In this study, glycerol was used to produce optically pure D-lactate by engineered Klebsiella pneumoniae strain. In the recombinant strain, D-lactate dehydrogenase LdhA was overexpressed, and two genes, dhaT and yqhD for biosynthesis of main byproduct 1,3-propanediol, were knocked out. To further improve D-lactate production, the culture condition was optimized and the results demonstrated that aeration rate played an important role in D-lactate production. In microaerobic fed-batch fermentation, the engineered strain accumulated 142.1 g/L optically pure D-lactate with a yield of 0.82 g/g glycerol, which represented the highest D-lactate production from glycerol so far. This study showed that K. pneumoniae strain has high efficiency to convert glycerol into D-lactate and high potentiality in utilization of crude glycerol from biodiesel industry. (C) 2014 Elsevier Ltd. All rights reserved.