In this study, the suitability of butyrate as substrate for polyhydroxyalkanoate (PHA) production by microbial enrichment cultures was assessed. Two sequencing batch reactors were operated under feast-famine conditions: one fed with butyrate, and another with mixed acetate and butyrate. The obtained results were compared to previous results with acetate as sole substrate. In all three reactors Plasticicumulans acidivorans dominated the enrichment culture. The carbon uptake rate and PHA yield were significantly higher on butyrate than on acetate, resulting in a higher PHA production rate. When both substrates were available the bacteria strongly preferred the uptake of butyrate. Only after butyrate depletion acetate was taken up at a high rate. The molar substrate uptake rate remained the same, suggesting that substrate uptake is the rate-limiting step. The results show that for optimized waste-based PHA production the pre-fermentation process should be directed towards butyrate production. (C) 2013 Elsevier Ltd. All rights reserved.