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Fluorescence Spectroscopy and Microscopy as Tools for Monitoring Redox Transformations of Uranium in Biological Systems.
[Thesis]. Manchester, UK: The University of Manchester; 2017.
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Abstract
The immobilisation of uranium is an important issue within the nuclear industrydue to contaminated land from accidental spillage, weapons testing or mining activities.Within the environment uranium is most commonly found in the +VI oxidation state asthe mobile uranyl cation [UO2]2+. Alternatively, the +IV oxidation state can also befound in the environment, forming either an insoluble crystalline uraninite phase, or amore soluble molecular uranium(IV) species. Many endogenous subsurface bacteria canbind and accumulate actinide ions through biosorption and can reduce mobileuranyl(VI) species down to immobile uranium(IV) compounds and mineral phases.This work presents an investigation into the bioreduction process by twoanaerobic Gram-negative bacteria, Geobacter sulfurreducens and Shewanellaoneidensis MR-1. Luminescence spectroscopy is used to monitor the intensity ofuranyl(VI) emission in situ over the course of a 24 hour bioreduction experiment withuranyl(VI) acetate as the electron acceptor and either acetate or lactate as the electrondonor. An increase in intensity of the emission around hour three or four during thereduction, followed by an overall decrease, is attributed as the disproportionation of anunstable uranyl(V) intermediate. The role of inner and outer membrane c-typecytochromes as well as flavin secretion is also investigated using three deletion mutantsof the S. oneidensis bacteria, which shows that in their absence, the reduction ofuranyl(VI) does not occur over the course of 24 hours. The emission of uranium(IV) isalso investigated during bioreduction in phosphate media and results show that emissioncan be observed in aqueous solutions at pH 7 pointing to the presence of a molecularproduct.One photon confocal and two photon fluorescence microscopy has been utilisedfor the very first time to directly optically image the bioreduction of uranyl(VI) incombination with luminescence lifetime mapping. The sorption of uranyl(VI) onto thesurface of the bacteria with differing lifetimes indicates a direct interaction betweenuranyl(VI) and surface bound c-type cytochromes, since this variation was not observedin mutant S. oneidensis strains where the cytochromes were not present. Combined,these results have established the applicability of opticalspectroscopy and microscopyin tracking the bioreduction of uranium in situ.