The efficient production of dihydroxyacetone (DMA) on biodiesel-derived glycerol based media was developed. A newly isolated strain, Gluconobacter frateurii CGMCC 5397, could convert crude glycerol to DHA with high yield and productivity. In shake-flask fermentation, the DMA concentration of 73.1 g l(-1) was attained at 48 h using an optimum medium containing biodiesel-derived crude glycerol. When fed-batch fermentation was carried out in a 7-l stirred bioreactor with crude glycerol, the DHA concentration, productivity, and yield were 125.8 g l(-1), 2.6 g l(-1) h(-1), and 90.5% at 48 h, respectively. This study suggests that the inexpensive biodiesel-derived crude glycerol could be utilized for efficient production of DHA by G. frateurii. (C) 2013 Elsevier Ltd. All rights reserved.