Azo dye is widely used in printing and dyeing process as one of refractory wastewaters for its high chroma, stable chemical property and toxicity for aquatic organism. Biocatalyzed electrolysis system (BES) is a new developed technology to degrade organic waste in bioanode and recover recalcitrant contaminants in cathode with effective decoloration. The ion exchange membrane (IEM) separate anode and cathode for biofilm formation protection. Azo removal efficiency was up to 60.8%, but decreased to 20.5% when IEM was removed. However, expensive ion exchange membrane (IEM) not suitable for further practical application, bioelectrochemical activity of bioanode is sensitive to the toxicity of azo dye. A gradient increase of azo dye concentration was used to acclimate anode biofilm to pollutant toxicity. The azo removal efficiency can be enhanced to 73.3% in 10 h reaction period after acclimation. The highest removal efficiency reached 83.7% and removal rates were increased to 8.37 from 3.04 g/h/L of dual-chamber. That indicated the feasibility for azo dye removal by single-chamber BES. The IEM cancellation not only decreased the internal resistance, but increased the current density and azo dye removal. (C) 2013 Elsevier Ltd. All rights reserved.