The aim of this study was to enhance the production of vitamin K-2 by using N-methyl-N-nitro-N-nitroso-guanidine (NTG) and low energy ion beam implantation and optimizing the fermentation medium. Mutation resulted in 1.66-fold higher production of vitamin K-2 than that of the parentl strain. The production by the mutant BN-P15-11-1 was increased 55% and reached 3.593 +/- 0.107 mg/L by using the Plackett-Burman and Box-Behnken designs to optimize the fermentation medium. The optimal fermentation culture medium was composed of (g/L) glycerol 69.6, sucrose 34.5, K2HPO4 4.0, peptone 20, yeast extract 25 and fermented at 37 degrees C and 150 rpm for 72 h. The results showed that the NTG and low energy ion beam implantation mutations and optimizing fermentation medium were effective methods to enhance vitamin K-2 production.