We synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3-HB-co-3-HV)] copolymer having different contents of 3-hydroxyvalerate (3-HV) units (16.04, 16.3, 24.95, 25.62, and 16.52 mol % 3-HV) with different yields of polyhydroxyalkanoates (PHAs) by feeding with different cooking oils and with Alkaliphilus oremlandii OhILAs strain. The PHA production efficiency of the Alkaiphilus strain was compared with that of the control strain, Bacillus cereus. The synthesis of each PHA biopolymer was performed with different toxic spent oils as the sole carbon source in an oil-in-water-based microemulsion medium. We observed that the productivity of the poly(3-hydroxybutyrate) [P(3-HB)] copolymer from the Alkaliphilus strain was higher than those of the PHAs isolated from B. cereus and the Escherichia coli XL1B strain. The synthesized PHA copolymers were characterized by 1H-NMR and Fourier transform infrared (FTIR) spectroscopy. In the 1H-NMR spectra, a doublet resonance peak at 1.253 ppm of the/ methyl protons of the 3-hydroxybutyrate (3-HB) side group and one at 0.894 ppm due to the methyl protons of the 3-HV side group indicated the presence of 3-HB and 3-HV units in the copolymer. The chemical shift values at 1.25 and 2.2 ppm, due to the resonance absorption peaks of the methyl protons and methylene protons, confirmed the synthesis of the P(3-HB) homopolymer. From the FTIR spectra, a strong CO stretching frequency in the range of 1745–1727 cm−1, together with strong CO stretching bands near 1200 cm−1 and a strong band near 3400 cm−1, confirmed the synthesis of P(3-HB-co-3-HV) and P(3-HB). Thus, waste cooking oil as a substrate provided an alternate route for the formation of P(3-HB-co-3-HV) and P(3-HB) by Alkaliphilus and E. coli strains, respectively. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 41080.