A microaerobic (2% O₂ v/v) biotrickle bed reactor supplied continuously with 2% methane to drive nitrate removal (MAME-D) was investigated using 16S rDNA and rRNA amplicon sequencing in combination with RNA-stable isotope probing (RNA-SIP) to identify the active microorganisms. Methane removal rates varied from 500 to 1000 mmol m^-3h^-1 and nitrate removal rates from 25 to 58 mmol m^-3h^-1 over 55 days of operation. Biofilm samples from the column were incubated in serum bottles supplemented with ¹³CH₄. 16S rDNA analysis indicated a simple community structure in which four taxa accounted for 45% of the total relative abundance (RA). Dominant genera included the methanotroph Methylosinus and known denitrifiers Nubsella and Pseudoxanthomonas; along with a probable denitrifier assigned to the order Obscuribacterales. The 16S rRNA results revealed the methanotrophs Methylocystis (15% RA) and Methylosinus (10% RA) and the denitrifiers Arenimonas (10% RA) and Pseudoxanthomonas (7% RA) were the most active genera. Obscuribacterales was the most active taxa in the community at 22% RA. Activity was confirmed by the Δ buoyant density changes with time for the taxa, indicating most of the community activity was associated with methane oxidation and subsequent consumption of methanotrophic metabolic intermediates by the denitrifiers. This is the first report of RNA stable isotope probing within a microaerobic methane driven denitrification system and the active community was markedly different from the full community identified via 16S-rDNA analysis.

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.