Figures and Tables

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Introduction

Methane is an important energy source, greenhouse gas, and potential biosignature on Earth and other planetary bodies including Mars and Enceladus (Schulte et al., 2006

Schulte, M., Blake, D., Hoehler, T., McCollom, T. (2006) Serpentinization and Its Implications for Life on the Early Earth and Mars. Astrobiology 6, 364–376. https://doi.org/10.1089/ast.2006.6.364

; Waite et al., 2017

Waite, H.J., Glein, C.R., Perryman, R.S., Teolis, B.D., Magee, B.A., Miller, G., Grimes, J., Perry, M.E., Miller, K.E., Bouquet, A., Lunine, J.I., Brockwell, T., Bolton, S.J. (2017) Cassini Finds Molecular Hydrogen in the Enceladus Plume: Evidence for Hydrothermal Processes. Science 356, 155–159. https://doi.org/10.1126/science.aai8703.

). The stable isotope composition of methane is often used to distinguish between abiotic, thermogenic, and microbial sources on Earth (Schoell, 1980

Schoell, M. (1980) The Hydrogen and Carbon Isotopic Composition of Methane from Natural Gases of Various Origins. Geochimica et Cosmochimica Acta 44, 649–661. https://doi.org/10.1016/0016-7037(80)90155-6

). However, there is ongoing debate about the isotopic range that is possible in biogenic methane (Etiope and Sherwood-Lollar, 2013)

Etiope, G., Sherwood-Lollar, B. (2013) Abiotic methane on earth. Reviews of Geophysics 51, 276–299. https://doi.org/10.1002/rog.20011

, complicating interpretation of methane sources in disciplines such as natural gas exploration, origin of life studies, and extraterrestrial life detection.

Hydrogenotrophic methanogenesis, or the oxidation of H₂ to reduce CO₂ to CH₄, is the autotrophic mode of growth and most relevant methanogenesis pathway regarding the study of early Earth and the search for extraterrestrial life (Schulte et al., 2006

Schulte, M., Blake, D., Hoehler, T., McCollom, T. (2006) Serpentinization and Its Implications for Life on the Early Earth and Mars. Astrobiology 6, 364–376. https://doi.org/10.1089/ast.2006.6.364

). Hydrogenotrophic methanogenesis usually results in methane with a δ¹³C range of −110 ‰ to −60 ‰ (Elvert et al., 1999

Elvert, M., Suess, E., Whiticar, M.J. (1999) Anaerobic Methane Oxidation Associated with Marine Gas Hydrates: Superlight C-Isotopes from Saturated and Unsaturated C 20 and C 25 Irregular Isoprenoids. Naturwissenschaften 86, 295–300. https://doi.org/10.1007/s001140050619

; Conrad et al., 2011

Conrad, R., Noll, M., Claus, P., Klose, M., Bastos, W. R., Enrich-Prast, A. (2011) Stable Carbon Isotope Discrimination and Microbiology of Methane Formation in Tropical Anoxic Lake Sediments. Biogeosciences 8, 795–814. https://doi.org/10.5194/bg-8-795-2011

). Factors including environmental stress, substrate availability, and metabolic reversibility control where biogenic methane plots within this range (Fuchs et al., 1979

Fuchs, G., Thauer, R., Ziegler, H., Stichler, W. (1979) Carbon Isotope Fractionation by Methanobacterium Thermoautotrophicum. Archives of Microbiology 120, 135–139. https://doi.org/10.1007/BF00409099

; Valentine et al., 2004

Valentine, D.L., Chidthaisong, A., Rice, A., Reeburgh, W.S., Tyler, S.C. (2004) Carbon and Hydrogen Isotope Fractionation by Moderately Thermophilic Methanogens 1. Geochimica et Cosmochimica Acta 68, 1571–1590. https://doi.org/10.1016/j.gca.2003.10.012

) with metabolic net fractionation (ɛ¹³C) estimated to range between 20 ‰ to −106 ‰ (Gropp et al., 2021

Gropp, J., Iron, M.A., Halevy, I. (2021) Theoretical estimates of equilibrium carbon and hydrogen isotope effects in microbial methane production and anaerobic oxidation of methane. Geochimica et Cosmochimica Acta 295, 237–264. https://doi.org/10.1016/j.gca.2020.10.018

). In contrast, abiotic environmental methane is more ¹³C enriched, with values as depleted as −47 ‰ to enriched positive values (Etiope and Sherwood-Lollar, 2013

Etiope, G., Sherwood-Lollar, B. (2013) Abiotic methane on earth. Reviews of Geophysics 51, 276–299. https://doi.org/10.1002/rog.20011

).

One example of environments where the source of methane remains ambiguous includes serpentinising systems, where water-rock reactions produce H₂ and CH₄ and cause extreme carbon limitation due to high alkalinity (Nothaft et al., 2021

Nothaft, D.B., Templeton, A.S., Rhim, J.H., Wang, D.T., Labidi, J., Miller, H.M., Boyd, E.S., Matter, J.M., Ono, S., Young, E.D., Kopf, S.H., Kelemen, P.B., Conrad, M.E., The Oman Drilling Project Science Team. (2021) Geochemical, Biological, and Clumped Isotopologue Evidence for Substantial Microbial Methane Production Under Carbon Limitation in Serpentinites of the Samail Ophiolite, Oman. Journal of Geophysical Research: Biogeosciences 126, e2020JG006025. https://doi.org/10.1029/2020JG006025

). The δ¹³C of CO₂/DIC found in natural serpentinites with high H₂ concentrations is −1.08 ‰ to −25.3 ‰ and methane detected in these systems can be relatively enriched in ¹³C when compared to most natural environments, with δ¹³C values as positive as +5 ‰ (Etiope and Sherwood-Lollar, 2013

Etiope, G., Sherwood-Lollar, B. (2013) Abiotic methane on earth. Reviews of Geophysics 51, 276–299. https://doi.org/10.1002/rog.20011

; Etiope et al., 2016

Etiope, G., Vadillo, I., Whiticar, M.J., Marques, J.M., Carreira, P.M., Tiago, I., Benavente, J., Jiménez, P., Urresti, B. (2016) Abiotic Methane Seepage in the Ronda Peridotite Massif, Southern Spain. Applied Geochemistry 66, 101–113. https://doi.org/10.1016/j.apgeochem.2015.12.001

; Miller et al., 2016

Miller, H.M., Matter, J.M., Kelemen, P., Ellison, E.T., Conrad, M.E., Fierer, N., Ruchala, T., Tominaga, M., Templeton, A.S. (2016) Modern Water/Rock Reactions in Oman Hyperalkaline Peridotite Aquifers and Implications for Microbial Habitability. Geochimica et Cosmochimica Acta 179, 217–241. https://doi.org/10.1016/j.gca.2016.01.033

; Nothaft et al., 2021

Nothaft, D.B., Templeton, A.S., Rhim, J.H., Wang, D.T., Labidi, J., Miller, H.M., Boyd, E.S., Matter, J.M., Ono, S., Young, E.D., Kopf, S.H., Kelemen, P.B., Conrad, M.E., The Oman Drilling Project Science Team. (2021) Geochemical, Biological, and Clumped Isotopologue Evidence for Substantial Microbial Methane Production Under Carbon Limitation in Serpentinites of the Samail Ophiolite, Oman. Journal of Geophysical Research: Biogeosciences 126, e2020JG006025. https://doi.org/10.1029/2020JG006025

). The seemingly abiotic signal of methane in these serpentinites has sparked debate regarding the potential contribution of microbial methanogenesis since autotrophic methanogens have also been found in these systems (Miller et al., 2016

Miller, H.M., Matter, J.M., Kelemen, P., Ellison, E.T., Conrad, M.E., Fierer, N., Ruchala, T., Tominaga, M., Templeton, A.S. (2016) Modern Water/Rock Reactions in Oman Hyperalkaline Peridotite Aquifers and Implications for Microbial Habitability. Geochimica et Cosmochimica Acta 179, 217–241. https://doi.org/10.1016/j.gca.2016.01.033

, 2018

Miller, H.M., Chaudhry, N., Conrad, M.E., Bill, M., Kopf, S.H., Templeton, A.S. (2018) Large Carbon Isotope Variability during Methanogenesis under Alkaline Conditions. Geochimica et Cosmochimica Acta 237, 18–31. https://doi.org/10.1016/j.gca.2018.06.007

; Nothaft et al., 2021

Nothaft, D.B., Templeton, A.S., Rhim, J.H., Wang, D.T., Labidi, J., Miller, H.M., Boyd, E.S., Matter, J.M., Ono, S., Young, E.D., Kopf, S.H., Kelemen, P.B., Conrad, M.E., The Oman Drilling Project Science Team. (2021) Geochemical, Biological, and Clumped Isotopologue Evidence for Substantial Microbial Methane Production Under Carbon Limitation in Serpentinites of the Samail Ophiolite, Oman. Journal of Geophysical Research: Biogeosciences 126, e2020JG006025. https://doi.org/10.1029/2020JG006025

). In theory, methanogens will produce methane with the same isotopic composition as the starting CO₂ at complete DIC consumption (Meister et al., 2019). However, the lack of net carbon isotope fractionation at complete DIC consumption has not been experimentally shown. Here, we aim to address the question whether hydrogenotrophic methanogens will actually produce isotopically heavy methane in a carbon limited system.

Lipids, which are utilised by all terrestrial life for energy storage and the construction of cellular membranes, are the most chemically stable polymer and can be preserved for over hundreds of millions of years within sediment (Sessions et al., 2004

Sessions, A.L., Sylva, S.P., Summons, R.E., Hayes, J.M. (2004) Isotopic Exchange of Carbon-Bound Hydrogen over Geologic Timescales 1 1Associate Editor: J. Horita. Geochimica et Cosmochimica Acta 68, 1545–1559. https://doi.org/10.1016/j.gca.2003.06.004

). Like methane, lipids have carbon isotope signatures that store metabolic and/or environmental information. Carbon limitation has been seen to cause a smaller depletion in δ¹³C of methanogen lipids when compared to carbon replete conditions, regardless of substrate (Londry et al., 2008

Londry, K.L., Dawson, K.G., Grover, H.D., Summons, R.E., Bradley, A.S. (2008) Stable Carbon Isotope Fractionation between Substrates and Products of Methanosarcina Barkeri. Organic Geochemistry 39, 608–621. https://doi.org/10.1016/j.orggeochem.2008.03.002

). δ¹³C of lipids analysed from carbon limited serpentinising systems have shown ¹³C isotopic enrichment, with an average δ¹³C of +2 ‰ and a high of +14 ‰ compared to typically depleted values of ∼−50 ‰ or less (Bradley et al., 2009

Bradley, A. S., Fredricks, H., Hinrichs, K., Summons, R.E. (2009) Structural Diversity of Diether Lipids in Carbonate Chimneys at the Lost City Hydrothermal Field. Organic Geochemistry 40, 1169–1178. https://doi.org/10.1016/j.orggeochem.2009.09.004

; Zwicker et al., 2018

Zwicker, J., Birgel, D., Bach, W., Richoz, S., Smrzka, D., Grasemann, B., Gier, S., Schleper, C., Rittmann S.K.-M.R., Kosun, E., Peckmann, J. (2018) Evidence for Archaeal Methanogenesis within Veins at the Onshore Serpentinite-Hosted Chimaera Seeps, Turkey. Chemical Geology 483, 567–580. https://doi.org/10.1016/j.chemgeo.2018.03.027

). It is unknown whether lipids follow the same trend as methane with respect to carbon isotope fractionation under carbon limitation.

This study thus investigates the carbon isotope composition of methane, biomass, and lipids of hydrogenotrophic methanogen Methanococcus maripaludis S2 in a closed system under DIC limitation with excess H₂. We hypothesise that ¹³C enriched methane should form from biological activity under extreme carbon limitation, giving rise to methane with δ¹³C values indistinguishable from abiotic methane.

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Methods

Experimental setup. Anaerobic batch cultures of Methanococcus maripaludis S2 were grown hydrogenotrophically in 160 mL serum vials in a modified DSMZ141 media devoid of yeast extract, cysteine, and other organic carbon sources. Cultures were given excess H₂ at 20 psi (7.4 mmol) as the electron donor and 1.08 mmol inorganic carbon (NaHCO₃) as the electron acceptor and sole carbon source. Cultures were continuously stirred at 625 rpm to increase the gas transfer rate between headspace and liquid. Cultures were grown at an initial pH of 6.7 and temperature of 37 °C. Optical density was measured continuously throughout growth at 630 nm. One vial was not inoculated, and the incubation was terminated immediately to capture initial DIC. Four incubations were terminated along various points of exponential growth phase. At termination, exponential cultures were injected with phosphoric acid to stop growth and release all remaining DIC into headspace. Three cultures were allowed to consume the entire DIC pool and sampled in early stationary phase. A small portion of stationary culture medium was acidified to determine if any DIC remained.

Headspace gas (CO₂, CH₄, and H₂) from acidified samples was transferred via gas-tight syringe to sealed serum vials filled with a 30 % NaCl solution for preservation until analysis (Gan et al., 1998

Gan, J., Papiernik, S., Yates, S.R. (1998) Static Headspace and Gas Chromatographic Analysis of Fumigant Residues in Soil and Water. Journal of Agricultural and Food Chemistry 46, 986–990. https://doi.org/10.1021/jf970735w

). After headspace sampling, biomass from stationary phase cultures was pelleted and freeze dried. Biomass pellets were divided in half for bulk biomass analysis and lipid extractions. Bulk biomass was oven dried to prepare for analysis. Lipids were extracted from biomass by acidic hydrolysis methanolysis (Zhou et al., 2020

Zhou, A., Weber, Y., Chiu, B.K., Elling F.J., Cobban, A.B., Pearson, A., Leavitt, W.D. (2020) Energy flux controls tetraether lipid cyclization in Sulfolobus acidocaldarius. Environmental Microbiology 22, 343–353. https://doi.org/10.1111/1462-2920.14851

). Phytane was extracted from the total lipid extract by ether cleavage and hydrogenation (Kaneko et al., 2011

Kaneko, M., Kitajima, F., Naraoka, H. (2011) Stable Hydrogen Isotope Measurement of Archaeal Ether-Bound Hydrocarbons. Organic Geochemistry 42, 166–172. https://doi.org/10.1016/j.orggeochem.2010.11.002

).

Sample analyses. Headspace gases (CO₂, CH₄, and H₂) were quantified on a gas chromatograph with flame ionisation and thermal conductivity detectors (SRI GC-FID/TCD Multi-Gas #5 Configuration). δ¹³C of CO₂ and CH₄ were measured on a Picarro Ring-Down Spectrometer G2201-I and δ²H of CH₄ on a gas chromatograph isotope ratio mass spectrometry system (Thermo Scientific GC TraceGas Ultra system connected to a Thermo Scientific Delta V Plus).

Bulk biomass δ¹³C was analysed on a Thermo Delta V continuous flow stable isotope ratio mass spectrometer attached to a Thermo Flash2000 Elemental Analyzer. Phytane was quantified on a GC flame ionisation detector (GC-FID Thermo TRACE 1310) and identified on a single quadrupole gas chromatography mass spectrometer (GC-MS Thermo ISQ LT with TRACE 1310). The δ¹³C of phytane was measured on a GC pyrolysis isotope ratio MS (GC IsoLink II + MAT253 Plus IRMS, Thermo Scientific).

All carbon and hydrogen isotope measurements were corrected using standards of known isotopic composition and are reported in the conventional delta notation vs. the Vienna Pee Dee Belemnite (VPDB)/Vienna Standard Mean Ocean Water (VSMOW) international scales respectively: δ¹³C = [¹³C/¹²C]_sample/[¹³C/¹²C]_VPDB − 1; δ²H = [²H/¹H]_sample/[²H/¹H]_VSMOW − 1. Observed isotope fractionation between two reservoirs is reported in alpha and epsilon notation: ¹³ɛ_a/b = ¹³α_a/b − 1 = [¹³C/¹²C]_a/[¹³C/¹²C]_b − 1. δ and ɛ values reported in per mille (‰) are implicitly multiplied by a factor of 1000 (Coplen, 2011

Coplen, T.B. (2011) Guidelines and Recommended Terms for Expression of Stable‐isotope‐ratio and Gas‐ratio Measurement Results. Rapid Communications in Mass Spectrometry 25, 2538–2560. https://doi.org/10.1002/rcm.5129

).

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Results and Discussion

During methanogenesis, CO₂ was fully consumed while H₂ remained in excess throughout the experiment. As CO₂ was consumed, the distillation process increased δ¹³C CO₂ from −2.8 ‰ initially to +78.5 ‰ in late exponential phase (see Table 1). Conversely, methane started relatively depleted with δ¹³C CH₄ −23.6 ‰ at the first measurement point (when ∼42 % of the CO₂ was consumed) and increased to −2.5 ‰ (i.e. slightly above the starting value of CO₂) at the end of the distillation when the methanogens reached stationary phase and had consumed >99 % of the CO₂.

Samples	% CO2 remaining	δ13CO2 (‰)	δ13CH4 (‰)	δ13C (‰) phytane	δ13C (‰) biomass	δ2H (‰) CH4
starting	100.0 ± 2.8	−2.8 ± 0.5
early-exponential	57.9 ± 2.3	+15.7 ± 0.6	−23.6 ± 0.5
early/mid-exponential	38.4 ± 2.3	+28.8 ± 0.9	−21.5 ± 0.7
mid/late-exponential	23.6 ± 2.3	+51.2 ± 1.4	−16.1 ± 0.7
late-exponential	13.2 ± 1.8	+78.5 ± 2.3	−12.0 ± 0.7
stationary	0.6 ± 1.4		−2.5 ± 0.4	−18.5 ± 0.4	−7.8 ± 0.4	−437 ± 14

The δ¹³C values for biomass and phytane measured in stationary phase were significantly more depleted than the methane that had accumulated at this point (Table 1), with ¹³ɛ_{biomass/methane} = −5.3 ‰ and ¹³ɛ_{phytane/methane} = −16 ‰. The biomass yield estimate from isotopic mass balance of the final methane and biomass is 6.4 %, assuming no other significant sinks of carbon. This yield is lower than other estimates with abundant substrate but comparable to those with limited substrate (Londry et al., 2008

Londry, K.L., Dawson, K.G., Grover, H.D., Summons, R.E., Bradley, A.S. (2008) Stable Carbon Isotope Fractionation between Substrates and Products of Methanosarcina Barkeri. Organic Geochemistry 39, 608–621. https://doi.org/10.1016/j.orggeochem.2008.03.002

).

Carbon isotope fractionation between methanogenesis products. The net carbon isotope fractionation between biomass and methane measured in stationary phase is shown in Figure 1 together with literature data.

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Overall, the data follows the trend that carbon limitation decreases the isotopic offset between biomass and methane. However, data from this study inverts the fractionation typically observed in laboratory experiments (Fuchs et al., 1979

Fuchs, G., Thauer, R., Ziegler, H., Stichler, W. (1979) Carbon Isotope Fractionation by Methanobacterium Thermoautotrophicum. Archives of Microbiology 120, 135–139. https://doi.org/10.1007/BF00409099

; Summons et al., 1998

Summons, R.E., Franzmann, P.D., Nichols, P.D. (1998) Carbon Isotopic Fractionation Associated with Methylotrophic Methanogenesis. Organic Geochemistry 28, 465–475. https://doi.org/10.1016/S0146-6380(98)00011-4

; Londry et al., 2008

Londry, K.L., Dawson, K.G., Grover, H.D., Summons, R.E., Bradley, A.S. (2008) Stable Carbon Isotope Fractionation between Substrates and Products of Methanosarcina Barkeri. Organic Geochemistry 39, 608–621. https://doi.org/10.1016/j.orggeochem.2008.03.002

; Nguyen et al., 2020

Nguyen, T.B., Topçuoğlu, B.D., Holden, J.F., LaRowe, D.E., Lang, S.Q. (2020) Lower hydrogen flux leads to larger carbon isotopic fractionation of methane and biomarkers during hydrogenotrophic methanogenesis. Geochimica et Cosmochimica Acta 271, 212–226. https://doi.org/10.1016/j.gca.2019.11.015

): methane in our cultures is more isotopically enriched than biomass (see Fig. 1; ɛ < 0 ‰/α < 1). Fractionation between lipids and methane exhibits the same pattern (Fig. S-1).

We thus observe that extreme carbon limitation in a closed system causes the isotopic signature of accumulated biomass to be more ¹³C depleted than methane. We hypothesise this could occur if biomass is primarily synthesised early on when there is abundant CO₂ available for both catabolism and anabolism. If methanogens prioritise the energy producing catabolic pathway of methanogenesis and reduce CO₂ fixation as CO₂ becomes limiting, the isotopic composition of biomass remains at relatively light values compared to the still accumulating methane that is produced from the increasingly enriched residual CO₂ (illustrated in Fig. 2 in the “anabolism stops” model). Although the impact of carbon limitation on biosynthesis inhibition has not been experimentally investigated in methanogens, inorganic carbon limitation has been found to increase energy producing metabolisms and decrease cell yield in ammonia oxidising bacteria to account for increased cellular maintenance energy requirements (Jiang et al., 2015

Jiang, D., Khunjar, W.O., Wett, B., Murthy, S.N., Chandran, K. (2015) Characterizing the Metabolic Trade-Off in Nitrosomonas Europaea in Response to Changes in Inorganic Carbon Supply. Environmental Science & Technology 49, 2523–2531. https://doi.org/10.1021/es5043222

; Mellbye et al., 2016

Mellbye, B.L., Giguere, A., Chaplen, F., Bottomley, P.J., Sayavedra-Soto, L.A. (2016) Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas Europaea. Applied and Environmental Microbiology 82, 3310–3318. https://doi.org/10.1128/AEM.00294-16

). Additionally, hydrogenotrophic methanogens have been found to uncouple biosynthesis from methanogenesis during stressful situations, including phosphate limitation (Leigh et al., 2008

Leigh, J.A., Whitman, W.B., Hackett, M. (2008) Hydrogenases of Methanococcus Maripaludis. U.S. Department of Energy. https://www.hydrogen.energy.gov/docs/hydrogenprogramlibraries/pdfs/review08/besp_1_leigh.pdf?sfvrsn=77852def_1

). As shown in Figure 2, such a decrease in biomass yield over time could cause the accumulated biomass in our experiments to be more isotopically depleted than final accumulated methane because it was predominantly produced from CO₂ early during the distillation when CO₂ was not yet as heavily ¹³C enriched. The accumulated methane is isotopically heavier than the biomass because methane generation continued when CO₂ was sparse and ¹³C enriched from the distillation process. This interpretation implies that the δ¹³C of lipids and biomass may not reflect the final environmental state but rather reflect a time of high CO₂ availability.

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Rayleigh distillation and CO₂ consumption. The observed isotopic compositions of methane and CO₂ fit a Rayleigh distillation model with increasing metabolic fractionation well. In its simplest form, this takes the shape of a two-step distillation process as illustrated in Figure 2 with an initial CO₂-CH₄ fractionation of −29.6 ± 2.3 ‰ that becomes more heavily fractionating (−44.0 ± 2.5 ‰) after ∼55 % of CO₂ is consumed (all 3 parameters estimated simultaneously by non-linear least squares NLS data fitting in R; RMSE 2.2 ‰). This model reflects a closed system that starts with all the carbon as 100 % reactant (CO₂) and ends with 100 % products (CH₄ and biomass) and both methanogenesis and biomass production stop once all CO₂ is consumed. The total biomass yield is assumed to be the 6.4 % inferred from overall isotope mass balance. As expected from a Rayleigh distillation process, residual CO₂ and accumulated CH₄ and biomass systematically get isotopically heavier as CO₂ is consumed. The offset between the biomass and methane curves (−5.3 ‰) results from the constraint posed by the isotopic composition of the final products. At full DIC consumption, the accumulated methane is slightly enriched compared to the initial δ¹³C of CO₂ (initial δ¹³C_CO2 = −2.8 ‰, final δ¹³C_CH4 = −2.5 ‰). This is the expected outcome from near complete consumption of CO₂ in a closed system for the majority product. By isotope mass balance, CH₄ will be necessarily enriched compared to CO₂ if biomass is more depleted than CO₂.

A modification of this model where anabolism stops as CO₂ becomes increasingly limiting can explain the observed isotopic composition of the accumulated biomass with a positive biomass CH₄ fractionation factor (ɛ > 0) that is consistent with literature data under non-carbon limiting conditions (Fig. 1). For example, if the methanogens stopped fixing carbon at a threshold of 15 % CO₂ remaining (example “anabolism stops” in Fig. 2), the metabolic fractionation factor between biomass CH₄ would be estimated at +8.9 ‰. The exact value of the fractionation factor depends on the threshold at which anabolism stops, which is not fully constrained by the available data (see Fig. S-2). However, based on the available literature data (Fig. 1), we hypothesise that this threshold model (“anabolism stops” in Fig. 2) is a more accurate representation of biomass fractionation, with biomass more enriched than the accumulated methane during initial metabolism until anabolism stops and the accumulated methane continuing to get more enriched from the distillation process. The resulting estimates of the methanogenesis fractionation fall into a narrow range regardless of when anabolism stops and closely match the estimates from the simpler model (“anabolism continues” in Fig. 2): the initial fractionation is −30.0 to 29.6 ‰ and increases to −45.5 to −43.7 ‰ after ∼55 % of CO₂ is consumed. These CO₂-CH₄ fractionation factors fall well within the possible range of methanogenic fractionation (−106 ‰ to −20 ‰, Gropp et al., 2021

Gropp, J., Iron, M.A., Halevy, I. (2021) Theoretical estimates of equilibrium carbon and hydrogen isotope effects in microbial methane production and anaerobic oxidation of methane. Geochimica et Cosmochimica Acta 295, 237–264. https://doi.org/10.1016/j.gca.2020.10.018

). See SI for all equations and github.com/KopfLab/2025_batther_et_al._C_limitation for implementation.

Environmental implications. These experimental findings and comparison to model predictions show that biological CH₄ can span the full range of δ¹³C CH₄ space depending on carbon availability and environmental conditions, such as hydrogen availability, rather than being constrained by the previously inferred microbial range (Fig. 3). The potential biogenicity of methane cannot be ruled out even if there is little isotopic fractionation between methane and the bulk carbon pool of any system. Rather, the lack of isotopic fractionation may be more indicative of extreme carbon limitation controlling the methane producing reaction pathway. Although such carbon limitation may be rare, environments such as serpentinising systems are an excellent example on Earth that may also be relevant to habitable rocky bodies in our solar system, and where the potential for microbial methanogenesis to produce ¹³C enriched methane has proven to be controversial. Therefore, the δ¹³C of CH₄ detected in the environment cannot be the sole measurement used to determine the source of CH₄, especially to rule out microbial activity. Given possible overlap with the large range of δ¹³C CH₄ formed from Fischer-Tropsch type reactions (Etiope and Sherwood-Lollar, 2013

Etiope, G., Sherwood-Lollar, B. (2013) Abiotic methane on earth. Reviews of Geophysics 51, 276–299. https://doi.org/10.1002/rog.20011

), multiple measurements will be required to determine the biogenicity of CH₄, including analysis of the Schultz-Flory distribution of higher hydrocarbons, gene sequencing, lipidomics, or clumped CH₄ measurements (Nothaft et al., 2021

Nothaft, D.B., Templeton, A.S., Rhim, J.H., Wang, D.T., Labidi, J., Miller, H.M., Boyd, E.S., Matter, J.M., Ono, S., Young, E.D., Kopf, S.H., Kelemen, P.B., Conrad, M.E., The Oman Drilling Project Science Team. (2021) Geochemical, Biological, and Clumped Isotopologue Evidence for Substantial Microbial Methane Production Under Carbon Limitation in Serpentinites of the Samail Ophiolite, Oman. Journal of Geophysical Research: Biogeosciences 126, e2020JG006025. https://doi.org/10.1029/2020JG006025

). However, if active biological CH₄ production can be confirmed, then the δ¹³C of CH₄ can be used to infer if an environment is carbon limited or carbon replete.

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Conclusion

This study experimentally demonstrates the impact of full carbon consumption on the carbon isotope signatures of CH₄, biomass, and lipids accumulated during purely autotrophic methanogenesis. The data show that carbon limitation causes accumulated CH₄ to isotopically reflect initial CO₂, which is supported by our Rayleigh Distillation model and has only been previously theorised. This can result in the production of biogenic methane tens of per mille more positive than traditionally assumed. Such ¹³C enriched biogenic methane contains valuable information about the extent of C limitation, which is rarely utilised in environmental assessments. Instead, the detection of ¹³C enriched methane has typically been attributed to abiotic sources, which may sometimes be erroneous. Additionally, we see a greater depletion of δ¹³C biomass/lipids relative to CH₄ than expected, which could be caused by the inhibition of anabolism by carbon limitation and is an important physiological response worthy of further investigation. In summary, the data support the concept that biogenic methane, biomass, and lipid δ¹³C values predicted for natural environments need to be significantly expanded to include the impact of extreme carbon limitation on isotope fractionation.

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Acknowledgements

This work was supported by NASA Exobiology Program award #21-EXO21-0055, “Methanogenic Activity And Isotopic Biosignatures Under Carbon Limitation”. The work at Lawrence Berkeley National Laboratory is supported by U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division, under Award Numbers DE-AC02-05CH11231. We acknowledge the analytical contributions of the CU Boulder Earth Systems Stable Isotope Lab (CUBES-SIL) Core Facility (RRID:SCR_019300). Publication of this article was funded by the University of Colorado Boulder Libraries Open Access Fund.

Editor: Eric H. Oelkers

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References

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Supplementary Information

The Supplementary Information includes:

• Methods
• Calculations
• Figures S-1 and S-2
• Supplementary Information References

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