Simultaneous nitrate and sulfate dependent anaerobic oxidation of methane linking carbon, nitrogen and sulfur cycles.

ANaerobic MEthanotrophic (ANME) archaea are critical microorganisms mitigating methane emission from anoxic zones. In previous studies, sulfate-dependent anaerobic oxidation of methane (AOM) and nitrate-dependent AOM, performed by different clades of ANME archaea, were detected in marine sediments and freshwater environments, respectively. This study shows that simultaneous sulfate- and nitrate-dependent AOM can be mediated by a clade of ANME archaea, which may occur in estuaries and coastal zones, at the interface of marine and freshwater environments enriched with sulfate and nitrate. Long-term (~1,200 days) performance data of a bioreactor, metagenomic analysis and batch experiments demonstrated that ANME-2d not only conducted AOM coupled to reduction of nitrate to nitrite, but also coupled to the conversion of sulfate to sulfide, in collaboration with sulfate-reducing bacteria (SRB). Sulfide was oxidized back to sulfate by sulfide-oxidizing autotrophic denitrifiers with nitrate or nitrite as electron acceptors, in turn alleviating sulfide accumulation. In addition, dissimilatory nitrate reduction to ammonium performed by ANME-2d was detected, providing substrates to Anammox. Metatranscriptomic analysis revealed significant upregulation of flaB in ANME-2d and pilA in Desulfococcus, which likely resulted in the formation of unique nanonets connecting cells and expanding within the biofilm, and putatively providing structural links between ANME-2d and SRB for electron transfer. Simultaneous nitrate- and sulfate-dependent AOM as observed in this study could be an important link between the carbon, nitrogen and sulfur cycles in natural environments, such as nearshore environments.