Phase Preference by Active, Acetate-Utilizing Bacteria at the Rifle, CO Integrated Field Research Challenge Site

Previous experiments at the Rifle, Colorado Integrated Field Research Challenge (IFRC) site demonstrated that field-scale addition of acetate to groundwater reduced the ambient soluble uranium concentration. In this report, sediment samples collected before and after acetate field addition were used to assess the active microbes via 13C acetate stable isotope probing on 3 phases [coarse sand, fines (8-approximately 150 μm), groundwater (0.2−8 μm)] over a 24-day time frame. TRFLP results generally indicated a stronger signal in 13C-DNA in the “fines” fraction compared to the sand and groundwater. Before the field-scale acetate addition, a Geobacter-like group primarily synthesized 13C-DNA in the groundwater phase, an alpha Proteobacterium primarily grew on the fines/sands, and an Acinetobacter sp. and Decholoromonas-like OTU utilized much of the 13C acetate in both groundwater and particle-associated phases. At the termination of the field-scale acetate addition, the Geobacter-like species was active on the solid phases rather than the groundwater, while the other bacterial groups had very reduced newly synthesized DNA signal. These findings will help to delineate the acetate utilization patterns of bacteria in the field and can lead to improved methods for stimulating distinct microbial populations in situ

Phylogenetic tree of active TRF’s resulting from the uranium additions (star) are shown with the nearest cultured relatives.

<p>The reconstruction was done using maximum likelihood methods on 438 aligned bases. Bootstrap values >50 for 100 iterations are indicated.</p

Ribosomal response to uranium additions in groundwater after 24h for 4 OTUs (TRF-212, 213, 214, 215 bp) from wells D02 (open square), D07 (closed square), sampled in 2008, D01 (open circle), D08 (closed circle), sampled in 2009 (note the difference in scales).

<p>Ribosomal response to uranium additions in groundwater after 24h for 4 OTUs (TRF-212, 213, 214, 215 bp) from wells D02 (open square), D07 (closed square), sampled in 2008, D01 (open circle), D08 (closed circle), sampled in 2009 (note the difference in scales).</p

Field Application of ²³⁸U/²³⁵U Measurements To Detect Reoxidation and Mobilization of U(IV)

Biostimulation to induce reduction of soluble U­(VI) to relatively immobile U­(IV) is an effective strategy for decreasing aqueous U­(VI) concentrations in contaminated groundwater systems. If oxidation of U­(IV) occurs following the biostimulation phase, U­(VI) concentrations increase, challenging the long-term effectiveness of this technique. However, detecting U­(IV) oxidation through dissolved U concentrations alone can prove difficult in locations with few groundwater wells to track the addition of U to a mass of groundwater. We propose the ²³⁸U/²³⁵U ratio of aqueous U as an independent, reliable tracer of U­(IV) remobilization via oxidation or mobilization of colloids. Reduction of U­(VI) produces ²³⁸U-enriched U­(IV), whereas remobilization of solid U­(IV) should not induce isotopic fractionation. The incorporation of remobilized U­(IV) with a high ²³⁸U/²³⁵U ratio into the aqueous U­(VI) pool produces an increase in ²³⁸U/²³⁵U of aqueous U­(VI). During several injections of nitrate to induce U­(IV) oxidation, ²³⁸U/²³⁵U consistently increased, suggesting ²³⁸U/²³⁵U is broadly applicable for detecting mobilization of U­(IV)

Percent total peak area from community DNA of 16S rRNA genes TRFLP profiles of pre-filters 2008 well D04 during field acetate injection: TRF 212-white, 213-light grey, 215-black, other <i>Geobacter</i>-like TRF’s-dark grey stipple, all other TRF peaks-light checked.

<p>Percent total peak area from community DNA of 16S rRNA genes TRFLP profiles of pre-filters 2008 well D04 during field acetate injection: TRF 212-white, 213-light grey, 215-black, other <i>Geobacter</i>-like TRF’s-dark grey stipple, all other TRF peaks-light checked.</p

Identification of Bacteria Synthesizing Ribosomal RNA in Response to Uranium Addition During Biostimulation at the Rifle, CO Integrated Field Research Site

<div><p>Understanding which organisms are capable of reducing uranium at historically contaminated sites provides crucial information needed to evaluate treatment options and outcomes. One approach is determination of the bacteria which directly respond to uranium addition. In this study, uranium amendments were made to groundwater samples from a site of ongoing biostimulation with acetate. The active microbes in the planktonic phase were deduced by monitoring ribosomes production via RT-PCR. The results indicated several microorganisms were synthesizing ribosomes in proportion with uranium amendment up to 2 μM. Concentrations of U (VI) >2 μM were generally found to inhibit ribosome synthesis. Two active bacteria responding to uranium addition in the field were close relatives of <i>Desulfobacter postgateii</i> and <i>Geobacter bemidjiensis</i>. Since RNA content often increases with growth rate, our findings suggest it is possible to rapidly elucidate active bacteria responding to the addition of uranium in field samples and provides a more targeted approach to stimulate specific populations to enhance radionuclide reduction in contaminated sites.</p></div

Electrode-Based Approach for Monitoring In Situ Microbial Activity During Subsurface Bioremediation

Current production by microorganisms colonizing subsurface electrodes and its relationship to substrate availability and microbial activity was evaluated in an aquifer undergoing bioremediation. Borehole graphite anodes were installed downgradient from a region of acetate injection designed to stimulate bioreduction of U(VI); cathodes consisted of graphite electrodes embedded at the ground surface. Significant increases in current density (≤50 mA/m2) tracked delivery of acetate to the electrodes, dropping rapidly when acetate inputs were discontinued. An upgradient control electrode not exposed to acetate produced low, steady currents (≤0.2 mA/m2). Elevated current was strongly correlated with uranium removal but minimal correlation existed with elevated Fe(II). Confocal laser scanning microscopy of electrodes revealed firmly attached biofilms, and analysis of 16S rRNA gene sequences indicated the electrode surfaces were dominated (67−80%) by Geobacter species. This is the first demonstration that electrodes can produce readily detectable currents despite long-range (6 m) separation of anode and cathode, and these results suggest that oxidation of acetate coupled to electron transfer to electrodes by Geobacter species was the primary source of current. Thus it is expected that current production may serve as an effective proxy for monitoring in situ microbial activity in a variety of subsurface anoxic environments

Sulfur Isotopes as Indicators of Amended Bacterial Sulfate Reduction Processes Influencing Field Scale Uranium Bioremediation

Aqueous uranium (U(VI)) concentrations in a contaminated aquifer in Rifle Colorado have been successfully lowered through electron donor amended bioreduction. Samples collected during the acetate amendment experiment were analyzed for aqueous concentrations of Fe(II), sulfate, sulfide, acetate, U(VI), and δ34S of sulfate and sulfide to explore the utility of sulfur isotopes as indicators of in situ acetate amended sulfate and uranium bioreduction processes. Enrichment of up to 7‰ in δ34S of sulfate in down-gradient monitoring wells indicates a transition to elevated bacterial sulfate reduction. A depletion in Fe(II), sulfate, and sulfide concentrations at the height of sulfate reduction, along with an increase in the δ34S of sulfide to levels approaching the δ34S values of sulfate, indicates sulfate limited conditions concurrent with a rebound in U(VI) concentrations. Upon cessation of acetate amendment, sulfate and sulfide concentrations increased, while δ34S values of sulfide returned to less than −20‰ and sulfate δ34S decreased to near-background values, indicating lower levels of sulfate reduction accompanied by a corresponding drop in U(VI). Results indicate a transition between electron donor and sulfate-limited conditions at the height of sulfate reduction and suggest stability of biogenic FeS precipitates following the end of acetate amendment

Chemical analysis of groundwater from well D04 during the 2008 field experiment-acetate (closed triangles) and bromide (open squares).

<p>The period of groundwater flush (shaded) and the times of biomass sampling (dotted line) are indicated. The chemical methods used in this analysis exhibit analytical variability in the femto-, nano-, and micro-molar range for uranium, iron/sulfide, and acetate respectively. All field measurements are beyond this range.</p

No Measurable Changes in ²³⁸U/²³⁵U due to Desorption–Adsorption of U(VI) from Groundwater at the Rifle, Colorado, Integrated Field Research Challenge Site

Groundwater samples were collected from the Integrated Field Research Challenge field site in Rifle, Colorado, over the course of a bicarbonate-induced U desorption–adsorption experiment. Uranium concentrations and high precision U isotopic compositions (²³⁸U/²³⁵U) of these groundwater samples were determined and used to assess the impact of bicarbonate-induced U­(VI) desorption from contaminated sediments on the ²³⁸U/²³⁵U of groundwater. The ²³⁸U/²³⁵U of groundwater was not significantly impacted by bicarbonate-induced desorption of U­(VI) from mineral surfaces or by adsorption of advecting U­(VI) from upgradient locations onto those surfaces after the treatment. Assuming this absence of a significant shift in U isotopic composition associated with desorption–adsorption applies to other systems, reduction of U­(VI) to U­(IV) is expected to be the dominant source of U isotopic fractionation associated with removal of U­(VI) from pore water as a result of natural and stimulated reductive pathways. Thus, changes in the ²³⁸U/²³⁵U composition of uranium-bearing fluids should be useful in quantifying the extent of reduction