Quantitation and Identification of Methanogens and Sulphate Reducers in Olkiluoto Groundwater

The GEOFUNC Project focuses on the microbiology connected to safety and risk
assessment of the final disposal of high radioactive nuclear waste. Methanogenic
archaea and sulphate reducing bacteria are significant groups of microorganisms in
anaerobic environments, and are of crucial concern for the safe long term storage of
nuclear waste in deep bedrock. The sulphate reducing bacteria are able to produce
sulphide which may cause corrosion of the copper in the radioactive waste storage
capsules. Methanogens, on the other hand, may produce quantities of methane from
various organic carbon compounds, CO2 and H2. Methane may both serve as carbon
source for methanotrophic microbial groups, and may also cause mobilization of
radionuclides, as a result of gas discharge through fractures in the bedrock. The
transition zones between the sulphate rich and methane rich waters are locations for
microbial processes where the methane may serve as carbon source for sulphate
reducing bacteria, which in turn would produce corrosive sulphides.

It has been estimated that only 1-10 % of all the microorganisms present in the
environment can be isolated and cultivated. Uncultured microorganisms can be
identified and their numbers in the environment quantified by identification of specific
marker genes that are essential for their functions by use of molecular methods.
Methanogens, for example, can be identified by their genes for methyl coenzyme M
reductase (mcrA), which is an essential enzyme involved in the production of methane.
The mcrA is specifically present only in methanogenic archaea. Sulphate reducers are
identified by their dissimilatory sulphite reductase genes (dsrB), which are present in
and essential for all microorganisms performing dissimilatory sulphate reducing. In the
GEOFUNC project, a quantitative PCR method (qPCR) was developed for the detection
of methanogens and sulphate reducers. This method is based on specific quantitative
detection of marker genes involved in methanogenesis (mcrA) or sulphate reduction
(dsrB) and enables the study of these microbial processes in the deep bedrock
groundwater.

In the GEOFUNC project, groundwater samples were studied from depths ranging from
-14.5 m to -581 m comprising samples from both drillholes in Olkiluoto and
groundwater stations in the ONKALO. Methanogenic archaea were present in almost all
samples. The highest number of methanogens was detected in the samples ONK-PVA1
(-14.5 m), OL-KR40 (-349 to -351 m and -545 to -553 m) and OL-KR47 (-334 to -338
m) and OL-KR23 (-347 to -376 m). In the samples, where the salinity exceeded 19 g/l
(Cl, Na, Ca), the number of methanogens was very low. Sulphate reducing bacteria
were present in all studied samples. The highest number of sulphate reducers were
detected in the samples ONK-PVA1 (-14.5 m), OL-KR23 (-347 to -376 m) and OLKR11 (-531 to -558 m) and OL-KR40 (-545 to -553 m), however, the last two samples are evident artefact mixtures of SO4- and CH4-rich groundwaters, thus they do not represent undisturbed in situ conditions in groundwater system.

The diversity of methanogens changed in relation to depth and a clear division into
phylogenetic groups containing either mcrA sequences from samples close to the land
surface or mcrA sequences from deeper samples. The sulphate reducers were different
in all studied samples. Despite their important ecological functions, both methanogens
and sulphate reducers were present as only small fractions of the total microbial
community in the Olkiluoto groundwater. The methanogens comprised at the most
0.44 % of the microbial community (-349 m) and the sulphate reducers 1.55 % (-14.5
m) and 1.3 % (-531 m).

The results presented in this report give indications to the diversity and quantity of the
populations of methanogenic archaea and sulphate reducing bacteria in the deep
groundwater of Olkiluoto. In spite of the small number of methanogens and sulphate
reducers in the environment there eological role is significant.