The deep biosphere refers to the vast ecosystem of life beneath the Earth’s surface, residing in the fractured bedrock and pores of rocks, largely isolated from solar energy. These fractures enclose an important reservoir of groundwater that contains microorganisms active in processes such as the uptake of inorganic carbon, sulfur cycling, or the degradation of organic matter. However, there is still much knowledge to be gained on the diversity and function of these subsurface microorganisms, and how the surface influences subsurface life. In this work, I explored interactions among subsurface microorganisms, studied subsurface microbial diversity in the light of surface recharge, and characterized microbial populations residing in biofilms.

Potential interactions among microorganisms were explored with anaerobic cultures using groundwaters from the Äspö Hard Rock Laboratory. By removal of larger cells (> 0.45 𝜇m in diameter), an inoculum enriched in ultra-small bacteria (nanobacteria) was obtained. Despite the presence of various sources of energy and nutrients, these nanobacteria did not grow over prolonged incubation times up to four months. Reconstructed genomes confirmed this group of bacteria to have a low metabolic potential, indicative of a symbiotic lifestyle.

Characterization of microbial communities in subsurface groundwaters and overlying environments on Äspö island revealed that a substantial proportion of the subsurface community was also detected in soil-hosted groundwaters. Considering the unidirectional water flow, this showed that part of the subsurface diversity between 70 and 460 m depth could originate from surface recharge, especially for the shallower groundwaters. In contrast to the high microbial diversity observed in Äspö groundwaters, characterization of a fracture fluid at 975 m depth in central Sweden revealed a microbial community dominated by a single population, adapted to the energy-limited conditions in the deep subsurface, namely the bacterium Candidatus Desulforudis audaxviator.

Furthermore, the activity (based on RNA transcripts) of attached microbial populations was measured using flow-cells that facilitated biofilm formation. An elevated number of genes involved in the transition from a planktonic to an attached lifestyle was observed. Interestingly, comparing the microbial activity in the biofilm to the planktonic community revealed Thiobacillus denitrificans to have a principal role in the biofilm formation. Combined, these findings help understand the magnitude of microbial diversity in the continental subsurface as well as how these microorganisms are adapted to cope with the energy limitations in this subsurface ecosystem.