Recombinant Escherichia coli JM109 harbouring the polyhydroxyalkanoate (PHA) biosynthesis gene (phaCABco) of Comamonas sp. EB172, an acid tolerant microbe, was examined for the production of PHAs from various carbon sources. The study demonstrated that the recombinant E. coli JM109 had the potential to utilize both sugar- and acid-based carbon sources, for the biosynthesis of both poly(3-hydroxybutyrate) P(3HB) and poly(3- hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) copolymers. In the shake flask experiments, the strain was capable of producing P (3HB-co-3HV) copolymer from mixed organic acids, and higher productivities were obtained using glucose compared to mixed acids. However, PHA accumulation was found to be similar, regardless of the carbon source used. Nitrogen supplementation in the medium was found to improve the cell dry weight, but negatively affected the 3HV formation in copolymer production. Maximum 3HV monomer (3 mol%) was obtained with C/N 42.1, using mixed acids as the carbon source. In the 2L bioreactor, the productivity and yield based on substrate utilization coefficient were found to be 0.16 g PHA/(L.h) and 0.41 g PHA/g substrate under C/N around 75, using 20 g/L glucose and 0.5 g/L ammonium sulphate, respectively. The polymer produced by the recombinant strain had molecular weight in the range of 8.5 x 105 to 1.4 x 106 Da. Overall, the ability of the recombinant E. coli JM109 to utilize both glucose and mixed acids, has widened its substrate selection for fermentation, including the opportunity to use renewable biomass.