The influences of three monovalent cations (Na+, K+, and NH4+) on bioconversion of glycerol to 1,3-propanediol by Klebsiella pneumoniae were investigated by designing three different media and pH control strategies in terms of metabolic control. Experimental results showed that glycerol dehydrogenase, one of the key enzymes of glycerol conversion, was inhibited by high concentration of Na+ under substrate-excess conditions, leading to a decline of yields of biomass and 1,3-propanediol on glycerol. It is feasible to adjust pH by using KOH in a medium consisting of enough NH4+ or using ammonia in a medium without NH4+ and Na+ in continuous cultures.