Microbiological analysis of the in situ Bitumen-Nitrate-Opalinus Clay interaction

Clay formations like the Opalinus Clay are foreseen to serve as the host rock for geological disposal of high- and intermediate-level long-lived radioactive waste in several countries, because of their favourable properties to delay the migration of radionuclides over time. However, bituminized intermediate-level long-lived radioactive waste may physico-chemically perturb the clay barrier properties because in time it will leach substantial amounts of nitrate and organic bitumen degradation products (BDP).
To study the physico-chemical impact of intermediate-level radioactive waste containing bitumen and nitrate, an in situ experiment in the Opalinus Clay (Saint Ursanne, Switzerland) named the Bitumen-Nitrate-Clay interaction (BN) experiment, is being performed at the Mont Terri Rock Laboratory. The in situ equipment of the BN-experiment consists of three separate packed-off intervals, supplied with a filter screen. Each interval is equipped with its own stainless steel water circulation unit. Such water circulation unit contains water sampling containers, circulation pumps and flow meters. One of the circulation units is equipped with an on-line UV spectrophotometer and pH electrode intervals, allowing a continuous monitoring of nitrate, nitrite concentrations, organic carbon level and pH.
In a first series of tests, the microbial and biogeochemical effect of a nitrate and/or acetate perturbation is studied. Acetate is used as it a good representation of BDP. Hereto, nitrate was injected in interval 1 while a mixture of nitrate and acetate is injected in interval 2. As an active microbial community can have a significant impact on the physical and (geo)chemical conditions of the clay surrounding the disposal gallery, microbial analyses were performed on samples taken from the interval solutions before, during and after this first series of tests. Our microbial investigations which included Scanning Electron Microscopy, molecular biology methods, ATP-measurements, and cultivation based techniques of the initial pore water samples, proved the presence and activity of bacteria.
Analysis of the 16S rDNA sequences obtained from the initial interval solutions, i.e. artificial pore water used to fill the intervals and which have been in contact with the surrounding clay for more than six months, indicates similar bacterial communities in all three solutions of the test intervals with the dominant population being Proteobacteria (81.5 – 94.9 %) and Firmicutes (3.4 – 11.1%). Actinobacteria (1.7 and 7.4%) have only been detected in the initial pore water of two intervals.
The first results of the Ribosomal Intergenic Spacer Amplification (RISA) analysis, using universal bacterial primers for 16S rDNA968-983 and 23S rDNA115-130, demonstrate that in both injection tests, i.e. nitrate (interval 1) or nitrate and acetate (interval 2), a strong shift in bacterial communities was induced. Just before the start of these injection tests the pore waters of the two intervals were strongly predominated by different Clostridial species most of them related to Desulfosporosinus species. In addition, smaller populations of Bacteroidetes and Beta– proteobacteria were found as well. Twenty-four hours later, a rapid and strong proliferation of Bacteroidetes, in interval 1, and of Alphaproteobacteria, in intervals 1 and 2, occurred. Specific for interval 1, a stimulation of Beta– and Deltaproteobacteria and a complete masking of the Clostridial groups had occured. In contrast, in interval 2, Gammaproteobacteria were stimulated and some Clostridia continued to persist. This shift may be due to bacterial contamination of the exchanged interval solutions and/or the drastic change of carbon– and/or electron acceptor source.