Archaeal ANaerobic MEthanotrophs (ANME) facilitate the anaerobic oxidation of methane (AOM), a process that is believed to proceed via the reversal of the methanogenesis pathway. Carbon isotopic composition studies indicate that ANME are metabolically diverse and able to assimilate metabolites including methane, methanol, acetate, and dissolved inorganic carbon (DIC). Our data support the interpretation that ANME in marine sediments at methane seeps assimilate both methane and DIC, and the carbon isotopic compositions of the tetrapyrrole coenzyme F430 and the membrane lipids archaeol and hydroxy‐archaeol reflect their relative proportions of carbon from these substrates. Methane is assimilated via the methyl group of CH3‐tetrahydromethanopterin (H4MPT) and DIC from carboxylation reactions that incorporate free intracellular DIC. F430 was enriched in 13C (mean δ13C = −27‰ for Hydrate Ridge and −80‰ for the Santa Monica Basin) compared to the archaeal lipids (mean δ13C = −97‰ for Hydrate Ridge and −122‰ for the Santa Monica Basin). We propose that depending on the side of the tricarboxylic acid (TCA) cycle used to synthesize F430, its carbon was derived from 76% DIC and 24% methane via the reductive side or 57% DIC and 43% methane via the oxidative side. ANME lipids are predicted to contain 42% DIC and 58% methane, reflecting the amount of each assimilated into acetyl‐CoA. With isotope models that include variable fractionation during biosynthesis for different carbon substrates, we show the estimated amounts of DIC and methane can result in carbon isotopic compositions of − 73‰ to − 77‰ for F430 and − 105‰ for archaeal lipids, values close to those for Santa Monica Basin. The F430 δ13C value for Hydrate Ridge was 13C‐enriched compared with the modeled value, suggesting there is divergence from the predicted two carbon source models.
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