Active anaerobic methane oxidation and sulfur disproportionation in the deep terrestrial subsurface.

Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawater-derived sulfate create a terrestrial sulfate-methane transition zone (SMTZ). We used chemical and isotopic data coupled to genome-resolved metaproteogenomics to demonstrate active life and, for the first time, provide direct evidence of active anaerobic oxidation of methane (AOM) in a deep terrestrial bedrock. Proteins from Methanoperedens (formerly ANME-2d) are readily identifiable despite the low abundance (≤1%) of this genus and confirm the occurrence of AOM. This finding is supported by 13C-depleted dissolved inorganic carbon. Proteins from Desulfocapsaceae and Desulfurivibrionaceae, in addition to 34S-enriched sulfate, suggest that these organisms use inorganic sulfur compounds as both electron donor and acceptor. Zerovalent sulfur in the groundwater may derive from abiotic rock interactions, or from a non-obligate syntrophy with Methanoperedens, potentially linking methane and sulfur cycles in Olkiluoto groundwater. Finally, putative episymbionts from the candidate phyla radiation (CPR) and DPANN archaea represented a significant diversity in the groundwater (26/84 genomes) with roles in sulfur and carbon cycling. Our results highlight AOM and sulfur disproportionation as active metabolisms and show that methane and sulfur fuel microbial activity in the deep terrestrial subsurface.

Active sulfur cycling in the terrestrial deep subsurface.

The deep terrestrial subsurface remains an environment where there is limited understanding of the extant microbial metabolisms. At Olkiluoto, Finland, a deep geological repository is under construction for the final storage of spent nuclear fuel. It is therefore critical to evaluate the potential impact microbial metabolism, including sulfide generation, could have upon the safety of the repository. We investigated a deep groundwater where sulfate is present, but groundwater geochemistry suggests limited microbial sulfate-reducing activity. Examination of the microbial community at the genome-level revealed microorganisms with the metabolic capacity for both oxidative and reductive sulfur transformations. Deltaproteobacteria are shown to have the genetic capacity for sulfate reduction and possibly sulfur disproportionation, while Rhizobiaceae, Rhodocyclaceae, Sideroxydans, and Sulfurimonas oxidize reduced sulfur compounds. Further examination of the proteome confirmed an active sulfur cycle, serving for microbial energy generation and growth. Our results reveal that this sulfide-poor groundwater harbors an active microbial community of sulfate-reducing and sulfide-oxidizing bacteria, together mediating a sulfur cycle that remained undetected by geochemical monitoring alone. The ability of sulfide-oxidizing bacteria to limit the accumulation of sulfide was further demonstrated in groundwater incubations and highlights a potential sink for sulfide that could be beneficial for geological repository safety.

Highly Diverse Aquatic Microbial Communities Separated by Permafrost in Greenland Show Distinct Features According to Environmental Niches.

The Greenland Analog Project (GAP) study area in the vicinity of Kangarlussuaq, Western Greenland, was sampled for surface water and deep groundwater in order to determine the composition and estimate the metabolic features of the microbial communities in water bodies separated by permafrost. The sampling sites comprised a freshwater pond, talik lake, deep anoxic groundwater, glacier ice and supraglacial river, meltwater river and melting permafrost active layer. The microbial communities were characterized by amplicon sequencing of the bacterial and archaeal 16S rRNA genes and fungal ITS1 spacer. In addition, bacterial, archaeal and fungal numbers were determined by qPCR and plate counts, and the utilization pattern of carbon and nitrogen substrates was determined with Biolog AN plates and metabolic functions were predicted with FAPROTAX. Different sample types were clearly distinguishable from each other based on community composition, microbial numbers, and substrate utilization patterns, forming four groups, (1) pond/lake, (2) deep groundwater, (3) glacial ice, and (4) meltwater. Bacteria were the most abundant microbial domain, ranging from 0.2-1.4 × 107 16S rRNA gene copies mL-1 in pond/lake and meltwater, 0.1-7.8 × 106 copies mL-1 in groundwater and less than 104 copies mL-1 in ice. The number of archaeal 16S and fungal 5.8S rRNA genes was generally less than 6.0 × 103 and 1.5 × 103, respectively. N2-fixing and methane-oxidizing Actinomycetes, Bacteroidetes and Verrucomicrobia were the dominant microorganisms in the pond/lake samples, whereas iron reducing Desulfosporosinus sp. dominated the deep anaerobic groundwater. The glacial ice was inhabited by Cyanobacteria, which were mostly Chloroplast-like. The meltwater contained methano- and methylotrophic Proteobacteria, but had also high relative abundances of the nano-sized Parcubacteria. The archaea composed approximately 1% of the 16S rRNA gene pool in the pond/lake samples with nano-sized Woesearchaeota as the dominating taxon, while in the other sample types archaea were almost negligent. Fungi were also most common in the pond/lake communities, were zoospore-forming Chytridiomycetes dominated. Our results show highly diverse microbial communities inhabiting the different cold Greenlandic aqueous environments and show clear segregation of the microbial communities according to habitat, with distinctive dominating metabolic features specifically inhabiting defined environmental niches and a high relative abundance of putatively parasitic or symbiotic nano-sized taxa.

Biogeochemical Cycling by a Low-Diversity Microbial Community in Deep Groundwater.

Olkiluoto, an island on the south-west coast of Finland, will host a deep geological repository for the storage of spent nuclear fuel. Microbially induced corrosion from the generation of sulphide is therefore a concern as it could potentially compromise the longevity of the copper waste canisters. Groundwater at Olkiluoto is geochemically stratified with depth and elevated concentrations of sulphide are observed when sulphate-rich and methane-rich groundwaters mix. Particularly high sulphide is observed in methane-rich groundwater from a fracture at 530.6 mbsl, where mixing with sulphate-rich groundwater occurred as the result of an open drill hole connecting two different fractures at different depths. To determine the electron donors fuelling sulphidogenesis, we combined geochemical, isotopic, metagenomic and metaproteomic analyses. This revealed a low diversity microbial community fuelled by hydrogen and organic carbon. Sulphur and carbon isotopes of sulphate and dissolved inorganic carbon, respectively, confirmed that sulphate reduction was ongoing and that CO2 came from the degradation of organic matter. The results demonstrate the impact of introducing sulphate to a methane-rich groundwater with limited electron acceptors and provide insight into extant metabolisms in the terrestrial subsurface.