Decrease of U(VI) immobilization capability of the facultative anaerobic strain Paenibacillus sp. JG-TB8 under anoxic conditions due to strongly reduced phosphatase activity

Abstract

Interactions of a facultative anaerobic bacterial isolate named Paenibacillus sp. JG-TB8 with U(VI) were studied under oxic and anoxic conditions in order to assess the influence of the oxygen-dependent cell metabolism on microbial uranium mobilization and immobilization. We demonstrated that aerobically and anaerobically grown cells of Paenibacillus sp. JG-TB8 accumulate uranium from aqueous solutions under acidic conditions (pH 2 to 6), under oxic and anoxic conditions. A combination of spectroscopic and microscopic methods revealed that the speciation of U(VI) associated with the cells of the strain depend on the pH as well as on the aeration conditions. At pH 2 and pH 3, uranium was exclusively bound by organic phosphate groups provided by cellular components, independently on the aeration conditions. At higher pH values, a part (pH 4.5) or the total amount (pH 6) of the dissolved uranium was precipitated under oxic conditions in a meta-autunite-like uranyl phosphate mineral phase without supplying an additional organic phosphate substrate. In contrast to that, under anoxic conditions no mineral formation was observed at pH 4.5 and pH 6, which was clearly assigned to decreased orthophosphate release by the cells. This in turn was caused by a suppression of the indigenous phosphatase activity of the strain. The results demonstrate that changes in the metabolism of facultative anaerobic microorganisms caused by the presence or absence of oxygen can decisively influence U(VI) biomineralization.

Figure 1. Normalized U(VI) luminescence spectra recorded from the uranium complexes formed under different pH and aeration conditions within 48 hours by the cells of Paenibacillus sp. JG-TB8.

For comparison, dotted vertical lines indicate the main luminescence emission maxima recorded for the sample incubated at pH 2. The dashed vertical line marks the position of the luminescence emission peak which was assigned to uranyl carboxylate complexes.

Figure 2. Uranium L _III-edge XANES spectra recorded from the uranium complexes formed by Paenibacillus sp. JG-TB8 at pH 2, 3, 4.5 and 6 under anoxic conditions.

In addition, the XANES spectra of two solutions, which serve as reference samples for the uranium oxidation states, one of U(VI) and another one of U(IV), each at a concentration of 4×10⁻² M in 1 M HClO₄, are shown in the figure. The dashed and the dotted line represent the position of the absorption edge of U(VI) and of the XANES peak resulting from the multiple scattering contribution of the axial oxygen atoms of U(VI), respectively.

Figure 3. U L _III-edge k ³-weighted EXAFS spectra (left) and the corresponding Fourier Transforms (right) (3.1 Å⁻¹<k<12.4 Å⁻¹) of the uranium complexes formed by Paenibacillus sp. JG-TB8 at pH 2, pH 3, pH 4.5, and pH 6 at different pH and aeration conditions within 48 hours.

For comparison, the spectra of three model compounds, namely uranyl succinate, uranyl-fructose(6)phosphate, meta-autunite are illustrated as well.

Figure 4. Structural models used for the fitting procedure of the EXAFS spectra obtained from the uranium complexes build by the cells of Paenibacillus sp. JG-TB8.

The left model created from the crystall structure of meta-autunite was used for the fitting procedure of the sample incubated at pH 6 under oxic conditions. The right model contains fragments of meta-autunite (monodentate coordination at phosphate groups) as well as uranyl triacetate (bidentate coordination to carboxylic groups) and was used for the fitting procedures of all other samples.

Figure 5. Transmission electron micrograph (left) of uranium precipitates deposited by the cells of Paenibacillus sp. JG-TB8 at pH 4.5 under oxic conditions.

Energy-dispersive X-ray spectra (right) of the sample points (S1–S6) are marked with arrowheads.

Figure 6. Representative microscopic pictures of Paenibacillus sp. JG-TB8, stained with the Live/Dead Kit after the treatment with uranium under aerobic (left) and anaerobic (right) conditions at pH 3 (A, B), pH 4.5 (C, D), and pH 6 (E, F) for 48 hours.

Pictures were taken in fluorescence mode using a fluorescence mirror unit (U-MSWB; Olympus Europa Holding GmbH, Hamburg, Germany) with excitation wavelengths between 420 and 460 nm.

Figure 7. Amount of orthophosphate in the supernatant of cells of Paenibacillus sp. JG-TB8 after an incubation of 48 hours in dependency on pH, the aeration conditions and the presence of uranium.