BACKGROUND: Copolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB) has gained importance in biotechnological processes due to its potential as an absorbable material in biopharmaceutical applications. The aim of the present study was to investigate the implementation of mixed substrate cultivation concept in single step production of copolymer P(3HB-co-4HB). Evaluation of the sole carbon sources was first carried out to elucidate the difference in the monomer content of 4HB using different types and concentrations of carbon sources. This is followed by the study of mixed-substrate cultivation on copolymer biosynthesis. Finally, the molecular weight of copolymers with various 4HB monomer compositions was also determined. RESULTS: Experimentation using the sole carbon source recorded the highest PHA content, PHA concentration and 4HB monomer composition of 57 wt%, 3.1 g L-1 and 48 mol%, respectively. Manipulation of the carbon source combination showed the synergistic influence of the substrates mixture towards single-stage production of P(3HB-co-4HB), which enhanced the yield of copolymer P(3HB-co-4HB), to a maximum PHA content and concentration of 74wt% and 6.9 g L-1. In addition, 4HB monomer composition up to a maximum of 70 mol% was obtained. Molecular weight characterization showed the increase of 4HB monomer composition which led to the decrease of molecular weight from 369.3 kDa to 34.0 kDa. CONCLUSION: The mixed substrate cultivation was proven to be a promising strategy to increase both polymer production and 4HB monomer composition in single stage production. This serves as a platform for further bioprocess development of copolymer with various 4HB monomer compositions. (C) 2013 Society of Chemical Industry