Using RNA-Stable isotope probing to investigate methane oxidation metabolites and active microbial communities in methane oxidation coupled to denitrification

The active denitrifying communities performing methane oxidation coupled to denitrification (MOD) were investigated using samples from an aerobic reactor (∼20% O₂ and 2% CH₄) and a microaerobic reactor (2% O₂, 2% CH₄) undertaking denitrification. The methane oxidation metabolites excreted in the reactors were acetate, methanol, formate and acetaldehyde. Using anaerobic batch experiments supplemented with exogenously supplied ¹³C-labelled metabolites, the active denitrifying bacteria were identified using 16S rRNA amplicon sequencing and RNA-stable isotope probing (RNA-SIP). With the aerobic reactor (AR) samples, the maximum NO₃^- removal rates were 0.43 mmol g^-1 d^-1, 0.40 mmol g^-1 d^-1, 0.33 mmol g^-1 d^-1 and 0.10 mmol g^-1 d^-1 for exogenously supplied acetate, formate, acetaldehyde and methanol batch treatments respectively, while with the microaerobic reactor (MR) samples, the maximum NO₃^- removal rates were 0.41 mmol g^-1 d^-1, 0.33 mmol g^-1 d^-1, 0.38 mmol g^-1 d^-1 and 0.14 mmol g^-1 d^-1 for exogenously supplied acetate, formate, acetaldehyde and methanol batch treatments respectively. The RNA-SIP experiments with ¹³C-labelled acetate, formate, and methanol identified Methyloversatilis, and Hyphomicrobium as the active methane-driven denitrifying bacteria in the AR samples, while Pseudoxanthomonas, Hydrogenophaga and Hyphomicrobium were the active MOD bacteria in the MR samples. Collectively, all the data indicate that formate is a key cross-feeding metabolite excreted by methanotrophs and consumed by denitrifiers performing MOD.