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Adding nutrient can accelerate diesel oil biodegradation in seawater. Response surface methodology (RSM) was employed to optimize the condition for increasing diesel oil degradation. Using a Plackett-Burman experimental design to aid in the first step of optimization, and nitrogen, phosphorus and pH were found to be significant factors affecting degradation process. To determine the optimal range of each significant variable, a central composite design was employed. Based on response surface and canonical analysis, the optimum concentrations of the nutrients were obtained as follows: nitrogen (NaNO3)0.143g, phosphorus (KH2PO4)0.022g, and the optimal pH was 7.4.