Roles Of Betaine In Vitamin B₁₂ Fermentation By Pseudomonas Denitrificans And Its Feeding Strategy Optimization

Vitamin B12(CV12) has become one of the most important biologically active substances for its wide utilization in medicine and food production. As an important precursor, betaine has significant effects on physiological characteristics of Pseudomonas denitrificans in VB12 fermentation. Therefore, it is important to investigate the mechanism of betaine and the optimal betaine feeding strategy for efficient VB 12 production.The catabolism of betaine was researched with the addition of U-13C glucose and natural labeling betaine simultaneously in the chemical defined medium. Through analyzing the enrichment of 45CO2 in the off-gas and the corrected fractiona labeling (FL) profiles of proteinogenic amino acids, it was showed that a great amount of betaine was decomposed and involved in cell formation as carbon source in exponential phase.Futhermore, the effects of betaine concentration under different feeding strategies on the VB12 production were studied in its biosynthesis phase. The results showed that maintaining a low betaine concentration at which enough methyl-group donor could be provided was beneficial to VB12 biosynthesis. Then, comparing distribution of VB12 outside and inside the cell in the late period of fermentation, it was indicated that extracellular VB12 accumulation could be accelerated by low betaine concentration.Betaine had a rapid stimulation on oxygen consumption of P. denitrificans through online analysis of off-gas with process mass spectrometer. The experimental results with addition of several betaine analogues revealed that this stimulation could probably be the result of protection of betaine on the cell from respiration inhibition of osmotic pressure. In shake falsks without betaine, the pH decreased obviously during fermentation with lactate formation.Based on the results above, a new betaine feeding strategy was employed by reducing the initial betaine concentration from 15g/L to 3g/L and maintaining the betaine concentration at 2-3g/L during fermentation. The biomass and VB12 production increased for 10.5% and 23.8%, respectively.