Biological treatment using sulfate reducing bacteria (SRB) has been found out to have the potential for treatment of wastewaters containing sulfate and heavy metals. In the present study, in addition to the investigation of carbon source effect on the growth and sulfate removal, a statistical model using the design of experiments methodology has been developed to optimize the removal of sulfate by the obtained consortium from the anaerobic digested. All parameters were selected in three levels. Investigating the data collected from experiments using response surface methodology proved that the interactions between parameters were insignificant and could be neglected. The optimum removal conditions were achieved at pH of 7.19, initial sulfate concentration of 2153.15 mg/L, COD (Chemical Oxygen Demand) to initial sulfate concentration ratio (COD/SO₄²⁻) of 2.72 and the COD related to ethanol to total COD ratio (COD_ethanol/COD_total) of 1 which led to sulfate removal of 98%. In five different batch experiments done in UASB reactor, the optimum conditions were applied to evaluate SRB performance in real and synthetic wastewater treatment mostly in all experiments. The differences of experiments were in real or synthetic wastewater, having an extra stage of treatment by H₂S, using organic wastewater as carbon source and presence of heavy metal in bacteria medium. The maximum obtained removal percentage for Zn and Ni were 99.996 and 96.87, respectively.