Promoting Uranium Immobilization by the Activities of Microbial PhosphatasesRobert J. Martinez1, Melanie J. Beazley2, Samuel M. Webb3, Martial Taillefert2 (co-PI) and Patricia A. Sobecky1 (PI)

1School of Biology, Georgia Institute of Technology, Atlanta Georgia2School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta Georgia

3Stanford Synchrotron Radiation Laboratory, Menlo Park, California

Abstract

The overall objective of this project is to examine the activity of nonspecific phosphohydrolases present innaturally occurring subsurface microorganisms for the purpose of promoting the immobilization ofradionuclides through the production of uranium [U(VI)] phosphate precipitates. Specifically, wehypothesize that the precipitation of U(VI) phosphate minerals may be promoted through the microbialrelease and/or accumulation of PO4

3- as a means to detoxify radionuclides and heavy metals. Anexperimental approach was designed to determine the extent of phosphatase activity in bacteria previouslyisolated from contaminated subsurface soils collected at the ERSP Field Research Center (FRC) in OakRidge, TN. Screening of 135 metal resistant isolates for phosphatase activity indicated the majority (75 of135) exhibited a phosphatase-positive phenotype. During this phase of the project, a PCR based approachhas also been designed to assay FRC isolates for the presence of one or more classes of the characterizednon-specific acid phophastase (NSAP) genes likely to be involved in promoting U(VI) precipitation.Testing of a subset of Pb resistant (Pbr) Arthrobacter, Bacillus and Rahnella strains indicated 4 of the 9 Pbrisolates exhibited phosphatase phenotypes suggestive of the ability to bioprecipitate U(VI). Two FRCstrains, a Rahnella sp. strain Y9602 and a Bacillus sp. strain Y9-2, were further characterized. TheRahnella sp. exhibited enhanced phosphatase activity relative to the Bacillus sp. Whole-cell enzyme assaysidentified a pH optimum of 5.5, and inorganic phosphate accumulated in pH 5.5 synthetic groundwater(designed to mimic FRC conditions) incubations of both strains in the presence of a modelorganophosphorus substrate provided as the sole C and P source. Kinetic experiments showed that thesetwo organisms can grow in the presence of 200 µM dissolved uranium and that Rahnella is much moreefficient in precipitating U(VI) than Bacillus sp. The precipitation of U(VI) must be mediated by biologicalactivity as less than 3% soluble U(VI) was removed either from the abiotic or the heat-killed cell controls.Interestingly, the pH has a strong effect on growth and U(VI) biomineralization rates by Rahnella.Thermodynamic modeling identifies autunite-type minerals [Ca(UO2)2(PO4)2] as the precipitate likelyformed in the synthetic FRC groundwater conditions at all pH investigated. Extended X-ray absorption finestructure measurements have recently confirmed that the precipitate found in these incubations is anautunite and meta-autunite-type mineral. A kinetic model of U biomineralization at the different pHindicates that hydrolysis of organophosphate can be described using simple Monod kinetics and thaturanium precipitation is accelerated when monohydrogen phosphate is the main orthophosphate species insolution. Overall, these experiments and ongoing soil slurry incubations demonstrate that thebiomineralization of U(VI) through the activity of phosphatase enzymes can be expressed in a wide range ofgeochemical conditions pertaining to the FRC site.

Acknowledgements

This research is supported by the Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG02-04ER63906. We thank Dave Watson (ORNL) for FRC samples, Dr. Adrian K. Arakaki (Georgia Tech) forphylogenetic computational analysis of NSAP genes, Hong Yi (Emory University) for electron microscopy imaging, Dr. Joan Hudson (Clemson University) for electron microscopy imaging and EDX analysis, DavidGiannantonio (Georgia Tech) and Alex Alverson (Georgia Tech) for technical assistance.

•U(VI)-phosphate speciation at equilibrium predicted byMINEQL+ as a function of pH in simulated groundwaterwith UO2

2+ = 200 µM, ΣPO43- = 200 µM , and PCO2 = 10-

3.5.

U(VI) – Phosphate Speciation as a Function of pH

Uranium Biomineralization at pH 5.5

Viable Cell Counts

U(VI) added

Phosphate Liberation

Rahnella sp.

Arthrobacter sp.

Bacillus sp.

Arthrobacter sp.

Rahnella sp. Bacillus sp. Rahnella sp.

Arthrobacter sp.

Bacillus sp.

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Tryptose MUP(visible light)

Tryptose MUP(UV light)

Lennox Broth(5mM Pb(NO3)2)

Microbial phosphatase screening and biomineralization

5

A. B. C.

Tryptose MUP (4-methylumbelliferyl phosphate ) agar plates and Tryptose Phosphate Methyl Green(TPMG) agar plates (not shown) were used to screen FRC isolates for phosphatase phenotypes (A andB). 4-methylumbelliferyl phosphate (MUP) fluorescence as well as methyl green precipitation (notshown) indicated phosphatase positive phenotypes (B). Lead (II) precipitation was also screened forisolates with and without phosphatase phenotype (C). Insoluble lead (II) phosphate (brown precipitate)may result from extracellular phosphatase activity and/or potentially from depolymerization ofcytoplasmic polyphosphate granules.

Conclusions

• Biomineralization may be complementary to bioreduction in the immobilization of uranium from contaminatedsites. The objective of this study was to determine if the phosphatase activity of subsurface microbes results inthe release of sufficient PO4

3- to complex and precipitate UO22+.

• For this study, the gram-negative Rahnella sp. Y9-602 and Bacillus sp. Y9-2 isolated from the FRC were studiedfor their phosphatase activity in the presence of U(VI).

• Kinetic studies were conducted in solutions containing the Rahnella sp. and Bacillus sp. isolates and the organo-phosphate compound G3P to determine if phosphatase activity would promote the precipitation of uranium.

•Both Rahnella sp. and Bacillus sp. hydrolyzed sufficient phosphate to precipitate ~95% and ~ 73%, respectively,of the initial uranium in biogenic incubations.

•Electron microscopy and EDX analysis reveal extracellular and cell associated U(VI) phosphate precipitates.

•Preliminary evidence of FRC soil phosphatase activity reveals the potential for stimulating sufficient phosphaterelease to allow for U(VI) phosphate mineral formation.

U(VI) Precipitation

(3). Subsurface geochemical parameters (pH, nitrate) will affect phosphatemineral formation by altering microbial phosphatase activity and/oraffecting the stability of the metal phosphate precipitates.

Hypotheses to be tested:

(1). Non-specific phosphophydrolases (acid phosphatases) provide subsurface microorganismswith resistance to heavy metals and lateral gene transfer has promoted the dissemination of thisphosphatase-mediated resistance.

(2). Phosphatase activities of the subsurface bacterial populations canpromote the immobilization of radionuclides via the formation ofinsoluble metal phosphate precipitates.

Image from http://public.ornl.gov/nabirfrc/photos_history.cfm Image from: http://www.inel.gov/initiatives/subsurface.shtml

B.A.

250 kDa150100

75

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MWM Y9602 Y9-2G.

Neighbor-joining analysis of acid phosphatase genes. Sequences were obtained by the aid of TASSER-Liteand the Enzyme Function Inference by Combined Approach (EFICAz) algorithm yielding Class A andClass B/Class C phylogenies, respectively (A) and (B). Alignments generated for each class of acidphosphatase proteins were used to generate phylum-specific and species-specific PCR primers. Type-strains were used to determine primer specificity for Class A (C), Class B (D), Class B (Kp) (E), and ClassC (F). Renatured whole cell lysates (100 µg/lane total protein) assayed for phosphatase activity in 100 mMNa acetate buffer at pH 5.5 with 5 mM phenolphthalein diphosphate substrate and 0.05 mg/ml methyl green(G).

1.00.90.80.7

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L - 1kb DNA ladderN - No DNA template control1 - FRC Y96022 - Enterobacter aerogenes (Class A control)3 - Klebsiella pneumoniae (Class A and B control)4 - Escherichia coli MG1655 (Class B control)5 - Salmonella enterica (Class B control)6 - Bacillus cereus (Class C control)7 - Bacillus thuringiensis (Class C control)8 - FRC Y9-2

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UO2(CO

3)

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UO2(OH)

2 (s)

Ca(UO2)

2(PO

4)

2 (s)

UO2SO

4 (aq)

UO2

2+ (aq)

% U

(VI)

pH

OH

O

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O

OH

HO

OH

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PO43-

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UO22+

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CF

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Time (h)

Rahnella sp. with U(VI) Rahnella sp. without U(VI) Bacillus sp. with U(VI) Bacillus sp. without U(VI) Arthrobacter sp. with U(VI) Arthrobacter sp. without U(VI)

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Cell-free(abiotic) control Rahnella sp. heat-kill control Rahnella sp. with U(VI) Rahnella sp. without U(VI) Bacillus sp. heat-kill control Bacillus sp. with U(VI) Bacillus sp. without U(VI) Arthrobacter sp. heat-kill control Arthrobacter sp. with U(VI) Arthrobacter sp. without U(VI)

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Cell-free(abiotic) control Rahnella sp. heat-kill control Rahnella sp. with U(VI) Rahnella sp. without U(VI) Bacillus sp. heat-kill control Bacillus sp. with U(VI) Bacillus sp. without U(VI) Arthrobacter sp. heat-kill control Arthrobacter sp. with U(VI) Arthrobacter sp. without U(VI)

0 12 24 36 48 60 72 84 96 108 120

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[U(V

I)]

- µM

Time (h)

Chemical Control Y9602Heat-kill Y9602U(VI) Y92Heatkill Y92U(VI) Arthrobacter Heatkill Arthrobacter wUVI

Electron Microscopy of FRC Rahnella sp. Exposed to U(VI)

•Electron microscopy of FRC Rahnella sp. revealedmembrane blebbing during incubation in simulatedgroundwater both prior to U(VI) addition and postU(VI) addition.

•Extracellular as well as cell associated electron-denseprecipitates were shown via EDX analysis (regionsmarked by arrows) to be composed of uranium andphosphorus.

•Imaging of cells 36 h post U(VI) addition revealed alow distribution of cell associated U(VI) precipitates.

Stimulation of FRC Microbial Community Phosphatase Activity

•Stimulation of FRC soils with 10 mM G3P revealsphosphatase activity in uncontaminated and Area 3(contaminated) soils.

•G3P hydrolysis in U(VI) contaminated soilsreveals lower phosphate accumulation relativeto uncontaminated soils.

•Future studies will examine the differences betweenmicrobial community structure and phosphatase activitybetween control and contaminated soils. In addition, theextent to which contaminated soils remove liberatedphosphate by adsorption and promote U(VI) precipitationwill be investigated.

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[PO43

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BckgdnoG3P BckgrdG3P Area3noG3P Area3G3P

Cells prior to U(VI) addition Extracellular U(VI) precipitation Cell associated U(VI) precipitation

400 nm667 nm

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Background soil without G3P

Area 3 soil with G3P

Background soil with G3PArea 3 soil without G3P