This study aimed to investigate the feasibility and technology to harvest poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) by mixed culture. Copolymer PHBHHx, usually fermented by pure strains, was reported to be synthesized from activated sludge for the first time. Sodium laurate was used as the sole carbon substrate for sludge acclimation and PHBHHx accumulation. Batch experiments were designed to look into the impact of the carbon, nitrogen, phosphorus and oxygen supply on PHBHHx production. The results showed that the acclimated excess sludge was able to produce PHBHHx, and the maximum output (505.6 mg/L PHBHHx containing 6.34 mol% HHx) was achieved with conditions of the continuous aeration, nitrogen and phosphorus limitation, and adequate carbon source implemented by pulse feeding 0.5 g/L sodium laurate every 4 h. Moreover, composition and structure of the PHBHHx from sludge were found similar to that from pure culture, according to literature, FTIR and NMR spectra. Finally, high-throughput sequencing technique characterized that phylum Chlorobi and genus Leadbetterella should be critical groups for PHBHHx synthesis in the sludge community. (C) 2015 Elsevier Ltd. All rights reserved.