Fluorescence spectroscopy and microscopy as tools for monitoring redox transformations of uranium in biological systems

Abstract

We report a study of redox reactions of uranium in model conditions using luminescence spectroscopy, which with its ease and wide availability has the potential to offer new insights into a bioremediation strategy of particular interest - the enzymatic reduction of U^VIO₂²⁺ by bacteria such as Geobacter sulfurreducens. The inherent luminescent properties of U^VIO₂²⁺ have been combined with confocal fluorescence microscopy techniques and lifetime image mapping to report directly on uranium concentration, localisation and oxidation state in cellular systems during uranium bioreduction, suggesting that localisation of uranyl species on the cell membrane surface plays an important role and that extracellular biogenic features form alongside uranyl sorbed cellular species during early stages of the bioreduction. The use of confocal microscopy in tandem with lifetime image mapping offers both improved temporal and spatial resolution (nanoseconds to microseconds and sub-micron respectively) than more conventional X-ray based techniques and offers the potential to image redox reactions occurring in situ. Together, these techniques provide an excellent and sensitive probe to assess the coordination environment of uranium during bioreduction processes that are currently being considered for remediation strategies of redox active radionuclides present in contaminated land.

Fig. 1. Emission spectra of the uranyl cation in a carbonate buffer solution (30 mmol) in the presence of Geobacter sulfurreducens at room temperature (solid line) and 77 K (dotted line), (λ _ex = 420 nm). Aliquot obtained immediately after introduction of UO₂(CH₃CO₂)₂ (5 mmol) to microcosm and brief agitation.

Fig. 2. The decrease in the intensity of uranyl emission over time in the steady-state emission spectrum of anaerobic uranyl solutions containing Geobacter sulfurreducens (λ _ex = 420 nm, 77 K).

Fig. 3. Phosphorescence lifetime image map focussing on a representative extracellular feature in samples taken immediately after the introduction of Geobacter sulfurreducens to uranyl acetate solution (left), a histogram showing the distribution of lifetime measurements (centre), and a representative, uranyl spectrum from one such extracellular feature (right) (λ _ex = 405 nm, room temperature).

Fig. 4. Bright-field microscopy image (top left), FLIM image (top right), Overlaid bright-field and FLIM image (bottom left) of Geobacter sulfurreducens and representative spectrum taken from one such bacterium (λ _ex = 405 nm, room temperature).