Over the last several decades, investigations of Earth's subsurface and other extremely low-biomass systems have refined our understanding of the environmental limits of life, driven by methodological advances that permit agnostic life detection of biology and their respective physical biosignatures and chemical biomarkers. These advances enable mission concepts centered on microbiological processes that facilitate identification of both active life and preserved biosignatures through measurements of metabolism and associated biochemical markers that, on Mars, are more likely to be retained below the surface. Terrestrially, although biological processes can exert a significant influence on Earth's crust, the presence of habitable conditions does not necessarily imply the existence of cellular life. The Viking missions constituted the first direct life-detection experiments on Mars but produced equivocal outcomes, prompting subsequent exploration strategies to emphasize surface habitability rather than direct biological testing. Leveraging progress in subsurface microbiology and planetary exploration, we contend that Mars missions are now poised to shift toward direct tests for extant microbial activity in the subsurface, with metabolic processes serving as a broadly applicable indicator of life.

In deep bedrock fractures of the Fennoscandian shield, pyrite with “superheavy” δ³⁴S values of up to 147‰ exists together with calcite. The extreme variability and ³⁴S-enrichment in pyrite indicate that microbial sulfate reduction (MSR) evolved to almost complete sulfate exhaustion in closed-system microenvironments. The bulk δ³⁴Spyrite of up to 81‰ implies a sulfate source with unusually high δ³⁴S, although such a source has not yet been identified. Here, we aim to decipher this source by analyzing carbonate-associated sulfate (CAS) of calcite related to superheavy pyrite. Our δ³⁴SCAS values reveal low to moderate ³⁴S enrichment, with δ³⁴SCAS between 2.3 and 28.2‰, which is far below the bulk δ³⁴Spyrite of up to 81‰ indicating a decoupled sulfate pool for CAS and pyrite origin. The extreme δ³⁴S fractionation recorded by pyrite implies very slow MSR rates, which typically promote calcite dissolution rather than precipitation. Calcite precipitation was thus inhibited during superheavy pyrite formation. Instead, the calcite shows signs for anaerobic oxidation of methane coupled to sulfate reduction (AOM-SR), reflected by ¹³C-depletion and moderate ³⁴S-enrichment. The AOM-SR process reduces sulfate concentration, in turn providing conditions suitable for the formation of superheavy pyrite. We propose that a limited supply of substrates (sulfate and organic matter) and Fe²⁺, their episodic input into the deep fracture system together with low diffusion rates of fracture fluids enabled a balance and completion of microbial methanogenesis, AOM-SR, and MSR. Extreme isotope signatures may thus represent diagnostic features of episodic, energy-limited deep biosphere ecosystems, with implications for understanding subsurface microbial ecology and biogeochemical cycling.

Schwertmannite and jarosite are naturally occurring iron (Fe) oxyhydroxysulfates with strong sorption capacities for hexavalent uranium [U(VI)] in various acidic sulfate-rich environments. These metastable minerals commonly undergo recrystallization, particularly in the presence of dissolved Fe2+ [Fe(II)aq], which may influence the fate of associated U(VI). Here, we quantified molecular-level changes in U repartitioning and speciation when U(VI)-sorbed schwertmannite and jarosite reacted with Fe(II)aq under near-neutral and anaerobic conditions over 2 weeks. The results show that Fe(II)aq additions promoted rapid mineral transformation to goethite via a dissolution–reprecipitation pathway, proceeding (near-completely) for schwertmannite but slowly and incompletely for jarosite. Importantly, even at early transformation stages when goethite likely only started forming on the surface of the transforming minerals, the recrystallization process led to near-complete retention of U, predominantly as U(VI), within the structure of the neo-formed goethite. Subsequent U reduction to U(V) increased with time but remained incomplete, even after extensive mineral transformation in the presence of 1–50 mM Fe(II)aq for 2 weeks. The results demonstrate that Fe(II)-promoted recrystallization of Fe-oxyhydroxysulfates can rapidly and persistently lock both U(VI) and U(V) into chemically stable goethite, with important implications for predicting U behavior and designing remediation strategies in various acidic and U-contaminated environments.

Glacial meltwater has been increasingly recognised as a potential source of atmospheric methane, yet its origin and variability in the High Arctic remain poorly constrained. In this study, we present measurements of methane concentration and carbon‑isotope composition in meltwater draining the northern Greenland Ice Sheet. Here we show that methane concentrations (12-20 nM) are significantly lower than those reported from other Greenland catchments, despite clear evidence of subglacial input. Isotopic signatures and regional geological context indicate that this methane is predominantly thermogenic, reflecting a geological source that is largely independent of subglacial microbial activity. These findings advance current understanding of methane sources beneath the Greenland Ice Sheet, revealing a thermogenic contribution alongside microbial methane in Arctic methane cycling. In this work we highlight the need to account for geological methane reservoirs when assessing present and future cryosphere–carbon feedbacks.

Microbial methane generation (methanogenesis) is an important metabolic process in the terrestrial deep biosphere and is an analog to early Earth as it is proposed to be one of the most ancient metabolisms on Earth. Signs of methanogenesis in meteorite impact craters are of particular interest in this respect as these settings are proposed hot spots for deep microbial colonization of the upper crust. Yet, reports of active deep rock-hosted methanogenesis are scarce, particularly for methylotrophic methanogenesis, while reports from terrestrial meteorite impact craters are completely lacking. Here, we used indigenous communities in cultures enriched from 400-m deep fluids to confirm and characterize active methane production from several carbon donors, including indigenous oil, in a terrestrial impact crater at Siljan, Sweden. Metagenomic and metatranscriptomic data of the methane-producing cultures revealed a consortium dominated by Acetobacterium sp. KB-1 and Candidatus Methanogranum gryphiswaldense, mediating methanogenesis solely via the methyl-reduction pathway, and resulting in a δ¹³Cmethanol-methane isotope enrichment of up to 98.6‰. These results provide insights into methylotrophic methanogenesis in deep subsurface environments in general, and in particular in fractured meteorite impact structures.

IMPORTANCE: This study revealed that microbes enriched from groundwater in a 380-m deep borehole within the Siljan meteorite impact crater in Sweden were capable of producing methane, a key greenhouse gas. This is especially significant because it is the first proof of active methanogens in an impact crater and showing a specific pathway of methane production—methylotrophic methanogenesis—is present in the deep terrestrial subsurface, an environment that is typically hard to study. These findings shed light on life in extreme conditions on Earth and show that meteorite craters can be biological hotspots, rich with ancient life processes.

The deep biosphere is thought to have dominated life on Earth for most of its history and played a crucial role in the early evolution of both prokaryotes and eukaryotes. Currently, an incoherent record of biosignatures such as body fossils, trace fossils and chemofossils stretching from present to ~ 3.5 Ga implies the presence of an abundant and rich, yet largely unexplored, record of an ancient deep biosphere in fractures, vesicles and vugs of the igneous oceanic and continental crust, and in meteorite impact structures therein. New multidisciplinary protocols combining detection of isotopic, molecular, morphological biosignatures and high spatial resolution geochronology, have been developed and successfully implemented to decipher the deep ancient biosphere in Precambrian cratons. Isotopic studies have suggested that ancient microbial activity can also be identified in fracture networks of meteorite impact structures, although the few studies applying coupled biosignature-geochronology protocols have reported microbial colonization records that considerably postdate the impact events. Robust geochronology-biosignature-petrographic correlations are thus needed to confirm impact event-related colonization. Here we summarize recent findings for detection of ancient subsurface microbial biosignatures in cratons, oceanic crust and impact structures and discuss future strategies for addressing current knowledge gaps. Such strategies should ideally involve biosignature detection protocols coupled with thermochronological constraints of habitable subsurface conditions as well as radiometric dating of ancient veins in the crystalline crust.

Deeply fractured rocks of meteorite impact structures have been hypothesized as hot spots for microbial colonization on Earth and other planetary bodies. Biosignatures of such colonization are rare, however, and most importantly, direct geochronological evidence linking the colonization to the impact-generated hydrothermal systems are completely lacking. Here we provide timing constraints to microbial colonization of the 77.85 ± 0.78 Ma old Lappajärvi impact structure, Finland, by using coupled microscale stable isotope biosignature detection and radioisotopic dating of vug- and fracture-filling assemblages in impactites. The first detected mineral precipitation at habitable temperatures for life (47.0 ± 7.1 °C) occurred at 73.6 ± 2.2 Ma and featured substantially ³⁴S-depleted pyrite consistent with microbial sulfate reduction. Later stages of vug-mineral precipitation occurred more than 10 Myr later, at gradually lower temperatures, and featured δ¹³Ccalcite values diagnostic for both anaerobic microbial consumption and production of methane. These insights confirm the capacity of medium-sized (and large) meteorite impacts to generate long-lasting hydrothermal systems, enabling microbial colonization as the crater cools to ambient conditions, an effect that may have important implications for the emergence of life on Earth and beyond.

Deeply fractured rocks of meteorite impact craters are suggested as prime niches for subsurface microbial colonization. Methane can be a product of such microbial communities and seeps of methane from impact craters on Earth are of strong interest as they act as analogs for Mars. Previous studies report signs of ancient microbial methanogenesis in the Devonian Siljan meteorite impact structure in Sweden, but the proportion of microbial methane, metabolic pathways, and potential modern activity remain elusive. In this study, gas composition, hydrochemistry, oil organic geochemistry, and microbial community analyses are reported in 400 m deep fractures of the Siljan impact structure. The results showed a dominantly microbial origin for methane, which was supported by highly negative delta 13CCH4 and positive delta 13CCO2 values along with multiply substituted isotopologues (Delta 13CH3D) that indicated disequilibrium fractionation due to microbial kinetic isotope effects. The presence of C2 to C5 hydrocarbons suggested a minor thermogenic input in the gas mix. Characterization of the microbial community via 16S rRNA gene amplicon sequencing and real-time PCR indicated a low abundance of several methanogenic archaeal populations, which is common for settings with active methanogenesis. Evidence of oil biodegradation suggested that secondary microbial hydrocarbon utilization was involved in the methanogenesis. Low sulfate and high alkalinity in the groundwaters also suggested a dominantly microbial methane formation driven by infiltration of freshwater that was coupled to sulfate reduction and secondary utilization of early mature thermogenic hydrocarbons.

The Anthropocene has been framed around humanity's impact on atmospheric, biologic, and near-surface processes, such as land use and vegetation change, greenhouse gas emissions, and the above-ground hydrologic cycle. Groundwater extraction has lowered water tables in many key aquifers but comparatively little attention has been given to the impacts in the deeper subsurface. Here, we show that fluid fluxes from the extraction and injection of fluids associated with oil and gas production and inflow of water into mines likely exceed background flow rates in deep (>500 m) groundwater systems at a global scale. Projected carbon capture and sequestration (CCS), geothermal energy production, and lithium extraction to facilitate the energy transition will require fluid production rates exceeding current oil and co-produced water extraction. Natural analogs and geochemical modeling indicate that subsurface fluid manipulation in the Anthropocene will likely appear in the rock record. The magnitude and importance of these changes are unclear, due to a lack of understanding of how deep subsurface hydrologic and geochemical cycles and associated microbial life interact with the rest of the Earth system.

Pyrite is a ubiquitous sulfide mineral found in diverse geological settings and holds great significance in the formation of Au deposits as well as the safe utilization of groundwater due to its remarkable ability to incorporate substantial amounts of As. However, despite its importance, there remains a dearth of fundamental data on the partitioning of As between pyrite and fluid, which is key for accurately modeling the As distribution in these environments.

Here, we present new insights into the partitioning behavior of As between pyrite and fluid at conditions that mimic natural fluid systems. Pyrite was synthesized by replacement of natural siderite in hydrothermal experiments at 200 °C and pH 5 applying a wide range of fluid As concentrations, spanning from 0.001 to 100 µg/g. The As distribution and concentration in synthetic pyrite was analyzed by quantitative LA-ICP-MS mapping providing a high spatial resolution and sensitivity at 2–3 µm image pixel size at a detection limit of ∼1 µg/g at the single pixel scale. Pyrite-fluid partitioning coefficients (D_As^(py/fluid)) between synthetic pyrite and experimental fluid agree with previously published data for high fluid As concentrations of 1 µg/g to 100 µg/g (D_As < 2000). However, at low As concentrations in the experimental fluid (<1 µg/g), a steep increase in the D_As^(py/fluid) values of up to ∼30,000 was detected, demonstrating even stronger As partitioning into pyrite. This is confirmed by the analyses of natural pyrite that precipitated from As-poor fluids (0.3–0.4 ng/g) within a deep anoxic aquifer in SE Sweden. The discovery holds significant implications for the mobility and scavenging of As, which in turn is important for understanding the formation and fingerprinting of mineral deposits as well as for the secure utilization of groundwater resources.