A pilot-scale NiiMi system for river landscape water treatment has been steadily operated for more than one year. The aim of the study was to investigate the spatial distributions of biofilm properties and water flow pattern in NiiMi system. Results showed that spatial distributions regularities of volatile suspended solid (VSS), respiration intensity, protein, polysaccharide, viable cell number and dehydrogenase activity were consistent with the aqueous rate. In addition, the viable cell, protein and polysaccharide specific dehydrogenase decreased with depth. Moreover, the protein: polysaccharide ratio increased with depth. Fourier transform infrared (FT-IR) spectroscopy indicated that protein was one of the components of extracellular polymeric substance (EPS). Gel filtration chromatography (GFC) analysis indicated that EPS had a broader MW distribution than that in the effluent water. Trace studies indicated that the occurrence of a dead zone volume of 18.7%, the top left section was the dead zone of the NiiMi system. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.