Abstract: In this paper, the fermentation processes of Pseudomonas denitrificans were investigated in 7L fermenter by using betaine and choline chloride as the typical methyl-group donors for vitamin B₁₂ biosynthesis, respectively.When using choline chloride as the methyl-group donor, the dissolved oxygen (DO) and pH respectively presented an obviously zooming and dropping phenomena, resulting in a slow cell growth and low vitamin B₁₂biosynthesis.However, the metabolism capability under betaine as methyl-group donor was significantly better than that of choline chloride, and the pH and DO could be maintained at a stable and appropriate level during the whole fermentation processes.As a result, the final cell growth and vitamin B₁₂ production were significantly higher than those obtained in choline chloride.