We recently discovered a neutral dicalcium uranyl tricarbonate complex, Ca₂UO₂(CO₃)₃(aq.), in uranium mining related waters [1]. We are now reporting a further validation of the stoichiometry and the formation constant of this complex using two analytical approaches with time-resolved laser-induced fluorescence spectroscopy (TRLFS) species detection: i) titration of a non-fluorescent uranyl tricarbonate complex solution with calcium ions, and quantitative determination of the produced fluorescent calcium complex via TRLFS; and ii) variation of the calcium concentration in the complex by competitive calcium complexation with EDTA^4-.Slope analysis of the log (fluorescence intensity)versuslog[Ca²⁺] with both methods have shown that two calcium ions are bound to form the complex Ca₂UO₂(CO₃)₃(aq.). The formation constants determined from the two independent methods are: i) logβ°₂₁₃=30.45±0.35 and ii) logβ°₂₁₃=30.77±0.25.A bathochrome shift of 0.35 nm between the UO₂(CO₃)₃^4-complex and the Ca₂UO₂(CO₃)₃(aq.) complex is observed in the laser-induced photoacoustic spectrum (LIPAS), giving additional evidence for the formation of the calcium uranyl carbonate complex.EXAFS spectra at the L_IIand L_III-edges of uranium in uranyl carbonate solutions with and without calcium do not differ significantly. A somewhat better fit to the EXAFS of the Ca₂UO₂(CO₃)₃(aq.) complex is obtained by including the U-Ca shell. From the similarities between the EXAFS of the Ca₂UO₂(CO₃)₃(aq.) species in solution and the natural mineral liebigite, we conclude that the calcium atoms are likely to be in the same positions both in the solution complex and in the solid.This complex influences considerably the speciation of uranium in the pH region from 6 to 10 in calcium-rich uranium-mining-related waters.

Bacillus sphaericus JG-A12 is a natural isolate recovered from a uranium mining waste pile near the town of Johanngeorgenstadt in Saxony, Germany. The cells of this strain are enveloped by a highly ordered crystalline proteinaceous surface layer (S-layer) possessing an ability to bind uranium and other heavy metals. Purified and recrystallized S-layer proteins were shown to be phosphorylated by phosphoprotein-specific staining, inductive coupled plasma mass spectrometry analysis, and a colorimetric method. We used extended X-ray absorption fine-structure (EXAFS) spectroscopy to determine the structural parameters of the uranium complexes formed by purified and recrystallized S-layer sheets of B. sphaericus JG-A12. In addition, we investigated the complexation of uranium by the vegetative bacterial cells. The EXAFS analysis demonstrated that in all samples studied, the U(VI) is coordinated to carboxyl groups in a bidentate fashion with an average distance between the U atom and the C atom of 2.88 ± 0.02 Å and to phosphate groups in a monodentate fashion with an average distance between the U atom and the P atom of 3.62 ± 0.02 Å. Transmission electron microscopy showed that the uranium accumulated by the cells of this strain is located in dense deposits at the cell surface.