Distinct microbial communities within and on seep carbonates support long-term anaerobic oxidation of methane and divergent pMMO diversity.

At methane seeps worldwide, syntrophic anaerobic methane-oxidizing archaea and sulfate-reducing bacteria promote carbonate precipitation and rock formation, acting as methane and carbon sinks. Although maintenance of anaerobic oxidation of methane (AOM) within seep carbonates has been documented, its reactivation upon methane exposure remains uncertain. Surface-associated microbes may metabolize sulfide from AOM, maintain carbonate anoxia, contribute to carbonate dissolution, and support higher trophic levels; however, these communities are poorly described. We provide insights into microbial diversity, metabolism, activity, and resiliency within and on seep carbonates through amplicon and metagenomic sequencing, incubations, and non-canonical amino acid tagging combined with fluorescence in situ hybridization (BONCAT-FISH). Ca. Methanophaga (ANME-1) dominated the carbonate interiors in active and low activity seeps, co-occurring with Ca. Desulfaltia as main sulfate reducer, potentially a new syntrophic partner in AOM. Single-cell BONCAT-FISH revealed variability in ANME-1 activity, suggesting potential dormancy in carbonates from low activity seep sites. However, incubations with carbonates from low activity seeps (≥24 months) showed exponential AOM reactivation (~44-day doubling), suggesting these carbonates retain the potential as long-term methane sinks under dynamic seepage conditions. Surface-associated microbial communities were heterogeneous and distinct from the carbonate interior and other seep habitats. Anaerobic methane-oxidizing biofilms and sulfide-oxidizing mats were associated with carbonates with high and intermediate AOM rates potentially influencing carbonate precipitation/dissolution. Shared aerobic methanotrophs between carbonate surfaces and invertebrates indicated carbonate surfaces may represent animal epibiont reservoirs. Recovered particulate methane monooxygenases included both aerobic methanotrophs and divergent forms associated with the Methylophagaceae, suggesting a new function in this group.