Microbial community analysis of deep clay formations and reconstruction of metabolic potential and physiological properties under extreme conditions

In Belgium, the Boom Clay layer located at 230 m depth under the Mol site is presently studied as reference host formation for the disposal of high-level radioactive waste. To study the physical, biochemical, and biotic influences on a deep subsurface repository, an underground research facility was constructed by SCK•CEN (HADES – High Activity Disposal Experimental Site). Experiments in the HADES underground laboratory indicated biochemical activity such as microbial sulfate reduction. Such reductive perturbations were also observed in other in situ experiments, or field works, performed abroad at the sites of the Mont Terri Rock Laboratory (Nagra, Switzerland), the Bure Underground Laboratory (Andra, France), and the Tournemire Tunnel (IRSN, France).

From a pure geological point of view, the survival of indigenous bacteria captured in clay since its sedimentation on the sea bed many millions of years ago (e.g. Boom Clay is estimated to be 35 My old) is really fascinating. But more specifically, a metagenomic analysis of community DNA extracted from the resident bacterial population in Boom Clay and gene orthologous comparison with extant metabolic genes will almost certainly lead to interesting observations in terms of microbial evolution and ancient ecology.

In frame of the R&D on the geological disposal of radioactive waste in clay we consider the autochthonous micro-organisms (thus those that were dormant for very extended periods of time and may become activated by the excavation) and also micro-organisms that were inadvertently introduced during the drilling and extension of the galleries. Clearly, the conditions in the closed environment of a high-level radioactive waste (HLW) repository will become quite extreme soon after waste emplacement. Not only will micro-organisms have to cope with elevated levels of radiation but they also will be subjected to a combination of high temperature (70 – 80 °C), due to the heat output of HLW, and extreme high pH (12.5 – 13.5), due to the extensive use of cement-based materials. In a medium-level radioactive waste (MLW) repository, in which no or very little heat is generated, only hyperalkaline conditions will prevail. However, in MLW storage, cellulose and nitrate may serve as nutritional sources for bacterial growth. In both disposal fields the high pH conditions will prevail for several thousands to tens of thousands of years while temperatures for HLW storage will very slowly decrease as a result of radioactive decay. The importance of microbial activity in such geological repositories is related to its possible impact on gas generation (e.g., hydrogen, methane, and nitrogen gas) and the corrosion of metallic canisters and overpacks by reduced-sulfur species (sulfate reduction). Geochemical conditions may also be disturbed, affecting water-rock interactions and radionuclide speciation and transport.

In this project we wish to characterise the indigenous bacterial community of the Boom Clay by resuscitation of individual isolates (allowing biochemical and chemotaxonomic analyses) and nucleic acid based identification schemes. The taxonomy of organisms present in clay water is determined by sampling the piezometers that are positioned at various locations in the HADES Underground Research Facility. Phylogenetic analyses are performed using a variety of target loci (rrn, apr, dsr) and metagenomic analyses will be undertaken to gain access to protein sequence data of Boom Clay communities. Metabolic reconstruction from sequence data will be done using the gene function predictions based on CHISEL results and conventional bioinformatic approaches. The predicted functions attributed to particular organisms or taxonomic groups will be projected onto the libraries of metabolic pathways from the PUMA and KEGG databases. Ideally this should allow us to forecast which metabolic end products (e.g. gasses, chemicals, etc.) might be generated by these communities. Finally, to assess adaptations of Boom Clay organisms to extreme conditions, mixed cultures taken from the piezometers but also lab cultures of purified strains (i.e. of the dominant species) will be subjected to successive rounds of high pH, high temperature, or a combination thereof. Surviving organisms will be further investigated at gene level by gene profiling and expression analysis.

Projects & Partners: ONDRAF/NIRAS, SCK•CEN Waste and Disposal expert group

Contacts and visits to microbial research groups of the Mont Terri Rock Laboratory (Switzerland) and of the Bure Underground Laboratory (France) are foreseen.

Contact: Janssen Paul