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Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) has been recognized as a sustainable process for simultaneous removal of nitrogen and methane. The metabolisms of denitrifying anaerobic methanotrophs, including Candidatus Methanoperedens and Candidatus Methylomirabilis, have been well studied. However, potential roles of heterotrophs co-existing with these anaerobic methanotrophs are generally overlooked. In this study, we pulse-fed methane and nitrate into an anaerobic laboratory sequencing batch bioreactor and enriched a mixed culture with stable nitrate removal rate (NRR) of ∼28 mg NO 3 −-N L−1 d−1. Microbial community analysis indicates abundant heterotrophs, e.g., Arenimonas (5.3%–18.9%) and Fimbriimonadales ATM1 (6.4%), were enriched together with denitrifying anaerobic methanotrophs Ca. Methanoperedens (10.8%–13.2%) and Ca. Methylomirabilis (27.4%–34.3%). The results of metagenomics and batch tests suggested that the denitrifying anaerobic methanotrophs were capable of generating methane-derived intermediates (i.e., formate and acetate), which were employed by non-methanotrophic heterotrophs for denitrification and biomass growth. These findings offer new insights into the roles of heterotrophs in n-DAMO mixed culture, which may help to optimize n-DAMO process for nitrogen removal from wastewater. [Display omitted] • Heterotrophic denitrification in a n-DAMO culture was investigated. • Denitrifying anaerobic methanotrophs generated formate and acetate from methane. • Heterotrophs employed methane-derived formate and acetate for denitrification. • Heterotrophs assimilated methane-derived formate and acetate for biomass growth. [ABSTRACT FROM AUTHOR]