本研究首先針對七株篩選自油品污染廠址之菌種（Rhodococcus erythropolis BC11, Bacillus pumillus CA20, Nocardioides simplex BC04, Comamonas testosterone CF3, Gordonia nitida JG39, Bacillus subtilis JG4, 及Pseudomonas aeruginosa RS1）與一株購買自美國菌種保存中心之菌種（Bacillus subtilis ATCC 21332），進行其生產生物界面活性劑之潛力評估。菌株生產生物界面活性劑之定性分析測試項目，包括表面活性、乳化指數、菌體表面疏水性、溶血活性等。其測試結果顯示，B. subtilis ATCC 21332具最佳之表面與乳化活性，可將純水之表面張力從72降低至27 mN m-1，與正己烷之界面張力從37.9降低至1.0 mN m-1，且對煤、柴油之乳化指數可以高達70%，其所生產出之生物界面活性劑－表面素（surfactin）為目前最具效果之生物界面活性劑之一，並有令人期待之商業應用價值，然其生產價格偏高，影響其實際應用之可行性。有鑑於此，本研究乃致力於發展更有效率且更經濟之表面素醱酵技術，以降低其商業化應用之門檻。

　　首先，本研究以重複批次操作策略評估以乳膠共聚物（polyurethane-polyurea copolymer）固定化B. subtilis ATCC 21332菌株生產surfactin之可行性。實驗結果顯示，在第一次批次操作時，surfactin之產量可達721 mg L-1，相較於懸浮細胞批次培養之產率（約100 mg L-1）有七倍以上之提升。同時，本研究亦發現培養基中添加適量之多孔性固體載體（如活性碳或發泡煉石）能夠顯著提升surfactin之產量，在培養基中加入25 g L-1之活性碳為載體時，可得最佳surfactin產值為3600 mg L-1，約為不添加固體載體控制組之36倍，顯示固體載體的存在能夠明顯地促進細胞生長，並提升surfactin產量。碳源濃度對於surfactin之生產亦相當重要，實驗結果顯示，最佳glucose初始濃度為40 g L-1。此外，適當之攪拌速率對於surfactin之生產也有正面之幫助，當控制於200 rpm時可得到最佳之產量。本研究利用一連串之酸沉澱與溶劑萃取將surfactin從醱酵液中分離並進一步純化，其產物之純度可達90%，且回收率亦有約72%。此產物可將純水之表面張力從72降低至27 mN m-1且具有相當低之臨界微胞濃度 (約10 mg L-1)。而此surfactin產物在100與600 mg L-1濃度下即分別可使煤、柴油之乳化指數達70%。

　　基於解決醱酵生產生物界面活性劑時所產生大量泡沫之問題及連續移除surfactin以誘發surfactin之大量生產考量下，本研究進一步開發改良式生物反應器。該改良式反應器乃在傳統醱酵槽外加裝泡沫收集器、細胞迴流系統與surfactin酸沉澱槽，同時在醱酵液中添加適當之固體載體（活性碳）藉以提高細胞濃度與surfactin產能。此反應器設計可以在嚴重發泡狀態下提供surfactin穩定、高效率之生產環境，且不須添加任何消泡劑。透過不同曝氣速率與攪拌速率之組合，本研究嘗試探討氧氣傳輸與質傳效率對surfactin生產之影響，實驗結果顯示其最佳組合為1.5 vvm與300 rpm，可得到最佳之surfactin最大生產速率、總生產速率、最高濃度與最大產量分別為190 mg L-1 h-1、106 mg L-1 h-1、6.45 g L-1以及161 mg surfactin (g glucose)-1。

　　最後，本研究嘗試利用重覆批次與饋料批次操作策略，企圖再提升surfactin之產量。搖瓶重覆批次實驗結果顯示，B. subtilis ATCC 21332之surfactin生產活性在三次重覆批次操作過程仍可以維持在75%以上，但在重覆批次時必須同時更換新鮮之培養基質與固體載體（活性碳）。然而，在一系列不同進料組成與進料體積之初步饋料批次實驗發現，除了在饋料批次時進料總濃度為2% glucose之實驗可以將總產量小幅度提高12%（12.90增加至14.47 g）以外，其他實驗則無法再提升surfactin之濃度與產量。整合所有饋料批次實驗之數據發現，造成饋料批次操作效果不彰之原因，可能是由於菌體細胞在surfactin合成時，受到quorum sensing控制機制之影響、或是代謝物與使用過固體載體對surfactin生產之抑制，或是pH高於6.5時所引起之嚴重起泡現象所致。因此，若要使饋料批次操作策略能夠成功的應用於surfactin之量產，必須對上述各可能之影響因素進行更深入之研究。

　　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.

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