Evidence for methanogenic Archaea and homoacetogenic Bacteria in deep granitic rock aquifers

The presence and diversity of methanogens and homoacetogens in deep granitic rock groundwater from Aspo Hard Rock Laboratory were studied. Concentrations of hydrogen and methane in Aspo groundwater were 45 nM-100 mu M and 19-1000 mu M, respectively. Groundwater-based media were used to count viable cells in the groundwaters from the subsurface. Different physiological groups of methanogens (autotrophic H-2/CO2 consuming and heterotrophic acetate, methanol and trimethylamine consuming) were found at depths ranging from 68 to 446 m below sea level in numbers from 12 to 4.5 x 10(5) cells/ml. Viable numbers of different physiological groups of methanogens and autofluorescent cells were counted repeatedly during a period of 1 year in the groundwater. The counts were reproducible in the same boreholes at different sampling times. Methane production was observed in the groundwater with added methanogenic substrates, at anaerobic conditions, at 17 degrees C, after 20-50 days of adaptation. The most active methane production took place in those groundwaters that were sources of methanogenic enrichment and pure cultures. Methanosarcina-like organisms were observed in groundwater from depths of 45-68 m and active enrichment cultures. 16S rRNA gene sequencing of enrichment cultures indicated the presence of a psychrophilic Methanohalophilus related organism at a depth of 414 m. A new species of alkaliphilic Methanobacterium was isolated from depths of 68, 409 and 420 m and studied. Homoacetogenic Bacteria were found in the granitic groundwater as viable cells from 10 to 3.6x10(4) cells/ml and produced acetate autotrophically. It appears that deep granitic groundwater is inhabited by autotrophic methanogens and acetogens, which may produce methane and acetate at the expense of subterranean hydrogen and bicarbonate.