This study investigated the effects of substrate composition (acetate and propionate) on synthesis of various mix of poly(hydroxybutyrate-hydroxyvalerate) (P(HB/HV)) from activated sludge, which was acclimated using a single carbon (acetate) and mixed carbons (acetate and propionate). Results of batch P(HB/HV) production tests indicated that the yield and synthesis rate of P(HB/HV) decreased as the proportion of propionate in the substrate increased. However, mixed-carbon-acclimated sludge with acetate and propionate exhibited better P(HB/HV) production performance than with acetate-acclimated sludge in terms of substrate utilization, yield of P(HB/HV) and HV fraction in P(HB/HV). The desired hydroxyvalerate (HV) fraction (0-74%) of the P(HB/HV) could be obtained based on the substrate composition and sludge acclimation. Furthermore, PCR-DGGE analysis indicated that specific species dominated the activated sludge used for P(HB/HV) production. Acidobacteria and Burkholderiales were the dominant bacterial populations and played an important role in HV synthesis. (C) 2013 Elsevier Ltd. All rights reserved.