The continental subsurface contains approximately 12 – 20% of Earth’s biomass. In deep rock environments this biomass dwells in aqueous spaces, fractures and pores of the rock, either attached in biofilms or free-living in the fluids. Meta-analyses have suggested a core deep biosphere microbiome from pre-collected data but face issues such as difference in sampling procedures, DNA extraction methods, negative control protocols, sequencing primers etc., which may introduce false variation. Addressing this issue and striving for consistent methodology, 7 sites of the Finnish Fennoscandian Shield was studied between 2009 – 2021 ranging in depth from 100 to 2300 m. Some of the sites have deep groundwater with more than 50 Ma residence times, whereas in other places the groundwater is considerably younger. All microbiomes contained bacteria, archaea and fungi, and the microbial community composition differed greatly between sites indicating that rock type and hydrogeochemistry play a great role in moulding the communities. Chemoheterotrophy was the universally dominant predicted metabolic strategy. The bacterial numbers in the groundwater were between < 1 to more than 5 x 10⁶ 16S rRNA gene copies mL^-1 but did not necessarily reflect sampling depth, but rather the concentration of DOC and DIC. In addition, dormant microbial communities activated within hours after introduction of CO₂ or methane. Finally, groundwater monitored at one site for 10 years showed that the microbial communities did dot remain static but varied in size (up to 100-fold) and composition over time in a cyclic manner, interchanging between mainly two distinct community compositions.

Nano-magnetite is a potential archive for biosignatures of iron-cycling microorganisms in hydrothermal systems, which are widely considered to be among the most ancient microbial habitats on Earth. Sulfidic diagenesis driven by hydrothermal fluids and microbial sulfur cycling potentially causes the rapid transformation of magnetite to iron sulfide minerals. Thus, identifying nano-magnetite and its transformation products in hydrothermal sulfide deposits is crucial for reconstructing iron- and sulfur-cycling microbial life in deep time. However, the identity and characteristics of iron sulfide minerals resulting from nano-magnetite sulfidation at hydrothermal conditions have previously not been constrained. Here we present experimental data on sulfidation reactions of synthetic and biogenic nano-magnetite at physical and chemical conditions relevant to microbial habitats in hydrothermal systems on early Earth (<121°C, anoxic, sulfidic). We characterize the resulting precipitates with analytical imaging techniques, mineralogical methods, and geochemical approaches (e.g., SEM-EDS, µXRD, Raman spectroscopy, sequential Fe extraction). Our results demonstrate a potential taphonomic bias against nano-magnetite in sulfidic hydrothermal habitats and suggest that biosignature records of iron- and sulfur-cycling microorganisms in ancient hydrothermal sulfides are affected by diagenetic fluid-mineral interactions.

Lake Towuti, Indonesia is a stratified ferruginous (iron-rich, sulfate-poor) system whose deep basin experienced dynamic changes in trophic and redox conditions since the Middle Pleistocene. As wet and dry periods alternated and sediment accumulated, microbial life sustained by metals and organic substrates became entombed in the subsurface. A 1 Ma stratigraphic archive retrieved by the International Continental scientific Drilling Program (ICDP) was sampled aseptically on site for microbiology analysis. Through taxonomic and functional analyses (16S rRNA amplicons, metagenomics), the BioMetArchive project aims at the first comprehensive characterization of the lacustrine subsurface biosphere in terms of diversity, abundance and metabolic functions.

Metagenomic data combined with high resolution cell counts and pore water geochemistry allowed to characterize the distribution of microorganisms throughout the core and to identify which microbial taxa and metabolic features are involved in the major biogeochemical cycles and organic matter remineralization during sediment burial. Results show a drastic decrease in the cell counts (from 10⁹ to 10⁴) as electron acceptors in the pore water chemistry become depleted within the upper 5 m of the sediment. Results of taxonomic and metagenomic analyses indicate that the sediment ferruginous conditions predominantly select for fermentative Bathyarchaeia. Metabolic features attributed to this entirely uncultivated phylum explaining their selective growth were indicative of sulfur transformations, organic matter fermentation and homoacetogenic dark carbon fixation. Thus, Lake Towuti shelters a deep biosphere displaying similarities to early life’s processes in ferruginous systems, which provides a direct link between cryptic sulfur cycling and redox conservative fermentations.

Seismic activity and consistently high CO₂ fluxes make the Eger Rift in Western Bohemia (CZ) a rare subsurface ecosystem and scientifically relevant location to study microbial behavior and assess how geologically derived compound are used in the deep subsurface. Studying microbial life in this ecosystem provides the opportunity to investigate how high CO₂ levels and mineralogy influence microbial community composition and metabolic activity. Seismic activity in this region can also release H₂, a process which may provide the basis for primary production through methanogenic archaea and should be explored.

To assess microbial processes associated with the Eger Rift subsurface we investigated diversity, community structure and metabolic attributes of bacterial and archaeal communities in drill core sediments and groundwater samples. We also analyzed the geochemical conditions in this subsurface system and studied the physiological responses of native Eger microbial communities to high CO₂ via enrichments

Genomic analysis of sediment and water samples, covering depths between 17m and 230m, provided novel insights into a CO₂ adapted microbial community. We detected strong Cyanobacteria and Proteobacteria signatures as well as unexpected archaeal diversity in sediments, and high abundances of acidophiles and sulfate reducers in water samples. Enrichment cultures from the recovered sediments suggested subsurface populations can actively utilize CO₂ and H₂, while reconstruction and annotation of MAGs provided insights into microbial processes driven by CO₂.

Going forward our data will be used to further investigate cellular processes under high CO₂ conditions and identify pathways and biomolecules which may be of industrial and biotechnological relevance.