The highly variable δ13C calcite values between different fractures and within single crystals point to spatiotemporal variation of the processes that lead to calcite precipitation. We focus our discussion on processes producing the youngest calcites, which feature large δ13C calcite excursions. The older type predates the impact and dates back to 600–400 Ma (Supplementary Fig. 2) and shows no δ13C signatures diagnostic for methane cycling. In order to link the mineral data to the present gas in the system, the discussion starts with interpretations of the gas compositions that exist from the new boreholes and from previous prospecting.

Interpretation of the origin of hydrocarbon gases is typically based on diagnostic geochemical signatures, normally by using discrimination diagrams (Fig. 7, based on a global compilation32) and a holistic approach including the geological context. The most widely used discrimination diagram is the ratio of methane to higher hydrocarbons, C 1 /(C 2 + C 3 ), versus δ13C CH4 (Fig. 7a). This differentiates microbial gas which usually has high C 1 /(C 2 + C 3 ) (>1000)26 from the typically lower ratios of thermogenic methane (<50)33,34. However, abiotic gas may also show high C 1 /(C 2 + C 3 )35,36 and cannot be excluded based on this ratio. For δ13C CH4 , there is typically a difference between methane sources, ranging from the substantially 13C-depleted microbial, through moderately 13C-depleted thermogenic to isotopically heavier abiotic methane26. Microbial methanogenesis can be divided into the carbonate reduction pathway and acetate (methyle-type) fermentation37, of which the former involves a larger kinetic carbon isotope effect26. At Siljan, a dominantly microbial gas fraction is suggested by the light δ13C values of the methane (−64 ± 2‰, Fig. 7a). However, the C 1 /(C 2 + C 3 ) values are slightly lower than expected from a pure microbial gas and therefore point to a mixed origin, as indicated by the position at the border between the microbial carbonate reduction and early mature thermogenic fields (Fig. 7a). Regarding the higher hydrocarbons, it has been demonstrated that microbial ethano- and propanogenesis occur in deeply buried marine sediments38, and the former also near gas wells in western Canada39. Presence of ethane and propane is thus not a definite marker for thermogenic gas. However, in a microbial gas, the presence of C 4+ gases (detectable n-C 4 , i-C 4 , i-C 5 , Supplementary Data 8) is not expected33,34, and the δ13C C2 values are typically not as heavy as those measured (−28 ± 2‰)40, in particular in comparison to the light δ13C CH4 values (−64 ± 2‰), which indicate thermogenic contribution. These relatively heavy δ13C C2 values and even heavier δ13C C3 values speak against a significant contribution from abiotic gas, which generally features decreasing δ13C values with higher carbon number of the homologues41. At other igneous rock sites in South Africa, Canada, and Scandinavia, abiotic methane shows lighter δ13C values (−50‰10,41) than the typically assigned values of abiotic methane (i.e. >−20‰42), although not as low as the methane at Siljan.

Fig. 7 Gas composition discrimination diagrams. (a) δ13C CH4 versus C 1 /(C2 + C 3 ). (b) δ2H CH4 versus δ13C CH4. (c) δ13C CH4 versus δ13C CO2 (adapted from32). Position of the gases from boreholes VM2 (diamonds = gas samples, triangle = water samples), VM5 (square) and drinking water well (circle) are shown. Genetic gas field abbreviations denote: CR CO 2 reduction, F methyl-type fermentation, SM secondary microbial, EMT early mature thermogenic, OA oil-associated thermogenic, LMT late mature thermogenic gas. Gas data were extracted from the database of AB Igrene (Supplementary Data 8), and from45 (drinking well at Gulleråsen close to Solberga, δ13C CH4 : −60.3‰, δ2H CH4 : −269‰). The sampling site of the gas data in borehole VM2 corresponds to the uppermost 13C-rich calcites in this borehole, whereas the other gas-sampled borehole (VM5) is just adjacent to other boreholes sampled for calcite (VM- and 01-boreholes). Errors (2σ) are within the size of the symbols Full size image

The isotopic hydrogen signature (δ2H CH4 ) of −240‰ SMOW for the VM5 borehole gas is, when plotted against δ13C CH4 (Fig. 7b) also in the microbial carbonate reduction field32, however, close to early mature thermogenic gas and fermentation type microbial gas. Additionally, the Siljan gas samples are overlapping with δ2H CH4 ranges of abiotic sources at other sites10, meaning that abiotic contribution cannot be ruled out based on the δ2H CH4 composition. Overall, the position of the gas samples at borders or within multiple empirically defined zones on the discrimination plots (Fig. 7a, b) shows that these plots alone are not diagnostic for any single process and/or gas origin.

The heavy δ13C CO2 (c. + 5–8‰, Fig. 7c, Supplementary Data 8) of the Siljan samples is typical for microbial methanogenesis through carbonate reduction32 and thus another feature supporting a dominantly microbial gas origin. These δ13C CO2 values are characteristic for secondary microbial methane43 formed following microbial utilization of primary thermogenic hydrocarbons (e.g. petroleum, seep oils and lighter hydrocarbons), which is supported by other biodegradation signatures. These signatures include high C 2 to C 3 ratios owing to that ethane is relatively resistant to biodegradation compared to the C 3+ homologues44. Biodegradation also discriminates against 13C C3 , leading to isotopically heavy residual propane44. In the Siljan gas, the anomalously heavy δ13C C3 values compared to the δ13C C2 values, the high C 2 to C 3 ratios that are far from the normal range for thermogenic gases, as well as other signatures presented in Supplementary Note 1, thus point to biodegradation, but to an unknown degree. The removal of the higher hydrocarbons during biodegradation increases the C 1 /(C 2 -C 3 ), which complicates the estimation of the mixing proportions between microbial and thermogenic gas.

All the gas data together thus point to a gas that is to a large extent microbial and to a significant extent thermogenic. Although abiotic gas contribution cannot be directly identified in the investigated (dominantly sedimentary) aquifer, it cannot be ruled out, at least not in the deep granite fracture system, because none of the borehole samples isolates gas from the crystalline aquifer alone. Theoretically, abiotic gas contribution from the granite fractures may thus be masked by gases from the sedimentary rock fractures. Variably depleted δ13C CH4 signatures from previous investigations16,45, summarized in Supplementary Note 2, are in accordance with the mixed gas of interpreted dominantly microbial and thermogenic origin detected in the present study (Fig. 7). Hence, although the results presented here and previously are not generally supportive of abiotic gas, such gas cannot be fully ruled out, at least not in the deeper, granitic system.

For the mineralogical record, the significantly 13C-enriched calcite observed in the fractures in limestone (δ13C calcite values as heavy as +21.5‰, Fig. 4b) and granite (up to +18.9) is evidence for formation following microbial methanogenesis in situ, owing to the discrimination that occurs against 13C during methanogenesis that leaves 13C-rich residual carbon behind25,27,46. However, the presence of 13C-rich calcite cannot completely rule out minor abiotic gas fractions. In addition, the FA n-C 12 to n-C 18 , particularly the odd chain and branched iC 15 , aiC 15 , n-C 15 , 12Me-C 16 , aiC 17 , and 12OH-C 18 as well as the n-alcohols and the 1-o-n-hexadecylglycerol preserved within 13C-rich, methanogenesis-related, calcite coatings are support for in situ microbial activity. These preserved FA can be tied to fermentation47, sulfate reduction by bacteria48 and other microbial processes (Supplementary Note 3), but not specifically to methanogens (archaea), which do not produce phospholipid fatty acids.

The U-Pb ages suggest that methanogenesis in the sedimentary and granite aquifers at Siljan led to precipitation of 13C-rich calcite on several occasions, from 80 ± 5 to 22.2 ± 2.5 Ma (Fig. 5). The distribution of this calcite marks microbial methanogenesis in the upper 214 m of the fractured crystalline rock in the crater structure (to depths of 620 m) and in the overlying sedimentary rock fractures over a depth span of more than 200 m. The substantial 13C calcite and 13C CO2 enrichments occurring in the limestone aquifer are noteworthy, because dilution by the C isotope signature of dissolved inorganic carbon (DIC) derived from limestone (δ13C: 0 to +2‰49) would be expected. To explain this feature, we propose local influence from kinetic microbial processes on the δ13C DIC signature in the Siljan aquifer, in common with observations from other deep energy-poor fracture system28,50 (Supplementary Note 4). This phenomenon should be particularly important in pore space infiltrated by gases, bitumen or seep oils, as shown by spatial relation of these features to significantly 13C-rich calcite (Fig. 3). Preserved hydrocarbon n-alkane pattern of calcite in bitumen-bearing fractures of the sedimentary rock and at the sediment-granite interface (VM2:212; VM1:251, Fig. 6) is indication for thermal- and biodegradation. It has previously been reported that biomarkers in bitumen in sedimentary rock fractures link its origin to shales and that mobilization and degradation of hydrocarbons have occurred on several events in the fracture systems22 (additional biomarker support in Supplementary Note 1).

Methanogenesis is commonly associated with sulfate-poor biodegraded petroleum reservoirs51 and initial steps of anaerobic utilization of organic matter (fermentation) involve hydrolysis followed by bacterial acetogenesis that converts volatile fatty acids into acetic acid, H 2 and CO 2 52. Alternatively, H 2 is produced by aromatization of compounds present in the seep oil51. Methanogenesis through CO 2 reduction, with H 2 as electron donor, has been proposed to be the dominant terminal process in petroleum biodegradation in the subsurface47, and this appears also to be the case at Siljan based on the widespread and pronounced heavy δ13C calcite and δ13C CO2 values (Figs. 4 and 7c). In sulfate-rich reservoirs, microbial sulfate reduction (MSR) can be involved in degradation of hydrocarbons. In the Siljan fractures, pyrite occasionally occurs together with 13C-enriched calcite. Pyrite formed by MSR is typically strongly depleted in 34S53. The very low minimum δ34S pyrite values (−40‰V-CDT, Supplementary Fig. 1) is thus proposed to reflect MSR. However, groundwater in granite fractures of adjacent boreholes show very low sulfate concentrations, 4.3–6.6 mg L−145, suggesting a generally low potential for MSR in that aquifer. Although anaerobic oxidation of organic matter by MSR can produce CO 2 that can be utilized by methanogens43, it did probably not result in large quantities of methane because sulfate reducers outcompete methanogens for H 2 and other substrates when sulfate concentrations are elevated54. Instead, fermentation likely dominated initial degradation steps of organics in the system providing H 2 for the methanogens to perform reduction of CO 2 . Furthermore, the low salinity in the deep granite aquifer45 is favorable for microbial methanogenesis55. Secondary methane formation following microbial utilization of primary thermogenic hydrocarbons typically involves large 13C-enrichment in carbonate32, as manifested by heavy δ13C calcite (Fig. 4) and δ13C CO2 (Fig. 7c).

Overall, the geological setting with relatively low temperatures and shallow reservoir with abundant seep oil/bitumen are, together with gas signatures and 13C-enriched calcite, in favor of formation of secondary microbial methane produced following biodegradation of thermogenic hydrocarbons (gas, seep oil and bitumen). The primary thermogenic gas remains as a minor biodegraded mixing fraction, in common with secondary methane reservoirs elsewhere32,43. In the deeper granite system, contribution from abiotic gas sources may also have been involved.

During AOM, a phenomenon where methanotrophs can act in syntrophic relationship with MSR, carbonate may precipitate and inherit the significantly 13C-depleted signatures of the methane56. The light δ13C calcite values detected at the sediment-granite contact at Siljan (−52.3‰, Fig. 4) are thus proposed to reflect AOM (Supplementary Note 5 describes more moderately 13C-depleted calcite). The U-Pb age of this calcite shows that AOM dates back at least 39 ± 3 Myr. The δ13C calcite values point to utilization of methane of dominantly microbial origin10,26, because thermogenic and abiotic methane are usually heavier57. The δ13C signature of carbonate originating from oxidized methane is typically diluted by other relatively 13C-rich dissolved carbon species prior to incorporation in calcite29. When taking such dilution into account, it is likely that the source methane was isotopically light, in line with the δ13C CH4 composition (−64 ± 2‰) of dominantly microbial origin (with a minor thermogenic and possibly a minor abiotic component) in boreholes VM2/5. Furthermore, in the sample with the most 13C-depleted (AOM-related) calcite, co-genetic pyrite has low minimum δ34S pyrite values (−18.7‰, Supplementary Fig. 1) reflecting large 32S enrichment characteristic for MSR-related sulfide53. This finding, together with MSR-related58,59 branched fatty acids (ai-C 15:0 , 12Me-C 16:0 , ai-C 17:0 , 12OH-C 18:0, Fig. 6b) mark coupled AOM-MSR at the sedimentary-granite rock contact. Another noteworthy feature is the overall large δ34S pyrite spans that mark MSR-related reservoir effects throughout the fracture system (Supplementary Fig. 5 and Note 5).

Several precipitation events are recorded by the intra-crystal variability of the C and Sr isotopes, and the U-Pb age groups. Our interpretation is that these precipitation events were caused by fracture reactivations, as presented in detail in Supplementary Note 6. In summary, there are tectonic events in the far-field and uplift events that temporally coincide with the ages of the methane related calcite at Siljan. Methane cycling can thus be related to these fracture reactivation events that are more than 300 million years younger than the impact.

A conceptual model for methane accumulation in the Siljan impact structure, as outlined in Fig. 8, has its basis in the isotopic inventory of secondary fracture minerals and gases. The microbial methanogenic processes date back at least to the Late Cretaceous (Fig. 5), although there are fractures in the granite that formed significantly earlier (Supplementary Fig. 2). The gas compositions corroborate that the gas in the sedimentary reservoir is microbial with contribution from a biodegraded thermogenic end-member linked to thermal maturity of black shales in the sedimentary pile, and perhaps a minor abiotic gas fraction. In the upper part of the sedimentary successions there is local seepage to the surface, as shown by methane in drinking water wells45. The isotopic composition of methane in such a well45 fits with both microbial and early mature thermogenic origin (Fig. 7b). Bitumen can be mobilized when thermally affected60. Bitumen and seep oil migration from the organic-rich shales into other sedimentary rock units and into the fractured granitic basement have thus likely been initiated when the sediments were thermally matured, either as a result of the heat from the impact23 or due to subsidence related to Caledonian foreland basin crustal depression61. The bitumen and seep oil occurrences (along with migrated thermogenic gas) provided energy for the indigenous microbial communities in the deep subsurface, as shown by the spatial relation to 13C-rich calcite. Deep abiotic gas contribution to the methane accumulations in the granite fractures cannot be ruled out. However, the apparently higher (but not yet quantified) abundance of gas beneath the sedimentary rock in the crater rim (Fig. 4) than in the central dome16, the input of shale-derived hydrocarbons to the granite fractures, and the similar 13C-enrichment of calcite in granite and sedimentary fractures point to similar formation and accumulation of methane in the granite fracture network as in the sedimentary rock. The dominantly Eocene–Miocene ages of the 13C-rich calcite indicate that the major microbial utilization of the hydrocarbons in the deep fractures occurred when temperatures were more favorable (<50 °C) for microbial activity, in line with the uplift and subsidence history of the south-central Fennoscandian shield62. The Eocene–Miocene microbial activity is proposed to be linked to regional re-opening of bitumen-bearing fracture sets. This enabled circulation of groundwater along flow paths with substrates accessible to the microbes in the form of bitumen/oil coatings, as well as facilitated circulation of biodegradable thermogenic gas in the deep reservoir. The spatial relation of 13C-enriched calcite and biodegraded bitumen/seep oil suggests secondary methane formation following anaerobic degradation of organic matter. This fermentation process produces H 2 for utilization by methanogens through reduction of CO 2 formed during biodegradation or occurring in the aquifer. The kinetic microbial processes producing methane resulted in large isotopic fractionations, as observed in the gases and secondary carbonates. Taken together, there are numerous lines of evidence in favor of long-term microbial methane formation in the Siljan crater, likely fueled by thermogenic gas, seep oil and bitumen mobilized from shales in the sedimentary successions and transported through fracture conduits to the deeper granite aquifer. The sedimentary successions, in turn, acted as cap rocks for the gas in the granite fractures.

Fig. 8 Conceptual model of the gas accumulation in the Siljan ring impact structure. Thermogenic gas formed in the (to varying degree) mature Silurian and Ordovician black shales in the sedimentary strata. This gas and related seep oil and bitumen dispersed in the adjacent sedimentary successions with local surficial seepage. Downward migration of these hydrocarbons has occurred into the granitic basement during fracture reactivation events. Biodegradation of the hydrocarbons has occurred in the fracture system and secondary methane has formed in situ. The mixed gas, of microbial (dominantly) and biodegraded thermogenic type, which also may have an abiotic end-member, has accumulated at the sedimentary-granite contact where anaerobic oxidation of methane has occurred Full size image

Input of hydrocarbons to the deep microbial communities has potential to result in accumulation of methane in basement fracture networks beneath sedimentary cap rocks. A relationship between 13C-rich calcite and bitumen like at Siljan occurs in deep crystalline rock fractures at Forsmark, Sweden25 and solid and gaseous hydrocarbon occurrences of sedimentary origin occur in fractured crystalline basement rocks on the British Isles63, Australia64, and the United States65. Whereas microbial generation of economic accumulations of methane within organic-rich shale are known from several locations66, the extent of gas accumulations in the upper crystalline continental crust buried beneath sedimentary successions and in fractured impact structures are less explored. The upper crystalline continental crust environment makes up one of the largest, but yet least surveyed, deep biosphere habitats on Earth. The extent, continuity and physicochemical prerequisites for gas accumulation here require more attention in order to assess the significance of this underexplored greenhouse gas source on a global scale.

In the Siljan impact structure, a relation between methane cycling and deep subsurface life is evident. The physical influence of the actual impact and the long-term effects are manifested by, first, abundant fracturing compared to surrounding rocks15 which is particularly important in igneous lithologies where colonization is restricted by the pore space2; second, dislocation of organic-rich sedimentary rocks that provide pathways for surficial organics to the deep endolithic communities; and third, development of a cap rock enclosing methane at depth. These effects collectively enable microbial colonization of the crater hundreds of millions of years after the impact. Our findings of widespread long-lasting deep microbial methane-forming communities in the Siljan crater support the hypothesis that impact craters are favorable for deep microbial colonization2,3. However, the link between microbial methanogenesis and organics from Paleozoic shale challenges the use of the Siljan crater as an analog for instantaneous extraterrestrial impact-related colonization. In an astrobiological context, the Siljan crater findings are nevertheless of large importance, as they display that multi-disciplinary micro-scale constraints for microbial activity (stable isotopes, geochronology, biomarkers) are needed to confirm that colonization and impact in ancient crater systems are coeval. This is particularly important because post-impact microbial colonization will likely occur in these favorable deep microbial settings and can thus easily be misinterpreted as impact related. Finally, the methods we have used here to provide the first evidence of long-term microbial methane formation and accumulation in a terrestrial impact crater would be optimal to apply to other impact-crater fracture systems, including methane emitting craters on Mars67, in order to enhance the understanding of microbial activity and gas cycling in this underexplored environment.