The culture variables were optimized to increase 1,3-dihydroxyacetone (DHA) production by Gluconohacter oxydans ZJB09112 in shake flasks and bubble column bioreactors. After fermentation in the optimized medium (g/l: yeast extract 5, glycerol 2.5, mannitol 22.5, $K_2HPO_4$ 0.5, $KH_2PO_4$ 0.5, $MgSO_4{cdot}7H_2O$ 0.1, $CaCO_3$ 2.0, pH 5.0), when five times of glycerol feeding were applied, $161.9{pm}5.9;g/l$ of DHA was attained at a $88.7{pm}3.2%$ conversion rate of glycerol to DHA.