A stepwise strategy was devised to optimize the bioconversion of residual glycerol from biodiesel synthesis into 1,3-propanediol (1,3-PD) by a new strain Klebsiella pneumoniae. This strain is unique in its ability to convert glycerol simultaneously into 1,3-PD. The influence of glycerol concentration, pH, temperature, nitrogen and mineral sources were first investigated using the Plackett-Burman (P-B) statistical design to screen the variables that significantly affect the production of 1,3-PD. Seven variables were statistically significant at 95% significance and a 2(7-3) fractional factorial design (FFD) was applied to further refine the optimization of culture conditions. Results showed that the ideal medium composition and culture conditions for the syntheses of 1,3-PD are: glycerol 65 g/L, yeast extract 5 g/L, peptone 5 g/L, (NH4)(2)SO4 7 g/L, K2HPO4 7 g/L, and temperature of 37 degrees C. Experiments in batch bioreactors under controlled pH produced up to 23.80 g/L of 1,3-PD and 12.30 g/L of ethanol, while in fed-batch cultivations a three-fold increase of 1,3-PD production (36.86 g/L) was obtained compared with the results of the FFD design. (C) 2013 Elsevier Ltd. All rights reserved.