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研究生:鄭捷倫
研究生(外文):Chieh-Lun Cheng
論文名稱:利用本土菌株PseudomonasaeruginosaJ16醱酵生產鼠李醣脂之探討
論文名稱(外文):Rhamnolipid production with an indigenous isolate Pseudomonas aeruginosa J16
指導教授:魏毓宏
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技暨生物資訊研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:101
中文關鍵詞:鼠李醣脂
外文關鍵詞:Rhamnolipid
相關次數:
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本研究使用自石化廢水篩選所獲得之台灣本土菌株Pseudomonas aeruginosa J16進行其代謝產物鼠李醣脂之定性、定量及醱酵生產策略上的討論。研究證實P. aeruginosa J16具代謝合成生物界面活性劑鼠李醣脂之能力,經高效液相層析儀、核磁共振光譜儀、快速原子撞擊質譜儀分析証實P. aeruginosa J16合成之鼠李醣脂主要為單鼠李醣脂及雙鼠李醣脂。其次經研究証實,P. aeruginosa J16最適合生產鼠李醣脂的碳、氮源分別為甘油及硫酸銨,其最佳產量為2.8 g/L。上述培養基進一步利用回應曲面實驗設計法求得最佳化培養基組成為Glycerol 3.5 %、(NH4)2SO4 33.25 mM、NaHPO4 40 mM、KH2PO4 40 mM、CaCl2 6 μM、 MgSO4.7H2O 577 μM、EDTA 4 μM、FeSO4.7H2O 4 μM。研究証實P. aeruginosa J16利用回應曲面實驗設計所得之最適化培養基,能將鼠李醣脂產量提升至4.2 g/L,為基礎培養基的7倍,最高產率為0.044 g/L/h。
於重複批次實驗研究上,可讓鼠李醣脂產率達0.086 g/L/h,為批次實驗之1.8倍,且可以連續操作三個批次以上,每批次產量皆可達4.3 g/L以上,並且將醱酵時間縮短兩倍。在饋料批次策略上,以菌體濃度作為添加營養源時機,成功的得到鼠李醣脂最佳產量8.8 g/L為批次實驗的1.9倍,為基礎培養基之17倍,在產率上也有明顯提升可達0.073 g/L/h,此結果顯示,以菌體濃度作為添加營養源時機,為一方便且具效率之醱酵方式。
Abstract
Pseudomonas aeruginosa J16 isolated from wastewater of a petrochemical factory located in southern Taiwan was used in the present work for qualitative、quantitative and production of rhamnolipid. Recent researchers reported that rhamnolipid is a biosurfactant primarily produced by Pseudomonas aeruginosa. The results of HPLC、 13C NMR and mass spectrometry analysis show that the biosurfactant consisted mainly of mono-rhamnolipid (RL1) and di-rhamnolipid (RL2). Preliminary tests indicated that a combination of glycerol and (NH4)2SO4 as the carbon and nitrogen source gave a good rhamnolipid production of 2.8 g/L. The optimal formulation was identified by response surface methodology (RSM) as follows: glycerol (3.51%), (NH4)2SO4 (33.3 mM), NaHPO4 (40 mM), KH2PO4 (40 mM), CaCl2 (6 μM), MgSO4.7H2O (577 μM), EDTA (4 μM), and FeSO4.7H2 (4 μM). Statistical experimental design methodology was not only applied to optimize the culture medium composition but further enhanced rhamnolipid production of 4.2 g/L and productivity of 0.044 g/L/h. The repeat-fed batch strategy not only abridges the time of fermentation about two times but also could repeat this process and get the same results at least three times. In each batch, this strategy could enhance rhamnolipid concentration and productivity were 4.3 g/L and 0.086 g/L/h, respectively. In fed batch strategy, fed timing is controlled by the change of optical density, and the best rhamnolipid production which successfully got was 8.8 g/L, approximately 1.9-fold higher than batch experiment and 17-fold higher than original medium. The results show that the fed batch strategy was convenient and efficient method for rhamnolipid production.
目錄
中文摘要 I
英文摘要 III
目錄 IV
表目錄 VII
圖目錄 VIII
第一章 前言 1
1-1 生物界面活性劑(Biosurfactants) 1
1-2 生物界面活性劑之生產菌株篩選及定量分析 5
1-3 生物界面活性劑之應用 6
1-4 醣脂類生物界面活性劑-鼠李醣脂 8
1-5 鼠李醣脂之醱酵生產 12
1-6 鼠李醣脂之應用 14
1-7 研究動機及目的 15
第二章、材料與方法 16
2-1 實驗藥品與分析儀器 16
2-1-1 實驗藥品: 16
2-1-2 實驗儀器: 18
2-2菌株及培養基 19
2-2-1 菌株: 19
2-2-2 培養條件: 19
2-2-3 培養基組成 20
2-3 鼠李醣脂(Rhamnolipid)的分離及純化 21
2-4 實驗步驟與分析方法 22
2-4-1鼠李醣脂(Rhamnolipid)之定性分析 22
2-4-2 鼠李醣脂(Rhamnolipid)之定量分析 22
2-4-3 甘油定量分析 23
2-4-4 硫酸銨定量分析 24
2-4-5 微生物生長之分析 24
2-4-6 酸鹼值測定 24
2-5 實驗設計法-回應曲面法 25
2-5-1 二水準因數設計(Two-Level Factorial Design) 25
2-5-2 陡升路徑法( method of path steepest ascent ) 26
2-5-3 中心混成設計(Central Composite Design,CCD ) 27
第三章、結果與討論 34
3-1 論文架構 34
3-2 鼠李醣脂之定性分析 34
3-3 鼠李醣脂之HPLC之定性及定量分析 40
3-4 培養基最適化 48
3-4-1 P. aeruginosa J16醱酵生產鼠李醣脂之培養基碳源探討 48
3-4-2 P. aeruginosa J16醱酵生產鼠李醣脂之培養基氮源探討 48
3-4-3 以實驗設計進行培養基組成之最適化探討 52
3-5 醱酵策略 66
3-5-1 批次醱酵 66
3-5-2 兩階段批次醱酵 68
3-5-3 饋料批次醱酵 74
3-6 鼠李醣脂之表面活性及乳化指數分析 85
3-7 鹽度、pH值對鼠李醣脂之影響 85
第四章、結論 91
第五章、未來展望 94
參考文獻 95
參考文獻
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