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HLC recombinant E.coli fermentation process of temperature-induced model, the sugar concentration and feeding operation optimization

Author: JinWei
Tutor: FanDaiZuo
School: Northwestern University
Course: Biochemical Engineering
Keywords: Human-Like Collagen the temperature induction model the transition temperature the glucose concentration the feeding operation
CLC: TQ936.2
Type: Master's thesis
Year: 2011
Downloads: 82
Quote: 2
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Abstract

In this study, the optimization was performed in the fermentation which Escherichia coli BL 21 products Human-Like Collagen:the temperature induction model, the feeding point, glucose concentration and the feeding operation.On the basis of the optimum culture temperature 34℃which was already optimized, this part was mainly about the induction temperature. Whether the transition temperature was necessary or not was studied when the OD (600nm) reached 85, it was showed that the percentage of resistant plasmid cell was decreased to 82% when there was no the transition temperature, and the final cell density and protein concentration were 69.7g/L and 6.3g/L respectively. The percentage of resistant plasmid cell was increased to 89% after the transition temperature, and the final cell density and protein concentration were 69.7g/L and 6.3g/L respectively, the protein concentration was increased by 11.9%. So the transition temperature was necessary for the high product density. Then the concrete induction model was investigated by setting three models:42℃for 3 hours firstly, then 39℃for the rest time; the temperature was decreased by 1℃from 42℃; always 42℃. It was showed that the final cell density and protein concentration were 65.5g/L and 7.3g/L in the first model,68.1g/L and 6.4g/L in the second model,62.2g/L and 9.7g/L in the third model. It was in the third model that the product concentration was the highest, so this model was used as the optimum induction model.The optimization of the feeding point and glucose density was performed on base of the organic acids. Firstly, from the analysis about DO, pH, organic acids and NH4+density, it was known that Escherichia coli BL 21 would use the organic acids and yeast extract’s C skeletons as the carbon source. The feeding point could be divided into three points:the glucose concentration was just zero; the density of total organic acids was just the lowest; the yeast extract’s C skeletons were used up. The results showed that the final cell density and product concentration of the second model were the highest, could reach 67.9g/L and 10.3g/L. Then, the glucose concentration was optimized after the feeding point. The final cell density and product concentration were the highest when the glucose concentration was controlled at 1.5g/L, could reach 68.7g/L and 12.1g/L, respectively. But the product concentration was lower when the glucose concentration was controlled at 0.5g/L, 1.0g/L and 2.0g/L, could only reach 5.1g/L,10.5g/L and 6.4g/L, respectively. The further study which the concrete feeding operation in 30L reactor was optimized was carried out on base of the operation in 12.8L reactor by the on-time and off-time. Before the induction, three patterns were set:1ml/22s; 3ml/66s; 5ml/110s. The same pattern was set after induction:1ml/8s. It was showed that the final cell density and product concentration were the highest when the third pattern was adopted, could reach 75.6g/L and 12.8g/L. After the induction, three patterns were also set:1ml/8s; 3ml/24s; 5ml/40s. The same pattern was set before induction:5ml/110s. The result showed that the final product concentration was the highest when the second pattern was adopted, could reach 14.6g/L, which was increased by 15.5% and 10.8% in comparison with the other two patterns. In conclusion, the final optimization results were as follows:5ml/110s before induction; 3ml/24s after induction.

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