Methane seepage intensity distinguish microbial communities in sediments at the Mid-Okinawa Trough

Shallow methane/sulfate transition zones in cold seeps are hotspots to study microbially mediated geochemical cycles due to high methane fluxes. However, our knowledge about the microbial communities in remote seafloor cold seep ecosystems with different methane seepage intensity is still sparse due to the challenge for sampling and visual obser-vations. In this work, three remotely operated vehicle (ROV) video-guided push sediment cores were sampled from cold seep fields with different methane seepage intensity (low-intensity seepage, R5-C1; moderate-intensity seepage, R6-C2; high-intensity seepage, R6-C3) at the western slope of Mid-Okinawa Trough (Mid-OT) and subjected to high throughput sequencing of 16S rRNA genes for bacteria and archaea. Vesicomyid clams and white microbial mats are visible by video at R6-C3 with methane bubbles. The high relative abundances of anaerobic methanotrophic archaea (ANME-1,-2, and-3), delta-Proteobacteriacea and Campylobacteria in R6-C3 indicated that the processes of anaerobic meth-ane oxidation (AOM), sulfate reduction and sulfur oxidation might occur in this active seeping site. In contrast, Bathyarchaeia, Nitrosopumilales, Sphingomonadales, and Burkholderiales were enriched in bubble-free sites, which com-monly involved in the degradation of organic compounds. Principal coordinate analysis showed that both bacterial and archaeal communities were clustered according to sampling sites, also indicating the impact of methane seepage intensity on microbial communities. The co-occurrence network analysis revealed that microbes at the site with high methane fluxes mainly cooperated with each other to sustain the ecosystems, whereas competition enhanced at sites with low methane fluxes. Detection of thermophiles Thermoanaerobaculia and Hydrothermarchaeota may indicate microbial transmission from nearby hydrothermal vents, suggesting potential interactions between cold seepage and hydrothermal vent ecosystems. These results expand our knowledge about the composition and distribution of bacteria and archaea with different methane seepage intensity in cold seep field at the Mid-OT, contributing to the on-going efforts in understanding carbon cycling in the cold seep ecosystems.