Microscopic and spectroscopic bioassociation study of uranium(VI) with an archaeal Halobacterium isolate.

The safe disposal of high-level radioactive waste in a deep geological repository is a huge social and technical challenge. So far, one of the less considered factors needed for a long-term risk assessment, is the impact of microorganisms occurring in the different host rocks. Even under the harsh conditions of salt formations different bacterial and archaeal species were found, e. g. Halobacterium sp. GP5 1-1, which has been isolated from a German rock salt sample. The interactions of this archaeon with uranium(VI), one of the radionuclides of major concern for the long-term storage of high-level radioactive waste, were investigated. Different spectroscopic techniques, as well as microscopy, were used to examine the occurring mechanisms on a molecular level leading to a more profound process understanding. Batch experiments with different uranium(VI) concentrations showed that the interaction is not only a simple, but a more complex combination of different processes. With the help of in situ attenuated total reflection Fourier-transform infrared spectroscopy the association of uranium(VI) onto carboxylate groups was verified. In addition, time-resolved laser-induced luminescence spectroscopy revealed the formation of phosphate and carboxylate species within the cell pellets as a function of the uranium(VI) concentration and incubation time. The association behavior differs from another very closely related halophilic archaeon, especially with regard to uranium(VI) concentrations. This clearly demonstrates the importance of studying the interactions of different, at first sight very similar, microorganisms with uranium(VI). This work provides new insights into the microbe-uranium(VI) interactions at highly saline conditions relevant to the long-term storage of radioactive waste in rock salt.

Impact of Haloarchaea on Speciation of Uranium-A Multispectroscopic Approach.

Haloarchaea represent a predominant part of the microbial community in rock salt, which can serve as host rock for the disposal of high level radioactive waste. However, knowledge is missing about how Haloarchaea interact with radionuclides. Here, we used a combination of spectroscopic and microscopic methods to study the interactions of an extremely halophilic archaeon with uranium, one of the major radionuclides in high level radioactive waste, on a molecular level. The obtained results show that Halobacterium noricense DSM 15987T influences uranium speciation as a function of uranium concentration and incubation time. X-ray absorption spectroscopy reveals the formation of U(VI) phosphate minerals, such as meta-autunite, as the major species at a lower uranium concentration of 30 µM, while U(VI) is mostly associated with carboxylate groups of the cell wall and extracellular polymeric substances at a higher uranium concentration of 85 µM. For the first time, we identified uranium biomineralization in the presence of Halobacterium noricense DSM 15987T cells. These findings highlight the potential importance of Archaea in geochemical cycling of uranium and their role in biomineralization in hypersaline environments, offering new insights into the microbe-actinide interactions in highly saline conditions relevant to the disposal of high-level radioactive waste as well as bioremediation.

Comparative analysis of uranium bioassociation with halophilic bacteria and archaea.

Rock salt represents a potential host rock formation for the final disposal of radioactive waste. The interactions between indigenous microorganisms and radionuclides, e.g. uranium, need to be investigated to better predict the influence of microorganisms on the safety assessment of the repository. Hence, the association process of uranium with two microorganisms isolated from rock salt was comparatively studied. Brachybacterium sp. G1, which was isolated from the German salt dome Gorleben, and Halobacterium noricense DSM15987T, were selected as examples of a moderately halophilic bacterium and an extremely halophilic archaeon, respectively. The microorganisms exhibited completely different association behaviors with uranium. While a pure biosorption process took place with Brachybacterium sp. G1 cells, a multistage association process occurred with the archaeon. In addition to batch experiments, in situ attenuated total reflection Fourier-transform infrared spectroscopy was applied to characterize the U(VI) interaction process. Biosorption was identified as the dominating process for Brachybacterium sp. G1 with this method. Carboxylic functionalities are the dominant interacting groups for the bacterium, whereas phosphoryl groups are also involved in U(VI) association by the archaeon H. noricense.

Multistage bioassociation of uranium onto an extremely halophilic archaeon revealed by a unique combination of spectroscopic and microscopic techniques.

The interactions of two extremely halophilic archaea with uranium were investigated at high ionic strength as a function of time, pH and uranium concentration. Halobacterium noricense DSM-15987 and Halobacterium sp. putatively noricense, isolated from the Waste Isolation Pilot Plant repository, were used for these investigations. The kinetics of U(VI) bioassociation with both strains showed an atypical multistage behavior, meaning that after an initial phase of U(VI) sorption, an unexpected interim period of U(VI) release was observed, followed by a slow reassociation of uranium with the cells. By applying in situ attenuated total reflection Fourier-transform infrared spectroscopy, the involvement of phosphoryl and carboxylate groups in U(VI) complexation during the first biosorption phase was shown. Differences in cell morphology and uranium localization become visible at different stages of the bioassociation process, as shown with scanning electron microscopy in combination with energy dispersive X-ray spectroscopy. Our results demonstrate for the first time that association of uranium with the extremely halophilic archaeon is a multistage process, beginning with sorption and followed by another process, probably biomineralization.

Influence of riboflavin on the reduction of radionuclides by Shewanella oneidenis MR-1.

Uranium (as UO2(2+)), technetium (as TcO4(-)) and neptunium (as NpO2(+)) are highly mobile radionuclides that can be reduced enzymatically by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble species. The redox chemistry of Pu is more complicated, but the dominant oxidation state in most environments is highly insoluble Pu(IV), which can be reduced to Pu(III) which has a potentially increased solubility which could enhance migration of Pu in the environment. Recently it was shown that flavins (riboflavin and flavin mononucleotide (FMN)) secreted by Shewanella oneidensis MR-1 can act as electron shuttles, promoting anoxic growth coupled to the accelerated reduction of poorly-crystalline Fe(III) oxides. Here, we studied the role of riboflavin in mediating the reduction of radionuclides in cultures of Shewanella oneidensis MR-1. Our results demonstrate that the addition of 10 µM riboflavin enhances the reduction rate of Tc(VII) to Tc(IV), Pu(IV) to Pu(III) and to a lesser extent, Np(V) to Np(IV), but has no significant influence on the reduction rate of U(VI) by Shewanella oneidensis MR-1. Thus riboflavin can act as an extracellular electron shuttle to enhance rates of Tc(VII), Np(V) and Pu(IV) reduction, and may therefore play a role in controlling the oxidation state of key redox active actinides and fission products in natural and engineered environments. These results also suggest that the addition of riboflavin could be used to accelerate the bioremediation of radionuclide-contaminated environments.