Initial Investigations into Microbial Dynamics and Biogeochemical Cycling in the Bedretto Tunnel

Over 70% of Earth’s bacteria and archaea live in the subsurface. These rock-dwelling microorganisms are capable of exerting considerable influence on their environment by altering and recycling nutrients, as well as inducing changes to fluid flow paths through bioclogging. Subsurface life, therefore, has considerable implications for both natural and engineered subsurface environments. The Bedretto tunnel, located within the Swiss Alps, is 5,218 meters long and is host to the Bedretto Underground Laboratory for Geosciences and Geoenergies (BULGG), which was built to study the feasibility of large-scale geothermal energy storage and extraction. The tunnel, with a maximum overburden of approximately 1,650m, is embedded within both gneiss and Rotondo granite and offers an ideal location to investigate the biogeochemical feedbacks associated with natural fluids as well as the effect that stimulation has on the biological and chemical properties of subsurface fluids. For these reasons, a multi-year, monthly survey of fracture fluids at over 20 locations across the entire length of the tunnel has been carried out since August 2020 with the goal of performing 16S rRNA sequencing of cells captured by 0.22µm Millipore Sterivex filters and cell enumeration by epifluorescence microscopy of cells fixed with ethanol. By studying the microorganisms inhabiting BULG, we will be able to understand how the physical-chemical heterogeneities of the subsurface influence microbial physiology and community structure. Preliminary results of DNA extractions from the cells concentrated on Sterivex filters show that there is a measurable amount of DNA found in the fluids of the Bedretto tunnel that correlates with pH, indicating the presence of microbial communities which may vary with changes in fluid chemistry. With continued monitoring through 2021, we will determine whether there is significant variability of microbial taxa at different locations within the tunnel and the relationship between the hydrochemical properties of the fluids and the microbial communities. Alongside the profiling survey, whole genome sequencing as well as targeted virome sequencing procedures will be developed and used to learn more about the genetic and metabolic capacity of the microbial communities and to better understand how viruses can influence their hosts in such an environment. These results will be compared to other subsurface environments around the globe to gain a more holistic understanding of microbial dynamics in the terrestrial subsurface. Together, these results provide a new and important tool for tracking subsurface processes.