Abstract:Objective Iron reduction-dependent anaerobic oxidation of methane (Fe-AOM) is an important pathway for methane emission reduction in anaerobic environments. However, it remains unclear how methane-oxidizing microbes perform Fe-AOM under nitrogen-limiting conditions.Methods Focusing on a methane-oxidizing consortium and ferrihydrite, this study employed nitrogen isotope tracing, three-dimensional fluorescence spectroscopy, electrochemical analysis, and high-throughput sequencing to investigate the Fe-AOM efficiency and the possibility of coupling Fe-AOM with biological nitrogen fixation under nitrogen-limiting conditions.Results The methane-oxidizing consortium was able to catalyze Fe-AOM under nitrogen-limiting conditions, reducing ferrihydrite to minerals such as siderite. The nitrogenase activity and 15N assimilation of the methane-oxidizing consortium in the presence of methane were significantly higher than those in the absence of methane, which demonstrated that the consortium could couple Fe-AOM with biological nitrogen fixation. Three-dimensional fluorescence spectroscopy and electrochemical analysis revealed that Fe-AOM promoted the production of dissolved protein-like substances, enhanced the redox activity of the methane-oxidizing consortium, and reduced ferrihydrite via direct electron transfer. Microbial community structure analysis showed significant enrichment of Methanobacterium (19.32%), iron-reducing bacteria such as Geobacter (6.14%) and Desulfovibrio (17.52%), as well as nitrogen-fixing bacteria like Azoarcus (1.69%) and Azospirillum (0.43%) during the Fe-AOM process. DNA-SIP analysis found that Azoarcus was significantly enriched in the heavy fraction of the labeled isotope group, confirming that it fixed isotope nitrogen.Conclusion It is thus hypothesized that the coupling of Fe-AOM with biological nitrogen fixation was primarily carried out by Methanobacterium which oxidized methane, Geobacter and Desulfovibrio responsible for the reduction of ferrihydrite, and Azoarcus catalyzing biological nitrogen fixation. Additionally, the positive correlations of the methane-oxidizing bacterium Methylocystis with iron-reducing bacteria and nitrogen-fixing bacteria suggested a certain contribution of Methylocystis to this process. These results provide new insights into understanding iron-dependent methane oxidation and nitrogen fixation in anaerobic environments.