Organic carbon concentrations in 3.5-billion-year-old lacustrine mudstones of Mars.

The Sample Analysis at Mars instrument stepped combustion experiment on a Yellowknife Bay mudstone at Gale crater, Mars revealed the presence of organic carbon of Martian and meteoritic origins. The combustion experiment was designed to access refractory organic carbon in Mars surface sediments by heating samples in the presence of oxygen to combust carbon to CO2. Four steps were performed, two at low temperatures (less than ∼550 °C) and two at high temperatures (up to ∼870 °C). More than 950 μg C/g was released at low temperatures (with an isotopic composition of δ13C = +1.5 ± 3.8‰) representing a minimum of 431 μg C/g indigenous organic and inorganic Martian carbon components. Above 550 °C, 273 ± 30 μg C/g was evolved as CO2 and CO (with estimated δ13C = -32.9‰ to -10.1‰ for organic carbon). The source of high temperature organic carbon cannot be definitively confirmed by isotopic composition, which is consistent with macromolecular organic carbon of igneous origin, meteoritic infall, or diagenetically altered biomass, or a combination of these. If from allochthonous deposition, organic carbon could have supported both prebiotic organic chemistry and heterotrophic metabolism at Gale crater, Mars, at ∼3.5 Ga.

Understanding the inventory of carbon on Mars is relevant to the question of whether Mars was ever habitable. In addition, constraining the cycling of carbon between atmospheric and crustal reservoirs on past and present Mars is crucial to understanding Martian climate and surface environmental evolution. Meteorite analyses and in situ pyrolysis performed on Mars indicates that Mars regolith is a significant reservoir of carbon. While carbonate is the dominant carbon phase, geologically labile and refractory organic carbon is also present. A large body of carbon abundance and isotopic information exists for Mars meteorites (1–4), which predominantly represent igneous rocks excavated from the subsurface. Surface sedimentary rocks, however, can only currently be studied using in situ measurement techniques enabled by landers or rovers. Constraining the relative abundance of each carbon phase can increase our understanding of potential cycling of carbon between reservoirs during early Martian history.

The Sample Analysis at Mars (SAM) instrument suite on the Mars Science Laboratory (MSL) Curiosity Rover designed an experiment to combust refractory organic carbon to CO2 to obtain its carbon isotopic composition (δ13C). This experiment was similar to laboratory stepped combustion of meteorites (2, 3, 5–7). SAM measured the abundances of evolved CO2 and water as well as δD, δ13C, and δ18O stable isotopic compositions for these volatiles. The δD results have been published elsewhere (8). Here we present carbon results of the combustion experiment, which represent the quantification of bulk refractory carbon in Mars surface materials.

Cumberland Mudstone

The Curiosity landing site at the base of Aeolis Mons (Mount Sharp) at Gale crater (Fig. 1) was chosen for ample orbital evidence of aqueous processes including layered phyllosilicate and sulfate deposits interpreted to represent global climate transitions on Mars (9). Crater count and stratigraphic relationships put the depositional timing of the lower layered sediments in Gale crater near the Hesperian-Noachian boundary (∼3.7 Ga), while the crater itself is Noachian in age (10, 11). Early in the mission, ∼444 m east of the Bradbury landing site, Curiosity encountered fine grain sedimentary rocks in the Sheepbed member of the Yellowknife Bay formation (Fig. 1), interpreted to be a mudstone associated with a lacustrine setting (12). Two holes were drilled at the John Klein and Cumberland drill sites in the Sheepbed member (Fig. 1). Drill fines of the Cumberland target (CB) were selected for combustion analysis.

Fig. 1.

(A) Landing ellipse of the Mars Science Laboratory Curiosity Rover at Gale Crater, Mars. (B) Rover Traverse from 2012 to 2022. Red dot shows the location of the Cumberland sample in the Yellowknife Bay formation. (C) Rover scale view of the Sheepbed mudstone member of the Yellowknife Bay formation, with locations of John Klein and Cumberland drill holes. Image Credit: NASA/JPL-Caltech/ASU, NASA/JPL-Caltech/Univ. of Arizona and Scott Rowland, NASA JPL-Caltech/MSSS.

The CB mudstone was extensively characterized by the MSL payload (13–15). Briefly, the Sheepbed member experienced at least two aqueous events post deposition with the nodules present being formed by trapped gases during early diagenesis. Fractures filled with Ca-sulfate indicated a subsequent aqueous event. Mineralogical analysis of the CB mudstone showed detrital basaltic minerals as well as Ca-sulfates, Fe-sulfides, and trioctahedral smectites (15, 16). The SAM instrument performed multiple evolved gas analysis (EGA) pyrolysis analyses on the CB mudstone, resulting in detection of chlorinated organics, hydroxyl water from smectite decomposition, low temperature CO2 evolution, and high levels of oxychlorine and nitrate (17). The Yellowknife Bay formation was ultimately deemed to represent a potentially habitable shallow lacustrine environment based on evidence for circumneutral pH (15), high water activity/low salinity (14), variable redox states indicated by mineralogy and volatile content (15, 17), and presence of biologically relevant elements (13, 17), making it a target of interest for measuring bulk carbon abundance and carbon isotope composition.

Methods

The SAM Combustion experiment used the SAM quadrupole mass spectrometer (QMS) and the SAM tunable laser spectrometer (TLS), which measures the abundance and isotopic composition of CO2. Both instruments, as well as the entire SAM suite, have been described in detail elsewhere (18, 19). The SAM Combustion experiment differed significantly from SAM’s nominal EGA pyrolysis experiment in which the QMS directly analyzed the gases released from samples heated to ∼870 °C at 35 °C/min under He flow (18). Combustion was performed in a closed oven in the presence of oxygen, which was either sourced from oxychlorine in the sample or added to the cup as pure O2 gas prior to the experiment using an onboard reservoir tank (18). Unlike pyrolysis, where gases evolved are continuously monitored using the QMS, gases produced during closed oven combustion were only released into the SAM manifold and analyzed by QMS and TLS upon completion of the heating step.

The combustion experiment was a four-step experiment performed in three separate experiment sequences executed on three sequential sols (). The experiment was performed on a single aliquot of the CB mudstone sample, described above. The sample was heated to 550 °C or 870 °C and held at temperature for 25 min in a closed oven with either oxychlorine from the sample (step 1) or O2 from the onboard tank (steps 2 to 4). Step 1 measured combusted CO2 below 550 °C. After step 1, the sample cup was returned to the sample manipulation system (SMS) for exposure to instrument sources of carbon overnight to characterize background in step 2. Step 3 was performed at 870 °C and was designed to combust refractory carbon. Step 4 was also performed at 870 °C to combust remaining refractory carbon. Full experimental details are available in .

Results

Abundance and isotope results are reported in Table 1. Error on TLS abundance and isotope values represent instrument precision. Error on QMS abundance values represents uncertainty associated with QMS calibration coefficients derived empirically during atmospheric runs on Mars during prelaunch calibration (). QMS performance on Mars has been carefully monitored throughout the mission, and revisions to calibration coefficients are made routinely (20) to maintain accuracy. Additional uncertainty associated with the sample mass estimate is incorporated into calculations of μg C/g values presented in Table 2. Reported values represent lower end CO2 abundance values, as several lines of evidence point to incomplete combustion, particularly in steps 2 to 4. While we do not have data for step 1, we predict that 24 to 30 μmol of O2 was present based on SAM EGA pyrolysis data for CB (17), which should have been enough to overcome oxygen limitation. Steps 2 to 4, however, each had 3.7 μmol O2. This amount was based on carbon abundance estimated using CO2 measured during pyrolysis EGA of CB samples and the fact that resource limitations precluded adding large excesses of oxygen. We note that the molar ratio of carbon in CO/CO2 was <3% except in step 4 where it was ∼30%, and CH4/CO2 was <1% except in step 2 where the QMS saturated on m/z 15, allowing us to only provide a lower limit value for methane. Because TLS precision is the same magnitude as uncertainties in the δ13C of CO2 associated with partial combustion reported in the literature (21), we do not add additional error to our isotope values.

Table 1.

Selected TLS and QMS data for each step

		Oxidant	TLS				QMS
		δ18O (‰)	CO2 μmol	μg C/g	CO2-δ13C (‰)	CO2-δ18O (‰)	CO2 μmol	CO nmol	O2 nmol	CH4 nmol
<550 °C	Step 1	+41‰ Mars O2	14 ± 0.1	957 ± 38	+1.5 ± 3.8	+55.0 ± 5.5	NA	NA	NA	NA
	Step 2	+20‰ Tank O2	5.1 ± 0.03	340 ± 13	−4.3 ± 4.3	+58.2 ± 7.0	5.4 ± 0.3	72 ± 95	61 ± 19	182 ± 16*
<870 °C	Step 3	+20‰ Tank O2	2.5 ± 0.01	166 ± 7	−16.2 ± 3.1	+28 ± 5.6	3.0 ± 0.7	93 ± 206	180 ± 28	15 ± 4
	Step 4	+20‰ Tank O2	1.2 ± 0.01	80 ± 3	+2.5 ± 4.4	+46 ± 4.5	1.0 ± 0.2	315 ± 49	24 ± 15	8 ± 1

Step 1 used oxychlorine native to Mars as the oxidant instead of tank O2. No QMS data are available for step 1 due to QMS shut down. Steps 2 to 4 used the SAM O2 tank carried for this purpose. The cup was returned to the sample manipulation system between steps 1 and 2 to estimate instrument background sources of carbon (). Step 3 was designed to access refractory carbon. Step 4 was designed to access refractory carbon not combusted in step 3

*CH4 was saturated in step 2 so value represents lower limit.

Table 2.

Comparison of refractory carbon abundance in various planetary materials

Material	Carbon concentration (μg C/g)	Comment/Ref
Combustion CO2 <550 °CSheepbed mudstone, step 1	957 ± 38	Combination of instrument C and at least ∼430 μg C/g Martian C
Combustion CO2 <550 °CSheepbed mudstone, step 2	340 ± 13	Mostly instrument C
Combustion CO2 + CO >550 °CSheepbed mudstone, steps 3 and 4	273 ± 30 (201–273)	Refractory and mineral-stablized organic C, includes C from both CO2 and CO. (Range reflects presence/absence of carbonate)
Pyrolysis <550 °CSheepbed mudstone–CO2	667–827	Sutter et al. (23)
Pyrolysis >550 °CSheepbed mudstone–CO2	72 ± 7	Sourced from carbonate, organics, or both
Estimated contribution from instrument sources to pyrolysis of CB mudstone	163–237	Calculated from MTBSTFA estimates from Freissinet et al. (24)
Pyrolysis >500 °C Sheepbed mudstone–hydrocarbon fragments	5.6–6.7	Eigenbrode et al. (26)
Pyrolysis >500 °C Murray mudstone–hydrocarbon fragments	8.8–24.4	Eigenbrode et al. (26)
Martian meteorite igneous organic carbon released by combustion >600 °C and <1,000 °C	1.3–150	Steele et al. (1); Grady et al. (2); Jull et al. (6, 7)
Estimates of total organic carbon concentration in Mars regolith due to exogenous inputs	10600.2–2.9 wt.%	Carrillo-Sanchez et al. (43)Steininger et al. (42)Schuerger et al. (41)

CO2 abundances measured by TLS and QMS (Table 1) for steps 2 to 4 allowed for cross-instrument validation of this data. The pressure of evolved volatiles in step 1 exceeded the safety limits set for the QMS, which shut itself down during analysis to prevent instrument damage. Therefore, a complete set of abundance data for steps 1 to 4 exists for TLS only, so all μg C/g data in the Discussion section was calculated using TLS values. TLS and QMS data were generally within error of one another for CO2 abundances (Fig. 2). TLS combustion data for all steps combined yielded 23.2 ± 0.1 μmol CO2 (0.5 ± 0.02 wt% CO2) for this triple portion of sample. For comparison, average CB pyrolysis concentrations (single portion of 45 ± 18 mg) contained ∼2.0 to 3.1 μmol CO2 (∼0.2 to 0.3 wt% CO2) based on QMS data (17). Step 1 had the largest evolutions of CO2. Step 3 evolved less CO2 (2.5 to 3 μmol) than either step 1 or 2, and step 4 evolved the least CO2 (1 μmol) of all steps.

Fig. 2.

TLS and QMS carbon abundances and TLS δ13C data. Bulk δ13C for steps 3 and 4 combined is −3.6 ± 3.1‰.

TLS δ13C and δ18O values are reported with respect to terrestrial standards Vienna Pee Dee Belemnite (VPDB, referenced to National Bureau of Standards NBS 19) and Vienna Standard Mean Ocean Water (VSMOW), respectively (Table 1). Because O2 from the oxygen tank was added in steps 2 to 4 for combustion, δ18O values for CO2 evolved in these steps was expected to be overprinted by the terrestrial tank value (∼20‰). Step 1, however, used only Martian oxychlorine [δ18O = 41 ± 4‰ (22)], as a source of oxygen.

Table 1 gives QMS abundance data for several additional species identified in combustion runs in steps 2 to 4 (additional QMS data in ). All carbon species other than CO2 were present at nanomolar concentrations. CO was most abundant in step 4. O2 was present in all steps at nanomolar abundance, although in step 3 all O2 can be attributed to sulfate decomposition based on observed SO2 abundance (see ), suggesting all added O2 was utilized during this step. The CO/CO2 ratio of 31% during step 4 is consistent with partial oxidation of a refractory phase. CH4 was present in all steps and saturated the QMS in step 2.

Discussion

Low Temperature: Below 550 °C.

Over the entire temperature range of the experiment (∼50° to 870 °C), the combustion run produced twice as much carbon in the form of CO2 (1543 ± 41 μg C/g) as EGA pyrolysis runs (∼667 to 827 μg C/g (23)) of the same CB mudstone. Low temperature (<550 °C) combustion (step 1) was responsible for most (957 ± 38 μg C/g) of this carbon.

Significant amounts of the carbon-bearing background contaminant, N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide (MTBSTFA) were present in both low temperature combustion steps due to elevated signals during prerun background scans for m/z 75, 127, and 147, diagnostic fragments derived from major byproducts of MTBSTFA reaction with water. Because the intent of step 2 was to characterize the amount of instrument carbon contributed by adsorption of MTBSTFA to the sample during overnight exposure in the SMS, MTBSTFA decomposition/combustion was expected the primary source of carbon in step 2. The amount of CO2 produced during combustion step 2 (340 ± 13 μg C/g) was only slightly more than the 163 to 237 μg C/g calculated to be sourced from MTSBTFA during pyrolysis EGA runs of the same CB mudstone material (24). Interestingly, more MTBSTFA byproducts were present in step 1 background than in step 2 background, suggesting that a significant amount of the CO2 evolved in step 1 was also due to combustion of MTBSTFA. However, it is notable that the 957 ± 38 μg C/g produced in step 1 represents at least 700 μg C/g in excess of calculated estimates for carbon (163-237 μg C/g) contributed by instrument sources during SAM EGA pyrolysis runs of CB (24). While this suggests a Martian source of low temperature carbon, we do not fully understand the long-term adsorption of MTBSTFA carbon to a sample while it sits in the SMS and cannot estimate instrument sources using concentration data alone.

Isotope ratios for low temperature steps are consistent with the presence of a 13C-enriched Martian end member (Fig. 3). If all the carbon detected during low temperature combustion in steps 1 and 2 was derived from MTBSTFA, then δ13C of CO2 in both low temperature steps would reflect this instrument source of carbon at δ13C = −35‰. Our step 1 and 2 combustion δ13C values were +1.5 ± 3.1‰ and −4.3 ± 4.3‰, respectively, and fall within the range of δ13C values calculated for the CO2 peak evolved between 180 to 450 °C in SAM EGA pyrolysis of CB samples, which show a weighted average δ13C of −6 ± 11‰ (22). While a mass balance approach involves too many assumptions to isolate individual contributions from different carbon sources, these δ13C values can put constraints on the maximum worst case contribution of MTBSTFA to the sample in step 1. Assuming a two-end member mixing model with a 13C depleted end member (MTBSTFA at δ13C = −35‰) and a 13C-enriched end member (carbonate at δ13C = +45‰), a maximum of 55% of the carbon could derive from MTBSTFA decomposition in step 1 (detailed calculation in S9). This leaves a minimum of 45%, or 431 μg C/g, of carbon derived from indigenous Martian or exogenous carbon. Interpretation of step 2 isotope data are complicated by the addition of instrument background carbon prior to combustion when the sample was returned to the SMS, coupled with the fact that any partial combustion in step 1 (while not expected) would have resulted in 13C enrichment of any step 1 residual carbon through Rayleigh distillation.

Fig. 3.

Carbon isotopic composition of carbon reservoirs on Mars. Our combustion data overlap with both igneous refractory carbon, meteoritic organics, and carbonate carbon. Atmospheric data from (29); Meteoritic EOM from (25, 26); Meteoritic IOM data from (34); Carbonate SNC range from (48); ALH84001 data from (7, 50); Igneous refractory carbon from (1); Gale Crater data from (13) Box and whiskers show the median, upper and lower quartiles, and maximum and minimum values, with outlier values shown as black dots.

The source of the ≥431 μg C/g non-instrument carbon could derive from a range of 13C enriched Martian and exogenous (meteoritic) reservoirs (Fig. 3). SAM EGA pyrolysis experiments of 13 samples demonstrated that CO2 evolved below 550 °C had a broad range of δ13C (−25 ± 20‰ to +56 ± 11‰) representing multiple carbon sources (22). In addition to the previously discussed instrument carbon source, carbon could come from oxidation or decarboxylation of Martian organics or exogenous carbon (17, 22–28). Recent work (28) showed that SAM-like pyrolysis of metastable organic salts such as acetates and oxalates produce CO2 and CO below 550 °C, putting upper limits for abundances of these compounds in SAM pyrolysis experiments as high as 2 to 3 wt%. These molecules may be present as radiolytic decomposition products of more complex organics (29, 30) or could represent simple molecules formed in situ by photolysis or electrochemistry (4, 22). Depending on formation mechanism, these molecules would likely be 10 to 30‰ depleted in 13C with respect to the Martian atmosphere at the time of their formation (22, 31), resulting in δ13C values of ∼+15 to 35‰ for photochemically created molecules in the current atmosphere. Carbonate decrepitation occurs over a broad temperature range (400° to 700 °C) in stepped combustion of meteorites (2). Carbonates in Noachian aged ALH84001 evolved CO2 with δ13C values of +32.5 to 40‰ between 430 and 600 °C in stepped combustion experiments (7). Although CheMin XRD did not identify carbonate above the 1% detection limit, the large amount of amorphous material detected by CheMin may indicate the presence of microcrystalline carbonate, which can evolve CO2 <550 °C. In addition, HCl evolved from oxychlorine decomposition can also drive CO2 evolution temperatures below 550 °C (17, 23). Finally, solvent extractable organic matter (EOM) in meteorites represents a less 13C enriched reservoir of carbon than carbonates or modern photochemical or electrochemical carbon, with values generally averaging around δ13C = +5‰ (32).

Oxygen isotopic compositions of CO2 evolved below 550 °C suggest contributions from an isotopically enriched component in the sample. The δ18O weighted average of O2 evolved from sand sample Rocknest and CB was ∼40‰ (22), which reflects the Martian oxychlorine source. δ18O of CO2 in step 1 was +55 ± 5.5‰, more enriched than oxychlorine and similar to atmospheric CO2 (δ18O = +50‰ (33)). Early thermal decomposition of siderite could also produce 18O enriched CO2; siderite precipitated at 25 °C would be 18O enriched by about 10‰ with respect to modern atmospheric CO2. In steps 2 to 4, δ18O of CO2 produced by combustion should have been approximately +20‰ (SAM tank value). Step 2 δ18O values were very similar to step 1, suggesting the addition of 18O enriched O2 to tank O2, along with the fact that the CO2 abundance in step 2 (5.41 μmol) was larger than the moles of O2 added (3.7 μmol). Based on mass balance calculations, the δ18O value measured in step 2 would require contribution from an O2 source with δ18O greater than +100‰, suggesting that instead isotopic exchange of CO2 with isotopically enriched O2 reservoirs in mudstone (e.g., silicates or oxides) may have occurred during the analysis.

High temperature: 550° to 870 °C.

More carbon was evolved as CO2 between 550° and 870 °C during the closed oven SAM combustion experiment than by SAM EGA pyrolysis alone. This additional CO2 is likely from combustion of a broad suite of refractory organic components in the sample. High temperature combustion (steps 3 and 4) yielded a total of 246 ± 7 μg C/g as CO2. In step 3 combustion appears to be nearly complete, with CO/CO2 ∼3% and CH4/CO2 < 1%. However, in step 4, 21 μg C/g was present as CO (CO/CO2 = 31%), suggesting that step 4 combustion did not go to completion. Adding CO carbon from steps 3 and 4 to CO2 carbon yields a total high-temperature carbon abundance of 273 ± 32 μg C/g (Table 2).

Some portion of this carbon may be from thermal decomposition of carbonate. Above 550 °C, EGA pyrolysis of triple-portion CB mudstone aliquots resulted in an average concentration of 72 ± 7 μg C/g based on CO2 QMS data. This release occurred at temperatures near those expected for calcite decrepitation (∼700 to 750 °C). However, CO2 coevolved with CO and m/z associated with decomposition of sulfates, which have been identified in this sample (17). These factors support either release of mineral-bound organic matter during sulfate decomposition or organic oxidation by O2 sourced from the decomposition of sulfates. In addition, Eigenbrode et al. (26) found organic carbon evolving from the same sample material at these temperatures, described below. Therefore, we report a range of 201 to 273 μg C/g for refractory organic carbon, as we cannot further constrain the carbonate abundance in the sample.

Eigenbrode, et al. (26) showed that masses consistent with aliphatic, aromatic, and organic-sulfur compounds were present above background at temperatures above 500 °C in samples of the same Yellowknife Bay mudstone. Total carbon concentration for these detected compounds was ∼40× less than that detected by high-temperature combustion (Table 2). The differences between carbon concentrations detected by combustion and pyrolysis are due to the nature of the experiments. At high temperatures above 500° to 550 °C, pyrolysis is limited to thermal cleavage or decarboxylation of macromolecules or mineral decrepitation/decomposition with subsequent release of compounds. As such, a significant fraction of more thermally recalcitrant organic matter is expected to be left undetected in the sample. In contrast, combustion leverages both thermal and oxidative processing of organic components including the most refractory macromolecular material, thus releasing more carbon detected as a single gas, CO2. Assuming the ratio of high-temperature combustion carbon to pyrolysis organic carbon for CB reported by Eigenbrode et al. (26), Murray mudstone could have refractory organic carbon concentrations up to 730 μg C/g, depending on carbonate abundance (see ).

High-temperature CO2 should represent carbon in the sample and not instrument background. SAM pyrolysis and laboratory testing have demonstrated that MTBSTFA decomposition occurs mostly below 550 °C, particularly in the presence of strong oxidants (see ). However, as previously mentioned, there is evidence for partial combustion in some steps. Because the combustion experiment was performed in a closed oven at 550 °C for 25 min in the presence of soil oxidants (step 1) and added O2 (step 2), most volatile compounds were likely released in these first two low-temperature steps by thermal desorption, oxidation, and some pyrolytic cleavage from macromolecules and organic mineral-bound or mineral-enclosed components. Upon oxidation at 870 °C, more thermal energy was available to break the stronger molecular bonds persisting in macromolecules and decomposed minerals that may be trapping organic components. Therefore, CO2 evolved at high temperatures reflects a more refractory carbon pool within this sample that is likely to include macromolecules and organic mineral-bound/enclosed components. This is consistent with the reported detections of complex organics evolved during high-temperature SAM pyrolysis (26).

The 13C enrichment in step 4 (δ13C = +2.5 ± 4.4‰) compared to step 3 (δ13C = −16.2 ± 3.1‰) may represent a more refractory reservoir of carbon that was released during the second heating step above 550 °C. Rayleigh type isotopic distillation may have resulted in the 13C enrichment of the remaining recalcitrant carbon in step 4. Alternatively, the difference in δ13C values between steps 3 and 4 may be due to isotopic heterogeneity. These values overlap with multiple carbon sources (Fig. 3) including carbonate (5, 34, 35), meteoritic insoluble organic matter (IOM) (36), and magmatic carbon (1), and likely represent a mixture of these carbon reservoirs. By mass balance, a bulk δ13C value for total carbon released in steps 3 + 4 is δ13C = −10.1‰. While we cannot constrain the amounts and isotopic compositions of every component of this mixture, a two end-member mixing model can help bound the isotopic composition of the organic carbon considering the case where no carbonate is present and the case where all 72 μg C/g CO2 detected in SAM EGA pyrolysis was from calcite of δ13C = +45‰. Such a calculation produces a range of δ13C values from −10.1‰ to −32.9‰, depending on how much carbonate is in the sample. Based on the behavior of both CO and SO2 in the sample, which support the present of some amount of organic material, it is likely that the value is somewhere in the middle. This is broadly consistent with refractory or macromolecular carbon (1, 2) but does not preclude contributions from other carbon sources.

With regards to δ18O composition, steps 3 (δ18O = +28 ± 5.6‰) and 4 (δ18O = +46 ± 4.5‰) showed enrichment in 18O with respect to the SAM tank value of δ18O = 20‰, suggesting the addition of O2 from a source in equilibrium with or enriched with respect to Martian atmosphere (δ18O = +50‰ (33)). To achieve the measured value in step 3, about one-third of the oxygen would have had to come from carbonate, theoretically possible if all CO2 detected in pyrolysis experiments was from carbonate. However, to achieve the heavier step 4 values, 75 to 90% of the oxygen in CO2 would have to come from carbonate, which is inconsistent with pyrolysis experiments. This suggests exchange with silicate oxygen or structural water, which is highly likely at these temperatures.

The source of the organic carbon evolving high-temperature CO2 during combustion is not easily resolved. Our δ13C values for combusted CO2 (δ13C = −10.1‰ to −32.9‰) do not show the same extreme 13C depletions as recently reported δ13C values for CH4 evolved during SAM EGA pyrolysis experiments (−96‰ to −71‰) on the same CB samples (37). This is likely because δ13C of combusted CO2 is a bulk measurement of all carbon in the sample, and CH4 only represents a very small portion (∼0.2‰) of the total carbon in the sample. Potential sources of refractory carbon include exogenous organic matter from meteoritic infall, geologic carbon produced by abiotic processes, geologically reworked biological organics, or a combination of these. Here we consider each possible source.

High-temperature combustion CO2 (δ13C = −10.1‰ to −32.9‰) has similar values to insoluble organic carbon evolved from meteorites (Fig. 3). Pyrolysis data for the isotopic composition of organic carbon evolved above 800 °C from carbonaceous meteorites gives values of around −16 to −1 ‰ (38). The range for IOM in meteorites is broader, with δ13C = −20 to −5‰ (32, 39). Mars surface sedimentary composition is greatly influenced by influx of meteoritic material (40) and the similarity between the distribution of organics found in the Murchison and Nahkla meteorites suggests that Mars meteorites contain a component of this exogenous carbon (1, 39). Concentration of total impact deposited organic carbon in Mars regolith is highly dependent on model assumptions such as influx, impact gardening depth, and total organic carbon content of exogenous materials, but estimates range anywhere from 10 μg C/g up to 2.9 wt.% C (41–43). It is therefore likely that the Sheepbed mudstone forming in the Yellowknife Bay Lake catchment would have been predisposed to accumulate exogenous carbon. However, studies predict that high rates of radiolytic destruction of light organics in Mars surface environments would outpace exogenous influx (44–46) and result in far lower concentrations of organics in the near surface. The abundance of exogenous organic carbon incorporated into the sediments lithified to form the Sheepbed mudstone would be determined by factors such as sedimentation rates and geochemical environment. The young surface exposure age dates for the Sheepbed mudstone (78 ± 30 Ma (47)), likely resulting from early burial and recent scarp retreat, suggest that irradiation and consequent destruction of organics in Sheepbed mudstone may have been limited compared to other Martian surfaces with older exposure ages. This supports the inclusion of meteoritic organics in the CB mudstone.

CO2 evolved during stepped combustion of many Mars meteorites above 600 °C is associated with igneous refractory carbon (2). Igneous Mars rocks contain 18 ± 26 ppm of refractory organic carbon synthesized in-situ, with an average δ13C of −19.1 ± 4.5‰ (1), also within the range of δ13C reported for combustion of CB sediments in step 3. However, the combustion values need to be considered in the context of the CB sample as a lacustrine mudstone, which has likely seen a different geological history than the deeply excavated igneous rocks that comprise most of the Martian meteorites reported to date. The only Mars meteorite sampling Martian surface breccia is NWA 7034, which gave organic carbon abundance of 22 ± 10 μg/g with δ13C of −23.4 ± 0.73‰, similar to the average for shergottites (48). Therefore, it is likely that some component of the total organics evolving CO2 in combustion of CB sample includes the refractory organic carbon component associated with basaltic minerals seen in Mars meteorites deposited as transported detritus to the Yellowknife Bay Lake and preserved in Sheepbed mudstone.

The δ13C of carbon evolved as CO2 at 870 °C in SAM combustion neither rules out nor confirms geological processing of biological organics, which could have been produced or deposited in lake sediments and converted to kerogen preserved in mudstones by exposure to heat from regional hydrothermal activity during burial (49) and sulfurization (26). Biological carbon fixation on Earth has a broad range of fractionations, with 13C depletion ranging from a few per mil to tens of per mil with respect to carbon source, depending on metabolism and organism. This, coupled with the wide range of isotopic compositions in carbon reservoirs on Mars (Fig. 3), complicates the use of isotopes to confirm or eliminate the potential for biological carbon fixation processes on Mars. Therefore, combustion δ13C isotopes cannot be used to support a biological origin for refractory carbon, but do not rule out this possible source.

Recent reports of a diverse array of organics released by SAM EGA pyrolysis at these temperatures in other Sheepbed mudstone samples, including sulfurized organics (26), highlight the preservation potential of these sediments. The mineralogy of the CB sample would have likewise supported preservation of organics through binding to phyllosilicates and Al-Fe oxyhydroxides, as well as iron sulfides, which could have provided a sink for oxygen during diagenesis (26). However, without additional isotopic or compositional data on these or other sources, it is difficult to further constrain the possibilities.

Conclusions

The SAM combustion experiment results support the presence of Martian carbon combusted or thermally evolved below and above 550 °C. The carbon isotopic composition of low-temperature CO2 is consistent with the presence of a 13C-enriched carbon phase of Martian origin in addition to carbon contributions from instrument background (MTBSTFA). Instrument background contributed a maximum of 55% of the total carbon detected in step 1. The remaining 431 μg C/g carbon could come from organics created by photochemistry, meteoritic organics, carbonate, or a mixture of these.

More CO2 was evolved above 550 °C in closed-oven combustion experiments than in previous pyrolysis experiments on the same sample, indicating that combustion released more mineral-bound and refractory carbon than pyrolysis of the same materials. The combustion experiment results above 550 °C revealed that 201 to 273 μg C/g of organic carbon was preserved in the 3.5-billion-year-old Yellowknife Bay lacustrine mudstones, roughly 40 times more organic carbon than previously reported by Eigenbrode et al. (26), and more organic carbon than reported for Martian meteorites (1). Isotopic compositions of this high-temperature organic carbon are consistent with the same igneous refractory or macromolecular carbon phase detected in Mars meteorites with contributions from meteoritic organics, but a biological origin for some amount of carbon cannot be ruled out.

Assuming a similar ratio of organic carbon released during combustion to pyrolysis organic carbon as seen in Yellowknife Bay samples, estimated organic carbon concentrations for the Murray mudstone could approach 800 μg C/g, depending on the amount of carbonate present. While results suggest this carbon was largely refractory or mineral-bound in nature, which attests to its preservation over billions of years, it remains to be determined if there is chemical information preserved within it that pinpoint how it was formed and what processing may have altered it since deposition.