Riboflavin Fermentation Process Optimization And Preliminary Study On Mechanism Of Metabolic Regulation

Riboflavin （vitamin B₂） is a kind of yellow, luminescent, water-soluble vitamin. Currently, there are three microorganisms, Ashbya gossypii, Candida famata and Bacillus subtilis used in riboflavin industry overproduction. In this study, a strain of Candida famata was selected to produce riboflavin. In order to study the effect of the growth of Candida famata and the riboflavin production, metabolic regulating inhibitors, oxygen-vector, precursors and growth factors were added to the shake-flask during the fermentation. A glucose-limited continuous culture in a 5L fermentor was conducted. The maximum riboflavin concentration was 13365 mg/L after 136 hours’ fermentation.The optimized fermentation of riboflavin with Candida famata was performed by statistical methods. Plackett-Burman design was carried out to evaluate the effect of ten variables of medium components and/or culture conditions on the riboflavin synthesis. The positive or negative effect of those variables was determined. Among those variables, three ones were found to be the most significant variables. The optimal values of the variables were determined by RSM analysis based on the central composite design （CCD） and were listed as follows: urea 3.1g/L, biotin 10.6μg/L, pH 5.7 respectively. The maximum riboflavin concentration was 6188.0 + 64.15 mg/L in the optimum medium, which was 144.3 % higher than that of the non-optimum medium.The relationship between riboflavin synthesis and yeast physiological characteristics, such as yeast morphology, reducing activity, acidification activity and oxidative stress, was pilotly studied during the fermentation. The results indicated that reducing activity and oxidative stress were both positively correlated with riboflavin synthesis; the acidification activity was the lowest for high-yield yeast at exponential growth phase. Organic acids in fermentation broth were determined by HPLC. The concentration of pyruvate and citric acid during the fermentation remained at relatively low level. However, the gluconic acid was accumulated, due to high HMP flux. The concentration variation of gluconic acid was consistent to that of the riboflavin. The SDS-PAGE electrophoresis of whole cell proteins showed that GTP cyclohydrolase II and riboflavin synthase, the two key enzymes, had high expression level.