AbstractStatistically based experimental designs were used to construct a mixed-culture community for maximizing the chemical oxygen demand (COD) degradation of pulping effluents by the use of six different strains, i.e., Agrobacterium sp., Bacillus sp., Enterobacter cloacae, Gordonia, Pseudomonas stutzeri, and Pseudomonas putida. Significant effects of single and mixed strains on COD degradation were quantified first by applying a fractional factorial design (FFD) of experiments, and four strains were selected as the main driving factors in the process of biodegradation of effluents. Then the Steepest Ascent method was employed to approach the experimental design space, followed by an application of response surface methodology to further optimize the proportion of cell concentration for different strains in pulping effluent. A quadratic model was found to fit COD removal efficiency. Response surface analysis revealed that the optimum levels of the tested variables for the degradation of COD, and optimized cells concentrations (OD600) of four strains in mixed-culture community were 0.35 Agrobacterium sp., 0.38 Bacillus sp., 0.43 Gordonia sp., and 0.38 P. putid., respectively. In a confirmatory experiment, three test runs were performed by using the optimized conditions, and a COD removal efficiency of (65.3 ± 0.5)% was observed, which was in agreement with the prediction.