Microorganisms have great potential to bind and thus transport actinides in the environment. Thus microbes indigenous to designated nuclear waste disposal sites have to be investigated regarding their interaction mechanisms with soluble actinyl ions when assessing the safety of a planned repository. This paper presents results on the pH-dependent sorption of U(VI) onto Pseudomonas fluorescens isolated from the granitic rock aquifers at Äspö Hard Rock Laboratory, Sweden. To characterize the U(VI) interaction on a molecular level, potentiometric titration in combination with time-resolved laser-induced fluorescence spectroscopy (TRLFS) were applied. This paper as a result is one of the very few sources which provide stability constants of U(VI) complexed by cell surface functional groups. In addition the bacteria-mediated liberation of inorganic phosphate in dependence on [U(VI)] at different pHs was studied to judge the influence of phosphate release on U(VI) mobilization. The results demonstrate that in the acidic pH range U(VI) is bound by the cells mainly via protonated phosphoryl and carboxylic sites. The complexation by carboxylic groups can be observed over a wide pH range up to around pH 7. At neutral pH fully deprotonated phosphoryl groups are mainly responsible for U(VI) binding. U(VI) can be bound by P. fluorescens with relatively high thermodynamic stability.
For the first time in the aqueous phase the existence of a U(VI)-benzoate complex with a 1:2 stoichiometry could be proven. Using UV-Vis spectroscopy and especially cryo time-resolved laser-induced fluorescence spectroscopy (TRLFS) it was possible to characterize this complex in detail.
Room temperature TRLFS measurements revealed a static as well as a dynamic ligand-initiated quench process in the U(VI)-benzoic acid system. At these conditions no luminescence emission resulting from complex formation was found. Consequently cryo TRLFS was applied to increase the maximum detectable benzoate:U(VI) ratio. By this for the first time a luminescence spectrum of the 1:2 U(VI)-benzoate complex could be determined. This species is characterized by emission bands at 467, 485, 505, 526, and 550 nm which are blue-shifted compared to the ones of the UO2
2+ ion. The luminescence lifetime of the 1:2 complex amounts to 9.21±0.01 μs at −18 ºC compared to 150.4±0.5 μs for UO2
2+.
The stability constant of the newly found species log β
120 has been calculated to be 4.48±0.24. The stability constant of the 1:1 complex was validated to amount to 2.64±0.19. UV-Vis spectroscopy combined with factor analysis yielded the molar absorption spectrum of the 1:2 U(VI)-benzoate species which is characterized by absorption bands at 406, 418, 432.5, 447, and 461 nm and a molar absorption coefficient of 22 L mol−1 cm−1.
The interaction between the Pseudomonas fluorescens biofilm and U(VI) were studied using extended X-ray absorption fine structure spectroscopy (EXAFS), and time-resolved laser fluorescence spectroscopy (TRLFS). In EXAFS studies, the formation of a stable uranyl phosphate mineral, similar to autunite (Ca[UO2]2[PO4]2•2-6H2O) or meta-autunite (Ca[UO2]2[PO4]2•10-12H2O) was observed. This is the first time such a biomineralization process has been observed in P. fluorescens. Biomineralization occurs due to phosphate release from the cellular polyphosphate, likely as a cell's response to the added uranium. It differs significantly from the biosorption process occurring in the planktonic cells of the same strain. TRLFS studies of the uranium-contaminated nutrient medium identified aqueous Ca2UO2(CO3)3 and UO2(CO3)3 (4-) species, which in contrast to the biomineralization in the P. fluorescens biofilm, may contribute to the transport and migration of U(VI). The obtained results reveal that biofilms of P. fluorescens may play an important role in predicting the transport behavior of uranium in the environment. They will also contribute to the improvement of remediation methods in uranium-contaminated sites.
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