A fractional factorial design (FFD) and a response surface methodology (RSM) were used to optimize the inoculum composition of six strains for treatment of synthetic domestic wastewater. The model predicted the highest overall specific substrate utilization rate (q) of 6.88 g TOC/(d-gVSS). The value is in accordance with the actual maximum q, and is 1.5 and 1.97 times greater than those without optimization for 4 and 6 strains respectively. Additionally, the shortest time to reach stationary phase (3.5 h) and highest maximum total organic carbon (TOC) removal efficiency (92%) were also achieved under the optimum condition. The results indicated that the FFD and RSM are powerful screening and optimizing tools for the microbial community. The experimental approaches enhance the overall specific rate of substrate utilization as well as other biodegradation parameters.