Active pathways of anaerobic methane oxidization in deep-sea cold seeps of the South China Sea

Anaerobic oxidation of methane (AOM) is critical for controlling methane emissions from deep-sea cold seeps. Microbial groups associated with different AOM processes have been briefly investigated, but the AOM activities associated with the utilization of different electron acceptors in the deep-sea methane seeps remain largely unknown. Here, surface sediments (0-4 cm) from two cold seeps (Site F/Haima) and the Xisha trough in the South China Sea (SCS) were incubated to measure AOM potentials driven by nitrite, nitrate, and sulfate as different electron acceptors with isotopically labeled methane gas ((CH4)-C-13), and the population shifts of microbial communities along the incubation were investigated by high-throughput sequencing and quantitative polymerase chain reaction based on both the 16S rRNA gene and functional genes. Nitrite and nitrate were preferentially used prior to sulfate during the incubation, and nitrate-/nitrite-dependent anaerobic methane oxidation (Nr-/N-DAMO) contributed more to the methane flux in both the cold seeps and the trough. The highest rates of DAMO and sulfate-dependent anaerobic methane oxidation (SAMO) were detected at Site F, consistent with the transcript abundance of mcrA and dsrB genes during the incubation, and might be explained by the in situ active functional groups. The former process might be explained by Campylobacteria, which could couple sulfide oxidation and nitrate consumption to further participate in the N-DAMO process, while the latter process might be due to the higher proportions of Desulfobacterota. This study elucidates the diversity and potential activities of major microbial groups in the DAMO and SAMO processes in the deep-sea cold seeps using isotopic tracing incubations and provides direct evidence for the occurrence of Nr-/N-DAMO as a previously overlooked microbial methane sink in the deep-sea hydrate-bearing sediments in the SCS.