| The strain XM023 producing CoQ10 was screened from strains preservated in the laboratory, its fermentation level of CoQ10 was up to 18.80mg/L. First through the observation of colony morphology, cell microscopy, liquid culture and biochemical characteristics, it was found that this strain was gram-negative bacteria with rich pigment in cell, which was preliminarily judged to be one typical of photosynthetic bacteria. The 16S rDNA of strain XM023 was sequenced and analyzed for similarities with related bacterial species. Results showed that strain XM023 was a member of the Rhodobacter sphaeroides.XM023 being a starting strain was mutagenized twice with UV and LiCl. By screeneing the mutants resisting PHB and roxithromycinid, a mutant XMⅡ136 was Obtained, the CoQ10 production reached to 31.79mg/L, which was 69.10% higher than the original strain. After five times of continuous culture, the yield of the mutant was remained at 29mg/L-33mg/L. Results showed that the obtained mutant XMⅡ136 was stable strain that was worthwhile to be studied further.The fermentation optimization of XMⅡ136 was studied. Through single factor test of mutant XMⅡ136, the result showed that the best carbon was glucose, the optimum nitrogrn was corn steep powder; the best fermentation condition was obtained based on the single factor test, it showed the optimal amount of inoculation was 10%, the optimum initial pH was 6.8 and the optimal liquid volume were 50mL culture in 250mL shake flasks. A Plackett-Bunnan design was used to select 3 important factors from 9 impact factors which affected CoQ10 fermentation in culture medium. They were glucose, NaCl and glutamic acid. The optimum components obtained by the response surface analysis method(RSM) were glucose34.2g/L, NaCl 2.42g/L, glutamic acid 1.98g/L. After these optimization, the fermentation level of XMⅡ136 reached 129.21mg/L, which was 306% higher than that of non-optimized fermenatation.Based on the studies of the metabolic characters of CoQ10 batch fermentation with mutant XMⅡ136, the kinetic model for cell growth, CoQ10 formation, sugar consumption were proposed according to the Logistic equation and Luedeking-Piret equation. The calculated results of models were compared satisfactorily with the experimental data. The model equations could really reflect the CoQ10 fermentation process and kinetic mechanism.The effect of vitamin on CoQ10 production were studied. The results showed that various vitamins had different effectence on CoQ10 production. The most high-yield was 179.85 mg/L when betacarotene was added,32.3% increase of the yields compared with not adding betacarotene. In additon, adding VB1, choline chloride, biotinhad could bring 11.2%,13.3%,10.8% increase of the yields respectively compared with the contrast. The optimum time to add different additives was also different, biotin, B1 were added better in early fermentation, whileβ-carotene, choline chloride added in the fermentation process showed effective. Soybean oil added to the culture medium could increased 7.6% CoQ10 production compared to the control.Under the conditions of shake-flask, the effects of adding time, adding volume and adding method of glucose on CoQ10 production were studied. The results showed that adding sugar at 36h was better than in the initial fermentation, the optimum adding amount of total glucose was 30g/L and using multi-addiing method accessed to the largest of CoQ10 production 218.78mg/L. On this basis, the amplification experiment of batch fermentation and fed-batch fermentation in 10L fermentation tank indicated that fed-batch fermentation was superior to batch fermentation for the synthesis of CoQ10. CoQ10 production by fed-batch fermentation was up to 704.9mg/L, increased 96.6% compared with batch fermentation. |
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