Uranium-contaminated Groundwater Research Articles

Characterization of uranium in the extracellular polymeric substances of anaerobic granular sludge used to treat uranium-contaminated groundwater

Characterization of the contents and forms (soluble ionic or particulate) of uranium reserved in AnGS's EPS.

New Ways to Neutralize Uranium in Contaminated Groundwater Uncovered

Researchers at Washington University have completed a series of experiments that expand the treatment possibilities for neutralizing uranium-contaminated groundwater and suggest long-term, stable solutions.

In situ mobility of uranium in the presence of nitrate following sulfate-reducing conditions

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

Biostimulation by Glycerol Phosphate to Precipitate Recalcitrant Uranium(IV) Phosphate.

Stimulating the microbial reduction of aqueous uranium(VI) to insoluble U(IV) via electron donor addition has been proposed as a strategy to remediate uranium-contaminated groundwater in situ. However, concerns have been raised regarding the longevity of microbially precipitated U(IV) in the subsurface, particularly given that it may become remobilized if the conditions change to become oxidizing. An alternative mechanism is to stimulate the precipitation of poorly soluble uranium phosphates via the addition of an organophosphate and promote the development of reducing conditions. Here, we selected a sediment sample from a U.K. nuclear site and stimulated the microbial community with glycerol phosphate under anaerobic conditions to assess whether uranium phosphate precipitation was a viable bioremediation strategy. Results showed that U(VI) was rapidly removed from solution and precipitated as a reduced crystalline U(IV) phosphate mineral similar to ningyoite. This mineral was considerably more recalcitrant to oxidative remobilization than the products of microbial U(VI) reduction. Bacteria closely related to Pelosinus species may have played a key role in uranium removal in these experiments. This work has implications for the stewardship of uranium-contaminated groundwater, with the formation of U(IV) phosphates potentially offering a more effective strategy for maintaining low concentrations of uranium in groundwater over long time periods.

Efficient removal of uranium from aqueous solution by zero-valent iron nanoparticle and its graphene composite

Zero-valent iron nanoparticle (ZVI-np) and its graphene composites were prepared and applied in the removal of uranium under anoxic conditions. It was found that solutions containing 24ppm U(VI) could be completely cleaned up by ZVI-nps, regardless of the presence of NaHCO3, humic acid, mimic groundwater constituents or the change of solution pH from 5 to 9, manifesting the promising potential of this reactive material in permeable reactive barrier (PRB) to remediate uranium-contaminated groundwater. In the measurement of maximum sorption capacity, removal efficiency of uranium kept at 100% until C0(U)=643ppm, and the saturation sorption of 8173mgU/g ZVI-nps was achieved at C0(U)=714ppm. In addition, reaction mechanisms were clarified based on the results of SEM, XRD, XANES, and chemical leaching in (NH4)2CO3 solution. Partially reductive precipitation of U(VI) as U3O7 was prevalent when sufficient iron was available; nevertheless, hydrolysis precipitation of U(VI) on surface would be predominant as iron got insufficient, characterized by releases of Fe2+ ions. The dissolution of Fe0 cores was assigned to be the driving force of continuous formation of U(VI) (hydr)oxide. The incorporation of graphene supporting matrix was found to facilitate faster removal rate and higher U(VI) reduction ratio, thus benefitting the long-term immobilization of uranium in geochemical environment.

Proteome of Geobacter sulfurreducens in the presence of U(VI).

Geobacter species often play an important role in the in situ bioremediation of uranium-contaminated groundwater, but little is known about how these microbes avoid uranium toxicity. To evaluate this further, the proteome of Geobacter sulfurreducens exposed to 100 µM U(VI) acetate was compared to control cells not exposed to U(VI). Of the 1363 proteins detected from these cultures, 203 proteins had higher abundance during exposure to U(VI) compared with the control cells and 148 proteins had lower abundance. U(VI)-exposed cultures expressed lower levels of proteins involved in growth, protein and amino acid biosynthesis, as well as key central metabolism enzymes as a result of the deleterious effect of U(VI) on the growth of G. sulfurreducens. In contrast, proteins involved in detoxification, such as several efflux pumps belonging to the RND (resistance-nodulation-cell division) family, and membrane protection, and other proteins, such as chaperones and proteins involved in secretion systems, were found in higher abundance in cells exposed to U(VI). Exposing G. sulfurreducens to U(VI) resulted in a higher abundance of many proteins associated with the oxidative stress response, such as superoxide dismutase and superoxide reductase. A strain in which the gene for superoxide dismutase was deleted grew more slowly than the WT strain in the presence of U(VI), but not in its absence. The results suggested that there is no specific mechanism for uranium detoxification. Rather, multiple general stress responses are induced, which presumably enable Geobacter species to tolerate high uranium concentrations.

Simultaneous adsorption and reduction of U(VI) on reduced graphene oxide-supported nanoscale zerovalent iron

The reduced graphene oxide-supported nanoscale zero-valent iron (nZVI/rGO) composites were synthesized by chemical deposition method and were characterized by SEM, high resolution TEM, Raman and potentiometric acid-base titrations. The characteristic results showed that the nZVI nanoparticles can be uniformly dispersed on the surface of rGO. The removal of U(VI) on nZVI/rGO composites as a function of contact time, pH and U(VI) initial concentration was investigated by batch technique. The removal kinetics of U(VI) on nZVI and nZVI/rGO were well simulated by a pseudo-first-order kinetic model and pseudo-second-order kinetic model, respectively. The presence of rGO on nZVI nanoparticles increased the reaction rate and removal capacity of U(VI) significantly, which was attributed to the chemisorbed OH− groups of rGO and the massive enrichment of Fe2+ on rGO surface by XPS analysis. The XRD analysis revealed that the presence of rGO retarded the transformation of iron corrosion products from magnetite/maghemite to lepidocrocite. According to the fitting of EXAFS spectra, the UC (at ∼2.9Å) and UFe (at ∼3.2Å) shells were observed, indicating the formation of inner-sphere surface complexes on nZVI/rGO composites. Therefore, the nZVI/rGO composites can be suitable as efficient materials for the in-situ remediation of uranium-contaminated groundwater in the environmental pollution management.

Model-based analysis of mixed uranium(VI) reduction by biotic and abiotic pathways during in situ bioremediation

Given the numerous unknowns and uncertainties in sediment systems, understanding of the mechanisms of U(VI) reduction is still at the stage of improvement. Recent studies have shown that reductive immobilization of U(VI) in the subsurface is not caused by purely biological or purely abiotic reactions but rather a group of interconnected abiotic–biotic pathways (e.g. via mackinawite and biomass). These new findings necessitate an update of the existing mathematical models that make simplifications typically involving a single reducing agent (e.g. indigenous bacteria) for in situ bioremediation of uranium-contaminated groundwater. In this study, a comprehensive model was constructed based on new experimental observations, including mixed U(VI) reduction by chemical and enzymatic reactions. Thermodynamic analysis was done to predict the feasibility of the potential pathways that affect mackinawite formation under field conditions. Model simulations indicate that low concentrations of the reactant species make the reaction of homogeneous Fe(II) oxidation coupled to U(VI) reduction unfavorable in the field. Instead, FeS precipitation is an important Fe(II) sequestration reaction once sulfate metabolism dominates. The subsequent reduction of U(VI) by FeS (mackinawite) contributes to the total U(VI) removal under a variety of U(VI) concentrations encountered at field sites, which is in accordance with experimental observations. The model suggests the potential for both competition and coordination between chemical and biological pathways on the cell surface, providing a possible explanation as to why U(VI) can be efficiently reduced at either low or high sulfate concentration during the process of in situ bioremediation. Further increase in the resolution of the model (e.g. across multiple scales such as genome-, micron- and pore-sale) is necessary for better understanding of the interactions between biotic and abiotic pathways.

Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater.

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100% of the sequences recovered during this phase of the bioremediation. These results suggest a prey-predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.

Remediation of Uranium-contaminated Groundwater by Sorption onto Hydroxyapatite Derived from Catfish Bones

Hydroxyapatite (HA) was prepared from catfish bones, identified as catfish HA (CFHA), using mechanical and chemical treatment methods. CFHA was characterized by x-ray diffraction (XRD) and scanning electron microscope (SEM) techniques to confirm the presence of HA. The ability of CFHA to remove uranium (U(VI)) from aqueous phase was investigated using both batch and column experiments. Adsorption experiments in batch experiments were carried by varying pH, preparation temperature, and particle size. The data shows that the maximum adsorption occurred between pH 5.5 and 7. The adsorption of U(VI) on CFHA was greater at 300 °C than at 100 °C. Batch data shows that the smallest particles, with maximum surface area, exhibited significant U(VI) removal efficiency. Column experiments were conducted using the smallest CFHA particles at different flow rates and breakthrough profiles were obtained. The scalability of the U(VI) removal process was tested by comparing the performances of columns packed with different CFHA. The results indicated that the reaction scales to the mass concentration of the reactants (CFHA and U(VI)). We also found that at pH 7, the CFHA packed in the column has the potential to remove about 3.9 mg of U(VI) per gram. Our study shows that CFHA may be used in permeable reactive barriers for remediating U(VI)-contaminated groundwater plumes.

Linking bacterial diversity and geochemistry of uranium-contaminated groundwater

To understand the link between bacterial diversity and geochemistry in uranium-contaminated groundwater, microbial communities were assessed based on clone libraries of 16S rDNA genes from the USDOE Oak Ridge Field Research Centre (FRC) site. Four groundwater wells (GW835, GW836, FW113-47 and FW215-49) with a wide range of pH (3 to 7), nitrate (44 to 23,400 mg L−1), uranium (0.73 to 60.36 mg L−1) and other metal contamination, were investigated. Results indicated that bacterial diversity correlated with the geochemistry of the groundwater. Microbial diversity decreased in relation to the contamination levels of the wells. The highly contaminated well (FW113-47) had lower gene diversity than less contaminated wells (FW215-49, GW835 and GW836). The high concentrations of contaminants present in well FW113-47 stimulated the growth of organisms capable of reducing uranium (Shewanella and Pseudomonas), nitrate (Pseudomonas, Rhodanobacter and Xanthomonas) and iron (Stenotrophomonas), and which were unique to this well. The clone libraries consisted primarily of sequences closely related to the phylum Proteobacteria, with FW-113-47 almost exclusively containing this phylum. Metal-reducing bacteria were present in all four wells, which may suggest that there is potential for successful bioremediation of the groundwater at the Oak Ridge FRC. The microbial community information gained from this study and previous studies at the site can be used to develop predictive multivariate and geographical information system (GIS) based models for microbial populations at the Oak Ridge FRC. This will allow for a better understanding of what organisms are likely to occur where and when, based on geochemistry, and how these organisms relate to bioremediation processes at the site.

Cooperation of Three Denitrifying Bacteria in Nitrate Removal of Acidic Nitrate- and Uranium-Contaminated Groundwater

In uranium-contaminated aquifers co-contaminated with nitrate, denitrifiers play a critical role in bioremediation. Six strains of denitrifying bacteria belonging to Rhizobium, Pseudomonas, and Castellaniella were isolated from the Oak Ridge Integrated Field Research Challenge Site (OR-IFRC), where biostimulation of acidic (pH 3.5–6.5), nitrate-contaminated (up to 140 mM) groundwater occurred. Three isolates were characterized in regards to nitrite tolerance, denitrification kinetic parameters, and growth on different denitrification intermediates. Kinetic and growth experiments showed that Pseudomonas str. GN33#1 reduced NO− 3 most rapidly (Vmax = 15.8 μmol e−·min−1·mg protein−1) and had the fastest generation time (gt) on NO− 3 (2.6 h). Castellaniella str. 4.5A2 was the most low pH and NO− 2 tolerant and grew rapidly on NO− 2 (gt = 4.0 h). Rhizobium str. GN32#2 was also tolerant of low pH values and reduced NO− 2 rapidly (Vmax = 10.6 μmol e−·min−1·mg protein−1) but was far less NO− 2 tolerant than Castellaniella str. 4.5A2. Growth of and denitrification by these three strains incubated together and individually were measured in OR-IFRC groundwater at pHs 5 and 7 to determine whether they cooperate or compete during denitrification. Mixed assemblages reduced NO− 3 more rapidly and more completely than any individual isolate over the course of the experiment. The results described in this article demonstrate 1) that this synthetic assemblage comprised of three physiologically distinct denitrifying bacterial isolates cooperate to achieve more complete levels of denitrification and 2) the importance of pH- and nitrite-tolerant bacteria such as Castellaniella str. 4.5A2 in minimizing NO− 2 accumulation in high-NO− 3 groundwater during bioremediation. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.

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.

Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism

The in situ stimulation of Fe(III) oxide reduction by Geobacter bacteria leads to the concomitant precipitation of hexavalent uranium [U(VI)] from groundwater. Despite its promise for the bioremediation of uranium contaminants, the biological mechanism behind this reaction remains elusive. Because Fe(III) oxide reduction requires the expression of Geobacter's conductive pili, we evaluated their contribution to uranium reduction in Geobacter sulfurreducens grown under pili-inducing or noninducing conditions. A pilin-deficient mutant and a genetically complemented strain with reduced outer membrane c-cytochrome content were used as controls. Pili expression significantly enhanced the rate and extent of uranium immobilization per cell and prevented periplasmic mineralization. As a result, pili expression also preserved the vital respiratory activities of the cell envelope and the cell's viability. Uranium preferentially precipitated along the pili and, to a lesser extent, on outer membrane redox-active foci. In contrast, the pilus-defective strains had different degrees of periplasmic mineralization matching well with their outer membrane c-cytochrome content. X-ray absorption spectroscopy analyses demonstrated the extracellular reduction of U(VI) by the pili to mononuclear tetravalent uranium U(IV) complexed by carbon-containing ligands, consistent with a biological reduction. In contrast, the U(IV) in the pilin-deficient mutant cells also required an additional phosphorous ligand, in agreement with the predominantly periplasmic mineralization of uranium observed in this strain. These findings demonstrate a previously unrecognized role for Geobacter conductive pili in the extracellular reduction of uranium, and highlight its essential function as a catalytic and protective cellular mechanism that is of interest for the bioremediation of uranium-contaminated groundwater.

Molecular Analysis of the Metabolic Rates of Discrete Subsurface Populations of Sulfate Reducers

Elucidating the in situ metabolic activity of phylogenetically diverse populations of sulfate-reducing microorganisms that populate anoxic sedimentary environments is key to understanding subsurface ecology. Previous pure culture studies have demonstrated that the transcript abundance of dissimilatory (bi)sulfite reductase genes is correlated with the sulfate-reducing activity of individual cells. To evaluate whether expression of these genes was diagnostic for subsurface communities, dissimilatory (bi)sulfite reductase gene transcript abundance in phylogenetically distinct sulfate-reducing populations was quantified during a field experiment in which acetate was added to uranium-contaminated groundwater. Analysis of dsrAB sequences prior to the addition of acetate indicated that Desulfobacteraceae, Desulfobulbaceae, and Syntrophaceae-related sulfate reducers were the most abundant. Quantifying dsrB transcripts of the individual populations suggested that Desulfobacteraceae initially had higher dsrB transcripts per cell than Desulfobulbaceae or Syntrophaceae populations and that the activity of Desulfobacteraceae increased further when the metabolism of dissimilatory metal reducers competing for the added acetate declined. In contrast, dsrB transcript abundance in Desulfobulbaceae and Syntrophaceae remained relatively constant, suggesting a lack of stimulation by added acetate. The indication of higher sulfate-reducing activity in the Desulfobacteraceae was consistent with the finding that Desulfobacteraceae became the predominant component of the sulfate-reducing community. Discontinuing acetate additions resulted in a decline in dsrB transcript abundance in the Desulfobacteraceae. These results suggest that monitoring transcripts of dissimilatory (bi)sulfite reductase genes in distinct populations of sulfate reducers can provide insight into the relative rates of metabolism of different components of the sulfate-reducing community and their ability to respond to environmental perturbations.

Monitoring the Metabolic Status of Geobacter Species in Contaminated Groundwater by Quantifying Key Metabolic Proteins with Geobacter-Specific Antibodies

Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of subsurface microorganisms are required before bioremediation practitioners will adopt molecular diagnosis of the bioremediation community as a routine practice for guiding the development of bioremediation strategies. Quantifying gene transcripts can diagnose important aspects of microbial physiology during bioremediation but is technically challenging and does not account for the impact of translational modifications on protein abundance. An alternative strategy is to directly quantify the abundance of key proteins that might be diagnostic of physiological state. To evaluate this strategy, an antibody-based quantification approach was developed to investigate subsurface Geobacter communities. The abundance of citrate synthase corresponded with rates of metabolism of Geobacter bemidjiensis in chemostat cultures. During in situ bioremediation of uranium-contaminated groundwater the quantity of Geobacter citrate synthase increased with the addition of acetate to the groundwater and decreased when acetate amendments stopped. The abundance of the nitrogen-fixation protein, NifD, increased as ammonium became less available in the groundwater and then declined when ammonium concentrations increased. In a petroleum-contaminated aquifer, the abundance of BamB, an enzyme subunit involved in the anaerobic degradation of mono-aromatic compounds by Geobacter species, increased in zones in which Geobacter were expected to play an important role in aromatic hydrocarbon degradation. These results suggest that antibody-based detection of key metabolic proteins, which should be readily adaptable to standardized kits, may be a feasible method for diagnosing the metabolic state of microbial communities responsible for bioremediation, aiding in the rational design of bioremediation strategies.

EVALUATION OF THE EFFICACY OF POLYPHOSPHATE REMEDIATION TECHNOLOGY: DIRECT AND INDIRECT REMEDIATION OF URANIUM UNDER ALKALINE CONDITIONS

A field-scale technology demonstration has been conducted to optimize polyphosphate remediation technology for enhanced monitored natural attenuation of the uranium plume within the 300 Area aquifer at the Hanford Site, southeastern Washington State. The objective was to evaluate the efficacy of polyphosphate to treat uranium-contaminated groundwater in situ. Focused application of polyphosphate was conducted in a source or 'hot spot' area to reduce the inventory of available uranium contributing to the groundwater plume through direct precipitation of uranyl-phosphate solids and secondary containment via precipitation of apatite which can serve as a long-term sorbent for uranium. The test site consisted of an injection well and 15 monitoring wells installed in the 300 Area near the process trenches that had previously received uranium-bearing effluents. The results indicated sequestration of uranium as insoluble phosphate phases appears to be a promising alternative for treating the uranium- contaminated groundwater at the Hanford Site 300 Area. However, the formation of the apatite during the test was limited due to two separate overarching issues: (1) formation and emplacement of apatite via polyphosphate technology, and (2) efficacy of apatite for sequestering uranium under the present geochemical and hydrodynamic conditions.

Acetate Availability and its Influence on Sustainable Bioremediation of Uranium-Contaminated Groundwater

Field biostimulation experiments at the U.S. Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, Colorado, have demonstrated that uranium concentrations in groundwater can be decreased to levels below the U.S. Environmental Protection Agency's (EPA) drinking water standard (0.126 μM). During successive summer experiments – referred to as “Winchester” (2007) and “Big Rusty” (2008) - acetate was added to the aquifer to stimulate the activity of indigenous dissimilatory metal-reducing bacteria capable of reductively immobilizing uranium. The two experiments differed in the length of injection (31 vs. 110 days), the maximum concentration of acetate (5 vs. 30 mM), and the extent to which iron reduction (“Winchester”) or sulfate reduction (“Big Rusty”) was the predominant metabolic process. In both cases, rapid removal of U(VI) from groundwater occurred at calcium concentrations (6 mM) and carbonate alkalinities (8 meq/L) where Ca-UO2-CO3 ternary complexes constitute >90% of uranyl species in groundwater. Complete consumption of acetate and increased alkalinity (>30 meq/L) accompanying the onset of sulfate reduction corresponded to temporary increases in U(VI); however, by increasing acetate concentrations in excess of available sulfate (10 mM), low U(VI) concentrations (0.1–0.05 μM) were achieved for extended periods of time (>140 days). Uniform delivery of acetate during “Big Rusty” was impeded due to decreases in injection well permeability, likely resulting from biomass accumulation and carbonate and sulfide mineral precipitation. Such decreases were not observed during the short-duration “Winchester” experiment. Terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes demonstrated that Geobacter sp. and Geobacter-like strains dominated the groundwater community profile during iron reduction, with 13C stable isotope probing (SIP) results confirming these strains were actively utilizing acetate to replicate their genome during the period of optimal U(VI) removal. Gene transcript levels during “Big Rusty” were quantified for Geobacter-specific citrate synthase (gltA), with ongoing transcription during sulfate reduction indicating that members of the Geobacteraceae were still active and likely contributing to U(VI) removal. The persistence of reducible Fe(III) in sediments recovered from an area of prolonged (110-day) sulfate reduction is consistent with this conclusion. These results indicate that acetate availability and its ability to sustain the activity of iron- and uranyl-respiring Geobacter strains during sulfate reduction exerts a primary control on optimized U(VI) removal from groundwater at the Rifle IFRC site over extended time scales (>50 days).

Biological reduction of uranium—From the laboratory to the field

The chemical and biological processes underlying in situ bioremediation of uranium-contaminated groundwater have been studied in the laboratory and in the field. This article focuses on the long-term stability of uraninite (UO 2) in the underground. A large tailings pond, ‘Dänkritz 1’ in Germany, was selected for this investigation. A single-pass flow-through experiment was run in a 100-liter column: bioremediation for 1 year followed by infiltration of tap water (2.5 years) saturated with oxygen, sufficient to oxidize the precipitated uraninite in two months. Instead, only 1 wt.% uraninite was released over 2.4 years at concentrations typically less than 20 μg/L. Uraninite was protected against oxidation by the mineral mackinawite (FeS 0.9), a considerable amount of which had formed, together with uraninite. A confined field test was conducted adjacent to the tailings pond, which after bio-stimulation showed similarly encouraging results as in the laboratory. Taking Dänkritz 1 as an example we show that in situ bioremediation can be a viable option for long-term site remediation, if the process is designed based on sufficient laboratory and field data. The boundary conditions for the site in Germany are discussed.

Genome-scale dynamic modeling of the competition between Rhodoferax and Geobacter in anoxic subsurface environments

The advent of rapid complete genome sequencing, and the potential to capture this information in genome-scale metabolic models, provide the possibility of comprehensively modeling microbial community interactions. For example, Rhodoferax and Geobacter species are acetate-oxidizing Fe(III)-reducers that compete in anoxic subsurface environments and this competition may have an influence on the in situ bioremediation of uranium-contaminated groundwater. Therefore, genome-scale models of Geobacter sulfurreducens and Rhodoferax ferrireducens were used to evaluate how Geobacter and Rhodoferax species might compete under diverse conditions found in a uranium-contaminated aquifer in Rifle, CO. The model predicted that at the low rates of acetate flux expected under natural conditions at the site, Rhodoferax will outcompete Geobacter as long as sufficient ammonium is available. The model also predicted that when high concentrations of acetate are added during in situ bioremediation, Geobacter species would predominate, consistent with field-scale observations. This can be attributed to the higher expected growth yields of Rhodoferax and the ability of Geobacter to fix nitrogen. The modeling predicted relative proportions of Geobacter and Rhodoferax in geochemically distinct zones of the Rifle site that were comparable to those that were previously documented with molecular techniques. The model also predicted that under nitrogen fixation, higher carbon and electron fluxes would be diverted toward respiration rather than biomass formation in Geobacter, providing a potential explanation for enhanced in situ U(VI) reduction in low-ammonium zones. These results show that genome-scale modeling can be a useful tool for predicting microbial interactions in subsurface environments and shows promise for designing bioremediation strategies.