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This study investigated the effects of using a combination of substrates (fructose, propanol, citric acid, acetic acid, propionic acid and beef extract) on the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyvalerate), which is also known as PHBV. The results of batch PHBV optimization by RSM, indicated that PHB was synthesized only when fructose was used as the sole carbon source. Fructose in combination with propanol and beef extract, exhibited better PHBV production performance, as compared to that of propionic acid which was used in previous studies. GC, (1)H and (13)C NMR spectroscopy revealed that the best copolymer composition was 54.1 mol% of 3-hydroxyvalerate (3HV) which was obtained in a basal medium combined with 0.3% (v/v) propanol and 0.0172% (w/v) beef extract. This is the first report on the production of PHBV with HV content of 54.1% by a wild-type Cupriavidus necator. The Fourier Transform Infrared Spectroscopy (FTIR) spectra of the PHAs indicated absorption bands at 1723, 1274, 1373, 1453, 2932cm(-1) corresponding to CO, CO stretching, CH3, -CH2 and -CH groups, respectively. This study showed that the recovered PHBV biopolymer from the optimized culture to have a melting temperature of 103.6°C, melting enthalpy of 55.67J/g and crystallization temperature of 76.1°C., (Copyright © 2016 Elsevier B.V. All rights reserved.)