Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

The in situ BN experiment performed at the international underground rock laboratory Mont Terry in Switzerland estimates the risks of release of nitrate and organic molecules into the opalinus clay due to the weathering of the bitumen containers, foreseen for disposal of long-living radioactive waste. The changes induced by the bitumen degradation products in the opalinus clay have many different bio-geo-chemical aspects which may facilitate migration of radionuclides. For this reason the BN experiment exploits a set of interdisciplinary (chemical, technical, and biological) approaches [1]. Response of bacteria present in the pore water of the interval 2 of the BN experiment to addition of nitrate, nitrite, and acetate is reported here.
Bacterial diversity in three samples collected from interval 2 of the BN experiment was studied by using ribosomal intergenic spacer amplification (RISA), cloning and sequencing of the 16S rRNA-gene of the RISA amplicons as described in [2]. The first sample was collected before the supplementation, the second sample - one day after the addition of 70 mg/L NO3-, 35 mg/L NO2- and 46 mg/L acetate, and the third sample - five days after the treatment.
The chemical analyses of the samples monitored by Bleyen at al. [1] demonstrated that three days after the supplementation the added nitrate was fully reduced to nitrite. Between the third and the fifth days from the beginning of the experiment fast reduction of nitrite occurred. After the fifth day - complete consumption of acetate was monitored and a process of slow nitrite reduction started which finished with complete removal of the latter two weeks after the supplementation.
The RISA molecular analyses performed in this work demonstrated strong shifting in bacterial community of the interval 2 only 24 hours after the addition of nitrate, nitrite and acetate. The strongly predominant in the untreated sample Firmicutes were almost completely overgrown by Alphaproteobacteria and by one particular population of Pseudomonas stutzeri (Gammaproteobacteria). The latter not identified in the non-supplemented samples. As P. stutzeri can use acetate as electron donor for nitrate reduction our results clearly demonstrate that this species is involved in the above reported [1] rapid reduction of nitrate to nitrite during the first 3 days after the treatment. The RISA analyses demonstrated that P. stutzeri was even more predominant in the sample collected five days after the treatment. On the basis of this result we suggested that this species plays also the major role in the fast reduction of nitrite. Our suggestion is in agreement with the published capability of P. stutzeri to perform fast reduction of nitrite to molecular nitrogen with acetate under conditions similar to ours [3]. The role (if any) of the rest of the bacterial community in the fast nitrate and nitrite reduction has to be cleared.
Analyses of additional samples collected between the 5th and the 14th days after the supplementation is needed in order to clear the role of the other members of the bacterial community of interval 2 in the slow nitrite reduction occurring after the 5th day of the treatment when acetate was completely utilized.

REFERENCES
[1] Bleyen et al. (2012) Clays in natural and engineered barriers for radioactive waste confinement. Montpellier, 22-25.10.2012, p.755.
[2] Selenska-Pobell et al (2001) Ant. Van Leeuvenhoek.79., 149-161.
[3] Strohm et al. (2007) Appl. Environ. Microbiol. 73, 1420-1424.