Resting cells of Skeletonema marinoi assimilate organic compounds and respire by dissimilatory nitrate reduction to ammonium in dark, anoxic conditions.

Diatoms can survive long periods in dark, anoxic sediments by forming resting spores or resting cells. These have been considered dormant until recently when resting cells of Skeletonema marinoi were shown to assimilate nitrate and ammonium from the ambient environment in dark, anoxic conditions. Here, we show that resting cells of S. marinoi can also perform dissimilatory nitrate reduction to ammonium (DNRA), in dark, anoxic conditions. Transmission electron microscope analyses showed that chloroplasts were compacted, and few large mitochondria had visible cristae within resting cells. Using secondary ion mass spectrometry and isotope ratio mass spectrometry combined with stable isotopic tracers, we measured assimilatory and dissimilatory processes carried out by resting cells of S. marinoi under dark, anoxic conditions. Nitrate was both respired by DNRA and assimilated into biomass by resting cells. Cells assimilated nitrogen from urea and carbon from acetate, both of which are sources of dissolved organic matter produced in sediments. Carbon and nitrogen assimilation rates corresponded to turnover rates of cellular carbon and nitrogen content ranging between 469 and 10,000 years. Hence, diatom resting cells can sustain their cells in dark, anoxic sediments by slowly assimilating and respiring substrates from the ambient environment.

Recurring volcanic winters during the latest Cretaceous: Sulfur and fluorine budgets of Deccan Traps lavas.

Two events share the stage as main drivers of the Cretaceous-Paleogene mass extinction-Deccan Traps volcanism, and an asteroid impact recorded by the Chicxulub crater. We contribute to refining knowledge of the volcanic stressor by providing sulfur and fluorine budgets of Deccan lavas from the Western Ghats (India), which straddle the Cretaceous-Paleogene boundary. Volcanic fluorine budgets were variable (400 to 3000 parts per million) and probably sufficient to affect the environment, albeit only regionally. The highest sulfur budgets (up to 1800 parts per million) are recorded in Deccan lavas emplaced just prior (within 0.1 million years) to the extinction interval, whereas later basalts are generally sulfur-poor (up to 750 parts per million). Independent evidence suggests the Deccan flood basalts erupted in high-flux pulses. Our data suggest that volcanic sulfur degassing from such activity could have caused repeated short-lived global drops in temperature, stressing the ecosystems long before the bolide impact delivered its final blow at the end of the Cretaceous.

Single cell dynamics and nitrogen transformations in the chain forming diatom Chaetoceros affinis.

Colony formation in phytoplankton is often considered a disadvantage during nutrient limitation in aquatic systems. Using stable isotopic tracers combined with secondary ion mass spectrometry (SIMS), we unravel cell-specific activities of a chain-forming diatom and interactions with attached bacteria. The uptake of 13C-bicarbonate and15N-nitrate or 15N-ammonium was studied in Chaetoceros affinis during the stationary growth phase. Low cell-to-cell variance of 13C-bicarbonate and 15N-nitrate assimilation within diatom chains prevailed during the early stationary phase. Up to 5% of freshly assimilated 13C and 15N was detected in attached bacteria within 12 h and supported bacterial C- and N-growth rates up to 0.026 h-1. During the mid-stationary phase, diatom chain-length decreased and 13C and 15N-nitrate assimilation was significantly higher in solitary cells as compared to that in chain cells. During the late stationary phase, nitrate assimilation ceased and ammonium assimilation balanced C fixation. At this stage, we observed highly active cells neighboring inactive cells within the same chain. In N-limited regimes, bacterial remineralization of N and the short diffusion distance between neighbors in chains may support surviving cells. This combination of "microbial gardening" and nutrient transfer within diatom chains represents a strategy which challenges current paradigms of nutrient fluxes in plankton communities.

Rapid shifting of a deep magmatic source at Fagradalsfjall volcano, Iceland.

Recent Icelandic rifting events have illuminated the roles of centralized crustal magma reservoirs and lateral magma transport1-4, important characteristics of mid-ocean ridge magmatism1,5. A consequence of such shallow crustal processing of magmas4,5 is the overprinting of signatures that trace the origin, evolution and transport of melts in the uppermost mantle and lowermost crust6,7. Here we present unique insights into processes occurring in this zone from integrated petrologic and geochemical studies of the 2021 Fagradalsfjall eruption on the Reykjanes Peninsula in Iceland. Geochemical analyses of basalts erupted during the first 50 days of the eruption, combined with associated gas emissions, reveal direct sourcing from a near-Moho magma storage zone. Geochemical proxies, which signify different mantle compositions and melting conditions, changed at a rate unparalleled for individual basaltic eruptions globally. Initially, the erupted lava was dominated by melts sourced from the shallowest mantle but over the following three weeks became increasingly dominated by magmas generated at a greater depth. This exceptionally rapid trend in erupted compositions provides an unprecedented temporal record of magma mixing that filters the mantle signal, consistent with processing in near-Moho melt lenses containing 107-108 m3 of basaltic magma. Exposing previously inaccessible parts of this key magma processing zone to near-real-time investigations provides new insights into the timescales and operational mode of basaltic magma systems.

A Late Paleocene age for Greenland's Hiawatha impact structure.

The ~31-km-wide Hiawatha structure, located beneath Hiawatha Glacier in northwestern Greenland, has been proposed as an impact structure that may have formed after the Pleistocene inception of the Greenland Ice Sheet. To date the structure, we conducted 40Ar/39Ar analyses on glaciofluvial sand and U-Pb analyses on zircon separated from glaciofluvial pebbles of impact melt rock, all sampled immediately downstream of Hiawatha Glacier. Unshocked zircon in the impact melt rocks dates to ~1915 million years (Ma), consistent with felsic intrusions found in local bedrock. The 40Ar/39Ar data indicate Late Paleocene resetting and shocked zircon dates to 57.99 ± 0.54 Ma, which we interpret as the impact age. Consequently, the Hiawatha impact structure far predates Pleistocene glaciation and is unrelated to either the Paleocene-Eocene Thermal Maximum or flood basalt volcanism in east Greenland. However, it was contemporaneous with the Paleocene Carbon Isotope Maximum, although the impact's exact paleoenvironmental and climatic significance awaits further investigation.

The rate and fate of N2 and C fixation by marine diatom-diazotroph symbioses.

N2 fixation constitutes an important new nitrogen source in the open sea. One group of filamentous N2 fixing cyanobacteria (Richelia intracellularis, hereafter Richelia) form symbiosis with a few genera of diatoms. High rates of N2 fixation and carbon (C) fixation have been measured in the presence of diatom-Richelia symbioses. However, it is unknown how partners coordinate C fixation and how the symbiont sustains high rates of N2 fixation. Here, both the N2 and C fixation in wild diatom-Richelia populations are reported. Inhibitor experiments designed to inhibit host photosynthesis, resulted in lower estimated growth and depressed C and N2 fixation, suggesting that despite the symbionts ability to fix their own C, they must still rely on their respective hosts for C. Single cell analysis indicated that up to 22% of assimilated C in the symbiont is derived from the host, whereas 78-91% of the host N is supplied from their symbionts. A size-dependent relationship is identified where larger cells have higher N2 and C fixation, and only N2 fixation was light dependent. Using the single cell measures, the N-rich phycosphere surrounding these symbioses was estimated and contributes directly and rapidly to the surface ocean rather than the mesopelagic, even at high estimated sinking velocities (<10 m d-1). Several eco-physiological parameters necessary for incorporating symbiotic N2 fixing populations into larger basin scale biogeochemical models (i.e., N and C cycles) are provided.

Age and composition of young basalts on the Moon, measured from samples returned by Chang'e-5.

Orbital data indicate that the youngest volcanic units on the Moon are basalt lavas in Oceanus Procellarum, a region with high levels of the heat-producing elements potassium, thorium, and uranium. The Chang'e-5 mission collected samples of these young lunar basalts and returned them to Earth for laboratory analysis. We measure an age of 1963 ± 57 million years for these lavas and determine their chemical and mineralogical compositions. This age constrains the lunar impact chronology of the inner Solar System and the thermal evolution of the Moon. There is no evidence for high concentrations of heat-producing elements in the deep mantle of the Moon that generated these lavas, so alternate explanations are required for the longevity of lunar magmatism.

Single-Cell Measurements of Fixation and Intercellular Exchange of C and N in the Filaments of the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

Under diazotrophic conditions, the model filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 develops a metabolic strategy based on the physical separation of the processes of oxygenic photosynthesis, in vegetative cells, and N2 fixation, in heterocysts. This strategy requires the exchange of carbon and nitrogen metabolites and their distribution along the filaments, which takes place through molecular diffusion via septal junctions involving FraCD proteins. Here, Anabaena was incubated in a time course (up to 20 h) with [13C]bicarbonate and 15N2 and analyzed by secondary ion mass spectrometry imaging (SIMS) (large-geometry SIMS [LG-SIMS] and NanoSIMS) to quantify C and N assimilation and distribution in the filaments. The 13C/12C and 15N/14N ratios measured in wild-type filaments showed a general increase with time. The enrichment was relatively homogeneous in vegetative cells along individual filaments, while it was reduced in heterocysts. Heterocysts, however, accumulated recently fixed N at their poles, in which the cyanophycin plug [multi-l-arginyl-poly(l-aspartic acid)] is located. In contrast to the rather homogeneous label found along stretches of vegetative cells, 13C/12C and 15N/14N ratios were significantly different between filaments both at the same and different time points, showing high variability in metabolic states. A fraC fraD mutant did not fix N2, and the 13C/12C ratio was homogeneous along the filament, including the heterocyst in contrast to the wild type. Our results show the consumption of reduced C in the heterocysts associated with the fixation and export of fixed N and present an unpredicted heterogeneity of cellular metabolic activity in different filaments of an Anabaena culture under controlled conditions. IMPORTANCE Filamentous, heterocyst-forming cyanobacteria represent a paradigm of multicellularity in the prokaryotic world. Physiological studies at the cellular level in model organisms are crucial to understand metabolic activities and qualify specific aspects related to multicellularity. Here, we used stable isotopes (13C and 15N) coupled to LG-SIMS and NanoSIMS imaging to follow single-cell C and N2 fixation and metabolic dynamics along the filaments in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. Our results show a close relationship between C and N fixation and distribution in the filaments and indicate that wild-type filaments in a culture can exhibit a substantial variability of metabolic states. This illustrates how some novel properties can be appreciated by studying microbial cultures at the single-cell level.

Characterizing the "fungal shunt": Parasitic fungi on diatoms affect carbon flow and bacterial communities in aquatic microbial food webs.

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed "fungal shunt" can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton-bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

Sunda arc mantle source δ18O value revealed by intracrystal isotope analysis.

Magma plumbing systems underlying subduction zone volcanoes extend from the mantle through the overlying crust and facilitate protracted fractional crystallisation, assimilation, and mixing, which frequently obscures a clear view of mantle source compositions. In order to see through this crustal noise, we present intracrystal Secondary Ion Mass Spectrometry (SIMS) δ18O values in clinopyroxene from Merapi, Kelut, Batur, and Agung volcanoes in the Sunda arc, Indonesia, under which the thickness of the crust decreases from ca. 30 km at Merapi to ≤20 km at Agung. Here we show that mean clinopyroxene δ18O values decrease concomitantly with crustal thickness and that lavas from Agung possess mantle-like He-Sr-Nd-Pb isotope ratios and clinopyroxene mean equilibrium melt δ18O values of 5.7 ‰ (±0.2 1 SD) indistinguishable from the δ18O range for Mid Ocean Ridge Basalt (MORB). The oxygen isotope composition of the mantle underlying the East Sunda Arc is therefore largely unaffected by subduction-driven metasomatism and may thus represent a sediment-poor arc end-member.

Combining SIMS and mechanistic modelling to reveal nutrient kinetics in an algal-bacterial mutualism.

Microbial communities are of considerable significance for biogeochemical processes, for the health of both animals and plants, and for biotechnological purposes. A key feature of microbial interactions is the exchange of nutrients between cells. Isotope labelling followed by analysis with secondary ion mass spectrometry (SIMS) can identify nutrient fluxes and heterogeneity of substrate utilisation on a single cell level. Here we present a novel approach that combines SIMS experiments with mechanistic modelling to reveal otherwise inaccessible nutrient kinetics. The method is applied to study the onset of a synthetic mutualistic partnership between a vitamin B12-dependent mutant of the alga Chlamydomonas reinhardtii and the B12-producing, heterotrophic bacterium Mesorhizobium japonicum, which is supported by algal photosynthesis. Results suggest that an initial pool of fixed carbon delays the onset of mutualistic cross-feeding; significantly, our approach allows the first quantification of this expected delay. Our method is widely applicable to other microbial systems, and will contribute to furthering a mechanistic understanding of microbial interactions.

Shocked quartz in distal ejecta from the Ries impact event (Germany) found at ~ 180 km distance, near Bernhardzell, eastern Switzerland.

Impact ejecta formation and emplacement is of great importance when it comes to understanding the process of impact cratering and consequences of impact events in general. Here we present a multidisciplinary investigation of a distal impact ejecta layer, the Blockhorizont, that occurs near Bernhardzell in eastern Switzerland. We provide unambiguous evidence that this layer is impact-related by confirming the presence of shocked quartz grains exhibiting multiple sets of planar deformation features. Average shock pressures recorded by the quartz grains are ~ 19 GPa for the investigated sample. U-Pb dating of zircon grains from bentonites in close stratigraphic context allows us to constrain the depositional age of the Blockhorizont to ~ 14.8 Ma. This age, in combination with geochemical and paleontological analysis of ejecta particles, is consistent with deposition of this material as distal impact ejecta from the Ries impact structure, located ~ 180 km away, in Germany. Our observations are important for constraining models of impact ejecta emplacement as ballistically and non-ballistically transported fragments, derived from vastly different depths in the pre-impact target, occur together within the ejecta layer. These observations make the Ries ejecta one of the most completely preserved ejecta deposit on Earth for an impact structure of that size.

Microbial Sulfur Isotope Fractionation in the Chicxulub Hydrothermal System.

Target lithologies and post-impact hydrothermal mineral assemblages in a new 1.3 km deep core from the peak ring of the Chicxulub impact crater indicate sulfate reduction was a potential energy source for a microbial ecosystem (Kring et al., 2020). That sulfate was metabolized is confirmed here by microscopic pyrite framboids with δ34S values of -5 to -35 ‰ and ΔSsulfate-sulfide values between pyrite and source sulfate of 25 to 54 ‰, which are indicative of biologic fractionation rather than inorganic fractionation processes. These data indicate the Chicxulub impact crater and its hydrothermal system hosted a subsurface microbial community in porous permeable niches within the crater's peak ring.

Exceptional sulfur and iron isotope enrichment in millimetre-sized, early Palaeozoic animal burrows.

Pyrite-δ34S and -δ56Fe isotopes represent highly sensitive diagnostic paleoenvironmental proxies that express high variability at the bed (< 10 mm) scale that has so far defied explanation by a single formative process. This study reveals for the first time the paleoenvironmental context of exceptionally enriched pyrite-δ34S and -δ56Fe in bioturbated, storm-reworked mudstones of an early Ordovician storm-dominated delta (Tremadocian Beach Formation, Bell Island Group, Newfoundland). Very few studies provide insight into the low-temperature sulfur and iron cycling from bioturbated muddy settings for time periods prior to the evolution of deep soil horizons on land. Secondary ion mass spectroscopy (SIMS) analyses performed on Beach Formation muddy storm event beds reveal spatially distinct δ34S and δ56Fe values in: (a) tubular biogenic structures and trails (δ34S ~ +40‰; δ56Fe ~ -0.5‰), (b) silt-filled Planolites burrows (δ34S ~ +40‰; δ56Fe ~ +0.5 to + 2.1‰), and (c) non-bioturbated mudstone (δ34S ~ +35‰; δ56Fe ~ +0.5‰). δ34S values of well above + 40.0‰ indicate at least some pyrite precipitation in the presence of a 34S-depleted pore water sulfide reservoir, via closed system (Raleigh-type) fractionation. The preferential enrichment of 56Fe in Planolites burrows is best explained via microbially-driven liberation of Fe(II) from solid iron parent phases and precipitation from a depleted 54Fe dissolved Fe(II) reservoir. Rigorous sedimentological analysis represents a gateway to critically test the paleoenvironmental models describing the formation of a wide range of mudstones and elucidates the origins of variability in the global stable S and Fe isotope record.

Metallomics in deep time and the influence of ocean chemistry on the metabolic landscapes of Earth's earliest ecosystems.

Modern biological dependency on trace elements is proposed to be a consequence of their enrichment in the habitats of early life together with Earth's evolving physicochemical conditions; the resulting metallic biological complement is termed the metallome. Herein, we detail a protocol for describing metallomes in deep time, with applications to the earliest fossil record. Our approach extends the metallome record by more than 3 Ga and provides a novel, non-destructive method of estimating biogenicity in the absence of cellular preservation. Using microbeam particle-induced X-ray emission (µPIXE), we spatially quantify transition metals and metalloids within organic material from 3.33 billion-year-old cherts of the Barberton greenstone belt, and demonstrate that elements key to anaerobic prokaryotic molecular nanomachines, including Fe, V, Ni, As and Co, are enriched within carbonaceous material. Moreover, Mo and Zn, likely incorporated into enzymes only after the Great Oxygenation Event, are either absent or present at concentrations below the limit of detection of µPIXE, suggesting minor biological utilisation in this environmental setting. Scanning and transmission electron microscopy demonstrates that metal enrichments do not arise from accumulation in nanomineral phases and thus unambiguously reflect the primary composition of the carbonaceous material. This carbonaceous material also has δ13C between -41.3‰ and 0.03‰, dominantly -21.0‰ to -11.5‰, consistent with biological fractionation and mostly within a restricted range inconsistent with abiotic processes. Considering spatially quantified trace metal enrichments and negative δ13C fractionations together, we propose that, although lacking cellular preservation, this organic material has biological origins and, moreover, that its precursor metabolism may be estimated from the fossilised "palaeo-metallome". Enriched Fe, V, Ni and Co, together with petrographic context, suggests that this kerogen reflects the remnants of a lithotrophic or organotrophic consortium cycling methane or nitrogen. Palaeo-metallome compositions could be used to deduce the metabolic networks of Earth's earliest ecosystems and, potentially, as a biosignature for evaluating the origin of preserved organic materials found on Mars.

Resting Stages of Skeletonema marinoi Assimilate Nitrogen From the Ambient Environment Under Dark, Anoxic Conditions.

The planktonic marine diatom Skeletonema marinoi forms resting stages, which can survive for decades buried in aphotic, anoxic sediments and resume growth when re-exposed to light, oxygen, and nutrients. The mechanisms by which they maintain cell viability during dormancy are poorly known. Here, we investigated cell-specific nitrogen (N) and carbon (C) assimilation and survival rate in resting stages of three S. marinoi strains. Resting stages were incubated with stable isotopes of dissolved inorganic N (DIN), in the form of 15 N-ammonium (NH4+ ) or -nitrate (NO3- ) and dissolved inorganic C (DIC) as 13 C-bicarbonate (HCO3- ) under dark and anoxic conditions for 2 months. Particulate C and N concentration remained close to the Redfield ratio (6.6) during the experiment, indicating viable diatoms. However, survival varied between <0.1% and 47.6% among the three different S. marinoi strains, and overall survival was higher when NO3- was available. One strain did not survive in the NH4+ treatment. Using secondary ion mass spectrometry (SIMS), we quantified assimilation of labeled DIC and DIN from the ambient environment within the resting stages. Dark fixation of DIC was insignificant across all strains. Significant assimilation of 15 N-NO3- and 15 N-NH4+ occurred in all S. marinoi strains at rates that would double the nitrogenous biomass over 77-380 years depending on strain and treatment. Hence, resting stages of S. marinoi assimilate N from the ambient environment at slow rates during darkness and anoxia. This activity may explain their well-documented long survival and swift resumption of vegetative growth after dormancy in dark and anoxic sediments.

Uncovering and quantifying the subduction zone sulfur cycle from the slab perspective.

Sulfur belongs among H2O, CO2, and Cl as one of the key volatiles in Earth's chemical cycles. High oxygen fugacity, sulfur concentration, and δ34S values in volcanic arc rocks have been attributed to significant sulfate addition by slab fluids. However, sulfur speciation, flux, and isotope composition in slab-dehydrated fluids remain unclear. Here, we use high-pressure rocks and enclosed veins to provide direct constraints on subduction zone sulfur recycling for a typical oceanic lithosphere. Textural and thermodynamic evidence indicates the predominance of reduced sulfur species in slab fluids; those derived from metasediments, altered oceanic crust, and serpentinite have δ34S values of approximately -8‰, -1‰, and +8‰, respectively. Mass-balance calculations demonstrate that 6.4% (up to 20% maximum) of total subducted sulfur is released between 30-230 km depth, and the predominant sulfur loss takes place at 70-100 km with a net δ34S composition of -2.5 ± 3‰. We conclude that modest slab-to-wedge sulfur transport occurs, but that slab-derived fluids provide negligible sulfate to oxidize the sub-arc mantle and cannot deliver 34S-enriched sulfur to produce the positive δ34S signature in arc settings. Most sulfur has negative δ34S and is subducted into the deep mantle, which could cause a long-term increase in the δ34S of Earth surface reservoirs.

In situ Rb-Sr dating of slickenfibres in deep crystalline basement faults.

Establishing temporal constraints of faulting is of importance for tectonic and seismicity reconstructions and predictions. Conventional fault dating techniques commonly use bulk samples of syn-kinematic illite and other K-bearing minerals in fault gouges, which results in mixed ages of repeatedly reactivated faults as well as grain-size dependent age variations. Here we present a new approach to resolve fault reactivation histories by applying high-spatial resolution Rb-Sr dating to fine-grained mineral slickenfibres in faults occurring in Paleoproterozoic crystalline rocks. Slickenfibre illite and/or K-feldspar together with co-genetic calcite and/or albite were targeted with 50 µm laser ablation triple quadrupole inductively coupled plasma mass spectrometry analyses (LA-ICP-MS/MS). The ages obtained disclose slickenfibre growth at several occasions spanning over 1 billion years, from at least 1527 Ma to 349 ± 9 Ma. The timing of these growth phases and the associated structural orientation information of the kinematic indicators on the fracture surfaces are linked to far-field tectonic events, including the Caledonian orogeny. Our approach links faulting to individual regional deformation events by minimizing age mixing through micro-scale analysis of individual grains and narrow crystal zones in common fault mineral assemblages.

Timing and origin of natural gas accumulation in the Siljan impact structure, Sweden.

Fractured rocks of impact craters may be suitable hosts for deep microbial communities on Earth and potentially other terrestrial planets, yet direct evidence remains elusive. Here, we present a study of the largest crater of Europe, the Devonian Siljan structure, showing that impact structures can be important unexplored hosts for long-term deep microbial activity. Secondary carbonate minerals dated to 80 ± 5 to 22 ± 3 million years, and thus postdating the impact by more than 300 million years, have isotopic signatures revealing both microbial methanogenesis and anaerobic oxidation of methane in the bedrock. Hydrocarbons mobilized from matured shale source rocks were utilized by subsurface microorganisms, leading to accumulation of microbial methane mixed with a thermogenic and possibly a minor abiotic gas fraction beneath a sedimentary cap rock at the crater rim. These new insights into crater hosted gas accumulation and microbial activity have implications for understanding the astrobiological consequences of impacts.