Abstract: A mutant strain of Klebsiella pneumoniae with improved tolerance to 1,3propanediol(1,3-PD) and acetate was isolated from medium containing selective pressure after 60Co irradiation. In this study, several fermentation processes, including different aeration time, anaerobic process, and glycerol concentration to be controlled during the midandlate fermentation phase, were conducted to investigate their effects on microbial metabolism. Results showed that the process without aeration and maintaining about 20 g/L glycerol concentration during the midandlate fermentation phase was suitable for 1,3-PD production. The fedbatch fermentation in a 5 L bioreactor showed that the production of 1, 3-PD reached 70.81 g/L, the mol yield of 1, 3-PD on glycerol was 0.649 and the productivity was 2.083 g/(L·h）, which were increased by 54.3%, 52.0% and 50.7% respectively compared with the results of parent strain.