Exploring fermentation strategies for enhanced production of a lipopeptide biosurfactant – surfactin

• Main Authors: Mao-Sung Yeh, 葉茂淞
• Other Authors: Jo-Shu Chang
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/78414182069637355293

博士 === 國立成功大學 === 化學工程學系碩博士班 === 94 === 　　Eight bacterial strains (purchased from American Type Culture Collection (ATCC) or isolated from oil-contaminated sites) were evaluated for their potential of biosurfactant production. The strains examined were Bacillus subtilis ATCC 21332, Rhodococcus erythropolis BC11, Bacillus pumillus CA20, Nocardioides simplex BC04, Comamonas testosterone CF3, Gordonia nitida JG39, Bacillus subtilis JG4, and Pseudomonas aeruginosa RS1. The results from batch experiments demonstrated that B. subtilis ATCC 21332 exhibited excellent surface and emulsion activity. The biosurfactant produced by the 21332 strain, surfactin, is recognized as one of the most effective biosurfactant available and possesses promising commercial applications. This motivated us to develop viable fermentation technology to produce surfactin in a more efficient and cost-effective way. 　　Surfactin production was first carried out by using immobilized cells of B. subtilis ATCC 21332 via matrix entrapment of polyurethane-polyurea copolymers. Repeated-batch experiments were conducted to evaluate the ability of surfactin production. The results show that the surfactin yield was about 721 mg L-1, which was 7 fold higher than that attained from fermentation with suspended culture of free cells. We also discovered that addition of a small quantity of solid porous carriers (e.g., activated carbon or expanded clay) into fermentation broth significantly increased surfactin production with B. subtilis ATCC 21332. Culture medium containing 25 g L-1 of activated carbon gave an optimal surfactin yield of 3600 mg L-1, which was approximately 36 fold higher than that obtained from carrier-free liquid culture. The marked increase in surfactin production was primarily attributed to stimulation of cell growth due to the presence of activated carbon carriers. Carbon source (glucose) concentration was essential to the production of surfactin with an optimal initial glucose concentration of 40 g L-1. An appropriate agitation rate also benefited surfactin production, as the best yield appeared at an agitation rate of 200 rpm. Surfactin was purified from fermentation broth via a series of acidic precipitation and solvent extraction. The resulting product was nearly 90% pure with a recovery efficiency of ca. 72%. The purified surfactin reduced the surface tension of water from 72 to 27 mN m-1 with a critical micelle concentration of ca. 10 mg L-1. Addition of 10 mg L-1 of surfactin also reduced the interfacial tension between water and normal hexane from 37.9 to 1.0 mN m-1. The surfactin product also attained an emulsion index of 70% for kerosene and diesel at a low concentration of 100 and 600 mg L-1, respectively. 　　Furthermore, an innovative bioreactor was tailored to solve the problems of severe foaming arising from production of surfactin. To cope with the rapid foam generation, a conventional jar fermentor was integrated with a foam collector, a cell recycler, and a surfactin precipitation unit. Meanwhile, solid carriers (e.g., activated carbon) were added into the fermentation broth to increase cell mass concentration and surfactin yield. The proposed bioreactor allowed stable and efficient surfactin fermentation under intensive foaming conditions without the need of adding antifoam agents. The effect of oxygen transfer rate and mass transfer efficiency on surfactin production was also explored by employing various combinations of aeration and agitation rates. The best combination was 1.5 vvm and 300 rpm, giving an excellent maximum production rate, overall production rate, surfactin concentration, and surfactin yield of 190 mg L-1 h-1, 106 mg L-1 h-1, 6.45 g L-1, and 161 mg surfactin (g glucose)-1, respectively. 　　Finally, we attempted to utilize repeated-batch and fed-batch operation strategies to further enhance the surfactin yield. The results show that the surfactin-producing activity of B. subtilis ATCC 21332 could maintain at a high level (> 75% during three operation runs) by replacing flash MSI medium and solid carriers when carrying out the repeated-batch experiments by carrier-added liquid culture in flask. However, with a series of fed-batch operations, the surfactin concentration was not significantly improved, while feeding of 2% glucose alone led to a 12% increase in total production of surfactin (from 12.90 to 14.47 g). The unsatisfactory outcome of fed-batch operation is thought to be due to be closely associated with the bacterial regulation mechanism (i.e., quorum sensing system) involved in surfactin synthesis by B. subtilis ATCC 21332. In addition, there are also some crucial factors influencing the production of surfactin, such as the inhibition of metabolites, the repression of used solid carriers, and the severe foaming that was pH sensitive. Apparently, it would require further systematic investigation to identify the role of those factors in the performance of surfactin production to elevate surfactin producing activity to a higher level.