Marine cold seep ANME-2/SRB consortia produce their lipid biomass from inorganic carbon

In cold seeps, anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) oxidize methane to inorganic carbon (IC) coupled to sulfate reduction. While catabolic pathways are well resolved, carbon flow into biomass remains 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-labeled 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. Remarkedly, IC assimilation in SRB lipids was eight times higher than in ANME lipids, suggesting that ANME either rely on fixation of internally generated CO 2 from 13 C-label-free methane before it can be exchanged with the environment or they utilize an additional, yet unknown carbon source for lipid biosynthesis. By examining the step-wise 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.