Biofilm displayed a quick development initially but slowed down during operation. Optimal DO level could be determined by different biofilm thicknesses and vice versa. Optimal N-removal was achieved at DO of 0.6 mg L−1 and biofilm thickness of 700 μm. AerAOB and AnAOB mixed with each other and showed no distinct niche. Specific cauliflower-like zooglea formed with the increase of biofilm thickness.