Methanogenic capabilities of ANME‐archaea deduced from 13C‐labelling approaches

Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME‐1 or ANME‐2. The ¹³C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate‐dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME‐1 and ANME‐2. Considerable methane production rates (MPRs) from CO₂‐reduction were observed, particularly from ANME‐2 dominated samples and in the presence of methane, which supports the hypothesis of a co‐occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through ¹³C‐assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the ¹³C‐uptake into lipids, ANME‐1 was found to thrive also under methane free conditions. Finally, C₃₅‐isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME‐1, most likely functioning as a hydrogen sink during methanogenesis.