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研究生:林詩耀
研究生(外文):Shih-Yao Lin
論文名稱:碳氫化合物降解與植物生長促進細菌之系統分類及分子生物偵測技術建立
論文名稱(外文):Characterization of hydrocarbon degrading and plant growth promoting bacteria: from systematic classification and molecular detection aspects
指導教授:楊秋忠楊秋忠引用關係
口試委員:譚鎮中簡宣裕劉瑞美沈佛亭
口試日期:2011-12-29
學位類別:博士
校院名稱:國立中興大學
系所名稱:土壤環境科學系所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:269
中文關鍵詞:根圈細菌分子生物偵測技術生物添加固氮螺旋菌屬
外文關鍵詞:rhizobacteriamolecular detectionbioaugmentationAzospirillum
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本研究針對碳氫化合物降解菌與促進植物生長之根圈細菌進行系統分類及分子生物偵測技術建立。分別自油污土壤等環境樣品中篩選出具有分解重油及芳香族碳氫化合物潛力之細菌,並探討菌株於環境中對污染物之降解因子及能力比較,研究期間共開發出多株新穎微生物資源。藉由菌種最佳化及生物添加策略,分析油污土壤中特定指標微生物族群數量、脫氫酵素 (dehydrogenase) 及脂肪分解酵素 (lipase) 以評估油污土壤之微生物活性,研究結果顯示以生物添加策略分解有機污染物時,Azospirillum picis CC-TAR-3T, Azospirillum rugosum CC-AFH-6T, Azospirillum formosense CC-Nfb-7T, Novosphingobium olei CC-TPE-1T, Sphingomonas formonsensis CC-Nfb-2T菌株可有效提昇土壤中脫氫酵素與脂肪分解酵素活性表現,分解菌株亦能於土壤中呈現穩定數量,屬於可行性之生物處理程序。
另一方面針對具有降解油污能力之PGPR進行生理生化及促進植物生長之特性研究。其中多株菌株除了具備有機污染物分解能力之外,另具備固氮、溶磷、蛋白分解、纖維素分解、載鐵物質分泌等能力,除了可應用於油污土壤之生物復育用途外,亦具有結合植生復育概念進行整合性生物復育之潛力。經由建立固氮螺旋菌屬之分子生物偵測系統,所設計之菌屬專一性引子對 (Azo494-F/Azo756-R) 可有效鑑別出演化分類地位極為相鄰的Rhodocista spp. 與Skermanella spp. 菌屬,此乃突破前人研究之限制。經由PCR-DGGE, FISH及real-time PCR等研究證實,偵測極限可達2.7 pg μL-1,相當於每克土壤可偵測6.6 × 102 CFU。此以PCR作為基礎之專一性菌屬偵測技術可應用於純系培養或土壤樣品中針對固氮螺旋菌屬之快速偵測與鑑定,且可應用於生態系統中固氮螺旋菌屬多樣性及新穎生物資源之開發研究。


In the present study, the systematic classification and molecular detection of hydrocarbon degrading and plant growth promoting rhizobacteria were established. The hydrocarbons degrading bacteria were isolated from oil contaminated samples and the degrading capability was analyzed. Dehydrogenase and lipase were used to estimate the microbial activity within oil contaminated soil. The strategy of bioaugmentation was demonstrated that isolates Azospirillum picis CC-TAR-3T, Azospirillum rugosum CC-AFH-6T, Azospirillum formosense CC-Nfb-7T, Novosphingobium olei CC-TPE-1T, and Sphingomonas formonsensis CC-Nfb-2T could increase the enzymatic activity and maintain the stable communities in biotreatmental processes. Besides, the biochemical characteristics and plant promoting ability of PGPR with oil degrading ability were studied. In addition to the abilities to degrade hydrocarbon pollutant of different strains, others have nitrogen fixating, tricalcium phosphate solubilizing, protein decompositing, cellulose decompositing and siderophore producing capabilities. These bacteria were able to be applied to oil-contaminated soil, and the phytoremediation strategy was potential to be combined to integrate the bioremediation processes. The molecular detection technique for the genus Azospirillum was established, the novel designed genus-specific primer pair (Azo494-F/Azo756-R) was successfully used to distinguish the closest related genus Rhodocista spp. and Skermanella spp. With PCR-DGGE, FISH, and real-time PCR technologies, the genus-specific primer was demonstrated with 2.7 pg μL-1 detection limits, containing 6.6 × 102 CFU per gram soil. The detection technique could be used to rapid determinate, identify and develop the novel nature bioresource in the environmental samples.

摘要 (I)
Abstract (II)
目錄 (III)
表次 (VIII)
圖次 (X)
壹、前人研究與文獻回顧 (1)
一、石油碳氫化合物之環境危害及生物降解機制 (1)
(一)有機污染物之生物毒性 (1)
(二)有機污染物於環境中之宿命 (1)
(三)有機污染物之生物可利用性 (4)
(四)污染物經生物分解之機制 (4)
二、有機污染物其降解指標與生物可利用性之影響因素 (11)
(一)有機污染物之化學組成及生物耐受性 (11)
(二)環境中微生物代謝活性及功能性酵素活性 (12)
三、促進植物生長之根圈細菌 (14)
(一)PGPR介紹 (14)
(二)藉由PGPR復育有機污染物 (15)
(三)PGPR與植生復育技術之結合 (16)
四、植生復育技術 (Phytoremediation) (18)
(一)優缺點及其進展 (18)
(二)植生復育技術與其用途 (19)
(三)污染物之生物可利用性 (22)
(四)植物吸收 (23)
五、固氮螺旋菌屬(Azospirillum spp.)之介紹 (26)
(一)發現與命名 (26)
(二)特性研究及其對植物生長所扮演的角色 (27)
(三)趨化性及根圈拓殖化機制 (28)
六、分子生物偵測技術 (31)
(一)變性梯度凝膠電泳 (32)
(二)螢光原位雜合 (35)
貳、研究大綱 (44)
論文研究流程圖 (45)
參、材料與方法 (46)
一、碳氫化合物污染降解細菌之篩選及降解因子探討 (46)
(一)菌株對環狀化合物毒性與濃度耐受性測試 (48)
(二)表面張力測定 (49)
(三)乳化指數測定 (50)
(四)親油指數分析 (50)
(五)酵素粗萃取與SDS-PAGE電泳分析 (51)
(六)Dehydrogenase脫氫酵素活性分析 (54)
(七)Lipase脂肪分解酵素活性分析 (56)
(八)微生物純系培養降解試驗 (57)
(九)環狀化合物降解基因表現研究 (59)
二、菌種鑑定與演化分類系統 (61)
(一)染色體DNA萃取 (61)
(二)16S rDNA序列增殖 (62)
(三)Agarose gel片段回收 (66)
(四)酒精沈澱與BigDye 螢光終止循環定序 (67)
(五)DNA序列校正與比對 (69)
(六)DNA序列檢索碼取得 (70)
(七)脂肪酸圖譜分析 (72)
(八)DNA/DNA hybridization雜合反應 (73)
(九)G+C mol% 分析 (76)
(十)多胺類 (polyamine) 萃取與分析 (77)
(十一)極性脂肪 (polar lipid) 萃取與分析 (79)
(十二)醌 (quinone) 萃取與分析 (81)
(十三)演化分類地位重建 (83)
(十四)演化距離與相似度計算 (83)
三、生理、生化試驗及PGPR特性研究 (84)
(一)生長溫度、鹽度及pH範圍之測定 (84)
(二)掃描式電子顯微鏡型態觀察 (84)
(三)穿透式電子顯微鏡型態觀察 (85)
(四)BIOLOG-GN2碳源利用性試驗 (87)
(五)API ZYM酵素活性試驗 (87)
(六)API 20NE基質利用與同化試驗 (88)
(七)固氮活性測定 (89)
(八)微生物溶磷能力測定 (93)
(九)吲哚乙酸 (indole-3-acetic acid, IAA) 生成量測定 (93)
(十)Decarboxylase脫羧反應測定 (96)
(十一)蛋白質分解酵素能力測定 (97)
(十二)羧甲基纖維素水解能力測定 (97)
(十三)載鐵物質 (siderophore) 生成能力測定 (98)
(十四)種子生物分析試驗 (bioassay) (100)
四、固氮螺旋菌屬 (Azospirillum) 專一性引子設計與測試 (101)
(一)標準菌株之16S rRNA基因序列檢索 (101)
(二)Azospirillum菌屬序列校正與資料庫更新 (102)
(三)DNA序列比對與特異性區域搜尋 (106)
(四)Azospirillum菌屬專一性引子設計 (106)
(五)設計引子之靈敏度測試 (107)
(六)設計引子之專一性測試 (108)
(七)菌屬專一性引子應用於土壤環境之靈敏度測試 (109)
(八)菌屬專一性引子應用於土壤環境之偵測極限試驗 (110)
五、分子生物偵測技術 (111)
(一)DGGE指紋圖譜分析 (111)
(二)Real-time PCR即時定量 (113)
(三)螢光原位雜合反應 (116)
肆、結果與討論 (118)
一、碳氫污染物降解菌篩選平台之建立 (118)
二、石油碳氫化合物降解因子分析 (124)
(一)菌株對環狀化合物毒性與濃度耐受性試驗 (124)
(二)水溶液相表面張力與乳化指數測定 (127)
(三)親油指數分析 (131)
(四)試驗菌株對有機污染物之SDS-PAGE蛋白分析 (133)
(五)微生物純系培養 - Phenanthrene降解試驗 (137)
三、生物添加對油污土壤中酵素活性及土壤參數變化 (139)
四、本土新穎性菌株生理、生化試驗特性研究 (144)
(一)Azospirillum picis CC-TAR-3T (144)
(二)Azospirillum formosense CC-Nfb-7T (146)
(三)Algoriphagus olei CC-Hsuan-617T (149)
(四)Novosphingobium soli CC-TPE-1T (151)
(五)Sphingomonas formosensis CC-Nfb-2T (153)
五、固氮螺旋菌屬之PGPR特性研究 (158)
(一)乙炔還原法之固氮活性 (158)
(二)微生物溶磷能力測定 (162)
(三)吲哚乙酸 (indole-3-acetic acid, IAA) 生成量 (164)
(四)蛋白酶、羧甲基纖維素水解酵素 (167)
(五)載鐵物質能力測定及脫羧酵素試驗 (170)
(六)種子生物試驗 (bioassay) (174)
六、固氮螺旋菌屬演化分類地位之探討 (176)
七、固氮螺旋菌屬專一性引子對設計 (184)
(一)Azospirillum菌屬序列校正與資料庫更新 (184)
(二)Azospirillum菌屬專一性引子設計與測試 (188)
八、固氮螺旋菌專一性引子於土壤樣品之偵測 (208)
(一)專一性引子應用於土壤之靈敏度試驗 (208)
(二)專一性引子應用於土壤偵之測極限探討 (209)
九、分子生物偵測技術於環境微生物之族群分析 (210)
(一)變性梯度凝膠電泳 (DGGE) (210)
(二)固氮螺旋菌數之螢光原位雜合反應 (219)
(三)Real-time PCR (qPCR) 定量分析 (225)
十、有機污染物降解菌與植物生長促進根圈菌整合特性 (228)
伍、結論 (230)
參考文獻 (233)
附錄 (258)
期刊發表 (Journal publication) (264)
專利申請 (Patent application) (265)
研討會發表 (Conference publication) (266)


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