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

<p dir="ltr">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.</p>