Bacterial and Archaeal Diversity in Groundwaters of the Siberian Deep-Well Radioactive Disposal Site TOMSK-7

Microbial diversity was studied in water samples collected at depths of 290 to 324 m from the Siberian deep-well radioactive disposal site S15 near the Russian city of Tomsk. For this molecular and cultivation approaches were applied. The biomass from the samples was concentrated via consequent filtration on filters with pore sizes of 1.2, 0.45, and 0.22 µm. For one of the samples 16S rDNA bacterial and archaea clone libraries were constructed from the total DNA (S15A) recovered from the biomass collected on the three filters. For another parallel sample two bacterial clone libraries S15B and S15D were generated from the DNA extracted individually from 0.45 and 0.22 µm filters, respectively. The analysis of the libraries demonstrated that the estimation of the diversity strongly depends on the way of collecting the biomass. About 65% of the clones in the S15A bacterial library were affiliated to ß-proteobacterial Dechlorosoma species. The rest of the clones represent very diverse bacterial groups. In the S15B library the Dechlorosoma sp. represented 30% of the total number of clones. This result indicates a possible dominance of Dechlorosoma sp. in the S15 samples [1]. Another 20% of the clones from the S15B library were affiliated with a low identity to a novel cyanobacteria-related lineage. Sequences representing the same “cyanobacteria-like” group were also found in the 0.22 µm library. However, populations of Cytophagales were the most predominant in the latter library and represented about 55% of the clones.
In addition, Euryarchaeota and Crenarchaeota 16S rDNA sequences were identified in the S15A total DNA as well. Most of them were affiliated to 16S rRNA genes of not jet cultured archaeons, found in different metal-rich habitats.

Autotrophic microorganisms were retrieved in the S15 samples by using primer pairs specific for the form I [2,3] and form II [4] of RubisCO gene. Some of the identified sequences from the form I were affiliated with RubisCO genes of Proteobacteria, which were related to those identified previously in the same samples via the 16S rDNA approach. Interesting is also the result that more than 80% of the form I sequences were affiliated with the RubisCO genes of different species from the β-subclass of Proteobacteria. The 16S rDNA approach indicated a dominance of β-proteobacterial species in the same samples as well. The retrieved form II sequences were affiliated with α-proteobacterial RubisCO genes.

Several oligotrophic bacteria were isolated under aerobic conditions from the 0.22 µm filter. They were related to -Proteobacterial and to Actinobacterial species. Three of the isolates, affiliated to Microbacterium oxydans possess microdiverse 16S rDNA stretches. The ability of these isolates to tolerate and sorb uranium and other heavy metals was studied. The results demonstrated that the resistance to and binding of heavy metals are strain-specific for these isolates.

Acknowledgements
This work was partly supported by grant FIKW-CT-2000-00105 (BORIS) from the European Community. We thank A. Zubkov, E. Zaharova, E. Kamenev and A. Rybalchenko for the supplying of the S15 samples.

References
[1] M. Nedelkova, G. Radeva and S. Selenska-Pobell. (2005) In: Underground Injection Science and Technology, Elsevier Science, (in press).
[2] A. Alfreider, C. Vogt, D. Hoffmann and W. Babel. Microbial Ecology 45 (2003), p. 17-328
[3] K. Nanba, G.M. King and K. Dunfield. Appl. Environ. Microbiology 70 (2004), p. 2245-2253
[4] H. Elsaied and T. Naganuma. Appl. Environ. Microbiology 67 (2001), p. 1751-1765