臺灣博碩士論文加值系統

本研究目的是採用抗 UV 的柴油分解混合菌群( UV12 與 UV24 )，以及單一柴油分解細菌(Ochrobactrum tritici B32)與酵母菌(Candida keelungensis sp.nov. SM22)來測試它們在 UV 及油汙染環境下胞外多醣與蠟酯的生產量及組成變化。其中 UV12 與 UV24 是將海水分別經 UV 照射 12 小時與 24 小時後，再添加 2% 柴油進行柴油分解菌之增富培養所得的混合菌株；SM22 及 B32 分別是分離自曾被油汙染過之海岸底砂與海水的酵母菌與細菌。這些混合菌株或單一菌經活化及培養後利用酒精沉澱萃取菌群或菌株所產生之胞外多醣並進行組成成分分析、物理化學特性以及油品乳化能力測試；並測試接種量、培養基組成(碳源、氮源及柴油含量)及不同培養條件(溫度、鹽度、pH 值及 UV )對胞外多醣產量、組成之影響；也利用上述列舉的方法來偵測這些菌群或菌株的蠟酯產量。培養基所含碳源及氮源是影響胞外多醣產量及乳化指數的重要因素，培養條件的改變除了使產量提高，也影響胞外多醣的組成及乳化油品的能力，這些菌群或菌株生產的胞外多醣組成成分含有碳水化合物、蛋白質、醣醛酸、還原醣及脂質。測試這些胞外多醣之溶解度時發現僅溶於極性較高的有機溶劑。在最適培養條件下培養可獲得之最大胞外多醣產量以 B32 產量最高 (16.8 mg/mL)，接著依序為 UV12 (16.1 mg/mL)、SM22 (15.6 mg/mL) 及 UV24 (14.0 mg/mL)。生物毒性測試的結果證實這些胞外多醣對豐年蝦幼體是無害的。在受柴油汙染的土壤樣品中測試這些菌群或菌株分解柴油能力的試驗時，發現在土壤環境，有光照較利於土壤中微生物生長，總石油碳氫化合物(total petroleum hydrocarbon, TPH ) 的降解效率也較高，經過 70 天的實驗期間，海水土壤樣品在光照條件下，控制組僅降解 71.9% 之 TPH，而有添加抗 UV 油降解菌之組別降解率則介於 80-95% 之間。培養基中添加柴油確實會增加微生物的蠟酯產量，柴油含量較低的時候(1-5%)；蠟酯產量隨柴油量增加而增加；當培養基所含柴油量較高的時候(10-20%)，蠟酯產量和柴油量則無相關。UV 會對大多數的微生物造成傷害，本實驗所選用之菌株對於 UV 具有耐受性，因此 UV 大幅增進胞外多醣的產量(1.1-5.2 倍)並且在一定的培養時間內產量能維持穩定，這對於未來應用於環境油汙染處理是有益的；且胞外多醣的乳化能力在不同溫度、pH 值及鹽度下能長時間維持穩定，也許未來除了應用在環境修復外還能應用在食品或是工業用途。

In this study, effect of UV and diesel oil on the production and composition of exopolysaccharides (EPS) and wax esters by the UV resistant oil degrading bacterium (Ochrobactrum tritice B32), yeast (Candida keelungensis SM22), and two microbial communities (UV12 and UV24) enriched from diesel oil were investigated. Effect of inoculum, medium composition (such as diesel oil concentration, carbon sources or nitrogen sources) and environmental factors (such as temperature, salinity, pH or UV radiation) on the yield and composition of the EPS were determined. Results showed that different carbon sources or nitrogen sources could change the composition (carbohydrate, protein, uronic acid, reducing sugar and lipid) and the emulsifying activity of EPS produced by the microorganisms. These EPSs were found to be completely soluble in distilled water but insoluble in chloroform, toluene and hexane. Maximum EPS production by UV12, UV24, SM22 and B32 were 16.1 mg/mL, 14.0 mg/mL, 15.6 mg/mL and 16.8 mg/mL, respectively.UV (A/B) radiation increased EPS production sharply (1.1-5.2 times) by these microorganisms. UV (A/B) radiation also changed composition of EPS produced by these microorganisms. These EPS were not toxic to Artemia salina nauplii.When testing their ability to remove diesel oil from the contaminated sediment, the results showed that UV radiation could increase marine diesel oil removal. Experimental groups removed 80~95% while the control removed only 71.9% TPH after 70 days inoculation.All the test microorganisms also produced wax esters. In most cases, higher concentration of oil (1-5%) produced higher concentration of wax esters (0.9-4.8 mg/mL). However, when oil concentration increased to 10-20%, wax ester production was not correlated to the oil concentration.The chemical nature and stability of the EPS suggest its potential application in bioremediation of marine environments. Their stability under diverse conditions, such as temperature, pH and salinity, also makes this biopolymer a versatile emulsifier for use in many food and pharmaceutical formulations.

目錄
壹、前言 -------------------------------------------------------------------------------- 1
貳、文獻回顧 -------------------------------------------------------------------------- 3
一、漏油污染處理 -------------------------------------------------------------- 3
二、紫外線對微生物的影響 -------------------------------------------------- 4
三、柴油的種類與性質 -------------------------------------------------------- 5
四、界面活性劑之作用機制 -------------------------------------------------- 6
五、界面活性劑種類 ----------------------------------------------------------- 7
六、生產界面活性劑的微生物 ----------------------------------------------- 8
七、微生物所產之胞外多醣 -------------------------------------------------- 9
八、影響胞外多醣產量及組成之因素 -------------------------------------- 10
九、微生物所產之蠟酯 -------------------------------------------------------- 15
參、實驗方法與材料 ----------------------------------------------------------------- 16
一、實驗藥品 -------------------------------------------------------------------- 16
二、實驗儀器 -------------------------------------------------------------------- 17
三、培養基組成 ----------------------------------------------------------------- 18
四、菌群與菌株來源 ----------------------------------------------------------- 19
五、菌群與菌株之活化與培養 ----------------------------------------------- 19
六、菌群與菌株保存 ----------------------------------------------------------- 20
七、實驗步驟與分析方法 ----------------------------------------------------- 20
1.接種量以及培養基組成對胞外多醣產量及組成之影響 --------- 20
2.培養基條件對胞外多醣產量及組成之影響 ------------------------ 21
3.胞外多醣萃取 ------------------------------------------------------------ 21
4.胞外多醣產量測定 ------------------------------------------------------ 21
5.胞外多醣乳化能力及穩定度測定 ------------------------------------ 22
6.胞外多醣蛋白質定量 --------------------------------------------------- 22
7.胞外多醣還原糖含量測定 --------------------------------------------- 22
8.胞外多醣醣醛酸含量測定 --------------------------------------------- 23
9.胞外多醣脂質含量測定 ------------------------------------------------ 24
10.胞外多醣之溶解度 ----------------------------------------------------- 24
11.胞外多醣之生物毒性測試 -------------------------------------------- 25
八、微生物分解柴油汙染土壤樣品試驗 ------------------------------------ 25
1.菌株之活化與培養 ------------------------------------------------------- 26
2.土壤處理 ------------------------------------------------------------------- 26
3.土壤環境因子 ------------------------------------------------------------- 27
4.土壤中之微生物菌數測定-平盤計數法 ------------------------------ 27
5.總石油碳氫化合物萃取及分析 ---------------------------------------- 27
九、蠟酯萃取 ---------------------------------------------------------------------- 28
十、統計分析方法 ---------------------------------------------------------------- 28
肆、結果與討論 ------------------------------------------------------------------------- 29
一、接種量對胞外多醣產量之影響 --------------------------------------------29
二、培養基添加不同濃度碳源對胞外多醣產量之影響 ------------------- 29
三、培養基添加不同濃度氮源對胞外多醣產量之影響 ------------------- 30
四、鹽度對胞外多醣產量之影響 ---------------------------------------------- 31
五、溫度對胞外多醣產量之影響 ---------------------------------------------- 32
六、pH 值對胞外多醣產量之影響 -------------------------------------------- 33
七、UV 及培養時間對胞外多醣產量之影響 ------------------------------- 34
八、柴油濃度對胞外多醣產量之影響 ---------------------------------------- 37
九、接種量對胞外多醣乳化指數之影響 ------------------------------------- 38
十、胞外多醣最佳產量及乳化能力 ------------------------------------------- 40
十一、胞外多醣對於不同油品之乳化穩定性 ------------------------------- 42
十二、溫度、鹽度及 pH 值對胞外多醣乳化柴油能力之影響 ---------- 44
1. 溫度 ------------------------------------------------------------------------- 45
2. 鹽度 ------------------------------------------------------------------------- 45
3. pH 值 --------------------------------------------------------------------- 45
十三、胞外多醣之組成 ----------------------------------------------------------- 51
十四、胞外多醣之溶解度 -------------------------------------------------------- 52
十五、胞外多醣之生物毒性測試 ----------------------------------------------- 53
十六、微生物分解柴油汙染土壤樣品試驗 ----------------------------------- 54
1.淡水土壤樣品之 TPH 降解及微生物菌數變化 --------------------- 54
2.海水土壤樣品之 TPH 降解及微生物菌數變化 --------------------- 58
十七、培養時間對胞外多醣及蠟脂產量之影響 ----------------------------- 61
十八、培養基柴油含量對蠟酯產量之影響 ----------------------------------- 63
伍、結論 ----------------------------------------------------------------------------------- 65
陸、參考文獻 ----------------------------------------------------------------------------- 66
附錄 ----------------------------------------------------------------------------------------- 75
附錄一、酚-硫酸呈色法分析之葡萄糖標準曲線 ---------------------------- 75
附錄二、蛋白質定量之 BSA 標準曲線 -------------------------------------- 75
附錄三、DNS呈色分析之葡萄糖標準曲線 ---------------------------------- 76
附錄四、Carbazole 顯色鑑定之葡糖醛酸標準曲線 ------------------------ 76
附錄五、GC /MS 分析所使用之柴油檢量線 -------------------------------- 77



表目錄
表一、柴油主要成分百分比----------------------------------------------------------- 6
表二、生物界面活性劑之微生物來源及特性-------------------------------------- 8
表三、由微生物生成之同質多醣---------------------------------------------------- 11
表四、由微生物生成之異質多醣---------------------------------------------------- 12
表五、不同微生物的胞外多醣產量------------------------------------------------- 13
表六、一些細菌胞外多醣的特性及功能------------------------------------------- 14
表七、GC/MS分析條件---------------------------------------------------------------- 28
表八、菌株之最適培養條件---------------------------------------------------------- 41
表九、最適及原始的培養條件下胞外多醣產量及乳化指數之變化---------- 42
表十、最適及原始的培養條件下胞外多醣產量及組成之變化---------------- 52
表十一、物質極性表------------------------------------------------------------------- 53
表十二、淡水土壤樣品中光照組之 TPH 降解及微生物菌數之變化------- 57
表十三、淡水土壤樣品中黑暗組之 TPH 降解及微生物菌數之變化------- 58
表十四、海水土壤樣品中光照組之 TPH 降解及微生物菌數之變化------- 61
表十五、海水土壤樣品中黑暗組之 TPH 降解及微生物菌數之變化------- 61










圖目錄
圖一、油料洩入海後之演變 ----------------------------------------------------------- 3
圖二、界面活性劑的濃度與表面張力及溶解度變化情形 ----------------------- 7
圖三、界面活性劑親水端與親油端示意圖 ----------------------------------------- 7
圖四、界面活性劑之離子型分類 ----------------------------------------------------- 8
圖五、緊密附著於菌體表面的莢膜胞外多醣 -------------------------------------- 10
圖六、不動桿菌屬細菌代謝正烷烴過程中蠟酯的產生途徑 -------------------- 15
圖七、微生物分解柴油汙染土壤樣品試驗分組示意圖 -------------------------- 26
圖八、接種量對胞外多醣產量之影響 ----------------------------------------------- 29
圖九、在培養基中添加不同濃度碳源對胞外多醣產量之影響 ----------------- 30
圖十、在培養基中添加不同濃度氮源對胞外多醣產量之影響 ----------------- 31
圖十一、培養基鹽度對胞外多醣產量之影響 -------------------------------------- 32
圖十二、不同培養溫度對胞外多醣產量之影響 ----------------------------------- 33
圖十三、培養基 pH 值對胞外多醣產量之影 -------------------------------------- 34
圖十四、UVA/B 照射及培養時間對 UV12 胞外多醣產量之影響 ------------ 35
圖十五、UVA/B 照射及培養時間對 UV24 胞外多醣產量之影響 ------------ 36
圖十六、UVA/B 照射及培養時間對 SM22 胞外多醣產量之影響 ------------ 36
圖十七、UVA/B 照射及培養時間對 B32 胞外多醣產量之影響 -------------- 37
圖十八、柴油濃度對胞外多醣產量之影響 ------------------------------------------ 38
圖十九、接種量對 UV12 胞外多醣油品乳化能力之影響 ----------------------- 39
圖二十、接種量對 UV24 胞外多醣油品乳化能力之影響 ----------------------- 39
圖二十一、接種量對 SM22 胞外多醣油品乳化能力之影響 -------------------- 40
圖二十二、接種量對 B32 胞外多醣油品乳化能力之影響 ----------------------- 40
圖二十三、於最適培養條件下所生產之胞外多醣對於不同油品的乳化能力 - 42
圖二十四、於最適培養條件下所產胞外多醣對乳化柴油之穩定性 ------------- 43
圖二十五、於最適培養條件下所產胞外多醣對乳化幫浦油之穩定性 ---------- 43
圖二十六、於最適培養條件下所產胞外多醣對乳化汽油之穩定性 ------------- 44
圖二十七、於最適培養條件下所產胞外多醣對乳化沙拉油之穩定性 ---------- 44
圖二十八、不同溫度對 UV12 胞外多醣乳化柴油能力之影響 ------------------ 46
圖三十九、不同溫度對 UV24 胞外多醣乳化柴油能力之影響 ------------------ 46
圖三十、不同溫度對 SM22 胞外多醣乳化柴油能力之影響 --------------------- 47
圖三十一、不同溫度對 B32 胞外多醣乳化柴油能力之影響 -------------------- 47
圖三十二、不同鹽度對 UV12 胞外多醣乳化柴油能力之影響 ------------------ 48
圖三十三、不同鹽度對 UV24 胞外多醣乳化柴油能力之影響 ------------------ 48
圖三十四、不同鹽度對 SM22 胞外多醣乳化柴油能力之影響 ------------------ 49
圖三十五、不同鹽度對B32胞外多醣乳化柴油能力之影響 ----------------------- 49
圖三十六、不同 pH 值對 UV12 胞外多醣乳化柴油能力之影響 -------------- 50
圖三十七、不同 pH 值對 UV24 胞外多醣乳化柴油能力之影響 -------------- 50
圖三十八、不同 pH 值對 SM22 胞外多醣乳化柴油能力之影響 -------------- 51
圖三十九、不同 pH 值對 B32 胞外多醣乳化柴油能力之影響 ---------------- 51
圖四十、胞外多醣對豐年蝦幼體之毒性測試結果 ---------------------------------- 54
圖四十一、在光照組之淡水土壤中 TPH 降解曲線 -------------------------------- 55
圖四十二、在黑暗組之淡水土壤中 TPH 降解曲線 -------------------------------- 56
圖四十三、在光照組之淡水土壤中微生物菌數變化 ------------------------------- 57
圖四十四、在黑暗組之淡水土壤中微生物菌數變化--------------------------------- 57
圖四十五、在光照組之海水土壤中 TPH 降解曲線 -------------------------------- 59
圖四十六、在黑暗組之海水土壤中 TPH 降解曲線 -------------------------------- 59
圖四十七、在光照組之海水土壤中微生物菌數變化 -------------------------------- 60
圖四十八、在黑暗組之海水土壤中微生物菌數之變化 ----------------------------- 61
圖四十九、培養時間對 UV12 胞外多醣及蠟酯產量之影響 ---------------------- 62
圖五十、培養時間對 UV24 胞外多醣及蠟酯產量之影響 ------------------------- 62
圖五十一、培養時間對 SM22 胞外多醣及蠟酯產量之影響 ---------------------- 63
圖五十二、培養時間對 B32 胞外多醣及蠟酯產量之影響 ------------------------ 63
圖五十三、在培養基添加柴油對蠟酯產量之影響 ----------------------------------- 64

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