High-purity propionate production from glycerol in mixed culture fermentation (MCF) induced by high ammonium concentration was investigated. Fed-batch experiments revealed that higher ammonium concentration (>2.9 g/L) had simultaneous negative effects on acetate and propionate degradation. Propionate production and yield was up to 22.6 g/L and 0.45 g COD/g COD glycerol, respectively, with a purity of 96%. Sequential batch experiments demonstrated that the yields of propionate were 0.3 +/- 0.05, 0.32 +/- 0.01, and 0.34 +/- 0.03 g COD/g COD at a glycerol concentration of 2.78, 4.38, and 5.56 g/L, respectively, and the purity of propionate was 91-100%. Microbial community analysis showed that the phylum Firmicutes dominated the bacterial community at different glycerol concentrations. However, the Methanosaeta population decreased from 46% to 6% when glycerol concentration increased from 2.78 to 5.56 g/L, resulting in lower acetate degradation rate. Thus, the present study might provide an alternative option for the production of propionate from glycerol via MCF. (C) 2016 Elsevier Ltd. All rights reserved.