Simultaneous enrichment of denitrifying methanotrophs and anammox bacteria

Interaction between denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (anammox) processes may play an important role in global carbon and nitrogen cycles. In this study, a coculture of denitrifying methanotrophs (DAMO archaea and DAMO bacteria) and anammox bacteria, initially sourced from the environment, was enriched with a supply of methane, nitrate, and ammonium. After a 4.5-month enrichment, simultaneous oxidation of methane and ammonium and reduction of nitrate were observed. The highest rate of nitrate reduction in the suspended DAMO culture was 4.84 mmol/L/day, and simultaneously, the highest ammonium removal rate was 4.07 mmol/L/day. Fluorescence in situ hybridization and analysis of 16S rRNA gene clone libraries revealed the coexistence of DAMO archaea, DAMO bacteria, and anammox bacteria. The development of anammox bacteria might reduce the enrichment time of DAMO microorganisms and promote the activity of DAMO archaea. The activity of the reactor fluctuated during the long-term operation, which might be caused by the formation of microbial clusters whereby DAMO archaea grew in aggregates that were surrounded by anammox and DAMO bacteria. This study is the first to demonstrate that it is feasible to establish a coculture of DAMO archaea, DAMO bacteria, and anammox bacteria from environmental inocula.