This study proposed a new method for determining the thicknesses of aerobic and anaerobic layers in a biofilm. The transport of dissolved oxygen (DO) or nutrients from the bulk solution to the biofilm was more closely related to the thicknesses of the aerobic and anaerobic layers than to the biofilm structure. The DO microdistribution of the biofilm from a sequencing batch biofilm reactor (SBBR) was measured using microelectrode. The measurements were used to speculate the biofilm structure. The average and maximum thicknesses were 365 and 640 mu m in the test biofilm, respectively. The mean thicknesses of the aerobic and anaerobic layers were 270 and 240 gm, respectively. The maximum thickness of the aerobic layer was 420 mu m. Aerobic oxidation mainly occurred at the top half of the biofilm during the SBBR aeration stage. The biofilm was heterogeneous. However, many holes were detected inside the biofilm. Some of these holes were connected to the bulk solution and allowed the bulk solution to enter and exit freely, whereas the others were isolated. (C) 2014 Elsevier B.V. All rights reserved.