Polyhydroxyalkanoate (PHA) is a microbial storage biopolymer that can be used in biodegradable plastics. This study investigates the effects of two carbon substrates (acetate and propionate) on PHA production using sludge from a wastewater treatment plant in terms of their microbial communities, PHA contents, polymer compositions and structures, and thermal characteristics of the PHA. Sludge was enriched aerobically with acetate or propionate in sequencing batch reactors (SBRs) under feast/famine feeding conditions. The sludge enriched with acetate produced a homopolymer of hydroxybutyrate (HB) and accumulated a maximum PHA of 44.78% gPHA/g mixed liquor suspended solids (MLSS). In contrast, the sludge enriched with propionate produced a copolymer of HB and hydroxyvalerate (HV) and accumulated a maximum PHA of 12.65% gPHA/gMLSS. The results from the microbial community analysis using 16S rRNA gene amplicon sequencing showed that bacteria belonging to the phylum Bacteroidetes and class Alphaproteobacteria appeared to be dominant in the system fed with acetate when PHA content was at its maximum. In contrast, Alphaproteobacteria and Gammaproteobacteria were dominant in the system fed with propionate when the PHA content was at its maximum. The PHA produced from acetate also had a higher melting temperature, higher glass transition temperature, and higher crystallinity than did that produced from propionate. © 2017 American Institute of Chemical Engineers Environ Prog, 36: 1754–1764, 2017