We investigated nitrogen removal performance and responsible microbial community in an anaerobic up-flow granular bed anammox reactor. The anammox reactor was operated more than 1 year. Biomass in the reactor formed granules after about 2 months of operation, and a sufficient amount of the granules was retained in the reactor with a metallic net to avoid biomass washout during the entire operation. The average diameter of the granules was 3.6mm at day 310. After 8 months of operation, stable nitrogen removal (60%) was achieved at an average total inorganic nitrogen removal rate of 14 kg-N m(-3)d(-1). The phylogenetic analysis and fluorescence in situ hybridization results revealed that the anammox granules consisted of mono species of anammox bacteria, "Candidatus Brocadia-like species", affiliated with "Candidatus Brocadia anammoxidans" with 16S rRNA gene sequence similarity of 95.7%. The relative abundance of the anammox bacteria in the granules was more than 80% of the total bacteria stained with 4',6-diamidino-2-phenylindole. The anammox bacteria were present throughout the granules whereas the other bacterial groups, Chloroflexi-like filamentous bacteria and betaproteobacterial ammonia-oxidizing bacteria, were mainly present on the surface of the anammox granules and around the anammox bacterial clusters. The in situ anammox activity was detected mainly from near the surface of granules to the upper 800 microm of the granules with microsensors. The granular anammox biomass tolerated higher concentrations of nitrite (400 mg-NL(-1)) than did the homogenized biomass (200 mg-NL(-1)) probably due to substrate diffusion limitation.
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