Development of High-Rate Anaerobic Ammonium-Oxidizing

1 To promptly establish anammox (anaerobic ammonium oxidation) reactors, appropriate 2 seeding sludge with high abundance and activity of anammox bacteria was selected by 3 quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative 4 PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into 5 up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier 6 and the reactor performances were monitored over one year. The anammox reaction was 7 observed within 50 days and a total nitrogen removal rate of 26.0 kg-N m-3 day-1 was obtained 8 after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic 9 retention time (HRT) and influent NH 4 + to NO 2-molar ratio could be important determinant 10 factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was 11 obtained at the shorter HRT and higher influent NH 4 + /NO 2-molar ratio. After anammox 12 reactors were fully developed, the community structure, spatial organization and in situ 13 activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S 14 rRNA approach and microelectrodes. In situ hybridization results revealed that the probe 15 Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm 16 (accounting for more than 70% of total bacteria). They were associated with 17 Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The 18 anammox bacteria present in this study were distantly related to the Candidatus Brocadia 19 anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed 20 that a high in situ anammox activity (i.e., simultaneous consumption of NH 4 + and NO 2