Coupling methanotrophic denitrification to anammox in a moving bed biofilm reactor for nitrogen removal under hypoxic conditions

Simultaneous removal of ammonium and nitrate was achieved in a methane-fed moving bed biofilm reactor (MBBR). In the reactor, methanotrophic microorganisms oxidized methane under hypoxic conditions likely to methanol, hence providing an electron donor to denitrifiers to reduce nitrate to nitrite that then allowed anaerobic ammonium oxidizing bacteria (Anammox) to remove excess ammonium as N2. The ammonium and nitrate removal rates reached 72.09 ± 5.81 mgNH4+–N/L/d and 62.61 ± 4.17 mgNO3−–N/L/d when the MBBR was operated in continuous mode. Nitrate removal by the methane-fed mixed consortia was confirmed in a batch test revealing a CH4/NO3− molar removal ratio of 1.15. The functional populations were unveiled by FISH analysis and 16S rRNA gene sequencing, which showed that the biofilm was dominated by Anammox bacteria (Candidatus Kuenenia) and diverse taxa associated with the capacity for denitrification: aerobic methanotrophs (Methylobacter, Methylomonas, and unclassified Methylococcaceae), methylotrophic denitrifiers (Opitutaceae and Methylophilaceae), and other heterotrophic denitrifiers (Ignavibacteriaceae, Anaerolineaceae, Comamonadaceae, Rhodocyclaceae and Thauera). Neither DAMO archaea nor DAMO bacteria were found in the sequencing analysis, indicating that more unknown community members possess the metabolic capacity of methanotrophic denitrification.