobile U(IV) species.

Feasibility of assessment of regulatory lipids in breath condensate as potential presymptomatic harbingers of pulmonary pathobiology.

Regulatory lipids from the airway surface readily form aerosols that can be recovered non-invasively by cooling expired breath to form breath condensate (BC). Regulatory lipids have been detected previously utilizing enzyme-linked-immunosorbent serologic assay (ELISA). Here we test the feasibility of assessment of regulatory lipids in BC by mass spectrometry so presently unknown lipid regulatory components can be detected without addition of specific antibodies as in the ELISA procedure. Baseline regulatory lipids were detected in >pg/mL BC in control animals or human lung tissue culture cells. In nearly every case animals exposed to toxins or infectious bacteria showed increases in the BC regulatory components. Lipids were recovered from BC by solid phase extraction. Phosphatidylcholine (PC) based lipids were detected as the progenitor (parent) ions of isomers that fragmented in producing product positive ions at m/z 184 (of phosphocholine) in tandem MS using capillary HPLC and electrospray ionization. BC eicosanoids such as prostaglandins, thromboxane, and isoprostanes require capillary gas chromatography for separation and detection that necessitates methoximation, pentafluorobenzyl (PFB) ester formation, and trimethyl silylation of hydroxyls prior to gas chromatography/ion trap tandem mass spectrometry of negative ions after chemical ionization (NICI). Tetradeuterated internal standards were utilized for quantitation with the GC/NICI/MS. Changes in concentrations of lipids and eicosanoids were observed in piglets, and rats exposed to aerosolized 100 mug/kg lipopolysaccharide (LPS), or 50 mug/kg and 150 mug/kg aerosolized Staphylococcal enterotoxin B (SEB) in BC as well as in human THP-1 cell culture cell supernatants and bronchoalveolar lavage (BAL) samples in rats. Responses of the molecular species of phosphatidylcholines (PCs), platelet activating factors (PAFs) and specific eicosanoids correlated to the toxin and bacterial infections suggesting that patterns of differential responses could be detected with further experimentation. Initial targets included prostaglandins (PGE(2), PGF(2alpha)), thromboxane (TXB2), and prostacyclin (as 6-Keto PGF(1alpha)) that show differential responses to inflammation, the leukotriene (LTB4) and PGD2 for allergic responses, isoprostanes (8-iso-PGF(2alpha)) for free radical oxidative stress responses, and HETEs for differential lipoxygenase activities. PAFs and lysoPAFs have been shown to increase with inflammation and in the feasibility experiments reported here. Preliminary studies show pulmonary responses of piglets to intrathecal exposure of toxicants (LPS and SEB) or infections with Actinobacillus pleuropneumoniae induce increased levels of lipids and two eicosanoids with the suggestion that differential patterns might be detected with expanded testing. Preliminary experience indicates numerous other eicosanoids were available for assay in BC. This suggests an important potential application of BC to observe a wide array of factors to establish comprehensive profiles for physiological and pathophysiological states. Ultimately this technique could be used as a non-invasive possibly presymptomatic assessment of pulmonary pathobiology.

Atmospheric pressure chemical ionization and atmospheric pressure photoionization for simultaneous mass spectrometric analysis of microbial respiratory ubiquinones and menaquinones.

An atmospheric pressure photoionization (APPI) source and an atmospheric pressure chemical ionization (APCI) source were compared for the selective detection of microbial respiratory ubiquinone and menaquinone isoprenologues using tandem mass spectrometry. Ionization source- and compound mass-dependent parameters were optimized individually for both sources, using the available quinone standards. Detection levels for the two ion sources were determined with ubiquinone-6 (UQ6) and menaquinone-4 (MK4, vitamin K2) standards using flow injection analysis and selected reaction monitoring (SRM). With APPI the calculated lower limit of detection (LLOD) was 1.7 fmol microl(-1) for UQ6 and 2.2 fmol microl(-1) for MK4 at a signal-to-noise ratio of 3. These LLODs were at least three times lower than with APCI. The selectivity of detection afforded by SRM detection reduced complex mixture analysis to 3 min per sample by eliminating the need for chromatographic separations. The detection method was successfully applied to quinone quantification in a variety of environmental samples and cell cultures. Adequate amounts of respiratory quinones can be extracted and quantified from samples containing as low as 2 x 10(7) cells.

Flash detection/identification of pathogens, bacterial spores and bioterrorism agent biomarkers from clinical and environmental matrices.

We propose to develop an integrated rapid, semiportable, prototype point microbial detection/identification system for clinical specimens that is also capable of differentiating microbial bioterrorism attacks from threats or hoaxes by defining the pathogen. The system utilizes "flash" extraction/analytical system capable of detection/identification of microbes from environmental and clinical matrices. The system couples demonstrated technologies to provide quantitative analysis of lipid biomarkers of microbes including spores in a system with near-single cell (amol/microl) sensitivity. Tandem mass spectrometry increases specificity by providing the molecular structure of neutral lipids, phospholipids, and derivatized spore-specific bacterial biomarker, 2,6-dipicolinic acid (DPA) as well as the lipopolysaccharide-amide-linked hydroxy-fatty acids (LPS-ALHFA) of Gram-negative bacteria. The extraction should take about an hour for each sample but multiple samples can be processed simultaneously.

High-density PhyloChip profiling of stimulated aquifer microbial communities reveals a complex response to acetate amendment.

There is increasing interest in harnessing the functional capacities of indigenous microbial communities to transform and remediate a wide range of environmental contaminants. Information about which community members respond to stimulation can guide the interpretation and development of remediation approaches. To comprehensively determine community membership and abundance patterns among a suite of samples associated with uranium bioremediation experiments, we employed a high-density microarray (PhyloChip). Samples were unstimulated, naturally reducing, or collected during Fe(III) (early) and sulfate reduction (late biostimulation) from an acetate re-amended/amended aquifer in Rifle, Colorado, and from laboratory experiments using field-collected materials. Deep community sampling with PhyloChip identified hundreds-to-thousands of operational taxonomic units (OTUs) present during amendment, and revealed close similarity among highly enriched taxa from drill core and groundwater well-deployed column sediment. Overall, phylogenetic data suggested that stimulated community membership was most affected by a carryover effect between annual stimulation events. Nevertheless, OTUs within the Fe(III)- and sulfate-reducing lineages, Desulfuromonadales and Desulfobacterales, were repeatedly stimulated. Less consistent, co-enriched taxa represented additional lineages associated with Fe(III) and sulfate reduction (e.g. Desulfovibrionales; Syntrophobacterales; Peptococcaceae) and autotrophic sulfur oxidation (Sulfurovum; Campylobacterales). Data implies complex membership among highly stimulated taxa and, by inference, biogeochemical responses to acetate, a nonfermentable substrate.

Evaluation and application of anion exchange resins to measure groundwater uranium flux at a former uranium mill site.

Laboratory tests and a field validation experiment were performed to evaluate anion exchange resins for uranium sorption and desorption in order to develop a uranium passive flux meter (PFM). The mass of uranium sorbed to the resin and corresponding masses of alcohol tracers eluted over the duration of groundwater installation are then used to determine the groundwater and uranium contaminant fluxes. Laboratory based batch experiments were performed using Purolite A500, Dowex 21K and 21K XLT, Lewatit S6328 A resins and silver impregnated activated carbon to examine uranium sorption and extraction for each material. The Dowex resins had the highest uranium sorption, followed by Lewatit, Purolite and the activated carbon. Recoveries from all ion exchange resins were in the range of 94-99% for aqueous uranium in the environmentally relevant concentration range studied (0.01-200 ppb). Due to the lower price and well-characterized tracer capacity, Lewatit S6328 A was used for field-testing of PFMs at the DOE UMTRA site in Rifle, CO. The effect on the flux measurements of extractant (nitric acid)/resin ratio, and uranium loading were investigated. Higher cumulative uranium fluxes (as seen with concentrations>1 ug U/gram resin) yielded more homogeneous resin samples versus lower cumulative fluxes (<1 ug U/gram resin), which caused the PFM to have areas of localized concentration of uranium. Resin homogenization and larger volume extractions yield reproducible results for all levels of uranium fluxes. Although PFM design can be improved to measure flux and groundwater flow direction, the current methodology can be applied to uranium transport studies.

Postbiostimulation microbial community structure changes that control the reoxidation of uranium.

This study evaluated the influence of changes in the microbial community structure on reoxidation of reduced uranium during a postbiostimulation period. Effluent groundwater from acetate-stimulated sediment flow-through columns was analyzed over 60 days after acetate amendment was discontinued. Only a small reoxidation of iron or uranium (17%) occurred in the presence of 1-2 mg L(-1) O(2) influent groundwater for the 2-month period. Most uranium reoxidation occurred during the first 2 weeks after biostimulation with acetate was discontinued. Groundwater and sediment microbial community compositions suggested that two processes played important roles immediately after the cessation of acetate addition. The first process was characterized by a predominance of both sediment-bound and planktonic microorganisms most closely related to Hydrogenophaga sp., Thiobacillus sp., and Gallionella sp., which could oxidize a variety of reduced compounds. The second process was characterized by organisms closely related to Lysobacter sp. and Sterolibacterium sp., with the potential to feed on complex organic compounds from biomass turnover. The presence of these bacteria and the lack of uranium oxidation implied that after acetate addition was stopped, reduced inorganic compounds and dead biomass became electron donors for a microbial community capable of using low ambient oxygen as a terminal electron acceptor, contributing to the preservation, at least temporarily, of biogenic U(IV).

Monitoring microbial community structure and dynamics during in situ U(VI) bioremediation with a field-portable microarray analysis system.

The objective of this study was to develop and validate a simple, field-portable, microarray system for monitoring microbial community structure and dynamics in groundwater and subsurface environments, using samples representing site status before acetate injection, during Fe-reduction, in the transition from Fe- to SO(4)(2-)-reduction, and into the SO(4)(2-)-reduction phase. Limits of detection for the array are approximately 10(2)-10(3) cell equivalents of DNA per reaction. Sample-to-answer results for the field deployment were obtained in 4 h. Retrospective analysis of 50 samples showed the expected progression of microbial signatures from Fe- to SO(4)(2-) -reducers with changes in acetate amendment and in situ field conditions. The microarray response for Geobacter was highly correlated with qPCR for the same target gene (R(2) = 0.84). Microarray results were in concordance with quantitative PCR data, aqueous chemistry, site lithology, and the expected microbial community response, indicating that the field-portable microarray is an accurate indicator of microbial presence and response to in situ remediation of a uranium-contaminated site.

Treatment of nitric acid-, U(VI)-, and Tc(VII)-contaminated groundwater in intermediate-scale physical models of an in situ biobarrier.

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.