Cycling of sulfur redox intermediates drives microbial activity in the sulfate-methane transition zone of cold methane seeps

Microbial sulfate reduction is a cornerstone of marine sediment biogeochemistry, driving carbon remineralization and fueling the anaerobic oxidation of methane (AOM). Yet in zones of high methane flux, sulfate limitations may constrain the sulfate-reducing bacteria (SRB) and anaerobic methanotrophic archaea (ANME) that typically perform AOM. Although often overlooked, sulfur redox intermediates are readily utilized by diverse microorganisms, potentially driving AOM in sulfate-limited zones. To resolve the microbial mechanisms underlying cryptic sulfur cycling in such sediments, Monterey Canyon cold methane seeps were investigated through an integrated geochemical, isotopic, and metatranscriptomic approach. High-resolution electrochemical measurements confirmed intense sulfide production in seep sites, and long-term anoxic incubations were conducted with sediment from the SMTZ amended with elemental sulfur, thiosulfate, or sulfate as the sole sulfur source, with or without methane. Over 650 days, sulfide accumulation was greatest in elemental sulfur treatments, followed by thiosulfate and sulfate; in all cases methane addition enhanced sulfide production. Isotopic measurements showed modest S-isotope fractionation indicating that the large fractionations typical of slow sulfate reduction were muted by additional sulfur transformations. In elemental sulfur treatments, isotopic and geochemical patterns suggested that disproportionation was unlikely. Metatranscriptomes revealed broad expression of sox genes and abundant dsr/apr across treatments, along with thiosulfate-linked phs upregulation. While ANME-2c and SEEP-SRB2 activity increased with methane, transcriptomic and isotopic data together highlighted the roles of Desulfocapsaceae, Desulfobulbaceae, and Sulfurovaceae lineages in mediating sulfur transformations. Taken together, these results demonstrate how cryptic sulfur cycling may sustain microbial communities in sulfate-depleted deep-sea sediments and contribute to AOM.