Combined genome-centric metagenomics and stable isotope probing unveils the microbial pathways of aerobic methane oxidation coupled to denitrification process under hypoxic conditions

•A stable hypoxic AME-D process was established in a methane-cycling bioreactor.•Genome-centric metagenomics with DNA-SIP were used to assess metabolic mechanism.•Methylomonas were actively prevalent at the enriched hypoxic AME-D community.•A new genome of Methylococcaceae with abundant denitrification genes was obtained.•Aerobic methanotrophs can accomplish partial denitrification independently. Obligate aerobic methanotrophs have been proven to oxidize methane and participate in denitrification under hypoxic conditions. However, this phenomenon and its metabolic mechanism have not been investigated in detail in aerobic methane oxidation coupled to denitrification (AME-D) process. In this study, a type of hypoxic AME-D consortium was enriched and operated for a long time in a CH4-cycling bioreactor with strict anaerobic control and the nitrite removal rate reached approximately 50 mg N/L/d. Metagenomics combined with DNA stable-isotope probing demonstrated that the genus Methylomonas, which constitutes type I aerobic methanotrophs, was the dominant member and contributed to methane oxidation and partial denitrification. Metagenomic binning recovered a near-complete (98%) draft genome affiliated with the family Methylococcaceae containing essential genes that encode nitrite reductase (nirK), nitric oxide reductase (norBC) and hydroxylamine dehydrogenase (hao). Metabolic reconstruction of the selected Methylococcaceae genomes also revealed a potential link between methanotrophy and partial denitrification.