Biosorption of uranium on the cells of the halophilic archaea Halobacterium noricense DSM 15987 under highly saline conditions

For the long-term storage of radioactive waste in salt rock formations it is important to know how microorganisms can affect the performance of a repository. It is possible that in salt rock indigenous microbes are able to influence the oxidation state, the speciation and hence the solubility of actinides. So the migration of actinides can be reduced or enhanced by different processes as for example by biosorption with cellular ligands [1].
The biosorption capacity of the archaea Halobacterium noricense DSM 15987, which was used as a reference strain due to its worldwide occurrence in salt rock, was investigated. The strain was isolated from an Austrian salt mine [2] and similar species were also found in the WIPP (Waste Isolation Pilot Plant, Carlsbad, New Mexico, USA) [3]. This rod shaped microorganism is a halophilic one which requires at least 2.1 M NaCl whereat the optimum is 3.0 M NaCl [2].
Independent of the uranium concentration (10 – 120 µM) Halobacterium noricense DSM 15987 shows good sorption capabilities at room temperature (RT). Constantly, 90 % of added uranium was bound on the cells. Looking at the time dependence of biosorption with a constant uranium concentration (100 µM) an asymptotic sorption curve was obtained. This accumulation kinetic indicates a two stage process.
Directly after the addition of uranium it is rapidly sorbed by the cells. After 3 h 35 % of uranium was accumulated. The second slower step is reached after 42 h, where 90 % of added uranium was bound to the cells. Under these conditions this corresponds to 37.5 ± 0.7 mg U(VI) per 1 g dry biomass. A slightly faster sorption can be seen at higher temperatures especially at 50 °C. There the maximal sorption of 90 % is already reached after 24 h.
Interestingly, with increasing incubation time, uranium concentration and also temperature an agglomeration of the cells occurred. Live/Dead staining showed that agglomerated cells were mostly alive but nearly all single cells were dead. So we conclude that this agglomeration process is a kind of stress response to protect the cells themselves for environmental challenges.
Furthermore, the supernatant and the washed cell suspension were analysed with TRLFS to characterize the binding of uranium to the cells. Despite the high quenching effect of chloride to uranium fluorescence a luminescence signal could be detected. The reason is the high quantum yield of some formed complexes. With parallel factor analysis (Parafac) we could assume that two uranium-cell-complexes were formed. Species 1 is difficult to interpret due to the unspecific form of the spectrum. Further investigations with reference substances have to be done. But comparing with literature we assign species 2 to a carboxylic cell-uranyl-complex [4]. These results were confirmed by IR spectroscopy where as well carboxylic vibrations could be detected.
The archaeum Halobacterium noricense DSM 15987 shows good uranium sorption efficiencies. Independent of the occurring agglomeration always 90 % of the added uranium was bound to the cells. The binding of uranium to carboxylic groups on the cell wall was proven with TRLFS and IR spectroscopy. A binding of uranium to phosphate groups is still under investigation.
Thanks to Sabrina Gurlit for analysing the samples with ICP-MS and Karsten Heim for recording the IR-spectra.

REFERENCES
1. Morris, K. et al., “Biogeochemical cycles and remobilisation of the actinide elements” Interactions of microorganisms with radionuclides, Volume 2, Page 101-141 (2002).
2. Gruber, C. et al., “Halobacterium noricense sp. nov., an archaeal isolate from a bore core of an alpine Permian salt deposit, classification of Halobacterium sp. NRC-1 as a strain of H. salinarum and emended description of H. salinarum” Extremophiles, 8, Page 431-439 (2004).
3. Swanson, J. S. et al., “Status Report on the Microbial Characterization of Halite and Groundwater Samples from the WIPP” Status report Los Alamos National Laboratory, Page 1ff. (2012).
4. Vogel, M. et al., “Biosorption of U(VI) by the green algae Chlorella vulgaris in dependence of pH value and cell activity” Science of the Total Environment , Page 384-395. (2010).