Investigation of the potential for microbial contamination of deep granitic aquifers during drilling using 16S rRNA gene sequencing and culturing methods

Total number of bacteria, viable counts of aerobic and anaerobic heterotrophic bacteria and 16S rRNA gene diversity were investigated during drilling of three boreholes in the walls of the Äspö hard rock laboratory tunnel, at depths ranging from 380 to 446 m below sea level. Water samples were taken from the drill water source, the drilling equipment and from the drilled boreholes. The drill water was kept under nitrogen atmosphere and all equipment was steam cleaned before the start of a new drilling. Total and viable counts of bacteria in the drilled boreholes were several orders of magnitude lower than in the samples from the drilling equipment, except for sulphate reducing bacteria. A total of 158 16S rRNA genes that were cloned from the drill water source, the drilling equipment and the drilled boreholes were partially sequenced. The drilled boreholes generally had a 16S rRNA diversity that differed from what was found in samples from the drilling equipment. Several of the sequences obtained could be identified on genus level as one of the genera Acinetobacter, Methylophilus, Pseudomonas and Shewanella. In conclusion, the tubing used for drill water supply constituted a source of bacterial contamination to the rest of the drilling equipment and the boreholes. The results show, using molecular and culturing methods, that although large numbers of contaminating bacteria were introduced to the boreholes during drilling, they did not establish in the borehole groundwater at detectable levels.