Medium optimization for production of polyb-hydroxyalkanoate (PHA) from Rhodobacter sphaeroides U7 cultivated in glutamate-acetate (GA) medium supplemented with 40 mM valeric acid as co-substrate under aerobic-dark condition was investigated. Studies on effect of nitrogen source and cultivation temperature by conventional and statistical methods illustrated that (NH 4 ) 2 SO 4 (0.2 g/l) had no effect and the optimal temperature was at 30°C. The optimum environmental conditions were found to be anaerobic-light (3000 lux) cultivation with aeration rate of 1.0 vvm and agitation speed of 200 rpm for PHA production (2.5 g/l) with the highest PHA content (65.15%) at 0.5 vvm, and 200 rpm. Under this optimized medium and condition, PHA production from R. sphaeroides U7 increased 3.86-folds (from 0.69 to 2.66 g/l) (PHA content increased 1.5-folds). The biopolymer was purified and characterized by using 13 C NMR, FTIR, DSC, X-ray diffraction and intrinsic viscosity techniques to be a copolymer poly(b-hydroxybutyrate-co-b-hydroxyvalerate) (PHBV) consisting of 84.8 mol% b-hydroxybutyric acid (HB) and 15.2 mol% b-hydroxyvaleric acid (HV).