Nitrogen and carbon flows in a microbial mat involving anaerobic oxidation of methane in northwestern Black Sea: perspectives from nitrogen and carbon isotopic composition of coenzyme F430 and its biosynthetic pathway

Abstract Coenzyme F430, a nickel-containing tetrapyrrole, serves as a cofactor for methyl-coenzyme M reductase (MCR), which catalyzes both the final step of methanogenesis and the initial step of anaerobic oxidation of methane (AOM). Due to its central role in these metabolic processes, coenzyme F430 can be used as a biomarker to investigate the distribution and activity of methanogens and anaerobic methane-oxidizing archaea (ANMEs) in various environments. In the northwestern Black Sea, chimney-like microbial mats dominated by a subcluster ANME-1 in the internal pink mat and ANME-2 in the outer black mat provide a sufficient quantity of coenzyme F430 for both stable carbon and nitrogen isotope analyses. In this study, we report the stable carbon and nitrogen isotopic compositions of coenzyme F430 and its derivatives from ANME-1 and ANME-2, as well as those of cell components, including bulk mat, amino acids, and membrane lipids. The carbon isotopic compositions of coenzyme F430 exhibit significant 13 C-depletion, reflecting the incorporation of methane-derived carbon. In contrast, coenzyme F430 from ANME-1 is enriched in 13 C and depleted in 15 N compared to that from ANME-2. These isotopic differences between ANME-1 and ANME-2 cannot be explained solely by biosynthetic isotope effects and likely reflect sequential methane and ammonium consumption within the microbial mat, which is influenced by surrounding seawater with low sulfate concentrations.