RESEARCH ARTICLE Enrichment of Denitrifying Methane- Oxidizing Microorganisms Using Up-Flow Continuous Reactors and Batch Cultures

Denitrifying anaerobic methane oxidizing (DAMO) microorganisms were enriched from paddy field soils using continuous-flow and batch cultures fed with nitrate or nitrite as a sole electron acceptor. After several months of cultivation, the continuous-flow cultures using nitrite showed remarkable simultaneous methane oxidation and nitrite reduction and DAMO bacteria belonging to phylum NC10 were enriched. A maximum volumetric nitrite consumption rate of 70.4¡3.4 mg-N?L21?day21 was achieved with very short hydraulic retention time of 2.1 hour. In the culture, about 68 % of total microbial cells were bacteria and no archaeal cells were detected by fluorescence in situ hybridization. In the nitrate-fed continuous-flow cultures, 58 % of total microbial cells were bacteria while archaeal cells accounted for 7 % of total cell numbers. Phylogenetic analysis of pmoA gene sequence showed that enriched DAMO bacteria in the continuous-flow cultivation had over 98 % sequence similarity to DAMO bacteria in the inoculum. In contrast, for batch culture, the enriched pmoA gene sequences had 89–91 % sequenc