The role of anaerobic respiration in the immobilization 1 of uranium through biomineralization of phosphate minerals

Although bioreduction of uranyl ions (U(VI)) and biomineralization of U(VI)-phosphate minerals are both able to immobilize uranium in contaminated sediments, the competition between these processes and the role of anaerobic respiration in the biomineralization of U(VI)-phosphate minerals has yet to be investigated. In this study, contaminated sediments incubated anaerobically in static microcosms at pH 5.5 and 7.0 were amended with the organophosphate glycerol-2-phosphate (G2P) as sole phosphorus and external carbon source and iron oxides, sulfate, or nitrate as terminal electron acceptors to determine the most favorable geochemical conditions to these two processes. While sulfate reduction was not observed even in the presence of G2P at both pHs, iron reduction was more significant at circumneutral pH irrespective of the addition of G2P. In turn, nitrate reduction was stimulated by G2P at both pH 5.5 and 7.0, suggesting nitrate-reducing bacteria provided the main source of inorganic phosphate in these sediments. U(VI) was rapidly removed from solution in all treatments but was not reduced as determined by X-ray absorption near edge structure (XANES) spectroscopy. Simultaneously, wet chemical extractions and extended X-ray absorption fine structure (EXAFS) spectroscopy of these sediments indicated the presence of U-P species in reactors amended with G2P at both pHs. The rapid removal of dissolved U(VI), the simultaneous production of inorganic phosphate, and the existence of U-P species in the solid phase indicate that uranium was precipitated as U(VI)-phosphate minerals in sediments amended with G2P. Thus, under reducing conditions and in the presence of G2P, bioreduction of U(VI) was outcompeted by the biomineralization of U(VI)-phosphate minerals and U(VI) sorption at both pHs

Microbial Community Responses to Organophosphate Substrate Additions in Contaminated Subsurface Sediments

<div><p>Background</p><p>Radionuclide- and heavy metal-contaminated subsurface sediments remain a legacy of Cold War nuclear weapons research and recent nuclear power plant failures. Within such contaminated sediments, remediation activities are necessary to mitigate groundwater contamination. A promising approach makes use of extant microbial communities capable of hydrolyzing organophosphate substrates to promote mineralization of soluble contaminants within deep subsurface environments.</p><p>Methodology/Principal Findings</p><p>Uranium-contaminated sediments from the U.S. Department of Energy Oak Ridge Field Research Center (ORFRC) Area 2 site were used in slurry experiments to identify microbial communities involved in hydrolysis of 10 mM organophosphate amendments [i.e., glycerol-2-phosphate (G2P) or glycerol-3-phosphate (G3P)] in synthetic groundwater at pH 5.5 and pH 6.8. Following 36 day (G2P) and 20 day (G3P) amended treatments, maximum phosphate (PO₄³⁻) concentrations of 4.8 mM and 8.9 mM were measured, respectively. Use of the PhyloChip 16S rRNA microarray identified 2,120 archaeal and bacterial taxa representing 46 phyla, 66 classes, 110 orders, and 186 families among all treatments. Measures of archaeal and bacterial richness were lowest under G2P (pH 5.5) treatments and greatest with G3P (pH 6.8) treatments. Members of the phyla <i>Crenarchaeota</i>, <i>Euryarchaeota</i>, <i>Bacteroidetes</i>, and <i>Proteobacteria</i> demonstrated the greatest enrichment in response to organophosphate amendments and the OTUs that increased in relative abundance by 2-fold or greater accounted for 9%–50% and 3%–17% of total detected <i>Archaea</i> and <i>Bacteria</i>, respectively.</p><p>Conclusions/Significance</p><p>This work provided a characterization of the distinct ORFRC subsurface microbial communities that contributed to increased concentrations of extracellular phosphate via hydrolysis of organophosphate substrate amendments. Within subsurface environments that are not ideal for reductive precipitation of uranium, strategies that harness microbial phosphate metabolism to promote uranium phosphate precipitation could offer an alternative approach for <i>in situ</i> sequestration.</p></div

Class-level distribution of enriched OTUs.

<p>Compiled OTUs enriched (i.e., 2-fold or greater increase in relative abundance) in replicate treatments representing the most responsive (A) archaeal and (B) bacterial classes from treatments amended with organophosphates at pH 5.5 and 6.8.</p

Dynamic archaeal and bacterial OTUs within sediment slurry treatments.

<p>Total detected archaeal (left column) and bacterial (right column) OTUs compiled from replicate treatments that significantly increased or decreased relative to incubations lacking organophosphate. Treatment conditions and total number of taxa plotted: (A) G2P (pH 5.5), (B) G3P (pH 5.5), (C) G2P (pH 6.8), and (D) G3P (pH 6.8). OTUs with a 2-fold or greater decrease in fluorescence were not detected.</p

DNA extractions from ORFRC subsurface sediments.

<p>DNA concentrations pre- and post-incubations (pH 5.5 and pH 6.8) amended with G2P, G3P, and without organophosphate. Error bars indicate standard deviation of replicate treatments (n = 3).</p

Archaeal and bacterial community structure.

<p>(A) Phylum-level richness of <i>Archaea</i> detected in sediments prior to treatments. (B) Archaeal richness detected in sediments pre- and post-treatments. (C) NMDS ordination of archaeal community distances present within replicate samples. (D) Phylum-level richness of <i>Bacteria</i> detected in sediments prior to treatments. (E) Bacterial richness detected in sediments pre- and post-treatments. (F) NMDS ordination of bacterial community distances present within replicate samples. Designations for all samples are as follows: <b>P</b>- sediments prior to treatment, <b>1</b>-pH 5.5 without organophosphate amendment, <b>2</b>-pH 5.5 amended with G2P, <b>3</b>-pH 5.5 amended with G3P, <b>4</b>-pH 6.8 without organophosphate amendment, <b>5</b>-pH 6.8 amended with G2P, and <b>6</b>-pH 6.8 amended with G3P. Phylum-level richness of <i>Archaea</i> and <i>Bacteria</i> prior to treatments represents the summation of total richness detected from replicate sediment DNA extractions.</p

Thermodynamic modeling of U(VI) in the presence of phosphate as a function of pH.

<p>ORFRC Area 2 groundwater concentrations of dissolved ions (GW-836 monitoring well), U(VI) = 4.5 µM, Ca²⁺ = 4.85 mM, (A) PO₄³⁻ = 500 µM and (B) PO₄³⁻ = 5 mM were used to model the distribution of U(VI) species. Dashed lines represent soluble species and solid lines represent insoluble species.</p

Study site.

<p>(A) Map of Point Aux Pins peninsula. Study site indicated by box in inset. (B) Sediment samples collected ∼25 m from shore outside the marsh stands (designated “Inlet”) and 2–4 m from shore within the marsh stands (designated “Marsh”).</p

Hydrocarbon degradation genes in Marsh sediments.

<p>Average abundances of hydrocarbon degradation genes in Marsh sediments compared between June and July. Significant changes indicated by *for p<0.05 and **for p<0.01.</p

GCMS analysis of MC252 oil and oil-contaminated sediment.

<p>(A) GCMS chromatogram of 10 ppm Macondo oil (MC252); (B) concentrations of <i>n</i>-alkanes in tar balls and sediment from July Marsh samples as determined by GCMS.</p