Production of Acinetobacter radioresistens lipase using Tween 80 as the carbon source

• Main Authors: Chen-You Li, 李承祐
• Other Authors: Teh-Liang Chen
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/yf257x

博士 === 國立成功大學 === 化學工程學系碩博士班 === 92 === Acinetobacter radioresistens produces an extracellular alkaline lipase, which has an optimal pH of 10.0. This enzyme has a potential application in the detergent industry, and the development of an economical process for mass production is necessary. Lipase of Acinetobacter radioresistens was produced using Tween 80 as the carbon source. Compared with olive oil, the hydrolysis rate of Tween 80 in the fermentation broth was much slower. Taking advantage of the slow hydrolysis rate, Tween 80 could provide oleic acid for cell growth and lipase production through a mode of controlled release, and the known repression of lipase synthesis by oleic acid could be avoided. The optimal lipase yield obtained with batch culture in a 2.5-L tank fermentor was 25 U/mL with a fermentation time of 6 h, when using 0.3 % (v/v) Tween 80. The existence of an optimal yield with respect to concentration of Tween 80 was a result of the compromise between cell growth and the repression of lipase synthesis. To further the lipase productivity, a fed-batch culture with the pH-stat feeding method was employed, and the lipase productivity achieved was 120 U/mL in 16 h. Various strategies for production of A. radioresistens lipase were compared. A fed-batch process for production of Acinetobacter radioresistens lipase was developed. Using Tween 80 as the carbon source, a series of pH-uncontrolled batch cultures with supplementation of yeast extract and/or tryptone were carried out to search for the balanced medium as well as the set point of pH to be used in the fed-batch phase. It was found that a medium containing 3 mL/L Tween 80 and 7.5 g/L yeast extract was optimal for the lipase production in batch culture (having a lipase yield of 24 U/mL), and the final culture pH, 7.1 (the pH at the beginning of the stationary phase), was a suitable set point for the pH-stat feeding. Using a medium of 10-fold concentration in the fed-batch phase, the culture was able to proceed with consistent yield coefficients of cell growth and lipase production, and a lipase yield of 138 U/mL was achieved after 600 mL of the medium was fed. Data obtained from fed-batch cultures with DO- and pH-stat feedings showed that specific growth rate μ was the intrinsic factor that determined the efficiency of lipase synthesis. The specific lipase productivity was maximal at μ= 0.1 h�{1, while the optimal lipase production rate occurred around μ= 0.2 h�{1. Based on a growth-associated pattern for lipase formation, the production of lipase with fed-batch culture could be simulated satisfactorily. In addition, Acinetobacter radioresistens lipase was produced with repeated fed-batch culture using a balanced medium based on Tween 80 as the carbon source. The feeding strategies examined were pH-stat and DO-stat methods. A constant cell concentration was shown to be a prerequisite to extend the number of repeated cycles, and an adequate cell growth rate was critical for a high lipase yield. With the pH-stat feeding, which was found to approximate an exponential feeding, the cell growth rate was too high to effect the lipase production. On the other hand, the DO-stat feeding, which approximated a constant-rate feeding, could provide an adequate cell growth rate for efficient lipase production. The lipase production rate obtained in a 2.5-L fermentor could reach 42,000 U h�{1.