Promoting uranium immobilization by the activities of microbial phophatases Page: 2 of 3
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for the FRC sites. Preliminary testing indicates that the several of the FRC isolates have an enhanced
tolerance to uranyl acetate relative to control strains.
II. Research on the second objective of our project, the determination of phosphatase activities in
promoting U immobilized, has progressed during this report period as follows. During this reporting period
we have conducted a series of kinetic studies in solutions containing the Y9 isolate and an
organophosphorus compound to determine if the phosphatase activity of Y9 would enhance the
precipitation of uranium. For these incubations two organophosphorus compounds with different chemical
structures were selected to determine how efficiently Y9 can hydrolyze these compounds. Phytic acid
(IP6) is a six-member carbon ring with six attached orthophosphate groups which can provide up to
twelve coordinate binding sites. Glycerol-3-phosphate (G3P) is a more labile compound with only one
orthophosphate attached to a three carbon chain. At neutral pH both molecules are negatively charged
providing reactive sites for cationic binding. At high concentrations of U022+ (300 microM) the solubility
of U(VI) as a function of (a) G3P and (b) IP6 after 24 hour abiotic equilibration was determined. Solubility
of U(VI) in G3P decreases sharply between 100 microM and 500 microM G3P and then increases at high
concentrations. U(VI) is highly insoluble at low concentrations of IP6 and precipitates completely. At
higher concentrations of IP6 the solubility of U(VI) is enhanced, possibly due to increased repulsive
negative charges around the uranyl ion. Preliminary results of the kinetic studies indicate that biotic
precipitation of uranium may be enhanced in the presence of G3P and phosphatase activity. U(VI)
analysis was determined by laser-induced fluorescence (LIF) with a pyrophosphate/hypophosphite
reagent. We have also determined U(VI)-phosphate speciation at equilibrium, predicted by MINEQL+, as
a function of phosphate concentration in solution and pH. Specifically, at low pH and low phosphate
concentration, uranium is mainly in the form of the uranyl ion (U022+). As the concentration of phosphate
approaches that of total uranium, highly insoluble uranium phosphate compounds are formed. In excess
phosphate, speciation calculations predict dissolution of this mineral and formation of soluble uranium-
phosphate complexes. At higher pH, uranium tends to be insoluble at low concentrations of phosphate
and soluble, as uranium phosphate complexes, at high phosphate concentrations. The solid phase
preferentially formed at pH > 7 is a uranium hydroxide mineral. These calculations were performed in the
absence of carbonate species to illustrate the effect of phosphate on uranium speciation. The addition of
carbonate does not change the behavior of either uranium or phosphate.
III. The third objective of our project is the determination of geochemical parameters affecting uranium
phosphate immobilization. For this objective we plan to focus on conditions relevant to the ORNL FRC
site. As the FRC sites contain high concentrations of nitrate and a low pH, we will investigate how tolerant
subsurface microorganisms are to nitrate and how pH changes may affect the biomineralization process.
Prior to selection of sites for requesting groundwater and soil samples from the FRC we will consult with
Dave Watson (Manager; ORNL FRC) for suitable locations (and avoiding sites that have undergone
recent biostimulation events). For these experiments, sediments and groundwater from nearby wells will
be collected from the FRC sites where characterized bacterial strains were previously isolated.
Specifically, we plan to: 1) characterize the chemical composition of the porewaters and sediments; 2)
establish that strains isolated from the FRC sites can immobilize U as uranium phosphate precipitate in
natural conditions with sediment slurries of the FRC sites; 3) determine the kinetics of precipitation of
uranium phosphate in sediments from the FRC sites with different nitrate and pH conditions with or
without the addition of U-precipitating FRC strain(s) in the presence of the natural subsurface populations.
DELIVERABLES: Publications and presentations:
Martinez, R.J. and Sobecky, P.A. Subsurface Uranium Immobilization Enhanced by Microbial
Phosphatases. Manuscript in preparation.
Sobecky, P., R. Martinez, M. J. Beazley, and M. Taillefert. Promoting Uranium Immobilization by the
Activities of Microbial Phosphatases. Poster presentation. Annual NABIR PI meeting, Warrenton, VA
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Sobecky, Patricia A. Promoting uranium immobilization by the activities of microbial phophatases, report, June 1, 2005; Atlanta, Georgia. (digital.library.unt.edu/ark:/67531/metadc884387/m1/2/: accessed September 19, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.