Seep sediments are dominated by intensive microbial sulfate reduction coupled to the anaerobic oxidation of methane (AOM). Through geochemical measurements of incubation experiments with methane seep sediments collected from Hydrate Ridge, we provide insight into the role of iron oxides in sulfate-driven AOM. Seep sediments incubated with 13 C-labeled methane showed cooccurring sulfate reduction, AOM, and methanogenesis. The isotope fractionation factors for sulfur and oxygen isotopes in sulfate were about 40‰ and 22‰, respectively, reinforcing the difference between microbial sulfate reduction in methane seeps versus other sedimentary environments (for example, sulfur isotope fractionation above 60‰ in sulfate reduction coupled to organic carbon oxidation or in diffusive sedimentary sulfate-methane transition zone). The addition of hematite to these microcosm experiments resulted in significant microbial iron reduction as well as enhancing sulfate-driven AOM. The magnitude of the isotope fractionation of sulfur and oxygen isotopes in sulfate from these incubations was lowered by about 50%, indicating the involvement of iron oxides during sulfate reduction in methane seeps. The similar relative change between the oxygen versus sulfur isotopes of sulfate in all experiments (with and without hematite addition) suggests that oxidized forms of iron, naturally present in the sediment incubations, were involved in sulfate reduction, with hematite addition increasing the sulfate recycling or the activity of sulfur-cycling microorganisms by about 40%. These results highlight a role for natural iron oxides during bacterial sulfate reduction in methane seeps not only as nutrient but also as stimulator of sulfur recycling.redox | anaerobic respiration | deep-sea | methanotrophy | ANME archaea M icrobial dissimilatory processes generate energy through the decomposition of substrates, whereas assimilatory processes use substrates for intracellular biosynthesis of macromolecules. The most known and energetically favorable dissimilatory process is the oxidation of organic carbon coupled to oxygen as terminal electron acceptor (Eq. 1). In sediments with a high supply of organic carbon, oxygen can be depleted within the upper few millimeters, leading to anoxic conditions deeper in the sediment column. Under these conditions, microbial dissimilatory processes are coupled to the reduction of a series of other terminal electron acceptors besides oxygen (1). The largest free-energy yields are associated with nitrate reduction (denitrification), followed by manganese and iron oxide reduction, and then sulfate reduction. Due to the high concentration of sulfate in the ocean, dissimilatory bacterial sulfate reduction (Eq. 2) is responsible for the majority of organic matter oxidation in marine sediments (2). Below the depth of sulfate depletion, traditionally the only presumed process is methanogenesis (methane production), where its main pathways are fermentation of organic matter, mainly acetate (Eq. 3), or the reduction of carbon di...