• Activated sludge;
  • Azo dye decolorization;
  • Plackett–Burman DOE;
  • Reactive Black 5;
  • Response surface methodology


In the present study both the synthetic media composition as well as some process parameters in the anaerobic decolorization of Reactive Black 5 (RB5) by activated sludge were studied and optimized using statistical design of experiments (DOEs). Statistical analysis of the results of Plackett–Burman DOE showed that the addition of Mn or Fe, or increase in concentration of Mg, had a positive effect on the anaerobic decolorization efficiency whereas the effect of increase in concentration of glucose, ammonium chloride, and calcium chloride was negative. The effect of change in the concentration of glucose, mixed liquor suspended solid (MLSS), and RB5 on the anaerobic decolorization efficiency and rate and chemical oxygen demand (COD) removal was studied using central composite design methodology. Statistical analysis of the data showed that all the factors had significant effect on both the dye decolorization efficiency and rate. The interaction of glucose with MLSS and with dye and the interaction of MLSS with glucose and with dye were significant when the response was decolorization efficiency and rate, respectively. When COD removal was the response, the effect of change in glucose and MLSS concentration and the interaction between these two factors had statistically significant effect on the response.