Some inorganic nitrogen sources and amino acids instead of yeast extract, which resulted in trouble of product purification, were introduced for 1,3-dihydroxyacetone (DHA) production by biotransformation with Gluconobacter oxydans. The results showed that urea is an optimal nitrogen source. Furthermore, the effects of glycerol- and urea-feeding strategies for DHA production by immobilized cells in a home-made bubble column bioreactor were optimized. Cells immobilization was prepared by cultivation in the bioreactor packed with porous ceramics, and then the broth was removed. Then, repeated biotransformation by continuous-feeding of glycerol and urea was developed. Up to 96.4 +/- 4.1 g/L of average DHA concentration with 94.8 +/- 2.2% of average conversion rate of glycerol to DHA was achieved after 12 cycles of run. Near colorless DHA solution with few impurities was obtained and the production cost could be decreased. (C) 2017 Elsevier Ltd. All rights reserved.