Chapter 15: An investigation into the role of c-type cytochromes and extracellular flavins in the bioreduction of uranyl(VI) by Shewanella oneidensis using fluorescence spectroscopy and microscopy

Optical spectroscopy and microscopy have been employed to examine the in situ bioreduction of uranyl(VI) acetate in bulk solution and at the microscopic level using lactate as the electron donor, over 24 hours by the Gram-negative metal-reducing bacterium Shewanella oneidensis. The wild type strain was used alongside three deletion mutants; JG 596 – lacking outer membrane (OM) c-type cytochromes, JG 1453 – lacking inner and outer membrane (IM, OM) c-type cytochromes and JG 1758 – lacking the ability to secrete extracellular flavins. Low temperature steady state emission spectroscopy of the uranyl(VI) supernatant monitored over 24 hours indicate a one-electron reduction pathway for the wild type whereas no significant bioreduction is observed for the deletion mutants lacking OM c-type cytochromes and lacking IM and OM c-type cytochromes confirming the crucial role of c-type cytochromes in the transfer of electrons to U(VI). One photon optical microscopy shows a variation in luminescence lifetimes across the surface of the bacterial cell, suggesting an interaction between the uranyl(VI) and a surface bound redox active species. As this variation is not as evident in the bacterial strains lacking the ability to produce cytochromes, it is inferred that the cytochromes are directly involved in electron transfer with the uranyl(VI) located on the cell surface. These techniques allow the direct visualisation of radionuclide-microbe interactions during the bioreduction of U(VI) at sub-micron spatial resolution, which has provided mechanistic insight into the mechanisms of in situ bioredmediation of uranium (and by extension other actinides) in microbial cultures and more complex environmental systems.