| Coenzyme Qio (CoQ10), as an essential electron carrier in the aerobic respiratory electron transfer system, has been widely applied in medical and food industry. The use of CoQ10 as a complementary therapy in heart failure is increasing every year. Economical production of CoQio by microbes will become more important due to the growing demands of CoQ10. In this article, mutation breeding, optimization of culture medium and technology of fed batch fermentation were studied using Rhodobacter sphaeroides as the starting strain.1.In this paper, the effects of four methods were studied on CoQio extraction. The methods were saponification method, ultrasonic method, freezing and melting repeatedly method, acid-heat method and acid-heat assisted ultrasonic method. The results showed that the acid-heat assisted ultrasonic method was feasible for CoQ10 extraction. By single factor experiment and orthogonal experiment, the optimum technological condition was obtained. The optimal condition for coenzyme Q10 extraction was as follows, the cells were harvested and ruptured at 80℃ in the presence of 300 μL HC1 and output power 400w, total time 20 min. Under the optimized condition, the yield of CoQ10 could reach to 3.738mg/g.2.By natural screening, R.spl-11 as starting strain was mutated by UV combined with LiCI and NTG based on VK3, NaN3 and PHB as the composite resistance. The strain R.sp3-7 was obtained and its CoQ10 yield reached 71.23mg/L, which was increased by 132.17% compared with the original strain. Then the fermentation condition of R.sp3-7 was optimized by the single factor experiment. The optimal temprature, initial pH, the amount of inoculum and the volume of culture medium were 32℃,7.0,8% and 40mL/250mL respectively. Under the optimized condition, the CoQ10 production could reach to 82.70mg/L.3.The culture medium of R.sp3-7 was optimized with Response Surface Analysis Method(RSM). The best carbon, nitrogen sources and adding metal ions were determined by single factor experiment. Three important factors from nine factors were selected by Plackett-Burman design, which were glucose, com steep liquor and MgSO4-7H2O respectively. The optimum components obtained by RSM were glucose 36.9g/L, corn steep liquor 5.3g/L and MgSO·7H2O 14.4g/L. After these optimizations, the fermentation level of CoQ10 reached 135.20mg/L, which was increased by 68.62% compared to the control.4. The influences of pH and dissolved oxygen(DO) on CoQ10 production were studied in 5L fermentor. The results showed that the optimal pH value for CoQ10 fermentation was 6.8. Its production and DCW reached 60.29mg/L and 16.32g/L respectively. The level of DO was kept at 1% by controlling aeration and stirring rate. The CoQio yield was 82.67mg/L under the optimum condition, which increased by 37.11% compared to non-changed dissolved oxygen. And DCW was 15.49g/L, which decreased by 4.5%。5.1n fed batch fermentation, glucose concentration was controlled at 5g/L..10g/L 15g/L respectively. And the highest CoQ10 production was 209.4 mg/L. Meanwhile, under this Glucose Fed-Batch Fermentation, adding 100mL ammonium sulfate solutions at 24h, 32h,48h,60h respectively, the highest biomass reached 14.61 g/L and CoQ10 production reached 252.88 mg/L, which increased by 14.61% and 14.61% respectively compared to batch fermentation. Thus CoQ10 production could be greatly improved by adding glucose and ammonium sulfate.These results of this study will lay a good foundation for further study and industrial-scale production. |
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