The High-cell-density Culture Fermentation And Metabolic Regulation Research Of Producing Coenzyme Q10 By Rhodobacter Sphaeroides

In this study, we using Rhodobacter sphaeroides RS-6 as the starting strain, the researth results included the shake flask fermentation conditions and culture medium is optimized, and on the basis of the optimization result of shake flask fermentation, further, we optimize the fed-batch cultivation fermentation process, finally we enlarge the optimized process to 50 L and 100 L.First of all, we optimize shake flask fermentation culture conditions and medium. Shake flask fermentation condition is optimized by single facter experiment, and the optimization fermentation condition is:seeding culture time 22 h (OD₆₆₀ is about 7), inoculation amount 16%, liquid volume 50 mL, shaker speed 210 rpm, temperature 32.5 and pH 6.5. Under the optimal culture conditions, the concentration of magnesium sulfate, ferrous shlfate, and sodium choloride is optimized by orthogonal experiment. We select 8 trace elements, and add them to culture medium at 0.05 g/L, then, we choice three which can increase the production of coenzyme Q₁₀ and effort their best concentration through single facter exoeriment. In the end we get the best combination of the three inorganic salts and trace elements:magnesium sulfate 8 g/L, ferrous sulfate 1.2 g/L, sodium chloride 4 g/L, calcium chloride 0.1 g/L, manganese sulfate 0.01 g/L, sodium perborate 0.1 g/L. after optimize the coenzyme Q₁₀ yield reached 197.67 mg/L is about 15% higher then before.Depend on the shake flask experiment, we optimize the coenzyme Q₁₀ fed-batch fermentation condition, rotate and aeriation rate was studied in a 15 L reactor, our results shows that when the rotate stay at 500 rpm and aeriation at 2 vvm has a higher coenzyme Q₁₀ production 1664.83 mg/L, about 15.5 times of batch culture. After analysis the effects of different rotate and aeriation rate on the growth of Rhodobacter sphaeroides and coenzyme Q₁₀ synthesis, we find that high level dissolved oxygen is contribute to the growth of Rhodobacter sphaeroides, but restrain the produce of coenzyme Q₁₀, thus, we continuously shift the rotate and aeriation rate in order to improve the produce of coenzyme Q₁₀, and obtain a optimized method from four strategy that is:stay a high rotate and aeriation rate when starting culture, and then after 48 h shift the rotate and aeriation rate to a low level, with that strategy we obtain a higher coenzyme Q₁₀ production to 1809.34 mg/L, increased about 9% further.Under the optimal rotate, aeriation rate adjusting strategy, we find that when the concentration of residual sugar in the broth stay at 5 to 10 g/L is better to achieve a high bacteria concentration and coenzyme Q₁₀ cotent. In the phosphorous source adding experiments, we find that when the potassium dihydrogen phosphate concentration in feeding medium is 3.9 g/L, the broth can stay a suitable phosphorous level which result in a high unit coenzyme Q₁₀ cotent of 16.67 ?mg/g DCW?, is about 15.2% higher than blank contral, and the maximam coenzyme Q₁₀ fermentation time is shorter, from 120 h to 96 h, the consumption of glucose is also decreased, from 2600 g to 2000 g, and the total coenzyme Q₁₀ yield has not been decreased, thus, decreased the production cost of coenzyme Q₁₀.Finally, we use equal volume constant stiring power amplification method enlarge the fermentation from 15 L to 50 L and 100 L. In 50 L reactor we use mass spectrometry detect exhaust components, then, we obtain CO2 release rate ?CER?,02 uptake rate ?OUR?, respiratory quotient ?RQ? and other parameters, combining with the offine datta such as, substrate concentration, DCW, coenzyme Q₁₀ production concentration, sugar consumption and sugar consumption accumulation data changes over time, we have a more comprehensive analysis. Further, the fermentation craft is enlarged to 100 L, the total coenzyme Q₁₀ yield is 94298.4 mg.