In order to improve the conversion rate of sugar and acid in Vc two-step fermentation,bacillus giant WS-05 was used as the starting strain,and the combined mutagenesis method of microwave and lithium chloride was used for mutagenesis selection,and the associated bacteria that were more conducive to the growth and metabolism of acid-producing bacteria were screened.The effects of dose and time of microwave-lithium chloride mutagenesis on the survival rate of Vc two-step associated bacteria were studied.Three groups of mutagenesis doses with survival rate of 55%~70%were selected for mutagenesis.Screening the associated bacteria that can ferment small bacteria to produce 2-keto-L-gulonic acid with high yield,The high-yielding strain D06 was obtained by shaking flask fermentation and re-screening.The conversion rate of the strain was 2%higher than that of the original strain.The effect of fermentation conditions on the new strains was studied.The results showed that the oxygen requirement of the bacteria for acid production could be fully met by appropriately increasing the ventilation volume of the new strains(20~25 mL liquid in 250 mL fermentation shaker),the optimal concentration of basal sugar was 7%,and the optimal growth temperature was 31℃.It was proved that the genetic character was stable,Its genetic character is stable,and the average conversion rate of 4 generations can reach 87.84%.