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標題: 根瘤菌胞外多醣對柴油乳化之研究
Studies on the Bioemulsification of Diesel by the Rhizobial Exopolysaccharides
作者: 林文中
Lin, Wen-Chung
關鍵字: Bioemulsifier
出版社: 生命科學系所
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摘要: 生物界面活性劑指的是微生物在一定條件下培養時,在代謝過程中分泌具有界面活性的代謝產物。由於化學合成界面活性劑在生產和使用過程中常會嚴重污染環境及危害人類健康。因此,近年來,生物界面活性劑的研究日益增多。根瘤菌之應用早已在農業上接種豆科作物普遍被施用,但對根瘤菌大量釋放胞外分泌物之界面活性及乳化能力則尚缺乏研究。本研究目的在探討根瘤菌胞外分泌物是否有界面活性功能特性與乳化能力,測試十七株根瘤菌以篩選對柴油具有高乳化能力之菌種,分離可乳化柴油的分泌物,並藉由調整pH值 (pH4 ~ pH10) 、生長溫度 (20 ~ 40℃) 、震盪速度 (90 rpm ~ 180 rpm) 以及培養基中的碳源 (葡萄糖、蔗糖、甘露醇、乳糖、半乳糖、果糖、阿拉伯糖、麥芽糖及木糖) 、氮源 (硫酸銨、氯化銨、硝酸鈉、硝酸鉀、穀氨酸、甘氨酸、天門冬氨酸及酪蛋白胺基酸) 、維生素 (生物素、維生素 B2及維生素 C) 的含量以及種類,以增加根瘤菌胞外多醣的產量及提升對柴油乳化能力。研究結果顯示, Rhizobium sp. CC-13H 所產生之胞外分泌物對柴油乳化指數可達到57.1% ,優於其他十六株根瘤菌。前人研究顯示根瘤菌胞外多醣溶於水但不溶於乙醇,因此利用乙醇萃取法可分離出乳化柴油的物質-胞外多醣,經冷凍乾燥後可得到根瘤菌胞外多醣初步產物6.82 g L-1 ,將根瘤菌胞外多醣初步產物回溶於1000 mL 去離子水中配製濃度0.682% 純化之胞外多醣溶液進行乳化實驗,研究結果發現可產生53.3% 乳化指數,証明根瘤菌胞外多醣對柴油確實具有乳化能力。本研究利用苯酚-硫酸法測試菌株CC-13H 胞外分泌物所含物為胞外多醣,並測得濃度0.682% 根瘤菌胞外多醣溶液之胞外多醣含量為398 μg mL-1 。本研究經調整pH值pH4 ~ pH10 ,固定培養溫度30℃及震盪速度150 rpm,研究發現培養條件達pH6 、 30℃ 及150 rpm ,此根瘤菌菌株CC-13H對柴油乳化指數為59.2% ,但胞外多醣產量可由原來398 μg mL-1 提升至411 μg mL-1 。調整震盪速度90 rpm ~ 180 rpm ,固定培養溫度30℃及pH值6,研究發現培養條件達pH6 、 30℃ 及125 rpm 此根瘤菌菌株CC-13H 對柴油乳化指數可由59.2% 提升至68% ,胞外多醣產量可由原來411 μg mL-1 提升至460 μg mL-1 。本研究經調整培養基中的碳源的種類以及濃度探討增加乳化能力以及胞外多醣產量之可能性,研究發現額外添加 1.5% 甘露醇對柴油乳化指數可提升至 81% ,胞外多醣產量可提升至591 μg mL-1 。再經調整培養基中的氮源的種類及濃度後發現發現並未提高對柴油之乳化指數及胞外多糖產量。調整培養基中的維生素的種類及濃度後發現額外添加1.5% 甘露醇 、 0.1% 硝酸鈉及維生素 B2 (0.001 g L-1) 對柴油乳化指數可達到 94% ,胞外多醣產量可達到658 μg mL-1 。以此配方進進行培養,另測試對煤油乳化指數為97.8% ,此結果和已知 Bacillus subtilis 生產生物界面活性劑 surfactin 對煤油乳化指數80% , Pseudomonas aeruginosa 生產的 rhamnolipid 僅達 75% ,以及 Acinetabacter calcoaceticus 生產的 emulsan 達 74% ,比較起來相當具有競爭力。本研究之成果將可提供應用於油品污染水體之生物復育,期能提高石油碳氫化合物污染之生物復育整治效能,縮短時間與降低成本,以提昇環保微生物界面活性劑之競爭力。
Sorption and sequestration of xenobiotic compounds within the soil matrix are critical processes affecting contaminant mobility, toxicity, and persistence. Slow desorption and release from the soil matrix to the aqueous phase represents a long-term contaminant source and hinders remediation efforts. Both synthetic and biological surfactants have been shown to enhance the apparent aqueous solubility of non-polar organic compounds resulting in increased bioavailability and biodegradation. However, there are also reports, which suggest that some synthetic surfactants inhibit biodegradation. This inhibition is generally attributed to toxicity or reductions in bioavailability due to partitioning of contaminant into surfactant micelles. Some microorganisms secrete biosurfactants under certain incubation conditions. Members of genus Rhizobia (an important genus of symbiotic bacteria) are being used extensively as inoculants for legumes. In addition to the improvements in plant N nutrition, they have characteristic exo-secretions. To study the surfactant activities of the Rhizobial exo-secretions and their emulsification ability 17 different strains of Rhizobia were tested in the present study under different growth conditions. Effect of pH (pH4 ~ pH10), temperature (20 ~ 40℃), agitation (90 rpm ~ 180 rpm), carbon source (glucose, sucrose, mannose, lactose, galactose, fructose, arabinose, maltose and xylose), nitrogen source ((NH4)2SO4, NH4Cl, NaNO3, KNO3, Glycine, L-Glutamic acid, L-Asparagine and Casamino acid), and vitamins (D-biotin, Ascorbic acid and Ca-pantothenate) were tested to optimize the ideal conditions for the increased production of exopolyasscharide by Rhizobia. The biosurfactnats produced were tested for their ability to emulsify diesel oil. Among the 16 Rhizobial species tested the biosurfactant produced by Rhizobium sp. CC-13H showed a comparatively higher diesel emulsification index of 57.1% than the others. The secreted exopolysaacharide was water-soluble but insoluble in alcohol. After freeze-drying the exopolyasscharide yield was 6.82 g L-1 (0.68%) from a 66 hr. grown medium. The major finding of this study was that the tested Rhizobial strain produced an exopolysaacharide, which showed an emulsification index of 53.3% . Quantification of the compound was performed by Phenol-sulfuric acid method wherein; hydrolysis of polysaccharides in presence of 95% sulfuric acid yields monosaccharides, and upon dehydration in presence of phenol an aldehyde-carbohydrate compound was formed. This compound showed absorption maximum at 490 nm. The amount of exopolyasscharide production was 398 μg mL-1 for the tested Rhizobium sp. CC-13H strain under optimum incubation temperature, pH and shaking rate with 30℃, 6.8 and 150 rpm respectively. Regulation of shaking rate between 90 rpm and 180 rpm, at pH 6 showed adaptation to produce the bioemuslifier. This strain produced 411 μg mL-1 exopolyasscharide with 59.2% emulsification index at 150 rpm ,while 460 μg mL-1 exopolyasscharide with 68% emulsification index was observed at 125 rpm. Different carbon and nitrogen source showed direct influence on the exopolyasscharide production. Among the tested carbon and nitrogen sources, the strain CC-13H produced a maximum of 591 μg mL-1 exopolyasscharide with emulsification index of 81% when the YEM media was supplemented with 1.5% mannitol and 592 μg mL-1 exopolyasscharide with 80% emulsification index for diesel oil when YEM was supplemented 1.5% mannitol and 0.1% NaNO3. Vitamins also showed to regulate the exopolyasscharide production. The strain CC-13H produced 658 μg mL-1 exopolysaacharide with 94% emulsification index when YEM media was supplemented with 1.5% mannitol, 0.1% NaNO3 and 0.001 g L-1 riboflavin. This component showed an emulsification index of 97.8% when tested against kerosene. Compared to Surfactin of Bacillus subtilis, with 80%, Rhamnolipid of Pseudomonas aeruginosa with 75% and Emulsan of Acinetabacter calcoaceticus with 74% emulsion index for kerosene, the present compound showed a higher emulsion index (97.8%) . From the results Rhizobia sp. CC-13H strain can be employed for the industrial scale biosurfactant production along with its use in bioremediation of oil polluted sites.
其他識別: U0005-2607200600180500
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