本研究所篩選TKU004這株生物界面活性劑生產菌，經由16SrDNA序列比對及API試驗鑑定為Bacillus licheniformis 。
    B. licheniformis TKU004 生產生物界面活性劑之較適培養條件為含有1%烏賊軟骨粉、0.1% K2HPO4 及0.05% MgSO4．7H2O之50 mL液態培養基 (pH 7)充填於250 mL 通氣錐形瓶並經滅菌20分鐘後，進行接菌並於37℃搖瓶(150 rpm)培養1天。所得醱酵上清液，經酸沈澱、乙酸乙酯萃取、甲醇萃取等步驟，進行生物界面活性劑之純化，其產量為0.551 g/L。TKU004生產之生物界面活性劑能降低水的表面張力至25.42 mN/m，乳化指數(E24)為70%，而且熱穩定性高，不會受高溫影響而失活。此生物界面活性劑之pH 安定性、鹽度安定性分別為pH＞7、鹽濃度＜2 %。
    此外，2 mg/mL之生物界面活性劑對Fusarium oxysporun生長具有40%抑制效果。

The biosurfactant producing strain, Bacillus sp. TKU004, was identified further as Bacillus licheniformis by 16S rDNA gene sequencing and API（Analyitcal Profile Index）tests.
    The optimized culture condition for biosurfactant production was composed of 1% squid pen powder (SPP), 0.1% K2HPO4, 0.05% MgSO4．7H2O (pH 7),with autoclave treatment for 20 min, afterward, TKU004 was incubated in 50 mL of above liquid medium in an areation flask (250 mL) and kept shaking at 37℃ for one days. 
    The biosurfactant was extracted from the culture supernatant by acid precipitation, extraction of ethyl acetate and methanol .The yields of biosurfactant was 0.551g/L. TKU004 biosurfactant could reduce surface tension of water from 72 to 29 dyne/cm.The emulsification index against machine oil was 80%.The biosurfactant had good thermal stability and was resistant to higher temperatures. The pH stability and salinity stability were pH＞7 and salinity ＜2%, respectively. 
    Besides, 2 mg/mL of the biosurfactant had 40％ antifungal activity against mycelia growth of Fusarium oxysporun.

目錄
                                                        頁次
授權書
簽名頁
誌謝
中文摘要..................................................Ⅰ
英文摘要..................................................Ⅲ
目錄......................................................Ⅴ
圖目錄....................................................Ⅹ
表目錄..................................................ⅩⅢ

第一章	 緒論............................................1
第二章	 文獻回顧........................................3
        2.1 地衣芽孢桿菌..................................3
        2.2 界面活性劑....................................4
2.3 界面活性劑種類........................................5
            2.3.1陰離子界面活性劑.........................5
            2.3.2 陽離子界面活性劑........................6
            2.3.3 非離子界面活性劑........................6
            2.3.4 兩性離子界面活性劑......................7
2.4 臨界微胞濃度..........................................7 
        2.5 生物界面活性劑................................9
        2.6 生物界面活性劑種類............................9
        2.7 幾丁質.......................................14
第三章 材料與方法........................................15
        3.1 實驗菌株.....................................15
        3.2 實驗材料.....................................15
        3.3 實驗儀器.....................................16
        3.4 生物界面活性劑生產俊之篩選...................17
        3.5 表面張力測量.................................17
        3.6 液滴擴散法...................................18
        3.7 生物界面活性劑較適生產條件探討...............18
            3.7.1碳/氮源濃度.............................18
            3.7.2培養通氣量..............................19
            3.7.3 培養溫度...............................19
            3.7.4培養基pH值..............................19
            3.7.5 較適培養條件...........................20
        3.8生物界面活性劑之分離純化......................20
        3.9生物界面活性劑之特性分析......................20
            3.9.1熱穩定性................................20
            3.9.2鹽安定性................................21
            3.9.3 pH安定性...............................21
            3.9.4 臨界微胞濃度...........................21
            3.9.5 乳化指數...............................22
            3.9.6 起泡能力...............................23
        3.10 真菌抑制試驗................................23
第四章 結果與討論........................................24
        4.1 界面活性劑生產菌株的篩選.....................24
        4.2 Bacillus sp.TKU004種名之鑑定.................26
        4.3 生物界面活性劑較適生產條件探討...............27
            4.3.1碳/氮源濃度探討.........................27
            4.3.2 通氣量培養探討.........................29
            4.3.3 培養溫度探討...........................31
            4.3.4 培養pH值探討...........................35
            4.3.5較適培養條件探討結果....................38
        4.4 生物界面活性劑製成研究.......................41
            4.4.1 生物界面活性劑純化程序.................43
            4.4.2 生物界面活性劑結構分析.................43
        4.5 生物界面活性劑特性分析.......................49
            4.5.1 熱穩定性...............................49
            4.5.2 鹽度安定性分析.........................51
            4.5.3 pH值安定性分析.........................53
            4.5.4 臨界微胞濃度分析.......................55
            4.5.5 乳化指數分析...........................57
            4.5.6 起泡能力分析...........................64
        4.6真菌抑制測試分析..............................67
        4.7鹽類測試分析..................................72
第五章 結論..............................................74
第六章 參考文獻..........................................75


圖目錄
                                                        頁次
圖2.1 界活性劑的濃度與表面張力、界面張力
       及溶解度變化之情形.................................8
圖2.2 鼠李醣脂化學結構...................................12
圖2.3 Sophorolipid化學結構...............................12
圖2.4 表面素化學結構.....................................13
圖2.5 Licheniformin化學結構..............................13
圖3.1 乳化指數計算之示意圖...............................22
圖4.1 API菌種鑑定........................................26
圖4.2 烏賊軟骨粉濃度對TKU004生物界面活性劑生產之影響.....28
圖4.3 醱酵體積對TKU004生物界面活性劑生產之影響...........30
圖4.4 一般錐形瓶中培養溫度對
      TKU004生物界面活性劑生產影響.......................32
圖4.5 通氣錐形瓶中培養溫度對
      TKU004生物界面活性劑生產影響.......................33
圖4.6 通氣量對TKU004生物界面活性劑生產之影響.............34
圖4.7 通氣錐形瓶中培養基PH值對
      TKU004生物界面活性生產影響.........................36
圖4.8 一般錐形瓶中培養基PH值對
      TKU004生物界面活性生產影響.........................37
圖4.9 TKU004生物界面活性劑分離程序.......................42
圖4.10 TKU004生產生物界面活性劑之純化分離流程圖..........44
圖4.11 TKU004生產生物界面活性劑之1H NMR..................45
圖4.12 TKU004生產生物界面活性劑之13C NMR.................46
圖4.13 TKU004生產生物界面活性劑之質量光譜................47
圖4.14 TKU004生產生物界面活性劑推測之化學結構............48
圖4.15 鹽度對於生物界面活性劑表面張力與乳化作用之影響....52
圖4.16 pH對於生物界面活性劑表面張力與乳化作用之影響......54
圖4.17 臨界微胞濃度值....................................56
圖4.18 TKU004生物界面活性劑與不同碳氫化合物乳化穩定度....59
圖4.19 TKU004生物界面活性劑與不同碳氫化合物
       振盪乳化穩定度....................................62
圖4.20 不同界面活性劑與廢機油、n-hexane、
       礦物油的乳化能力..................................63
圖4.21 起始泡沫高度與生物界面活性劑濃度示意圖............65
圖4.22 起始泡沫高度......................................66
圖4.23 TKU004生物界面活性劑抗真菌特性....................68
圖4.24 TKU004生物界面活性劑抑制F. oxysparun..............69
圖4.25 TKU004生物界面活性劑對F. oxysparun抑制活性........70
圖4.26 不同界面活性劑對F. oxysparun抑制活性..............71
圖4.27 添加鐵離子對微生物與生物界面活性劑的影響..........73


表目錄
                                                        頁次
表2.1 微生物及其生產的界面活性劑.........................11
表4.1 生物界面活應劑生產菌株.............................25
表4.2 TKU004生物界面活性劑之較適生產條件.................39
表4.3 B. licheniformis TKU004,B. subtilis 20B and   
      B. licheniformis MS3 表面張力之比較................39
表4.4 醱酵液之pH值變化...................................40
表4.5 高壓滅菌對此生物界面活性劑一些性質之影響...........50
表4.6 微生物生產之生物界面活性劑特性比較.................60

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