Cold seep nitrogen fixation and its potential relationship with sulfur cycling

ABSTRACT Dinitrogen (N 2 ) fixation is a crucial source of bioavailable nitrogen in carbon-dominated cold seep systems. Previous studies have shown that diazotrophy is not necessarily dependent on sulfate-dependent anaerobic oxidation of methane for energy, and diverse catabolism can fuel the high-energy-demanding process in sediments. However, it remains unclear whether diazotroph can obtain energy by sulfur oxidation in sulfur-rich cold seep water column. Here, field investigations and in situ experiments were conducted in Haima cold seep to examine the effects of diverse sources of dissolved organic matter (DOM) on N 2 fixation, specifically containing sulfur, carbon, nitrogen, and phosphorus. We found that active N 2 fixation occurred in the water column above the Haima cold seep, with the Dechloromonas genus dominating the diazotroph community as revealed by nifH gene using high-throughput sequencing. In situ experiments showed an increased rate of N 2 fixation (1.15- to 12.70-fold compared to that in control group) and a greater relative abundance of the Dechloromonas genus following enrichment with sulfur-containing organic matter. Furthermore, metagenomic assembly and binning revealed that Dechloromonas sp. carried genes related to N 2 fixation ( nifDHK ) and sulfur compound oxidation ( fccAB and soxABCXYZ ), implying that the genus potentially serves as a multifunctional mediator for N 2 fixation and sulfur cycling. Our results provide new insights regarding potential coupling mechanism associated with sulfur-driven N 2 fixation in methane- and sulfide-rich environments. IMPORTANCE N2 fixation is an important source of biologically available in carbon-dominated cold seep systems as little nitrogen is released by hydrocarbon seepage, thereby promoting biological productivity and the degradation of non-nitrogenous organic matter. Cold seeps are rich in diverse sources of dissolved organic matter (DOM) derived from the sinking of photosynthetic products in euphotic layer and the release of chemosynthesis products on the seafloor. However, it remains unclear whether N2 fixation is coupled to the metabolic processes of DOM, as determined by e.g., carbon, nitrogen, phosphorus, and sulfur content, for energy acquisition in sulfur-rich cold seeps. In this study, diazotroph community structure and its response to DOM compositions were revealed. Moreover, the metagenomics analysis suggested that Dechloromonas genus plays a dominant role in potential coupling N2 fixation and sulfur oxidation. Our study highlighted that sulfur oxidation in deep-sea cold seeps may serve as an energy source to drive N2 fixation.