Estructura y diversidad de las comunidades microbianas de bentonitas y sus interacciones con radionucleidos.

Resum

Radioactive wastes are hazardous materials of serious environmental concern which must be safely stored for many years for the radiotoxicity to decrease to non dangerous levels (Hedin, 1999). For this reason, the deep geological disposal of radioactive wastes, encapsulated in corrosion-resistant metal containers, has been internationally considered as the safest option for their disposal (IAEA, 2003). A number of potentially suitable host rock types have been being studied in different countries (Thury and Bossart, 1999; Stroes-Gascoyne et al., 2007; Alonso et al., 2008). In Spain, the geological formations considered are granitic rocks, clay formations and salt deposits (Astudillo Pastor, 2001). In addition, the bentonite formations from Cabo de Gata National Park, Almeria, were selected as the best characterized from different physicochemical points of view, as engineered barriers (Villar et al., 2006). However, the safety of this long-term geological disposal could be compromised not only by physical and chemical factors, but also by the biogeochemical activity of either indigenous microorganisms of the host rock or microorganisms introduced during the construction of the repository. Microbial processes can affect the security of the deep geological repository through three different mechanisms: transformation of clay minerals, degradation of the metal containers, and mobilization of radionuclides. So far, it is of great importance to study the microbial occurrence in the selected Spanish bentonite formations and the interaction mechanisms of their microbial populations with radionuclides. The main aims of this doctoral thesis are to study the microbial diversity in bentonite formations selected as reference material for safety barriers in a deep geological repository, and to elucidate the interaction mechanisms of selected microbial isolates with uranium(VI) and curium(III) as representatives of hexavalent and trivalent actinides, respectively. In this study, three different bentonite formations were sampled in Cabo de Gata National Park: El Cortijo de Archidona (sample BI-2, collected from the surface and sample BI-3, taken from 20 cm depth), El Toril (BII-2, surface) and Los Trancos (BIV-2, surface and BIV-3, 20 cm depth). The mineralogy of the studied bentonite samples is dominated by the presence of montmorillonite, with quartz and feldspars as minor phases. The most relevant difference between the studied samples is the presence of jarosite, an iron sulphate mineral, in sample BII. The first step was to study the bacterial diversity of the bentonite samples by molecular approaches based on the 16S ribosomal RNA gene analysis (Illumina sequencing platform and traditional clone libraries). Proteobacteria, Bacteroidetes, Actinobacteria and Acidobacteria were the main phyla identified. Some of the identified clones were described for their effects on the biogeochemical cycle of elements including Fe(III) (e.g. Herbaspirillium, Janthinobacterium and Massilia), U(VI) through enzymatic reduction (e.g. Acidovorax). In addition, some clones were reported to tolerate high concentrations of heavy metals (e.g. Ralstonia and Variovorax). The microbial diversity, analyzed by culture dependent methods, was dominated by Alphaproteobacteria (50% of the isolates) and Actinobacteria (44%) in sample BI. In the case of sample BII, Actinobacteria was the most abundant phylum (75%), with a heterogeneous distribution. In addition to bacteria, one yeast strain, affiliated to Rhodotorula mucilaginosa, was isolated from sample BII. Moreover, the uranium tolerance of the microbial isolates was evaluated by the determination of the minimal inhibitory concentration and by flow cytometry technique. These results indicated that in sample BII, two strains presented a high uranium tolerance: the bacterial strain Stenotrophomonas sp. BII-R7 and the yeast strain R. mucilaginosa BII-R8. The high uranium tolerance exhibited by the yeast cells is a biological mediated process since under the same experimental conditions, the yeast cells BII-R8 and those of the bacterium BII-R7 exhibited different levels of uranium tolerance. For instance, whereas at 2 mM of uranium concentration, almost 74% of the yeast cells are alive, 100% of the Stenotrophomonas cells are not viable. The interaction mechanisms of the yeast BII-R8 with uranium(VI) was studied using a combination of spectroscopic (XAS and TRLFS) and microscopic (STEM-HAADF) techniques. XAS and TRLFS analysis indicated that the U-complexes formed by the yeast cells are similar to those of organic phosphate groups, showing a local coordination similar to that of meta-autunite, a uranium-phosphate mineral phase. STEM-HAADF analysis of the U-treated yeast cells revealed the presence of metal accumulates at the cell surfaces and intracellularly. The interaction between curium(III) and the yeast strain R. mucilaginosa BII-R8 demonstrated that the biosorption of Cm(III) is a reversible process where two Cm(III) species were identified. Cm(III)-Rhodoturula species 1 is characterized by an emission maximum at 599 ± 1 nm and an average luminescence lifetime of 215 ± 36 µs. Whereas Cm(III)-Rhodoturula species 2 shows a more red shifted emission maximum at 602.0 ± 0.5 nm and a shorter average luminescence lifetime of 124 ± 15 µs. These TRLFS data indicated that the microbial strain sorbs this radionuclide making it more mobile and more likely to reach the biosphere. The last step was the elaboration of long-term uranyl-nitrate-treated and not-treated microcosms of the Spanish bentonite samples BI and BII, to evaluate the response of the subsurface bacterial community of these bentonites to the addition of uranium. Significant changes were observed in the structure of the bacterial population of the uranyl-treated microcosms, such as the enrichment of bacterial strains (e.g. Bacillus, Pseudomonas) described for their ability to precipitate uranium through phosphatase activity, in comparison to the non-treated samples. Interestingly, the total acid phosphatase activity was increased in the uranium-treated microcosm of sample BII. Therefore, a novel approach was applied to create a database of acid phosphatase catabolic genes, with the aim of fulfilling the uranium biomineralization process. So far, it can be concluded that this is the first study describing the microbial diversity of bentonite formations in Cabo de Gata National Park (Almeria, Spain) used as reference material for engineering barriers in the concept of the deep geological repository of radioactive wastes. The microbial diversity found in the bentonite samples might be able to interact efficiently with radionuclides, affecting the safety of the deep geological repository of radioactive wastes, and could be used in the bioremediation of radionuclide contaminated sites. Moreover, this study takes one step further in the possibility of using the taxonomic information of a studied ecosystem to get a better understanding of its catabolic potential. References: Alonso et al., 2008. Geotech Geol Eng 26:817-826 Astudillo Pastor, J., 2001. ENRESA S.A. ISBN:84-931224-4-0. Hedin, A., 1999. Report: TR-99-06. IAEA, 2003. TRS 413, IAEA, Vienna. Stroes-Gascoyne et al., 2007. Phys Chem Earth 32: 219-231. Thury, M. and Bossart, P., 1999. Eng Geol, 52(3): 347-359. Villar et al., 2006. J Iber Geol 32: 15-36.