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X-ray absorption spectroscopy reveals the transient oxidation state during microbial uranium(VI) reduction by a sulfate-reducing microorganism

High-energy-resolution fluorescence-detected X-ray absorption near-edge structure (HERFD-XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy were used to investigate the reduction of U(VI) by the sulfate-reducing bacterium Desulfosporosinus hippei DSM 8344T, confirming the partial reduction of U(VI) and the presence of U(V).

Clay formations are potentially used as host rocks for deep geological repositories storing high-level radioactive waste with bentonite as backfill material. Such repositories require careful consideration of various factors to ensure long-term safety. Among these, natural microbial communities can influence the behaviour of the waste, particularly in scenarios involving water intrusion. Microorganisms may interact with released radionuclides and affect their mobility through processes like the bioreduction of mobile, water-soluble U(VI) to less soluble U(IV).

Several studies indicate the presence of Desulfosporosinus, among other sulfate-reducing microorganisms, in both clay rock and bentonite [1]. Desulfosporosinus hippei DSM 8344T, an anaerobic, spore-forming microorganism, is a closely related phylogenetic relative to the isolated species [2]. Therefore, this strain was selected to gain deeper insights into its interactions with U, the main

component of spent nuclear fuel. The bioreduction of U(VI) by this microorganism was investigated using various spectroscopic methods (Figure 116), with a specific focus on the transient oxidation state U(V), known for its relative instability due to disproportionation into U(IV) and uranyl(VI). Nevertheless, different studies reported at least an intermediate stabilisation of this oxidation state by special ligands or in different mineral phases [3].

HERFD-XANES and EXAFS spectroscopy were conducted at beamline BM20 at the U M4 and the U L3 edges, respectively, while the latter technique was used to detect various U-species associated with the cells. Time- dependent experiments in synthetic Opalinus Clay pore water [4] (100 µM U(VI), pH 5.5) revealed a fast removal of up to 80% of the U from the supernatants within 48 h, which is a first hint for an ongoing U(VI) reduction. The formed U(IV) has a lower solubility and precipitates from solution. The composition of the U oxidation states was assessed at different incubation times using HERFD- XANES spectroscopy.

The HERFD-XANES spectra of cell pellets at varying incubation times, alongside reference spectra of U(VI), U(V), and U(IV), demonstrate the differences in the spectra recorded after 4 h, 24 h, 48 h, and 168 h (Figure 117). Notably, the intensity of the first post-edge feature (labeled as d in Figure 117) following the white line (c) decreases gradually with longer incubation times. The second post-edge feature (e) exhibits a similar trend.

The data collected from cell pellets after various incubation times revealed that U(VI) is the dominant oxidation state in all samples, but minor differences

Fig. 116: Schematic setup of the investigation procedure.