Microbial characterization of deep-waters from a borehole within the Ivrea-Verbano Zone

The deep subsurface is a dynamic and biologically active environment that harbors a vast array of microbial communities, accounting for a substantial fraction of Earth’s biomass. Most of the available information about subsurface ecosystems in continental regions is derived from studies on sedimentary rock formations and the analysis of groundwater and deep fluids accessed through boreholes and mines. Research on microbial life within crystalline bedrock has historically been more limited, primarily focusing on rocks such as granites, schists, and serpentinized ophiolites. Nevertheless, several studies have demonstrated that fractured crystalline rocks can host unique and diverse microbial ecosystems. In this study, we present the microbiological characterization of a water overflow at the borehole 5071_1_B (IGSN: ICDP5071EH30001) in the context of the ICDP-sponsored DIVE (Drilling the Ivrea-Verbano zonE) project (expedition number 5071) aiming for a full geophysical and petrological characterization of the continental lower crust in the Ivrea-Verbano Zone and for the identification of microbial communities inhabiting the different lithologies encountered in borehole 5071_1_B. During the drilling operation, a water overflow was observed at a depth of 300-316 m below current surface, due to the presence of a deep aquifer. The fluids were analysed geochemically and through a combination of 16S rRNA gene amplicon sequencing, metagenomic analysis, and epifluorescence microscopy. By using the waters of the nearby Toce River and on-site contamination tracking procedures we provide hypotheses on the origins of the rising fluids, as well as insights into the microbial taxonomic and functional diversity within the deep aquifer fluids.