Distinct Microbial Communities Within and On Seep Carbonates Support Long-term Anaerobic Oxidation of Methane and Novel pMMO Diversity

At methane seeps worldwide, syntrophic anaerobic methane-oxidizing archaea and sulfate-reducing bacteria (ANME-SRB) promote carbonate precipitation and rock formation, acting as methane and carbon sink.

While maintenance of active anaerobic oxidation of methane (AOM) within seep carbonates has been documented, the ANME-SRB reactivity to methane exposure remains uncertain. Surface-associated microbes may metabolize AOM-derived sulfide, maintain carbonate anoxia, and contribute to carbonate dissolution and higher trophic levels; however, these microbial communities are poorly described thus far.

Here we provide new insights into microbial diversity, metabolic potential, activity, and resiliency within and on Southern Californian methane seep carbonates, by combining 16S rRNA and metagenomic sequencing, laboratory incubations, and BONCAT-FISH. Ca. Methanophaga (ANME-1) dominated the carbonate interiors across different seepage activities, based on sequencing, while the dominant SRB was Ca. Desulfaltia, potentially a new ANME partner. BONCAT-FISH revealed differences in ANME-1 cell activity, suggesting cell dormancy or DNA preservation at less active seep sites. Carbonate incubations from low activity seeps (≥24 months) showed an exponential AOM reactivation (44-day doubling time), suggesting seep carbonates remain potential methane sinks over dynamic seepage conditions.

The surface-associated communities were distinct from the carbonate interior and other seep habitats, and highly heterogeneous. Surface ANME-SRB biofilms and sulfide-oxidizing bacterial mats were associated with high and intermediate AOM carbonates, potentially influencing carbonate precipitation/dissolution.

Carbonate surfaces shared diverse aerobic methanotrophs with invertebrates, potentially serving as pool for animal epibionts. Besides particulate methane monooxygenases from aerobic methanotrophs, we found divergent forms including within a Methylophagaceae (GCA-002733105) MAG suggesting a new function within Methylophagaceae.

Anaerobic methane oxidation activity of methane seep carbonates from Del Mar and SMM800. In situ AOM indicators and CH₃D rate measurements characterize low to high AOM activity carbonates. A) Seep carbonate collection sites Del Mar (light green marker) and Santa Monica Mound 800 (SMM800, dark green marker) are located 129 km apart. Map obtained from Google Maps. B) Biogeochemical seep carbonate setting. In situ images of C) the Del Mar outcrop, R1 and R2 originated from the top, R3 and R4 from closer to the sediment. D) R9, from a nearby Del Mar area with sulfide oxidizing mats. E) Chimlet and F) Protochimney are two chemoherm-like structures and were collected from different sides of the Santa Monica Mound 800. Chimlet actively bubbled with methane upon recovery. For scale, the red laser points in the images are 29 cm apart. G) Anaerobic methane activation rates (nmol D cm^-3 d^-1) measured in anoxic incubations with monodeuterated methane based on: CH₃D + SO₄ ^2-  HCO^{3 –} + HS- + HDO. We measured δD of water over five timepoints and calculated the rate from a linear increase unless stated otherwise. Error bars show the standard error of k calculated from the linear regression. Two subsamples of R9, R9.1 and R9.2 with different color, light grey and dark grey, respectively, were incubated for AOM rates. The orientation of the R9 piece dedicated for rates could not be reconstructed. At the last time point (t4) sulfide was measured and was detectable in R9.1, Chimlet top, middle, bottom, and Protochimney surface. *Deuterium above background was only detected at t4 indicating a nonlinear increase in R2 and R3., b.d. below detection, surf. surface, int. interior, btm. bottom. — biorxiv.org

Distinct Microbial Communities Within and On Seep Carbonates Support Long-term Anaerobic Oxidation of Methane and Novel pMMO Diversity, biorxiv.org

Astrobiology