Continental scientific drilling and microbiology: (extremely) low biomass in crystalline bedrock of central Sweden

Abstract. Scientific drilling expeditions offer a unique opportunity to characterize the microbial communities in the subsurface that have been long-term isolated from the surface. With subsurface microbial biomass being low in general, biological contamination from the drilling fluid, sample processing, or molecular work is a major concern. To address this, characterization of the contaminant populations in the drilling fluid and negative extraction controls are essential for assessing and evaluating such sequencing data. Here, crystalline rock cores down to 2250 m depth, groundwater-bearing fractures, and the drilling fluid were sampled for DNA to characterize the microbial communities using a broad genomic approach. However, even after removing potential contaminant populations present in the drilling fluid, notorious contaminants were abundant and mainly affiliated with the bacterial order Burkholderiales. These contaminant microorganisms likely originated from the reagents used for isolating and amplifying DNA despite stringent quality standards during the molecular work. The detection of strictly anaerobic sulfate reducers such as Candidatus Desulforudis audaxviator suggested the presence of autochthonous deep biosphere taxa in the sequenced libraries, yet these clades represented only a minor fraction of the sequence counts (< 0.1 %), hindering further ecological interpretations. The described methods and findings emphasize the importance of sequencing extraction controls and can support experimental design for future microbiological studies in conjunction with continental drilling operations.