Martian Methanotrophy?

The observed ¹³C-depleted EGA CH₄ was presumably released from organic materials in the solid sample during pyrolysis. The canonical explanation on Earth for highly depleted carbon associated with a paleosurface would be microbial methanotrophy converting methane into biomass when the methane was already ¹³C depleted because of its biological production during methanogenesis (e.g., ref. 16). The Archean Tumbiana Formation has highly ¹³C-depleted biomass preserved along with abundant lacustrine stromatolites (e.g., refs. 17 and 18). The bulk biomass found in the Tumbiana Formation has δ¹³C values as low as −60‰ (19), the origin of which is often attributed to widespread methanotrophy during a period of abundant methane in the Earth’s atmosphere and shallow basins (16, 20). Highly depleted biomass is also found in some sediments in and around active modern marine methane seeps (e.g., refs. 21⇓⇓–24), and there are several examples of highly ¹³C-depleted biomass preserved in paleo seeps deposits (25, 26).

Because of both the overall oxidizing state of the Martian atmosphere and the presence of ferric iron and sulfate minerals, during times of large methane releases, methanotrophy would be energetically favorable. Based on observations of apparent methane plumes from the Martian interior and the growing understanding of the ways methane can be microbially oxidized on Earth, microbial methanotrophy has been proposed as a possible metabolism for recent and ancient Mars (27, 28). This explanation would also potentially explain the observation of reduced sulfur in many of the same samples in which negative δ¹³C values were found (29) because marine methane oxidation is coupled to sulfate reduction. If the highly ¹³C-depleted carbon and reduced sulfur are indeed found predominantly together on a paleosurface, the co-occurrence could result from the coproduction of ¹³C-depleted microbial biomass and sulfides during the microbial anaerobic oxidation of methane based on sulfate as an electron acceptor.

In order to get the observed magnitude of ¹³C depletion in the observed EGA methane released from organic materials, the CH₄ that was originally consumed by microorganisms in this model would need to already be highly ¹³C depleted (perhaps −40 to −100‰) because the observed depletion on Earth during anaerobic methanotrophy is on the order of about 30‰ (30), assuming fractionations associated with possible Martian metabolisms are similar to those observed on Earth. Therefore, this explanation requires multiple steps with the CH₄ consumed by the methanotrophs to be biological CH₄ from microbial methanogenesis. Furthermore, the inorganic CO₂ fueling this microbial ecosystem would need to have a δ¹³C composition similar to Martian magmatic carbon (−20‰; refs. 12 and 31) rather than the highly ¹³C-enriched values observed in the present Martian atmosphere (+46‰; ref. 8).

For this model to work, either the deposition of the ¹³C-depleted carbon would have needed to occur before the loss of significant carbon from the Martian atmosphere or there would need to have been CO₂ reservoirs in the Martian subsurface that were isolated from the atmosphere. In either scenario, the initial carbon could have had an isotopic composition down to −20‰. The first option, however, appears inconsistent with Gale crater sedimentology, given the relatively late emplacement of the Stimson formation that is cut by the paleosurface of interest here. The other option is possible, but it is hard to evaluate with our limited knowledge of the hydrodynamics of subsurface Mars. Potentially supporting this model is the observation of long, straight alkanes in the CB sample (32). Overall, there is, however, no supporting sedimentological evidence for microbial methanotrophy on the paleosurface discussed here. This is in sharp contrast to analog methanotrophic environments on Earth, for which there is often plenty of textural evidence for microbial processes in deposited layers. It is plausible that such evidence once existed at Gale crater as an authigenic carbonate (e.g., refs. 33 and 34) that was later dissolved by acidic fluids. While sedimentary structures may be destroyed by dissolution, dissolution can concentrate molecular biosignatures. The Curiosity rover will again encounter the Stimson formation sandstone on the Greenheugh pediment, providing an opportunity to search for specific organic molecules (e.g., refs. 35 and 36) in this rock unit. The lack of sedimentary evidence for microbial surface activity and the need to avoid influence from a ¹³C-enriched Martian atmosphere cast enough doubt on this biological explanation of surface methanotrophy to tentatively dismiss it pending further exploration for either evidence of surface-associated microbial activity or microbial-influenced sediments whose organic material could have been redeposited to the paleosurface.

Interstellar Dust?

The solar system passes through an average-sized giant molecular cloud (GMC) once every 100 million years, providing a mechanism for triggering cooling events on terrestrial planets through the influx of particles to planetary atmospheres that is substantially higher (20 to 100 times) than the ongoing flux of interplanetary dust (37). The solar system passing through a dense molecular cloud would inevitably also result in an influx of ¹³C-depleted carbonaceous particles (38), as about 1% of such interstellar clouds is dust (39). The δ¹³C value for interstellar dust in the Allende meteorite has been shown to be as low as −260‰ (40), demonstrating that interstellar dust is a potential source of highly depleted carbon for periodic deposition on the surface of Mars. It is also plausible, but presently uncertain, that these particles would similarly be associated with sulfides having negative δ³⁴S values, as observed in the Curiosity data. The flux of such particles would be quite low and typically diluted by other sources of carbon. However, because the arrival of such particles is predicted to trigger a global cooling event, it is reasonable that particles would accumulate on the surface of glacial ice largely absent of typical sedimentary carbon. Glacial melt during the glacial period and ice retreat after should leave the interstellar dust particles on the glacial geomorphological surface. The existing sedimentological evidence for the paleosurface that has cut the Stimson formation does not rule out glacial processes, with Curiosity having only visited the Greenheugh pediment surface briefly at EB, and some studies across Mars support such an interpretation for similar outflow channels (41⇓⇓–44).

This explanation of interstellar dust as a source of the observed ¹³C-depleted values at Gale crater largely fits the observations if the isotopic signatures are not diluted by other geological processes and with the understanding that this explanation relies on a rare event, making it perhaps coincidental that the mission should find this signature preserved. Finally, the SAM results presented here suggest that the most ¹³C-depleted values are liberated during relatively low oven temperatures, with the high-temperature examples typically being tails of lower-temperature methane release. These observations might be understood to indicate that the organic precursor molecules being converted into methane in the SAM oven are not recalcitrant kerogen-like molecules, as pyrolysis of kerogen usually occurs at a higher temperature. If this interpretation is correct, this constraint can be consistent with an interstellar dust origin, as there are a wide variety of known interstellar molecules, including many that are highly volatile (45). Overall, this explanation is plausible, but it requires additional research outside the scope of this report to verify, including the δ³⁴S values and the nature and relative amounts of carbon forms in GMC dust.

Abiotic Reduction of CO₂?

There is growing recognition of the possible abiotic production of organic carbon on Mars through either electrochemical reduction (46) or photochemical reduction (10). In both cases, CO₂ would be converted into organic material abiotically and therefore could, in principle, lead to ¹³C-depleted organic material associated with a paleosurface. For electrochemical reduction, sulfides are one of the various reduced minerals that thermodynamically drive such carbon fixation reactions, and, similarly, sulfides are possible mineral catalysts that can promote photochemical reduction of CO₂. Thus, if abiotic reduction of CO₂ is an important process on Mars, it is likely to be associated with reduced sulfur, as observed at Gale crater. The present atmosphere of Mars, as previously noted, is highly enriched in ¹³C (+46‰), and so either the abiotic reduction would need to have been occurring in subsurface environments (which does not match our observations) or would need to have occurred prior to the loss of much of the Martian atmosphere (which appears somewhat inconsistent with the timing of the erosional paleosurface with which the depleted carbon appears to be associated). While the timing of the erosional paleosurface is poorly constrained to the Hesperian, deuterium enrichment of clays at Gale crater indicated significant loss of atmospheric volatiles by this time (47), and the implications of that loss for the overall history of Martian volatile loss is an area of active research (48). Overall, though, the carbon isotopic fractionations expected during abiotic reduction are likely similar to those measured for abiotic hydrothermal reduction (up to ∼50‰; SI Appendix, Fig. S9B). Fractionation of this magnitude would not produce organic material as depleted as the observed TLS values for the EGA methane reported in Table 1, and therefore, this explanation does not appear to explain the observations of highly ¹³C-depleted evolved methane during pyrolysis of samples at Gale crater. However, carbon isotopic fractionation during photochemical reduction of CO₂ on a mineral surface has not been experimentally evaluated, and so, further research is needed before we can fully evaluate the abiotic reduction as the source of the highly ¹³C-depleted carbon isotopic values reported here. To be clear, there are a number of other CH₄ isotope values reported in Table 1 that could reflect the reduction of CO₂ on the Martian surface, including the most ¹³C-enriched values (−1 ± 6‰ to +22 ± 10‰). These ¹³C-enriched results match what would be expected for processes, biotic or abiotic, that produce organic material from the modern Martian atmosphere, for example.

Photolysis of CH₄ or CO and SO₂?

Photochemical reactions are attractive as a source for the observed phenomenon because photochemical reactions are known to have influenced Mars’s volatiles, including the sulfur isotopic values found in Martian meteorites (49⇓⇓–52). Also, the fallout of products from atmospheric photochemistry might be expected to accumulate on an erosional surface, such as the proposed paleosurface discussed throughout this paper. There are several different overall schemes by which photochemistry in the Martian atmosphere might result in the large δ¹³C depletions observed by the MSL-TLS component of the SAM instrument.

A growing number of studies have reported variable CH₄ abundances in the Martian atmosphere (53⇓–55), including recent reports of seasonal and diurnal variation (56, 57) and plumes (55) detectable on the surface at Gale crater. The photolysis of atmospheric CH₄ in a relatively dry, anaerobic atmosphere can produce organic aerosols and particles (e.g., Ref. 58). Perhaps when the postulated paleosurface was exposed, CH₄ photolysis resulted in the deposition of such organic material. This model would explain why the highly depleted δ¹³C values seem to be associated with a possible paleosurface, similar to a model proposed for the Late-Archean Earth (59). Further, the model could potentially also explain the observation of ³⁴S-depleted reduced sulfur in the same deposits if there was also SO₂ photolysis along with the CH₄ photochemistry producing sulfur-containing organic molecules. For example, Halevy (60) suggested that photoexcitation of SO₂ can lead to the production of methanesulfonic acid on the Archean Earth. Additional work is needed to explore the possibility that CH₄ photolysis during Martian plume events would deposit ³⁴S-depleted sulfur-containing organic material. However, past studies indicate that the magnitude of carbon isotopic fractionation during CH₄ photolysis is relatively small (<15‰; refs. 61⇓–63). Fractionation during CH₄ photolysis appears insufficient to produce δ¹³C values as depleted as some of those observed at Gale crater even when starting from bulk Mars δ¹³C values (−20‰) representative of carbon from the interior of Mars. In order to produce organic material that is highly ¹³C depleted (considerably more depleted than −70‰), another step with significant δ¹³C fractionation would need to have occurred during the formation of the methane. In principle, this could be abiotic CO₂ reduction in the Martian crust during serpentinization or microbial methanogenesis. As noted previously, the abiotic reduction of CO₂ results in ∼50‰ ¹³C depletion. If the source CO₂ had a δ¹³C composition of −20‰, subsequent abiotic reduction to CH₄, and subsequent photochemistry, then the resultant organic material deposited could be as depleted as perhaps −85‰, a value that does not adequately include all of the values observed at Gale crater. If microbial methanogenesis were producing the observed atmospheric CH₄, photolysis of the biologically produced CH₄ would result in isotopic compositions consistent with the strongly depleted ¹³C values observed. At a minimum, however, new observations would be needed to confirm a biological origin for Mars CH₄ plumes before this explanation can be accepted.

Alternatively, photochemical reduction of CO₂ to formaldehyde (CH₂O) via CO as an intermediate (e.g., ref. 64) might be responsible for producing a ¹³C-depleted organic material because the photochemical partitioning of CO₂ and CO can lead to ¹³C depletion in the CO relative to CO₂ (65⇓–67). However, most past studies have considered Martian CO to be isotopically similar to CO₂, with any ¹³C depletion relative to CO₂ to be on the order of about 30‰ (68). Recently, however, photodissociation of CO has been shown to yield hundreds of per mil ¹³C enrichments in the resultant CO₂ at 70 K using vacuum ultraviolet (VUV) radiation around 100 nm from a synchrotron source (69), while photodissociation of CO₂ has been shown to yield tens of per mil ¹³C enrichments in the resultant CO (70, 71). While these experiments use different ultraviolet (UV) wavelengths and different temperatures, together they illustrate how photochemical partitioning in the Martian atmosphere might result in anomalously ¹³C-depleted CO that could influence geochemistry observed at the Martian surface (72). In fact, CO photolysis has been calculated to have large carbon isotopic fractionation (with a fractionation factor of 0.6) at the top of the Martian atmosphere (73). VUV radiation would interact with the upper Martian atmosphere, and the presence of CO in the Martian atmosphere is maintained by CO₂ photolysis. If Martian photochemistry resulted in ¹³C-depleted CO throughout the atmosphere in the past, photoproduction of formaldehyde and other organics from CO could have accumulated on the exposed surface at Gale crater, resulting in the isotope results reported here (67, 74). Obstacles to the deposition of organic material from the photolysis of CO₂ have been the low yield and photochemical instability of the formaldehyde produced (75). Photochemically produced formaldehyde on Mars might react quickly with SO₂ to yield hydroxymethanesulfonate, protecting it from photolysis back to CO (76), and it is plausible that elevated levels of atmospheric SO₂ would directly shield formaldehyde because of the spectral overlap of SO₂ and CH₂O photoabsorption cross sections combined with the greater strength of SO₂ absorption (77, 78). After major eruptions, when there were higher levels of CO, SO₂, and H₂, the deposition of organics should be maximized, including perhaps formaldehyde, hydroxymethanesulfonate, carbonyl sulfide (79), and thioformaldehyde (80). Because of a relatively long photochemical equilibrium lifetime of SO₂ of ∼1 Mars year (75, 81) and photodissociative shielding of CH₂O afforded by SO₂, the likelihood of such reactions increases. Incorporation of ¹³C-depleted organics into frost or glacial ice at the Martian surface could also concentrate these photochemical products and lead to their preservation in the local soil, protected from further UV reactions. However, the VUV wavelength radiation shown to produce large fractionation is too short for most of the Martian atmosphere and has yet to be explored at various temperatures, limiting any conclusions that can be drawn here. Also, direct observation of CO in the Martian atmosphere by spectroscopy from Earth has not revealed a large ¹³C depletion in CO, with the results appearing to be within 250‰ of the telluric value (82) and probably slightly ¹³C enriched relative to Earth (66, 68). Without further information with which to deconvolve the details of where and how carbon was fractionated, we can conclude the photochemical production of organic material from ¹³C-depleted CO is a possible scenario on Mars for deposition of highly depleted organic material on to an exposed surface (74) that should not be rejected without further investigation of Martian CO and the photochemical processes that influence its carbon isotopic composition.

Samples.

The first drilled MSL samples were collected from the Sheepbed member mudstones in Yellowknife Bay. The Yellowknife Bay region in Gale includes portions of high thermal inertia rocks toward the distal end of the alluvial fan from Peace Vallis. The first and second samples of Sheepbed mudstones—John Klein and CB, respectively—were taken to investigate rocks deposited in this fluvial-lacustrine context. Analyses of these samples indicated the presence of an ancient habitable environment on Mars (2).

After analysis of the Sheepbed mudstones, the rover continued its traverse through Aeolis Palus and then began its ascent of Mt. Sharp. Starting in September 2014, the rover transitioned from Bradbury group rocks to Mt. Sharp group rocks, which consist of hundreds of meters of sedimentary stratigraphy subdivided into the Murray formation and the Carolyn Shoemaker formation. The Murray formation comprises the basal unit of Mt. Sharp and is divided among several distinct lithologic members dominated by mudstones. The Carolyn Shoemaker formation conformably overlies the Murray formation and has prevalent sandstone lithology. To date, more than 20 drilled samples of Mt. Sharp group rocks have been collected and analyzed from the various members. Included among these samples are analyses of regions that show unique properties from orbit such as the VRR, the Glen Torridon region, and the lower sulfate-bearing unit. VRR is a prominent topographic high on lower Mount Sharp that had orbital evidence for elevated hematite and was determined to have formed from several diagenetic events (14). South of the ridge is the Glen Torridon region, which was notable for its elevated orbital spectral signature of phyllosilicates. The most recent samples (as of July 2021) have been documenting the transition from clays to hydrated sulfates.

In a few locations along the rover’s traverse, samples have been taken from the Siccar Point group, which is comprised of the Stimson formation sandstones. The Stimson formation unconformably overlies the Mt. Sharp group and represents the remnants of an ancient, lithified dune field. The first four samples of Stimson formation rocks were taken to study both the parent rock and areas of localized alteration. The fifth sample of Stimson formation was taken later in the mission, during the Glen Torridon campaign, when the rover ascended the Greenheugh pediment.

In addition to the drilled samples, MSL has analyzed scooped samples of sand. The fine sand-sized GB and Ogunquit Beach samples were taken from the active Bagnold Dune field and represent local modern aeolian processes.

MSL EGA and MSL Carbon Isotopic Analysis.

The SAM consists of a suite of instruments including two pyrolysis ovens, a QMS, six gas chromatography columns, and a TLS (3). Together, SAM can measure the volatile composition of solid samples on Mars through thermal decomposition experiments. EGA–mass spectrometry (EGA-MS) heats samples to ∼850 °C and sends evolved volatiles directly to the QMS for identification with a constant flow of He. In this paper, QMS was used for the quantification of sulfur gases, as well as BSW (see SI Appendix, Supplementary Text for additional details). As explained further in the supplement, quadratic discriminant analyses of the QMS gas results provided posterior probabilities for whether each drill sample contained reduced sulfur (6). Gases during EGA can be sampled by the TLS within a specified temperature range. The TLS uses several specific infrared wavelengths to quantify H₂O, CO₂, and CH₄ as well as the isotopes of these gases when present in great enough abundance.

For SAM EGA runs, the solid sample is contained in an oven whose temperature is increased at 35 °C/min to ∼850 °C while helium (0.8 sccm, 25 mbar) is flowed through the pyrolysis oven. After evacuation of the TLS sample (Herriott) cell to record empty cell values, the TLS cell is opened to receive a “temperature cut” ingest (e.g., 200 to 350 °C) during EGA that introduces He and evolved volatiles into the cell, which is then sealed off for analysis. Typically, empty-cell methane abundances are extremely low (parts per billion by volume) compared to the full-cell EGA values (tens of parts per million by volume [ppmv]) so that empty-cell corrections are not needed. Full-cell pressures (mainly helium) are typically 5 to 15 mbar.

As further discussed in the supplement, three strong ¹²CH₄ lines and four strong ¹³CH₄ CH₄ lines are identified in the recorded TLS spectra, although only two of the ¹³CH₄ lines are chosen for analysis to minimize water interferences (SI Appendix, Fig. S1). With the cell closed, the laser scans over the methane lines every second, and on-board, TLS captures average spectra over sequential 2.7-min periods that are downloaded. Analysis of each of these 2.7-min spectra is done on a line-by-line basis through comparison with HITRAN 2016 calculations (that employ terrestrial δ¹³C values, so that volume mixing ratios in ppmv are retrieved for each of the three ¹²CH₄ and two ¹³CH₄ lines. In some cases of very high mixing ratios, the strongest ¹²CH₄ is very deep and not used. Then, the average values of the retrieved volume mixing ratios for ¹²CH₄ and for ¹³CH₄ are calculated from each of the 2.7-min spectra. During the run, 26 full-cell spectra are recorded, so that the 26 values retrieved of average abundance values can be statistically analyzed to find the mean value with SEs of either 67% CI or 95% CI. Ratioing the ¹³CH₄ mean abundance with that determined for ¹²CH₄ provides the δ¹³C values reported here normalized to the terrestrial PDB reference expectation such thatδ13C=((C13C12) Sample(C13C12) PBD std.−1) × 1,000 ‰,where PDB std. is Pee Dee Belemnite standard, which is equal to 0.0112372.

Supporting Laboratory Analyses.

As discussed in the SI Appendix, Supplementary Text, laboratory analyses were conducted (SI Appendix, Figs. S3 and S4, and Table S2) in support of this paper, including an NMR-based investigation (83) of the intramolecular isotopes of methyl-trifluoroacetamide, part of the MTBSTFA molecule. Laboratory studies were also used to study carbon isotopic fractionation during pyrolysis to CH₄ under conditions similar to that of the SAM instrument. Various carbon-containing materials were placed in silver boats and dropped into an oven at 400 °C under flowing helium. The oven temperature then increased to >850 °C, and evolved gases, including CH₄, were trapped out of the He flow between 455 °C and 755 °C to simulate a high-temperature TLS cut.