Multistage bioassociation of uranium to a halophilic archaeon under highly saline conditions

For the final disposal of radioactive waste in a deep geological repository, salt rock is considered as a potential host rock formation. A lot of research has been conducted regarding the physical and geochemical properties and retention suitability of this potential host rock. However, information about the indigenous microorganisms and their impact on the migration behavior of radionuclides in a worst-case scenario – the release of radionuclides – are widely missing in particular. In this work, we studied the interactions between the radionuclide uranium and the extreme halophilic archaeon Halobacterium (Hbt.) noricense. Extensive investigations were performed with an isolate originating from an Austrian salt mine, Hbt. noricense DSM-15987 [1]. Surprisingly, the obtained kinetics of the sorption experiments showed that bioassociation is not only a sorption process; i.e. fast sorption within the first hours until reaching a stable equilibrium state. The obtained kinetics showed a multistage process with a fast sorption phase during the first two hours of exposure. For the next hours an increasing amount of uranium was detectable (ICP-MS) in the supernatant, implying that the sorbed uranium was released from the cells. Subsequently, the amount of bioassociated uranium was found to increase very slowly until a maximum sorption of 80% was reached after 48 h. For more molecular information of these, hitherto unknown, bioassociation processes on archaeal cells, several spectroscopic and microscopic methods were applied. In situ Attenuated Total Reflection Fourier-transform Infrared (ATR FT-IR) spectroscopy provided evidence that uranium simultaneously binds to carboxylic and to phosphate groups within the initial sorption process of uranium to cells of Hbt. noricense DSM-15987. Despite of the high chloride concentration (3 M) required for the experiments and of the resulting quenching effect of chloride on uranium luminescence, we were able to detect weak signals by Laser-induced Fluorescence spectroscopy. The obtained results support the bioassociation kinetics where a higher amount of sorbed uranium was found after 2 hours of exposure time than after 5 hours. From parallel factor analysis, a preference for uranium to carboxylic groups could be verified. Furthermore, the impact of uranium on archaeal cells over time was monitored microscopically, and the viability was proven using the LIVE/DEAD® Bac LightTM Bacterial Viability Kit from Molecular probes. Moreover, it was shown that with increasing uranium concentration the cells tend to form biofilm-like agglomerates. We assume that this effect might reflect a stress reaction to protect the cells from environmental challenges like the presence of uranium. The heavy metal ions could be localized on the cell surface of the halophilic archaeon within the first sorption phase and later on in the biofilm-like agglomerates by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy.
Due to the fact that Hbt. noricense is an archaeon commonly found in salt rock, the kinetics of uranium bioassociation was investigated as well with the Hbt. noricense WIPP strain isolated from the Waste Isolation Pilot Plant (WIPP), NM, USA. The obtained kinetic curve showed the same shape as the one from the Hbt. noricense DSM-15987 strain. Additionally, Hbt. noricense WIPP formed similar but smaller biofilm-like structures. In summary, independent of the origin of both strains, both halophilic archaea obviously interact with dissolved uranium within a unique bioassociation process. This process, including the subsequent biofilm formation, will be investigated in more detail in the near future.
[1] Gruber, C. et al. (2004) Extremophiles, Vol. 8, 431-439.