The influences of trace metals in the wastewater and shear stress by aeration were particularly examined to clarify the formation mechanism of nitrifying granules in an aerobic upflow fluidized bed (AUFB) reactor. It was found that Fe added as a trace element to the inorganic wastewater accumulated at the central part of the nitrifying granules. Another result obtained was that suitable shear stress by moderate aeration (0.07-0.20 L/min/L-bed) promoted granulation. Furthermore, it was successfully demonstrated that pre-aggregation of seed sludge using hematite promoted core formation, leading to rapid production of nitrifying granules. From these results, a nitrifying granulation mechanism is proposed: 1) as a first step, nitrifying bacteria aggregate along with Fe precipitation, and then the cores of granules are formed; 2) as a second step, the aggregates grow to be spherical or elliptical in form due to multiplication of the nitrifying bacteria and moderate shear stress in the reactor, and then mature nitrifying granules are produced. Fluorescence in situ hybridization (FISH) analysis successfully visualized the change in the spatial distribution of nitrifying bacteria in the granules, which supports the proposed granulation mechanism.

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