Extreme 13C depletion of carbonates formed during oxidation of biogenic methane in fractured granite

Precipitation of exceptionally 13C-depleted authigenic carbonate is a result of, and thus a tracer for, sulphate-dependent anaerobic methane oxidation, particularly in marine sediments. Although these carbonates typically are less depleted in 13C than in the source methane, because of incorporation of C also from other sources, they are far more depleted in 13C (δ13C as light as −69‰ V-PDB) than in carbonates formed where no methane is involved. Here we show that oxidation of biogenic methane in carbon-poor deep groundwater in fractured granitoid rocks has resulted in fracture-wall precipitation of the most extremely 13C-depleted carbonates ever reported, δ13C down to −125‰ V-PDB. A microbial consortium of sulphate reducers and methane oxidizers has been involved, as revealed by biomarker signatures in the carbonates and S-isotope compositions of co-genetic sulphide. Methane formed at shallow depths has been oxidized at several hundred metres depth at the transition to a deep-seated sulphate-rich saline water. This process is so far an unrecognized terrestrial sink of methane.

In contrast to shallow marine sediments where sulphate is generally depleted with depth, numerous granitic shield areas worldwide host sulphate-rich water at great depth, in brines derived from a proposed marine source such as basinal brines from overlying sedimentary rock successions [19][20][21] . When the basement rock eventually has been re-exposed after erosion of the sedimentary successions, repeated infiltrations of surface water, including glacial melt water, marine water and meteoric water, are possible and controlled by factors such as location and topography. Fresh water (meteoric) therefore typically occupies the upper bedrock fracture volume, a mixture of brackish and glacial water is found at intermediate depth, and saline sulphaterich water is generally restricted to great depth, such as described for the crystalline bedrock of southeastern Sweden in the Baltic Shield 22,23 . Owing to changing hydrochemical conditions during infiltration of occasionally carbon-rich water from the terrestrial environment above and increased alkalinity in response to microbial breakdown of organic matter and methane, carbonates (as a rule calcite) can become oversaturated and precipitate on the fracture walls. The stable carbon isotope composition of these calcites can thus be used to identify biological processes in the fractured upper crust, especially across depth-related hydrochemical boundaries, at which microbial communities thrive 14 . However, low-temperature calcite has only been sparsely used to reveal microbial processes in deep bedrock fractures because of its' rare, fine-grained and finely zoned nature, as well as because of challenges and costs of sampling representative calcite crystals deep in the Earth's crust 3,24-28 . In the present study we have examined more than 40 cored boreholes drilled in one of the most extensively studied granitic bedrock sites in the world, the Laxemar area, Sweden, and sampled fine-grained-zoned calcite crystals throughout the upper 1,000 m of the bedrock. These calcites postdate fluid-inclusionrich calcite formed at 450°C (refs 25,29) and have single-phased fluid inclusions suggesting, although few in numbers, formation at o50°C (ref. 30). The stable isotopic composition of carbon and oxygen was determined in transects across the numerous growth zones present within the calcite crystals, in a total of more than 430 analyses by secondary ion mass spectrometry (SIMS; 10 mm wide, 1-2 mm deep spots). The sulphur isotope inventory, 34 S/ 32 S presented as d 34 S, of co-genetic pyrite crystals were explored by similar SIMS analyses (n ¼ 101), which can reveal coeval SRB activity 31 . The organic inventory of the calcites and the groundwater chemistry are also explored. Thereby, the hydrochemical and biological evolution in situ in the fractures could be revealed in detail.
Here we report extremely 13 C-depleted carbonates precipitated in fractures deep within granitoid rocks. The d 13 C values are by far the lowest ever reported for carbonates (d 13 C: À 125%) and are suggested to be related to microbial sulphate-dependent anaerobic oxidation of biogenic methane. Methane oxidation occurring in the energy-limited and carbon-poor groundwater systems deep within Earth's terrestrial landscape evidently results in a unique carbon isotope variability compared with other environments, such as the well-characterized sedimentary AOM settings.

Results
Extreme carbon isotope variation of calcite in rock fractures. Within the individual calcite crystals (Fig. 1) there is large d 13 C variation, of up to almost 110% between different growth zones, thus revealing large temporal variation of the processes in the fractures (Figs 2b and 3 Supplementary Fig. 1). The range of the d 13 C values depends on the depth at which the calcites were precipitated and reside and, therefore, mark different biological processes at different depths within the fracture network (Fig. 2b). In the upper 200 m several calcites have zones with positive d 13 C values (for example, in Fig. 3a). Such 13 C-enriched carbonate pools develop where methanogenesis occurs 32,33 , leading to a 12 C-rich methane and 13 C-rich residual CO 2 from which calcite formed. Although measurements of methane are relatively scarce in this area and the concentrations are generally low (o0.2 ml l À 1 ), anomalies with elevated concentrations (40.6 ml l À 1 , related to waters with abundant cultivatable methanogens and high concentrations of dissolved organic carbon, DOC 34 ), are indeed restricted to shallower depths than the sulphate-rich saline waters ( Supplementary Table 2). The organic input to the system in the form of descending DOC, which fuels the methanogenesis, evidently has a surficial origin.
Extreme 13 C depletion of À 125 to À 50% was observed in calcites between 200 and 730 m. This 13 C depletion is so strong that it cannot originate from any other source compound than methane. For methane to be incorporated in calcite, it first has to be oxidized, in this particular setting by anaerobic mechanisms because of consistently reducing conditions (absence of O 2 ; Eh ranges from À 210 to À 380 mV (ref. 22)), far below the redox transition at 20 m depth 35 . In addition, aerobic methane oxidation would have resulted in calcite dissolution rather than precipitation 36 . The oxidation of methane causes a decrease in 13 C in the produced CO 2 , with fractionation (a CH4-CO2 ) commonly in the order of 1.009-1.024 (refs 17,37,38). The calcites with the lightest d 13 C must therefore have been produced from a source methane with d 13 C lighter than À 100%.
Consequently, the C-isotope data suggest a dominantly biological origin of the source methane because thermogenic methane rarely exceed values lighter than À 50% (ref. 17). Thirteen fractures from boreholes spread out over a 3 Â 3 km area featured AOM-related calcite, which in seven fractures had lighter d 13 C than À 90% (four lighter than À 100%), marking that extraordinary 13 C depletions are widespread in this environment and not single occurrences. All of these 13 fractures carried calcite with minimum d 13 C values that were similar to or lighter than reported for AOM-related authigenic marine carbonate ( À 69% (ref. 5)). Minimum sulphur isotope values of pyrite (d 34 S pyrite , full results in Supplementary Table 3) co-genetic with AOM calcite are mainly 22-33% lighter, and in the rare case 46.8% lighter, than the anticipated source sulphate ( þ 25% (ref. 31)). This is evidence of activity of SRB, which preferentially use the lighter S isotope in their metabolism, with large related fractionation between sulphate and produced sulphide as a consequence 39 . Large fractionation of the sulphur isotopes characterizes AOMrelated microbial sulphate reduction, particularly when methane concentrations are low, such as at SMTZ 40 . These findings are in accordance with previous observations of SRB-related pyrite in the deep fracture system at Laxemar 31 ( Fig. 2d) and at nearby Ä spö 3 . In the latter case, pyrite was accompanied by 13 C-depleted calcite (at lightest À 46.5% V-PDB; Vienna Pee Dee Belemnite), although not specifically interpreted to be AOM-related 3 .
Other d 13 C values than those related to methanogenesis (heavy) or AOM (light) within the crystals reflect a dominance of inorganic C ( À 10 to À 3%) observed at all depth, and dominance of C from microbial degradation of organic matter (down to À 30±5, potentially reflecting mixing of different C sources, including a minor AOM part). The latter d 13 C signatures appear down to À 730 m, which thus represent a depth limit for the descent of DOC from the terrestrial ecosystem above.
Organic inventory of the calcite crystals. In addition to the diagnostic 13 C-depleted carbonate, the presence of typically 13 C-depleted specific lipid biomarker signatures of ANME and its SRB partner frequently serve as diagnostic tracers for active or fossil AOM in marine settings 14,18,41 . In the tiny calcite crystals from bedrock fractures studied here, it is impossible to determine d 13 C of specific organic compounds. Nevertheless, our coupled gas chromatograph mass spectrometer (GC/MS) and Time-of-Flight (ToF)-SIMS analyses revealed signatures of methylated anteiso-(ai) and iso-(i) fatty acids (ai-C 15:0 , 10Me-C 16:0 , i-and ai-C 17:0 , Fig. 4), hopanes (norhopane and hopene) and several non-isoprenoidal dialkyl glycerol diethers with branched alkyl, cyclohexyl or cyclopropyl units (DAGE, Fig. 4, Supplementary Table 4), in the AOM-related calcites. These organic molecules clearly originate from in situ microbial activity because they are mostly SRB-specific, and in the case of DAGEs, and ai-C 15:0 highly AOM-specific 8,9,18 . This suggests that a similar twomembered ANME-SRB microbial consortium as described at other AOM sites 13,18 operated here as well. The organic material within the calcites generally clusters along intracrystal grain boundaries (the borders between different growth zones), as shown by ToF-SIMS analyses of crystal interiors. Raman spectroscopy and ToF-SIMS revealed that there is no abundant organic material in the calcite matrix apart from along these grain boundaries. The GC/MS and ToF-SIMS analyses of calcite leachates (from up to 220 mg large calcite samples), which represent accumulated organic material from grain boundaries of several crystals showed more pronounced signatures of organic compounds compared with spot analyses of the crystals. Raman spectroscopy and scanning electron microscopy (SEM) investigations showed, however, that fine-grained clay minerals   [42][43][44] . Significant, but not complete, fossilization of the microorganisms in biofilms at the grain boundaries is therefore proposed on the basis of the observations of the interiors of the AOM crystals.
General mechanisms of methane formation and oxidation. Taken together, the d 13 C data of the calcites suggest a mechanism whereby methane is formed in shallow fractures (Fig. 2a, 22,46 , and heavier than all other potential source water types). The AOM-and methanogenesisrelated calcite generally do not make up the whole crystals but can be both preceded and succeeded by calcite with distinctly different d 13 C-and d 18 O-signatures (Fig. 3b-e). This reflects that methanogenesis and AOM have been initiated but also terminated in response to changing hydrochemical conditions in the fracture system. The AOM-related calcite growth is frequently accompanied by relative enrichment in 18 O in the calcites, indicative of increased proportion of marine water relative to fresh waters (Fig. 3b,c), further supported by brackishmarine salinities (2.4 wt.% NaCl) estimated in the only AOM sample with measurable fluid inclusions (at À 642 m, Supplementary Table 6, Supplementary Note 2, Supplementary  Figs 3 and 4). This is in line with the sequence of infiltration of surficial water during the repeated Pleistocene deglaciation cycles in this area, involving high hydraulic heads that pressed glacial melt water several hundred metres into the fracture network and subsequent marine transgressions over the depressed land masses 22 . During the marine transgression, dense marine (brackish) water was brought on top of light fresh water in the fractures. In such a system advection can occur, under ideal conditions, with a velocity of up to the order of 1 cm s À 1 (see Supplementary Note 3), corresponding to a travel time of about a single day for a vertical downward distance of 500 m. In contrast to methane diffusion, which is too slow (Supplementary Note 3), advective transport, particularly favourable after a glaciation but certainly possible also in other situations, is thus fully capable of carrying dissolved methane deep into the crust, down to the deep sulphate-rich brines, within a short period of time.
Marine waters descending through organic-rich sediments will have delivered to the superficial fractures dissolved organic matter that favour methanogenesis but also dissolved sulphate that will lead to methane oxidation. In near-surface fractures, however, pyrite crystals with extremely heavy d 34 S values of up to more than þ 90% V-CDT (Vienna Canyon Diablo Troilite) exist 31 . This is evidence for extensive sulphate consumption by SRB because values as heavy as these can have been formed only where the dissolved sulphate pool was nearly exhausted in the fracture. Consequently, because sulphate was exhausted, methane concentrations were allowed to build up and descend downwards.    Supplementary Table 4.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8020 ARTICLE Dominantly pre-Holocene methane oxidation. The Holocene post-glacial marine transgression generally did not reach as far inland as the investigated boreholes. Hence, the groundwater captured by these is still made up of a significant portion of glacial meltwater on top of the deep saline water. This C-poor Holocene glacial meltwater mixed with and/or replaced an older marine water, to which AOM is possibly related. Therefore, ongoing AOM is not believed to be significant at this site. This scenario is supported by (1) AOM-related and methanogenesis-related calcite being succeeded by calcite without AOM d 13 C signature of fresh water type (dominantly glacial or meteoric water replacing the marine/saline water, Fig. 3d,e), (2) the overall heavier d 18 O of the AOM-and methanogenesis-related calcites (dominantly marine) than those expected for calcite precipitated from the current groundwater with a large glacial component at great depth and a large meteoric component at shallow depth (Fig. 2c) and (3) (Fig. 2c) indicate potential but limited methanogenesis-related (and minor deeper AOM-related) calcite formation from the current DOC-rich meteoric waters at these depths, in accordance with the observed scattered enhanced methane concentrations (Fig. 2a) and presence of methanogens and SRB 34 .

Discussion
Our study shows that AOM-related calcite precipitation was established at the border between a descending sulphateexhausted water, carrying dissolved methane, and a deeper sulphate-rich old saline water. The processes are outlined in Fig. 5 and include reduction of sulphate to 34 S-depleted sulphide and oxidation of 13 C-depleted biogenic methane to 13 C-depleted bicarbonate by a syntrophic consortium of ANME and SRB. These reactions cause an increased alkalinity invoking precipitation of 13 C-depleted calcite on the fracture wall. This calcite formed from reaction of the produced bicarbonate with the abundant dissolved Ca 2 þ in the deep saline water (up to 740 mg l À 1 (ref. 24)). The most likely explanation why the calcites are considerably more 13 C-depleted than the numerous marine AOM calcites observed worldwide is that the latter form in DOC-and DIC-rich sediment porewaters and thus more readily incorporate carbonate from other carbon sources than oxidized methane 9,48 . In strong contrast, the deep groundwater in crystalline rocks carries low concentrations of DIC and DOC (in Laxemar, each less than 2 mg l À 1 compared with maximum values of 330 and 21 mg l À 1 , respectively, in the upper 200 m), indicating that methane has been an almost exclusive carbon source for the 13 C-depleted calcites. The low concentrations of DOC in the deep groundwater were likely also partly refractory to microbial degradation in similarity with the limited availability and poor reactivity of organic substrates in deep-sea sediments 49 . Pyrite (depleted in 34 S compared with sulphate) is formed by reaction of the SRB-produced dissolved sulphide and Fe 2 þ (present at 0.1-0.8 mg l À 1 (ref. 24)). The large range in d 34 S pyrite of 68.9% ( À 22.1 to þ 46.8%, for example, in Fig. 3f) and frequent progressively heavier d 34 S with growth clearly reflect bacterial sulphate reduction in a system where the reduction rate has exceeded the supply of sulphate by advection and diffusion 50 . Increase in d 34 S values by up to almost 40% over a crystal growth distance of 50 mm (Fig. 3f) strongly suggests very local microscale isotope systematics at the fracture wall, in spatial relation to microbial communities 31 . Similarly, the contrasting d 13 C values in the calcites compared with those in the bulk fracture water (Fig. 3b) support local incorporation of 13 C-depleted bicarbonate formed by AOM at the fracture wall. However, the generally irregular d 13 C evolution within these calcites rules out similar closed system evolution of the carbon system as for the sulphur system. The irregular d 13 C variation of up to 45% in the AOM-related zone within the calcite crystals instead reflects either (1) local variability of incorporation of carbonate into calcite from other carbon sources than methane and/or (2) substantial variation of the d 13 C values of the oxidized biogenic methane as a result of methanogenesis during substratelimited conditions 51 occurring in the deep biosphere, or because of other factors that influence the kinetic carbon isotope effect during methanogenesis, including sulphate availability (cf. ref. 52), substrate utilized and temperature 17,51 . As the d 13 C value of the oxidized methane is unknown, we cannot exclude that the source methane of the o À 100% calcites has been extremely depleted in 13 C. It has recently been demonstrated that low sulphate concentrations (o50 mg l À 1 ) can lead to microbially mediated carbon isotope equilibration between methane and carbon dioxide resulting in some degree of reversibility of the methane oxidation (that is, back-flux) 53,54 . This causes progressively decreased d 13 C values of the residual methane. An extraordinary 13 C-depleted methane, affected by partial and reversible oxidation in a sulphate-limited system, such as in most of the current shallow and intermediate waters (Fig. 2a), can therefore indeed have been the source of the extremely 13 C-depleted calcites.
Reactions at the methanesulphate transition zone: At this transition AOM occurs, involving bacterial sulphate reduction promoting pyrite precipitation and increased alkalinity triggering calcite formation. AOM occurs preferentially in microbial communities (black, degraded over time) at the fracture walls, and the incorporation of carbon into calcite is therefore dominated by products of the local AOM process, as shown by both the d 13 C values, and by the closed system conditions of the sulphate reduction (evidenced by the d 34 S evolution within the pyrites).
The setting described here is not unique for the Baltic Shield, but is found on all continents in a variety of regions. Therefore, the identified methane-consumption mechanism in bedrock fractures and similar 13 C-depleted carbonates can occur worldwide. Our finding of AOM at a transition between biogenic methane formed at shallow depth and a deeper sulphate-rich saline water is, however, completely reverse to the SMTZ observed in typical well-characterized sedimentary AOM settings and at another well-characterized crystalline rock site (Olkiluoto, Finland) 1,2 , where deep-seated methane instead intersects with sulphate from superficial layers. Deep sulphate-bearing brine incursions that fuel AOM below the SMTZ have, however, been reported from a few marine sediments as well 55 . Although hydrochemical data are scarce from great depth in crystalline rocks, deep sulphate-rich waters, capable of ultimately producing the reversed SMTZ as described here, have been reported elsewhere 19,20,56,57 , offering opportunities for similar biogeochemical processes on a global scale. Although SRB-and methanogenesis-related isotopic signatures have been reported from groundwater and minerals in fractured crystalline rock in for example Sweden, Finland and USA 3,26,31,33,[58][59][60] , and a few scattered possible AOM indications of calcite (bulk samples) from Sweden exist 3,59,61 , more detailed and comprehensive in situ studies of low-temperature calcite are needed to better constrain the extent of past and present AOM deep in crystalline rocks globally. The episodic nature of the processes indicates dependency of certain conditions for initiation of AOM that may vary from site to site.
In conclusion, our study reveals a previously unknown methane-sulphate transition zone with consumption of biogenic methane at the border to sulphate-rich deep saline water at great depth in crystalline rocks. The relatively energy-limited and C-poor environment at great depth within these granite fractures has resulted in utilization and almost exclusive incorporation of C from biogenic methane into the extremely 13 C-depleted calcites precipitated on the fracture walls during AOM by ANME-SRB consortia. These findings greatly expand the observed range of carbon isotope variation in carbonates in natural environments, even when compared with sediment-hosted AOM occurrences, which previously were considered to host the most 13 C-depleted carbonates globally. The conditions associated with methane oxidation deep within Earth's terrestrial landscape is evidently substantially different compared to methane oxidation in other environments.

SIMS.
Calcite and pyrite crystals sampled from 18 drill cores were mounted in epoxy, polished to expose crystal cross-sections and examined using SEM (to trace zonations). Intracrystalline SIMS analysis (10 mm lateral beam dimension, 1-2 mm depth dimension) of carbon, oxygen and sulphur isotopes were performed on a Cameca IMS1280 ion microprobe. Analytical transects were made within several crystals from each sample. Settings follow those described in ref. 31, with some differences; O was measured on two Faraday cages (FC) at mass resolution 2,500, whereas C used a FC/EM combination, with mass resolution 2,500 on the 12 C peak and 4,000 on the 13 C peak to resolve it from 12 24,64 ). Significant influence of organic carbon was avoided in the SIMS analyses by careful spot placement to areas in the crystals without microfractures or inclusions, at a sufficient distance from grain boundaries where fine-grained clusters of other minerals and remnants of organic material may appear. The uncertainty associated with potential organic inclusions and matrix composition is therefore considered to be insignificant compared with the isotopic variations.
ToF-SIMS and GC/MS. Extraction of calcites for analyses of organic compounds was carried out accordingly. Calcite powder was extracted with 2 ml of predistilled dichloromethane in a Teflon-capped glass vial (ultrasonication, 35 min, 50°C). The supernatant was decanted after centrifuging. Extraction was repeated three times. After evaporation of the combined extracts and re-dissolution in pure dichloromethane, the solvents were dried with N 2 . Extracts were re-dissolved with 20 ml of n-Hexane and derivatized by adding 20 ml BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide)/pyridine and heated (40°C, 1.5 h). Remnant calcite powder was decarbonized with BSTFA, TMCS (trimethylchlorosilane)/methanol and derivatized by heating (80°C, 1.5 h). The lipid fraction was separated by mixing with hexane and decanting the supernate. Extraction was repeated three times. The samples were dried with N 2 , redissolved with 50 ml of n-Hexane and analysed with GC/MS and ToF-SIMS. One microlitre of each sample extract was analysed with a Varian CP-3,800 GC/1,200-quadrupole MS (settings in ref. 64). ToF-SIMS analyses of single calcite grains and organic extracts were conducted using a ToF-SIMS IV (ION-TOF GmbH, with liquid bismuth cluster ion source; settings in ref. 65). Compounds and corresponding characteristic fragments detected with ToF-SIMS and GC/MS within the AOM calcites are listed in Supplementary Table 4.
Raman spectrometry. The Raman spectrometry analyses were performed on seven samples with a confocal laser Raman spectrometer (Horiba instrument LabRAM HR 800), equipped with a multichannel air-cooled ( À 70°C) 1,024 Â 256 pixel CCD (charge-coupled device) array detector. Acquisitions were obtained with a 1,800-line per mm grating. The excitation source was provided by a 514-nm Argon laser (Melles Griot 543) with a laser power at the sample surface of 8 mW. An Olympus BX41 microscope was coupled to the instrument. The laser beam was focused through a Â 100 objective to obtain a spot size of B1 mm. The spectral resolution was B0.3 cm À 1 per pixel. The accuracy of the instrument was controlled by repeated use of a silicon wafer calibration standard with a characteristic Raman line at 520.7 cm À 1 . The Raman spectra were achieved with the LabSpec 5 software. Results are briefly presented in Supplementary Note 1.
Fluid inclusion microthermometry. Fluid inclusions were analysed in handpicked calcite crystals (0.5-1.5 mm in size) and in the epoxy-mounted crystals used for SIMS analysis following polishing of the mount to a 150-mm double-polished section. Microthermometric analyses of fluid inclusions were made with a Linkam THM 600 stage mounted on a Nikon microscope utilizing a Â 40 long workingdistance objective. The working range of the stage is from À 196 to þ 600°C (for details see ref. 66). The thermocouple readings were calibrated by means of SynFlinc synthetic fluid inclusions and well-defined natural inclusions in Alpine quartz. The reproducibility was ± 0.1°C for temperatures below 40 and ± 0.5°C for temperatures above 40°C.
Scanning electron microscopy. The zonation and inclusions of other minerals within polished hand-picked calcite crystals mounted in epoxy were examined using a Hitachi S-3,400 N SEM equipped with an integrated energy-dispersive spectroscopy system. The acceleration voltage was 20 kV, and the working distance 9.2 mm. During semiquantitative energy-dispersive spectroscopy analyses of other minerals within the calcites, oxide and element calibration standards (Smithsonian mineral standards) were used, linked to a cobalt drift standard (calibrated twice every hour) and a stable specimen current. Spot size was B5 mm.
Hydrochemistry. Groundwater from water-conducting fractures were sampled in 3-to 10-m packed-off boreholes by SKB, and results were extracted from their database (SICADA). Only sections with o1% drilling water are used. Quality control of the analyses and details of methods are described in ref. 67.