臺灣博碩士論文加值系統

本研究為獲得產界面活性劑之高效生成菌，應用於油品店家內排水管之生物處理與資源化，研究方法以生活周遭常見使用油品店家，其附近下水道採集污泥作為菌源，經分離與純化後，篩選出優勢菌種EBL1~6並進行特性分析，發現EBL2、3、6具有產界面活性劑能力；進行菌種鑑定與親緣樹狀圖分析認定，EBL6為Pseudomonas aeruginosa。最後將EBL6進行單一碳源與雙碳源測定最佳培養添加量，其代謝物萃取出鼠李醣脂並純化，即可進行定性與定量分析。
將EBL1~6進行CTAB實驗，其均可不同程度地形成暈圈，其中EBL6暈圈形成能力為最高，測定表面張力最低可達37dyn/cm且具有乳化柴油之能力，碳源以MSB添加1g/L Gluocse為最佳生長添加量，菌體乾重達1.1g/L，生長速率為0.0393(h-1)。雙碳源生長MSB添加Glucose培養第50小時加亞油酸油脂發酵為顯著，菌體乾重達1.74 g/L。定性分析中EBL6代謝物進行FTIR的分析結構具有鼠李醣脂疏水性與親水性。HPLC進行梯度流洗得知RhaC10C10、RhaC10C10、RhaC10H2與RhaC10C12四個組合物。定量分析以蔥酮-硫酸法檢測鼠李醣脂總糖含量為204mg/L，每批MSB添加1g/L之Glucose發酵培養，每200mL之培養液搖瓶試驗均可得1.02g/L之鼠李醣脂產物。
上述結果顯示使用油品店家下水道分離之EBL6具有良好產生物界面活性劑之特性，未來可加入發酵罐增加產量與應用在污染場址模擬處理環境。

In this study, we used the highly effective bacteria for the production of surfactant, which was used in the biological treatment and resourceization of the drain pipe in the oil . The method to carried out was using the oil store in the surrounding life, and collected the sludge as the bacteria source. After isolation and purification, the dominant strains EBL1~6 were screened and analyzed. The results showed that EBL2,3 and 6 had the ability of producing surfactant. The results showed that EBL6 was Pseudomonas aeruginosa. Finally, the optimal amount of EBL6 was determined by single carbon and double carbon sources. The metabolites were extracted for purified, qualitative and quantitative analysis.
After that,the EBL1~6 was subjected to CTAB experiment, which could form halo in different degrees. The ability of EBL6 halo was the highest, the minimum surface tension was 37dyn / cm and the ability of emulsified diesel oil was determined. The carbon source was added with MSB 1g / L Gluocse was the best growth. The dry weight of the cells was 1.1 g / L and the growth rate was 0.0393 (h-1). The growth of stain was increased by the addition of Glucose to the 50th day, and the dry weight of the cells was 1.74 g / L. Qualitative analysis of EBL6 metabolites for FTIR analysis of the structure with rhamnolipid hydrophobicity and hydrophilicity. RhaC10C10, RhaC10C10, RhaC10H2 and RhaC10C12 were obtained by gradient elution. Quantitative analysis The total sugar content of rhamnolipid was 204mg / L by scallion-sulfuric acid method. Glucose fermentation was carried out with 1g / L of each MSB. 1.02g / L mice were obtained for each 200mL culture flask Li sugar and fat products.
The above results show that the use of oil shop sewer separation of EBL6 has a good product of surfactant characteristics and amount, the future can be added to the fermentation tank to increase production and application in the contaminated site simulation processing for the environment.

明志科技大學碩士學位論文口試委員審定書i
致謝 ii
摘要 iii
Abstract iv
目錄 vi
表目錄 ix
圖目錄 x
第一章 前言 1
1.1研究動機 1
1.2研究目的與架構 2
第二章 文獻回顧 5
2.1界面活性劑之特性 5
2.2界面活性劑的種類 7
2.2.1化學界面活性劑 7
2.2.2生物界面活性劑 10
2.3生物界面活性劑特性解析 13
2.3.1生物界面活性劑產生的機制 14
2.3.2現有的生物界面活性劑產生菌 15
2.3.3產生的種類 16
鼠李醣脂(RhamnoLipid) 17
枯草素(Surfactin) 21
2.4生物界面活性劑分析的方法 23
2.4.1表面張力 23
2.4.2微生物動力成長 26
2.4.3臨界微胞濃度(CMC) 29
2.4.4 起泡性 31
2.4.5油品乳化與排油圈現象 31
2.5生物界面活性劑萃取與分析方法 32
2.5.1分析方法 33
2.6國內外文獻探討整理 34
第三章 材料與方法 35
3.1實驗目的 35
3.2實驗材料與設備 36
3.2.1實驗藥品 36
3.2.2 實驗設備 38
3.3 實驗方法與架構 39
3.4優勢菌種篩選純化 41
3.5生物界面活性劑特性分析 43
3.5.1 CTAB培養基油圈測定 44
3.5.2表面張力測定 44
3.5.3臨界微胞濃度(CMC) 45
3.5.4乳化性與發泡性測試 45
3.6生物界面活性劑產生菌之菌種鑑定 46
3.6.1 生物界面活性劑產生菌序列及特性分析 47
3.7微生物動力生長測定 47
3.7.1微生物生長最佳碳源添加 47
3.8 生物界面活性劑定性與定量分析 48
3.8.1生物界面活性劑(鼠李醣酯)萃取 48
3.8.2 FTIR官能基結構分析 49
3.8.3 HPLC定量分析 50
3.8.4蔥銅-硫酸法定量測定 51
第四章 結果與討論 52
4.1生物界面活性劑特性分析 52
4.1.1CTAB培養基暈圈測定 52
4.1.2表面張力測定 54
4.1.3臨界微胞濃度(CMC) 56
4.1.4乳化性與發泡性測試測試 57
4.2 EBL2、EBL3、EBL6 16S rDNA菌種鑑定 59
4.1.2EBL2、3、6序列及特性分析 63
4.1.5 EBL2、3、6之生長曲線測定 64
4.2 Pseudomonas aeruginosa EBL6之碳源最佳添加量 66
4.2.1MSB添加Glucose 66
4.2.2 MSB添加Succinate 72
4.2.3MSB添加甘油 73
4.2.4MSB添加亞油酸油脂 79
4.3雙碳源生長曲線測定 85
4.4 FTIR定性之分析 87
4.5 HPLC定量分析 89
4.6 蔥酮-硫酸法定量分析 91
第五章 結論與建議 93
5.1結論 93
5.2建議 96
參考文獻 97

1.王正雄，2002，用途廣泛、環境友善之生物界面活性劑，環檢雙月刊第44期。
2.林昇慧，陳婉伶，郭武東，2007，微生物發酵產業，第415期。第22-27頁。
3.林清安，2004，界面活性劑與紡織工業的應用，台灣區絲織工業同業公會，第47-112頁。
4.曹恒光，連大成，2001，淺談微乳液，日用化學工業，第42卷。
5.陳億全，林裕明，林松池，1999，建立高效液相層析技術以分析與純化生物界面活性劑之方法，興大工程學報，第25-33頁。
6.陳藹然，2010，解析化學清潔劑，化學專刊，第10期，第1-8頁。
7.趙承琛，1993，工業升級之特用化學品-界面活性劑，復文書局，台南。
8.趙承琛，1999，界面科學基礎，復文書局。台南。
9.蔡尚芳，2003，表面張力，科學教育月刊，第21-30頁。
10.张翠坤，常冬妹，杨洪江，2015，铜绿假单胞菌高产鼠李糖脂菌株的筛选. 生物技术通报，第31期。第177-183頁。
11.鄭捷倫，2007，利用本土菌株PseudomonasaeruginosaJ16醱酵生產鼠李醣脂之探討，碩士論文，元智大學生物科技暨生物資訊研究所。
12.Abbasi, H., Hamedi, M. M., Lotfabad, T. B., Zahiri, H. S., Sharafi, H.,Masoomi, F., . . . Noghabi, K. A. Biosurfactant-producing bacterium,Pseudomonas aeruginosa MA01 isolated from spoiled apples: Physicochemical and structural characteristics of isolated biosurfactant. Journal of Bioscience and Bioengineering, 2012, 113(2), 211-219.
13.Ahmed M. Elazzazy , T. S. A., O.A. Almaghrabi. Isolation and characterization of biosurfactant production under extreme environmental conditions by alkali-halo-thermophilic bacteria from Saudi Arabia. Saudi Journal of Biological Sciences, 2015.
14.Aparna, A., Srinikethan, G., & Smitha, H. Production and characterization of biosurfactant produced by a novel Pseudomonas sp. 2B. Colloids and Surfaces B: Biointerfaces, 2012, 95, 23-29.
15.Banat, I. M. Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: a review. Bioresource Technology, 1995, 51(1), 1-12.
16.Cameotra, S., & Makkar, R. Synthesis of biosurfactants in extreme conditions. Applied Microbiology and Biotechnology, 1998, 50(5), 520-529.
17.Cooper, D. G., & Goldenberg, B. G. Surface-active agents from two Bacillus species. Applied and environmental microbiology, 1987, 53(2), 224-229.
18.Das, M., Das, S., & Mukherjee, R. Surface active properties of the culture filtrates of a Micrococcus species grown on n-alkanes and sugars. Bioresource Technology, 1998, 63(3), 231-235.
19.Desai, J. D., & Banat, I. M. Microbial production of surfactants and their commercial potential. Microbiology and Molecular biology reviews, 1997, 61(1), 47-64.
20.Doo, M.-H., Li, H., Jang, H.-I., Song, I.-S., Chung, S.-J., & Shim, C.-K. Effect of nonylphenol ethoxylates (NPEs) on barrier functions of epithelial cell membranes: opening of tight junctions and competitive inhibition of P-gp-mediated efflux. International journal of pharmaceutics, 2005, 302(1), 145-153.
21.El-Sheshtawy, H., & Doheim, M. Selection of Pseudomonas aeruginosa for biosurfactant production and studies of its antimicrobial activity. Egyptian Journal of Petroleum, 2014, 23(1), 1-6.
22.Glover, R. E., Smith, R. R., Jones, M. V., Jackson, S. K., & Rowlands, C. C. An EPR investigation of surfactant action on bacterial membranes. FEMS microbiology letters, 1999, 177(1), 57-62.
23.Inès, M., & Dhouha, G. Lipopeptide surfactants: Production, recovery and pore forming capacity. Peptides, 2015, 71, 100-112.
24.Jorge F.B. Pereira, E. J. G., Rita Costa, Rui Vitorino, José A. Teixeira,, & João A.P. Coutinho, L. R. R. Optimization and characterization of biosurfactant production by Bacillus subtilis isolates towards microbial enhanced oil recovery applications. Fuel, 2013, 111, 259-268.
25.Kearns, D. B., & Losick, R. Swarming motility in undomesticated Bacillus subtilis. Molecular microbiology, 2003, 49(3), 581-590.
26.Lan, G., Fan, Q., Liu, Y., Chen, C., Li, G., Liu, Y., & Yin, X. Rhamnolipid production from waste cooking oil using Pseudomonas SWP-4. Biochemical Engineering Journal, 2015, 101, 44-54.
27.Lépine, F., Déziel, E., Milot, S., & Villemur, R. Liquid chromatographic/mass spectrometric detection of the 3-(3-hydroxyalkanoyloxy) alkanoic acid precursors of rhamnolipids in Pseudomonas aeruginosa cultures. Journal of Mass Spectrometry, 2002, 37(1), 41-46.
28.Lin, S. C. Biosurfactants: recent advances. Journal of Chemical Technology and Biotechnology, 1996, 66(2), 109-120.
29.Loo, T. W., & Clarke, D. M. Nonylphenol ethoxylates, but not nonylphenol, are substrates of the human multidrug resistance P-glycoprotein. Biochemical and biophysical research communications, 1998, 247(2), 478-480.
30.Marcia Nitschke , G. M. P. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresource Technology, 2006, 97, 336-341.
31.Mata-Sandoval, J. C., Karns, J., & Torrents, A. High-performance liquid chromatography method for the characterization of rhamnolipid mixtures produced by Pseudomonas aeruginosa UG2 on corn oil. Journal of Chromatography A, 1999, 864(2), 211-220.
32.Nathália Maria P. Rocha e Silva , R. D. R., Juliana M. Luna ,, & Valdemir A. Santos , L. A. S. Screening of Pseudomonas species for biosurfactant production using low-cost substrates. Biocatalysis and Agricultural Biotechnology, 2014, 3, 132-139.
33.Rahim, R., Ochsner, U. A., Olvera, C., Graninger, M., Messner, P., Lam, J. S., & Soberón‐Chávez, G. Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di‐rhamnolipid biosynthesis. Molecular microbiology, 2001, 40(3), 708-718.
34.Rakeshkumar M. Jain, Kalpana Mody, Nidhi Joshi, Avinash Mishra, & Jha, B. Production and structural characterization of biosurfactant produced by an alkaliphilic bacterium, Klebsiella sp.: Evaluation of different carbon sources. Colloids and Surfaces B: Biointerfaces, 2013, 108, 199-204.
35. Rocha e Silva, N. M. P., Rufino, R. D., Luna, J. M., Santos, V. A., & Sarubbo, L. A. Screening of Pseudomonas species for biosurfactant production using low-cost substrates. Biocatalysis and Agricultural Biotechnology, 2014, 3(2), 132-139.
36.Robin Geys, W. S. a. I. V. B. Biotechnological opportunities in biosurfactant production. Current Opinion in Biotechnology, 2014, 30, 66-72.
37.Rocha e Silva, N. M. P., Rufino, R. D., Luna, J. M., Santos, V. A., & Sarubbo, L. A. Screening of Pseudomonas species for biosurfactant production using low-cost substrates. Biocatalysis and Agricultural Biotechnology, 2014, 3(2), 132-139.
38.Sakthipriya, N., Doble, M., & Sangwai, J. S. Biosurfactant from Pseudomonas species with waxes as carbon source – Their production, modeling and properties. Journal of Industrial and Engineering Chemistry.
39.Sakthipriya, N., Doble, M., & Sangwai, J. S. Biosurfactant from Pseudomonas species with waxes as carbon source – Their production, modeling and properties. Journal of Industrial and Engineering Chemistry, 2015, 31, 100-111.
40.Silva, S. N. R. L., Farias, C. B. B., Rufino, R. D., Luna, J. M., & Sarubbo, L. A. Glycerol as substrate for the production of biosurfactant by Pseudomonas aeruginosa UCP0992. Colloids and Surfaces B: Biointerfaces, 2010, 79(1), 174-183.
41.Sira Pansiripat, O. P., Ratana Rujiravanit, Boonyarach Kitiyanan,, & Pastra Somboonthanate, S. C. Biosurfactant production by Pseudomonas aeruginosa SP4 using sequencing batch reactors: Effect of oil-to-glucose ratio. Biochemical Engineering Journal, 2010, 49, 185-191.
42.SKAKKEBÉK, N. E., MEYTS, E. R. D., JØRGENSEN, N., CARLSEN, E., PETERSEN, P. M., GIWERCMAN, A., . . . MÜLLER, J. Germ cell cancer and disorders of spermatogenesis: an environmental connection? Apmis, 1998, 106(1‐6), 3-12.
43.Soberón-Chávez, G., Lépine, F., & Déziel, E. Production of rhamnolipids by Pseudomonas aeruginosa. Applied Microbiology and Biotechnology, 2005, 68(6), 718-725.
44.Van Hamme, J. D., Singh, A., & Ward, O. P. Physiological aspects: Part 1 in a series of papers devoted to surfactants in microbiology and biotechnology. Biotechnology Advances, 2006, 24(6), 604-620.