Optimization Of Nutrients And Environmental Conditions For Vitamin C Fermentation

Ketogulonigenum vulgare and Bacillus megaterium were co-existed and co-growth in the established industrial process of vitamin C production. Among of them, K. vulgare, a gram-negative bacteria, could convert L-sorbose into high titer of 2-keto-L-gulonic acid (2-KLG), the precursor of vitamin C in the culture broth, with the help of B. Megateriu. In this thesis, the nutritional and environmental conditions for high titer, high yiels and high productivity of 2-KLG accumulation were carefully investigated, and the main results were described as follows:(1) In the first optimization step the influence of six metal ions was evaluated using a fractional factorial design. Fe³⁺, Mg²⁺, Mn²⁺influenced vitamin C production positively. Then, the path of steepest ascent and the Box-Benhnken design were used for further optimization on these three factors. The optimal concentrations of the variables were determined as: Fe³⁺: 0.21 mmol/L, Mn²⁺: 9.98 mmol/L, Mg²⁺: 3.93 mmol/L. Under the optimal culture condition, the yield of the 2-keto-L-gulonic acid increased by 144.4%.(2) The mode of oxygen concentration control is an important factor for batch 2-KLG fermentation. It was shown that high level of oxygen concentration improved cell growth but inhibited 2-KLG yield. Low level of oxygen concentration was opposite to the high level.(3) The effect of oxygen concentration, varied from DO50 % to DO90 %, on the batch fermentation of 2-KLG was investigated. Owing to the difference occurred in the optimal oxygen concentration for cell growth and 2-KLG production by thoroughly analyzing the kinetics of batch 2-KLG production under different DO, a two-stage oxygen concentration control strategy, in which the DO was kept at 90 % during the first 20 hours of cultivation, followed by a shift from 90 % to 50 %, then maintained at 50 % until the end of the cultivation was developed in order to enhance the production of 2-KLG. The result indicated that the 2-KLG production was 58.7 g/L, which was respectively increased 1.46, 1.23 and 1.17 times than that at DO90 %, 70 % and 50 %, and 2-KLG productivity was increased 0.96, 1.06 and 1.23 times than that at above three oxygen concentration. Determined kLa under two-stage oxygen concentration control strategy, provided a certain degree of amplification theory for industry.(4) The optimal individual cell age of K. vulgare and B. megaterium were determined, the stock incubation time of former one and later one was 31-32 h and 8 h separately. The optimal condition was as follow: inoculum size 10 %, inoculum ratio 1:1.(5) A two-stage pH control strategy was developed, in which the pH was kept at 8.0 during the first 16 hours of cultivation, followed by a shift from 8.0 to 6.0, then maintained at 6.0 until the end, in order to enhance the production of 2-KLG. It was showed that the pH control strategy could improved cell growth, 2-KLG yield and shorten fermentation period.