U(VI) Sequestration in Calcium-Phosphate Minerals P ... · 6 National Laboratory. Area 2 groundwater contains high concentrations of dissol ved calcium (ca. 7 4 mM), and thus release

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U(VI) Sequestration in Calcium-Phosphate Minerals Produced by Microbial 1

Glycerol 3-Phosphate Metabolism 2

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U(VI) CAPTURE IN BIOGENIC Ca-P MINERALS 4

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Evgenya S. Shelobolina, Hiromi Konishi, Huifang Xu, and Eric E. Roden* 6

Department of Geology and Geophysics 7

1215 W. Dayton St. 8

University of Wisconsin 9

Madison, WI 53706 10

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*Corresponding Author: 21

Phone: 608-890-0724; fax: 608-262-0693; email: [email protected] 22

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00628-09 AEM Accepts, published online ahead of print on 24 July 2009

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ABSTRACT 1

Previous studies have demonstrated the potential for removal of U(VI) from solution via 2

precipitation of U(VI)-bearing calcium-phosphate (Ca-P) minerals coupled to microbial 3

hydrolysis of glycerol phosphate compounds. We evaluated this process in circumneutral pH 4

groundwater from Area 2 of the U.S. Department of Energy Field Research Center at Oak Ridge 5

National Laboratory. Area 2 groundwater contains high concentrations of dissolved calcium (ca. 6

4 mM), and thus release of phosphate during glycerol phosphate metabolism has the potential to 7

create conditions favorable for U(VI) sequestration in Ca-P minerals. Microbial enumeration 8

and isolation studies verified the presence of aerobic and nitrate-reducing glycerol 3-phosphate 9

(G3P)-metabolizing microorganisms in Area 2 sediments. Coprecipitation of U(VI) with Ca-P 10

minerals coupled to microbial G3P hydrolysis was demonstrated in artificial groundwater under 11

aerobic and nitrate-reducing conditions. TEM analysis and mineral washing experiments 12

demonstrated that U(VI) was incorporated into the structure of the insoluble Ca-P mineral 13

hydroxyapatite (Ca5(PO4)3OH). Our results support the idea that U(VI) can be effectively 14

removed from solution in contaminated aquifers through stimulation of microbial 15

organophosphate metabolism. 16

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INTRODUCTION 1

Reductive precipitation of U(VI) through stimulation of anaerobic, dissimilatory metal-2

reducing microbial activity is a demonstrated method for in situ immobilization of uranium in 3

subsurface environments [3, 41]. This is an attractive approach for aquifers that are naturally 4

low in dissolved O2 and NO3-, e.g. the uranium contaminated aquifer adjacent to the Colorado 5

River in Rifle, CO [3]. Alternative uranium remediation approaches may be called for in 6

uranium contaminated subsurface environments that contain significant concentrations of O2 and 7

NO3- entering through vertical recharge or lateral transport. One such potential strategy is 8

immobilization of uranium through precipitation of U(VI)-bearing calcium-phosphate (Ca-P) 9

phases [6, 39]. Uranium incorporation into Ca-P minerals is a naturally occurring process. For 10

example, U(VI)-phosphate phases at a weathered portion of the Coles Hill, VA uranium ore 11

deposit maintain groundwater U(VI) concentrations at ca. 0.06 µM [11, 12]. The shallow portion 12

of this deposit exhibits a sharp Fe redox front with U(IV) assemblages (containing uraninite 13

UO2) located on the reduced side, and U(VI) assemblages (containing autunite, 14

(Ca(UO2)2(PO4)3, and other uranium-bearing Ca-P phases), on the weathered oxidized side. 15

Interestingly, uranium concentrations on both oxidized and reduced sides of the redox front are 16

similar, suggesting no significant uranium loss as the result of oxidation and weathering. 17

A major limitation for engineering uranium immobilization through coprecipitation with 18

Ca-P minerals is the potential decrease in hydraulic conductivity of sediments due to rapid 19

mineral precipitation at or near the point of phosphate (hereafter abbreviated as Pi) injection. In 20

order to avoid this, Pi should be delivered into the subsurface at a relatively slow rate. A potential 21

amendment for in situ uranium remediation allowing slow Pi liberation is glycerol phosphate. 22

Release of Pi from glycerol phosphate is promoted by microbial activity. 23


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Glycerol phosphate can facilitate microbial growth in two ways. First, it can serve as a P 1

source for phosphatase-positive heterotrophic microorganisms. Phosphatases are enzymes that 2

mediate hydrolysis of G3P and other phosphate-containing compounds, resulting in a release of 3

Pi for use in cell biosynthesis [33]. Second, glycerol phosphate can serve as a combined source 4

of phosphorus, carbon, and energy for growth by microorganisms. Such organisms may either 5

carry-out an initial extracellular glycerol phosphate hydrolysis step followed by glycerol and Pi 6

uptake and utilization [33], or take up glycerol phosphate directly without hydrolysis in exchange 7

for Pi [5, 16, 17]. Phosphatase positive organisms are often isolated from metal-contaminated 8

sediments [13, 21, 25, 27, 31]. One possible reason for this is that phosphatase activity decreases 9

metal toxicity to the cells by causing precipitation of metal-phosphate phases [13, 14, 24, 27, 28, 10

31, 35]. 11

Previous studies have demonstrated the efficacy of glycerol phosphate-hydrolyzing 12

microorganisms in removal of uranium and other radionuclides from solution through formation 13

of metal-phosphate mineral phases. Macaskie and co-authors [19, 20, 22-24] isolated a 14

Citrobacter species from metal contaminated soil that hydrolyzed glycerol 3-phosphate 15

(hereafter abbreviated as G3P) and accumulated heavy metals (as insoluble metal phosphates) on 16

the cell wall. This finding served as a basis for proposed ex situ processes to remove uranium and 17

other metals from contaminant waste streams. Sobecky and colleagues [6, 25] determined the 18

phosphatase activity of 135 strains isolated from acidic sediments at the U.S. Department of 19

Energy Oak Ridge National Laboratory Field Research Center (ORFRC), and demonstrated that 20

activity of these bacteria can promote uranium immobilization through formation of 21

autunite/meta-autunite group minerals. 22


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The purpose of this study was to evaluate the potential for in situ immobilization of 1

U(VI) in Area 2 subsurface sediments at the ORFRC. The circumneutral pH groundwater at Area 2

2 contains high concentrations of dissolved calcium (ca. 4 mM). Hence release of Pi during 3

microbial G3P metabolism has the potential to create conditions favorable for Ca-P mineral 4

precipitation and U(VI) sequestration. The key questions examined were the abundance of G3P-5

metabolizing microorganisms in Area 2 sediments, and the nature and stability of the uranium-6

bearing Ca-P precipitates formed during microbial G3P metabolism. G3P-metabolizing 7

organisms, capable of utilizing G3P as combined phosphorus, carbon and energy source, are 8

likely to be preferentially stimulated in organic matter-poor subsurface sediments such as those 9

at the ORFRC. G3P was therefore provided as the sole carbon and energy source in all our 10

experiments. 11

MATERIALS AND METHODS 12

Sediment Sampling. Sediment was obtained from Area 2 at the ORFRC (see 13

http://www.esd.ornl.gov/orifrc). The Area 2 site is a shallow pathway for migration of 14

contaminated groundwater to seeps in the upper reach of Bear Creek at ORNL. Samples for 15

enumeration and isolation of G3P-metabolizing microorganisms were obtained from the zone of 16

maximum U(VI) contamination (ca. 18 ft depth) in a sediment core collected with a pneumatic 17

hammering device (GeoProbe

). Detailed description of the stratigraphy and 18

sediment/groundwater characteristics of Area 2 are available elsewhere [30]. 19

Media and solutions. Standard PIPES-buffered culture medium contained (mM): PIPES (10.0), 20

NH4Cl (5.0), NaH2PO4⋅⋅⋅⋅H2O (0.5), KCl (1.3), and vitamin/trace element solutions [18] (10 mL 21

per L each). Pi was added to the medium in order to facilitate recovery of microorganisms with 22

Pi-irrepressible phosphatases that would remain active in the presence of Pi, some of which 23


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would be expected to be released during in situ organophosphate metabolism. For aerobic 1

cultivations, 9 mL PIPES medium was dispensed into 16 × 150 mm Kimble culture tubes capped 2

with KimKap caps (Kimble Glass & Plastic Labware LLC) and autoclaved. For cultivation of 3

nitrate-reducing organisms, 9 mL of PIPES medium containing 5-10 mM sodium nitrate was 4

dispensed into 27-mL anaerobic pressure tubes (Bellco Glass Inc) and bubbled with N2. The 5

tubes were capped with butyl rubber stoppers and sterilized by autoclaving. 6

The potential for co-precipitation of U(VI) with Ca-P minerals was examined in a PIPES-7

buffered artificial groundwater medium designed to mimic the groundwater in well 835 at Area 2 8

(PBAGW835). PBAGW835 contained (mM): CaCO3 (2.75), CaSO4⋅2H2O (0.9), 9

Ca(NO3)2⋅4H2O (0.25); K2SO4 (0.1), MgCO3 (1.1), Na2CO3 (6.0), and H2PIPES (10.0). The 10

final pH was 6.8. For anaerobic incubations the medium was bubbled with N2. In all cases U(VI) 11

was added in the form of uranyl acetate. 12

Glycerol 3-phosphate (DL-alpha-glycerol phosphate) was provided to aerobic and nitrate-13

reducing media to enumerate and isolate microorganisms that use G3P as a combined 14

phosphorous, carbon, and energy source. G3P was added from a 100 mM filter-sterilized stock 15

solution after the medium was autoclaved. 16

Enumeration and isolation of G3P-metabolizing microorganisms. G3P-metabolizing bacteria 17

were enumerated by the Most Probable Number (MPN) technique [40] using standard PIPES-18

buffered culture medium amended with 10 mM G3P. Dilutions from 10−1

to 10−10

were 19

inoculated into triplicate tubes. A direct isolation approach was applied to recover aerobic G3P-20

metabolizing microorganisms. Aerobic plates solidified with 1.5% BBL agar (Becton Dickinson, 21

Cockeysville, MD) were inoculated at the same time as MPN series. The inoculated tubes and 22

plates were incubated at room temperature (22 °C) for 2 weeks. The positive tubes were 23


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determined by measuring optical density at 600 nm. Strains M1, N51, and O25 were recovered 1

from individual colonies in the last positive dilution (10-3

) plates. 2

Ca-P-U precipitate washing experiments. Ca-P-U coprecipitates were recovered by 3

centrifugation at 10,000 × g for 10 min. The precipitates were washed (in triplicate) with an 4

equal volume of fresh PBAGW835 at different intervals over a 28 hr period. The supernatant 5

from each washing was analyzed for U(VI) as described below. 6

16S rRNA gene sequencing and phylogenetic analysis of isolates. The16S rRNA gene 7

sequences of strains M1, N51, and O25 were obtained using standard methodologies as 8

previously described [34]. The 16S rRNA gene was amplified using the bacterial forward primer 9

8F (AGAGTTTGATCMTGGCTCAG) and the universal reverse primer 1525R 10

(AAGGAGGTFWTCCARCC). These primers and the conserved internal primers 338F 11

(ACTCCTACGGGAGGCAGC), 519F (CAGCACGCCGCGGTAATFWC), 519R 12

(GWATTACCGCGGCKGCTG), 907R (CCGTCAATTCMTTTRAGTTT), and 1392R 13

(ACGGGCGGTGTGTRC) were used to obtain the nearly complete sequence. The 16S rRNA 14

gene fragments were compared to the Genbank nucleotide database using BLASTN and 15

BLASTX algorithms [2]. 16

Analytical techniques. Filtered samples were passed through a 0.2 µm syringe filter prior to 17

acidification with 1% HNO3. G3P, Pi, nitrate and nitrite concentrations were measured with 18

Dionex DX-100 ion chromatography (Dionex Corp., Sunnyvale, CA) with a Dionex AS4-SC 19

IonPac column. Ca and P were quantified by ICP-OES. U(VI) was quantified via Kinetic 20

Phosphorescence Analysis (KPA) (Chemchek Corp., LaBrea, CA). Cells were counted with 21

acridine orange staining and epifluorescence microscopy as previously described [10]. 22


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Transmission electron microscopy (TEM). Ca-P-U coprecipitates were recovered by 1

centrifugation at 10,000 × g for 10 min. The minerals were washed 7 times with deionized 2

water. Samples were collected on copper grids (mesh size, 200 µM) covered with a carbon-3

coated Formvar film and examined with a transmission electron microscope Philips CM 200UT 4

(Spherical aberration coefficient = 0.5mm; Point to point resolution = 0.19 nm) equipped with 5

NORAN Voyager X-ray energy-dispersive spectroscopy (EDS) at the Materials Science Center 6

of the University of Wisconsin - Madison. The acceleration voltage was 200 kV. 7

RESULTS 8

Enumeration and isolation of G3P-metabolizing microorganisms. MPN analyses showed that 9

both aerobic and nitrate-reducing G3P-metabolizing organisms are present in Area 2 sediments. 10

The sediment appeared to contain greater numbers of nitrate-reducing G3P-metabolizing bacteria 11

(3.4 × 104 ± 1.3 × 10

4 cells g

-1) than aerobic G3P-metabolizing bacteria (2.3 × 10

3 ± 0.9 × 10

3 12

cells g-1

). Several pure cultures were obtained from the highest positive aerobic dilutions 13

including strains M1, N51 and O25. Partial sequencing (ca. 1400 bp fragment) of the 16S rRNA 14

gene from these organisms indicated that all three isolates belong to the Gammaproteobacteria. 15

Strain M1 was 99% similar to Aeromonas hydrophila, strain N51 was 99% similar to Pantoea 16

agglomerans, and strain O25 was 99% similar to Pseudomonas rhodesiae. 17

U(VI) immobilization during microbial G3P metabolism and Ca-P mineral formation. 18

When grown aerobically on a standard PIPES-buffered culture medium, all three pure cultures 19

(strains M1, N51 and O25) utilized G3P and released Pi at a ratio close to one (data not shown). 20

In contrast, when Ca-rich PBAGW835 medium was used, the ratio of Pi released to G3P utilized 21

declined to ca. 0.75, indicating precipitation of Ca-P minerals. G3P hydrolysis triggered parallel 22


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Ca-P mineral formation (indicated visually) and U(VI) immobilization (Fig. 1A-C). No changes 1

in Pi or uranium concentration were detected in abiotic control incubations with G3P (Fig. 1D). 2

Aeromonas hydrophila strain M1 was used for detailed studies of Ca-P mineral 3

precipitation and U(VI) immobilization. The experiments were carried out under nitrate-reducing 4

conditions at both relatively high G3P and U(VI) concentrations (Fig. 2) and at more field-5

relevant lower concentrations (Fig. 3). In cultures containing 8 mM G3P and 100 µM U(VI), 6

virtually all the dissolved U(VI) (final U(VI) ca. 1 µM) was removed from solution during G3P 7

metabolism (Fig. 2A,D). Precipitation of Ca-P mineral was verified by the loss of Ca from 8

solution (Fig. 2C). A smaller but significant degree of Ca and U(VI) loss was observed in 9

cultures containing ca. 0.8 mM G3P and 10 µM U(VI) (Fig. 3C, D). In all cases, nitrate was 10

reduced to nitrite. No change in G3P, Ca, Pi, or U(VI) concentration took place in abiotic 11

controls. 12

In order to estimate how much U(VI) was incorporated (e.g. by adsorption) into 13

microbial biomass versus co-precipitation with Ca-P minerals during G3P metabolism, glycerol 14

(8 or 0.8 mM with 100 or 10 µM U(VI), respectively) was used as a carbon and energy source 15

instead of G3P. Aerobic oxidation of 8 mM glycerol resulted in generation of 4.5 × 107 cells 16

mL-1

and in the loss of ca. 16 µM U(VI) from solution (Fig. 2F). Only ca. 0.7 × 107 cells mL

-1 17

were generated in the 0.8 mM glycerol cultures, to which only a minor fraction (< 15%) of the 10 18

µM U(VI) became associated (Fig. 3E, F). 19

TEM analysis of Ca-P-U precipitates. TEM analysis of Ca-P-U precipitates produced by strain 20

M1 showed that U(VI) was preferentially incorporated into aggregates of nanocrystals of 21

hydroxyapatite Ca5(PO4)3OH (hereafter abbreviated as HA) (Fig. 4A). Selected area electron 22

diffraction (SAED) showed no diffraction peaks from either uraninite or autunite, but diffuse 23


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diffraction from HA (Fig. 4B). X-ray energy-dispersive spectra (EDS) clearly showed U, Ca, P, 1

and O peaks (Fig. 4C). It is important to note that the mineral phases were washed extensively 2

prior to examination in order to eliminate surface-bound U(VI). Thus, the uranium detected by 3

EDS was present as part of the mineral structure. 4

Stability of Ca-P-U precipitates formed during microbial G3P metabolism. U(VI)-bearing 5

precipitates formed during metabolism of 8 mM G3P/100 µM U(VI) by strain M1 were collected 6

and washed repeatedly with PBAGW835. Five consecutive washing cycles resulted in 7

solubilization of ca. 16 % of the U(VI) content of the precipitate; after that no measurable U(VI) 8

was released (Table 1). 9

DISCUSSION 10

Pure culture isolates. Strains Aeromonas hydrophila M1 (family Aeromonadaceae), Pantoea 11

agglomerans N51 (family Enterobacteriaceae) and Pseudomonas rhodesiae O25 (family 12

Pseudomoinadaceae) were isolated from the highest positive aerobic G3P-metabolizing MPN 13

dilutions. All three strains belong to Gammaproteobacteria and were capable of metabolizing 14

G3P and removing U(VI) from solution under aerobic and nitrate-reducing conditions. 15

Gammaproteobacteria have been identified in multiple clone libraries from Area 2 ORFRC 16

sediments [1, 29]. Akob et al. [1] also found that the predominant Gammaproteobacteria under 17

nitrate-reducing conditions were members of Aeromonadaceae, Enterobacteriaceae, and 18

Pseudomonadaceae families. Therefore our three isolates provide reasonable pure culture 19

models for studying potential in situ G3P metabolism in Area 2 ORFRC sediments. 20

Nature and stability of U(VI)-bearing precipitates. Strain M1 incubations with either 8 mM 21

glycerol/100 µM U(VI) or 0.8 mM glycerol/10 µM U(VI) were used to assess how much U(VI) 22

immobilization could be attributed to adsorption to cell biomass as opposed to Ca-P mineral 23


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formation. Only a minor fraction (≤ 16 %) of the U(VI) became associated with biomass in both 1

cultures (Figs. 2F, 3F). This result indicates that most of the U(VI) lost from solution during 2

G3P metabolism (Figs. 1-3) was sequestered in Ca-P minerals. The amount of U(VI) taken up 3

during growth on glycerol was similar to the amount released during repeated washing of 4

precipitates from a 8 mM G3P culture (Table 1), which suggests (but does not prove) that cell 5

lysis might have been responsible for the small amount of U(VI) solubilized during washing of 6

the precipitate. To our knowledge this is first study to report on the stability of a U-bearing Ca-P 7

mineral produced via microbial organophosphate metabolism. 8

Speciation calculations were conducted with the geochemical modeling software 9

PHREEQC [32], using published solubility products for HA [26] and autunite [15], optimized 10

stability constants (log K values) for aqueous U(VI)-carbonate complexes [9], and provisional 11

stability constants for Ca-U(VI)-carbonate complexes (CaUO2(CO3)32-

and Ca2UO2(CO3)3) [7]. 12

The calculations indicated that virtually all of U(VI) exists in artificial groundwater PBAGW835 13

medium as U(VI)-carbonate and Ca-U(VI)-carbonate complexes. The speciation calculations 14

also demonstrated that the Pi enriched artificial groundwater became highly oversaturated with 15

respect to Ca-P mineral phases such as brushite (CaHPO4⋅2H2O) and HA. In our experiments 16

HA was the main mineral phase produced during microbial G3P metabolism. This is in contrast 17

to formation of specific Ca-P-U mineral phases such as autunite or meta-autunite that have been 18

documented in other recent studies of P-based uranium sequestration [6, 38]. This difference 19

may be related to the higher abundance of dissolved Ca in the PBAGW835 (ca. 4 mM) compared 20

the medium used in these studies (0.2-0.5 mM), and to the lower solubility of HA compared to 21

autunite (see below). In addition, speciation calculations showed that the pH dependence of HA 22

vs. autunite solubility is such that autunite would be expected to form at the pH value of 5.5 23


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employed by Beazley et al. [6], whereas HA precipitation is favored at circumneutral pH. 1

Precipitation of HA could remove U(VI) from solution directly via coprecipitation, or by 2

formation of surfaces that have a high affinity for U(VI) sorption [4, 8]. These two mechanisms 3

are likely to act in concert, since sorption of U(VI) to HA surface would be expected to lead to 4

incorporation of uranium into growing mineral crystallites by the phenomenon of surface 5

enrichment and entrapment [37]. 6

Implications for in situ uranium bioremediation. Measurements of groundwater geochemistry 7

in Area 2 sediments [30] indicate that this is a circumneutral environment in which aerobic 8

metabolism and nitrate reduction are the dominant microbial respiratory pathways. We therefore 9

tested whether U(VI) could be effectively removed from Ca-containing groundwater at Area 2 10

through co-precipitation with Ca-P minerals under aerobic or nitrate-reducing conditions. G3P 11

was chosen as a P amendment based on the idea that microbial activity could provide slow but 12

sustained release of Pi for in situ bioremediation. Enumeration and isolation studies verified the 13

presence of substantial numbers of aerobic and nitrate-reducing G3P-metabolizing 14

microorganisms in uranium-contaminated Area 2 sediments. Mineral washing experiments 15

together with TEM analysis demonstrated that uranium was associated with insoluble HA in 16

microbially-mediated Ca-P-U coprecipitation experiments. Our findings confirm that U(VI) can 17

be sequestered by Ca-P minerals in aqueous solutions analogous to natural Ca-bearing 18

groundwater, and thus support the concept of Ca-P based field-scale strategies for 19

immobilization of U(VI) [6, 39] in subsurface environments where maintenance of long-term 20

anaerobic conditions is difficult or impossible. 21

Coprecipitation of U(VI) with HA observed in our experiments may be advantageous for 22

in situ U(VI) immobilization. HA is inherently less soluble than autunite at circumneutral pH: 23


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equilibrium speciation calculations made using PHREEQC (see Tables S1 and S2) indicated that 1

the extent of dissolution of an initial 10 mmol L-1

of autunite, in P- and U-free solution 2

approximating the composition of Area 2 groundwater, would be ca. 100-fold greater than 3

dissolution of 10 mmol L-1

of HA. Thus, uranium sequestered into HA should be more stable 4

than uranium contained in autunite. In addition, rates of U(VI) release after cessation of 5

bioremediation would be controlled by the slow dissolution of a large pool of relatively insoluble 6

HA, as opposed to more rapid dissolution of a much smaller pool of more soluble autunite or 7

meta-autunite, the size of which would be dictated by the initial abundance of U(VI) in the 8

groundwater. Although many other factors could influence Ca-P mineral dissolution kinetics 9

(e.g. mineral particle size, surface area, the presence of defects and impurities [36]), these simple 10

considerations suggest that U(VI) incorporation into HA via coprecipitation is likely to be more 11

favorable for long-term U immobilization compared to formation of discrete Ca-P-U phases such 12

as autunite. Controlled studies of the partitioning and long-term fate of U(VI) in Ca-P mineral 13

precipitation systems that approximate those present in situ at Area 2 (and other potential Ca-P 14

based uranium bioremediation sites) are required to evaluate this assertion. 15

ACKNOWLEDGMENTS 16

This work was supported by grants DE-FG02-06ER64184 and ER64172-1027487-001191 from 17

the Environmental Remediation Science Program, Office of Biological and Environmental 18

Research, U.S. Department of Energy. 19


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Table 1. Release of U(VI) during repeated washing of solids from aerobic 8 mM G3P/100 µM 1

U(VI) M1 cultures with PBAGW835. Data represent the mean ± SD of triplicate samples. 2

Washing step

U(VI) released

(µM)

U(VI) released

(% total)

#1 after 2 hours of equilibration 2.3 ± 0.7 2.5





#6 after 2 hours of equilibration 0 0

#7 after 2 hours of equilibration 0 0

Remaining U(VI) recovered by 2M HNO3 77.2 ± 0.8 83.9

Total 92.0 100

3


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FIG 1. G3P metabolism with concomitant U(VI) precipitation in aerobic PBAGW835 medium. 1

(A) Aeromonas hydrophila strain M1; (B) Pantoea agglomerans strain N51; (C) Pseudomonas 2

rhodesiae strain O25; (D) abiotic control. Data points show the mean ± SD of triplicate cultures. 3

Symbols: () G3P, (�) Pi, (�) U(VI). 4

5

FIG. 2. Aeromonas hydrophila strain M1 metabolism in nitrate-reducing PBAGW835 medium 6

containing 100 µM U(VI) and either 8 mM G3P (A-D) or 8 mM glycerol (E, F). Data points 7

show the mean ± SD of triplicate cultures. Solid and dashed lines show results of inoculated and 8

uninoculated systems, respectively. Symbols: () G3P, (�) Pi, (�) nitrate, (�) nitrite, (�) Ca, 9

(�) U(VI), (º) cell numbers. 10

11

FIG. 3. Aeromonas hydrophila strain M1 metabolism in nitrate-reducing PBAGW835 medium 12

containing 100 µM U(VI) and either 0.5 mM G3P (A-D) or 0.5 mM glycerol (E, F). Data points 13

show the mean ± SD of triplicate cultures. Solid and dashed lines show results of inoculated and 14

uninoculated systems, respectively. Symbols as in Fig. 2. 15

16

FIG. 4. (A) Low-magnification TEM image showing aggregates of U(VI)-bearing HA 17

nanocrystals. (B) Selected area electron diffraction pattern showing diffuse peaks consistent 18

with HA; no diffraction peaks from either uraninite or autunite were observed. (C) EDS 19

spectrum of HA nanocrystals showing peaks for uranium. 20


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FIG. 11

Hours

0 20 40 60 80

0

25

50

75

100

125

150

175

G3P

, P

i, m

M

0

1

2

3

4

5

6

7

8

9

U(V

I), µ

M

Hours

0 20 40 60 80

0

25

50

75

100

125

150

175

G3P

, P

i, m

M

0

1

2

3

4

5

6

7

8

9

U(V

I), µ

M

Hours

0 20 40 60 80

0

25

50

75

100

125

150

175

G3P

, P

i, m

M

0

1

2

3

4

5

6

7

8

U(V

I), µ

M

Hours

0 20 40 60 80

0

25

50

75

100

125

150

175

G3P

, P

i, m

M

0

1

2

3

4

5

6

7

8

9

U(V

I), µ

M

A

DC

B


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FIG. 2 1

G3P

, P

i, m

M

0

2

4

6

8

nitra

te, nitrite

, m

M

0

2

4

6

8

10

12

0

20

40

60

80

100

120

Ca

2+,

mM

3.0

3.5

4.0

4.5

5.0

U(V

I), µ

M

Hours

0 10 20 30 40 50

0

20

40

60

80

100

120

Hours

0 10 20 30 40 50

10

7cells

ml-1

0

1

2

3

4

5

6

nitra

te, m

M

4

5

6

7

8

BA

FE

DC

U(V

I), µ

M


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FIG. 3 1 G

3P

, P

i, m

M

0.0

0.2

0.4

0.6

0.8

nitra

te, nitrite

, m

M

0

2

4

6

8

0

2

4

6

8

10

12

14

Ca

2+,

mM

2.5

3.0

3.5

4.0

U(V

I), µ

M

Hours

0 10 20 30 40 500

2

4

6

8

10

12

14

Hours

0 10 20 30 40 50

10

7cells

ml-1

0.0

0.5

1.0

1.5

2.0

nitra

te, m

M

1

2

3

FEU

(VI)

, µ

M

DC

BA


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FIG. 4 1

2

O

P

Ca

Ca

Cu

U

U

U U

Cu

Cu U

U UU

0

1000

2000

3000

Co

unts

0 5 10 15 20

Energy (keV)

C


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Documents

U(VI) Sequestration in Calcium-Phosphate Minerals P ... · 6 National Laboratory. Area 2 groundwater contains high concentrations of dissol ved calcium (ca. 7 4 mM), and thus release