Aims: Statistical approach, central composite design (CCD) was used to investigate the complex interaction among temperature (25-37 °C), initial medium pH (5-9), inoculum size (4-10 % (v/v)), concentration of (NH4)2SO4 (0-1 g/L) and concentration of mixed organic acids (5-10 g/L) in the production of polyhydroxyalkanoates by Comamonas sp. EB172.Methodology and Results: Mixed organic acids derived from anaerobically treated palm oil mill effluent (POME) containing acetic:propionic:butyric (ratio of 3:1:1) were used as carbon source in the batch culture of Comamonas sp. EB172 to produce polyhydoxyalkanoates (PHAs). The analysis of variance (ANOVA) showed that all five factors were significantly important in the batch fermentation by shake flask with a P value of less than 0.001. The optimal temperature, initial medium pH, inoculum size, concentration of (NH4)2SO4 and concentration of mixed organic acids were 30 °C, 7.04, 4.0 % (v/v), 0.01 g/L and 5.05 g/L respectively.Conclusion, significance and impact of study: Optimization of the production medium containing mixed organic acids has improved the PHA production for more than 2 folds. Under optimal condition in the shake flask fermentation, the predicted growth is 2.98 g/L of dry cell weight (DCW) with 47.07 wt % of PHA content. The highest yield of PHA was 0.28 g of PHA per g mixed organic acids.