Abstract
In cold seeps, anaerobic methanotrophic archaea (ANME) and sulphate‐reducing bacteria (SRB) oxidise methane to inorganic carbon (IC) coupled to sulphate reduction. While catabolic pathways are well resolved, carbon flow into biomass as well as the functional roles of lipid biomarkers remain unclear. We conducted lipid stable isotope probing (lipid‐SIP) experiments with Astoria Canyon sediments dominated by ANME‐2/SRB consortia and incubated samples with either 13 C‐labelled methane ( 13 CH 4 ) or dissolved IC (DI 13 C). Lipid‐specific δ 13 C analysis showed higher 13 C incorporation from DI 13 C than from 13 CH 4 . After 30 days, δ 13 C values were up to +417‰ in SRB‐specific fatty acids (e.g., C 16:1ω5c , cyC 17:0ω5,6 ) and +126‰ in ANME‐2‐specific isoprenoid lipids (e.g., archaeol, crocetane). Based on these values, we calculated carbon assimilation rates and found that both partners primarily assimilate IC. Remarkably, IC assimilation in SRB lipids was eight times higher than in ANME lipids, suggesting that ANME may use additional yet‐to‐be‐identified carbon sources, potentially produced by their partner SRB. By examining the stepwise 13 C‐enrichment of ANME‐ and SRB‐derived lipids, we further delineate biosynthetic pathways for archaeal and bacterial diether lipid formation and highlight crocetane as a bilayer‐modulating isoprenoid hydrocarbon potentially affecting membrane fluidity and proton permeability.