Characterization of hydrocarbon degrading and plant growth promoting bacteria: from systematic classification and molecular detection aspects

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

Phenotypic and genotypic characterization of heavy oil degrading bacteria

Common industrial pollution, such as electroplating discharging the waste water, leaking from storage tanks and pipeline released accidental spills, making the pollutant enter into soil and natural environment. In the past decade, bioremediation technology have been developed and improved to clean up soils polluted with hazardous chemicals. The aim of this study is to isolate heavy oil-degrading bacteria from heavy oil-contaminated soil, and to characterize the heavy oil-degrading capabilities of these isolates. In the preliminary work, fourteen bacteria were isolated from the contaminated soil, and after screening for the degradative traits, eight isolates were obtained. The 16S rRNA gene sequencing results showed that these isolates belong to Bosea thiooxidau、Brevibacillus parabrevis、Exiguobacterium aestuarii、Gordonia alkanivoran、Lysobacter concretionis、Microbacterium terrae、Pseudomonas putida and Pseudomonas stutzeri. Their physiological and biochemical properties were characterized. Surface tension assay results showed the reduction of surface tension from 68 to 45 dyne cm-1 by strain CC-SR1. By using hexadecane as the hydrophobic substance, strain CC-JG39 show the maximum hydrophobicity (96.25%). In the heavy oil-degradation experiment, strain CC-PF degraded 52.48% heavy oil, strain CC-SR1 degraded 41.79% heavy oil and strain CC-JG39 degraded 34.92% heavy oil when using the heavy oil as the sole carbon source. Biostimulation and bioaugmentation approaches were carried out in the remediation of heavy oil contaminated soil. The results showed that the heavy oil residual concentration was 8680.32 mg Kg-1soil without treatment, while the heavy oil residual concentration reduced to 5066.90 mg Kg-1soil by inoculating the consortium only, and the heavy oil residual concentration reduced to 4326.21 mg Kg-1soil by amending of consortium and inorganic salts. The maximum degradation happened in the treatment of combination of inorganic salts and rhamnolipid, which showed 2569.82 mg Kg-1soil in heavy oil residual concentration. Gene encoding for catechol dioxygenase of strain CC-SR1 was identified and blast with NCBI GenBank database, and the result showed the sequence similarity of 90% (296/326) with nahH gene in Brevibacillus parabrevis. Bacterial population in the consortium was monitored in the heavy oil containing medium by DGGE technique, which showed stable growth of strain CC-LSH2, CC-LSH6, CC-SR7 and CC-PF in the culture medium. Whereas, strain CC-LSH7 and CC-JG39 became the dominant population during the degradation process. Bacterial population was also monitored in the soil contaminated with heavy oil, and strain CC-LSH2, CC-LSH6, CC-JG39 and CC-PF presented during the first six days. Only strain CC-JG39 and CC-PF were monitored after incubation for nine days by DGGE analysis.常見的工業污染，如電鍍工廠的任意排放廢水、石化燃料儲存槽老舊所發生的洩漏事件、運輸管線的破裂等，均使污染物進入到土壤與自然環境中。過去十年來，生物技術蓬勃發展，常用來清除被污染土壤中的危險性化合物。本研究之目的為由受重油污染之土壤中，分離出具備降解能力之細菌並觀察其降解能力。本研究經由篩選後所得的八株試驗菌株。初步以16S rRNA基因序列鑑定菌種分別為 Bosea thiooxidau、Brevibacillus parabrevis、Exiguobacterium aestuarii、Gordonia alkanivoran、Lysobacter concretionis、Microbacterium terrae、Pseudomonas putida、Pseudomonas stutzeri八個菌種。經研究其生理生化測試之特性，其中菌株CC-SR1能夠有效降低表面張力自68至45 dyne cm-1，疏水指數則以菌株CC-JG39最高96.25%。降解能力方面，以重油作為唯一碳源時，菌株CC-PF降解率為52.48% 、CC-SR1降解率為41.79%、CC-JG39降解率為34.92% 。生物促進法方面，以單獨添加混合菌株、添加無機鹽類及添加界面活性劑與無機鹽類相互比較，結果得知未處理的樣品重油濃度為8680 mg Kg-1soil ；單獨添加混合菌株油品殘餘量為5067 mg Kg-1soil (降解率為41.62%) ；添加混合菌株與無機鹽類所得濃度為4326 mg Kg-1soil (降解率為50.16%)。若添加界面活性劑與混合菌株、無機鹽類搭配則可達到最佳降解效果，殘餘油品濃度為2570 mg Kg-1soil (降解率為70.39%)。catechol dioxygenase 基因片段分析方面，CC-SR7 菌株 PCR 產物經定序後與 NCBI 資料庫相比對，證實其具有組成 catechol 2,3-dioxygenase 之nahH 基因 (accession number: AB234618, 296/326= 90%) 。在40-60% 變性梯度膠體電泳 (DGGE) 監測菌相變化方面，混菌測試結果得知菌株 CC-LSH2、CC-LSH6、CC-SR7、CC-PF於混菌期間均能維持穩定生長，不受彼此抑制導致生長效果不彰的情形，而CC-LSH7、CC-JG39等菌株於降解期間也隨時間增長逐漸成為優勢族群，土壤中經由添加混合降解菌處理後，於30天試驗期中以DGGE分生監測發現第一天及第二天可明顯監測到CC-LSH2、CC-LSH6、CC-JG39與CC-PF降解菌，而第九天之後僅發現菌株CC-JG39與CC-PF之存在。目錄 中文摘要...................................................................................................I 英文摘要................................................................................................III 表次.........................................................................................................XI 圖次......................................................................................................XIII 前言...........................................................................................................1 一、研究緣起與動機..............................................................................1 二、研究目的與內容..............................................................................2 前人研究...............................................................................................3 一、 石油碳氫化合物...........................................................................3 (一) 石油簡介與油品特性................................................................3 (二) 重油............................................................................................5 (三) 生物毒性....................................................................................6 (四) 碳氫化合物污染統計................................................................7 (五) 有機污染物於環境中之宿命....................................................7 (六) 有機污染物之生物可利用性..................................................12 二、 石油分解菌................................................................................14 (一) 降解碳氫化合物之微生物種類..............................................14 (二) 生物分解污染物機制..............................................................15 三、 界面活性劑 (surfactants) .........................................................22 (一) 界面活性劑之種類..................................................................22 1. 化學合成界面活性劑 (chemical synthetic surfactants) ...........23 2. 生物界面活性劑 (biosurfactants) ............................................23 (二) 生物界面活性劑之特性與分類..............................................24 四、 土壤受碳氫化合物污染之復育技術........................................24 (一) 物化處理法...............................................................................26 (二) 生物復育法...............................................................................26 五、 影響生物降解碳氫化合物之限制因子....................................31 六、 分子生物技術於污染整治上的重要性與應用........................31 (一) 變性梯度膠體電泳 (denaturing gradient gel electrophoresis, DGGE) ....................................................................................31 (二) 螢光原位雜合技術 (fluorescence in-situ hybridization, FISH) ............................................................................36 (三) 即時定量聚合酶連鎖反應 (real-time quantitative PCR) .....37 材料與方法........................................................................................38 一、 土壤樣品來源............................................................................38 二、 重油分解菌之分離與純化........................................................38 (一) 增殖培養..................................................................................38 (二) 選擇性培養基..........................................................................44 三、 菌種保存....................................................................................44 四、 菌株之生理生化特性分析........................................................45 (一) 菌株存活試驗..........................................................................45 (二) 菌株產酸與產鹼試驗..............................................................45 (三) 菌株耐酸與耐鹼試驗..............................................................46 (四) 菌株耐鹽性試驗......................................................................46 (五) DNase Test Agar測試..............................................................46 (六) 表面張力測定..........................................................................48 (七) 疏水性指數分析 (hydrophobicity determination) ................49 (八) BTEX 毒性物質耐受性分析.................................................50 (九) 酵素反應 (API ZYM®, bioMérieux, France) ........................51 (十) 二氧化碳產量 (環檢所公告 NIEA A448.10C) ...................53 (十一) Lipase 脂肪分解酵素定量分析.........................................54 (十二) 土壤中catechol dioxygenase 活性測試.............................56 五、 菌種鑑定....................................................................................57 (一) 染色體 DNA 的製備.............................................................57 (二) 土壤 DNA 製備.....................................................................59 (三) 瓊脂醣膠體電泳......................................................................61 (四) BOX-PCR................................................................................63 (五) 16S rRNA gene PCR................................................................66 (六) Amplified 16S rDNA restriction analysis (ARDRA) ..............70 (七) 切膠回收..................................................................................72 (八) BigDye 螢光終止循環定序...................................................73 (九) 酒精沈澱..................................................................................75 六、 細菌對重油之降解試驗............................................................76 (一) 水樣中重油的檢測方法..........................................................76 (二) 土樣中重油檢測方法..............................................................81 (三) 生物優殖 (Bioaugmentation) 降解試驗...............................82 (四) 生物促進 (Biostimulation) 降解試驗...................................82 七、 catechol 1,2-dioxygenase與catechol 2,3-dioxygenase基因之偵測................................................................................................83 八、 變性梯度膠體電泳監測法........................................................85 (一) 樣品製備..................................................................................85 (二) 組合水平式電泳膠片配件......................................................87 (三) 膠片灌注..................................................................................87 (四) 聚丙烯醯膠膠體電泳..............................................................88 (五) 膠片染色、退染及照膠............................................................89 結果與討論...........................................................................................90 一、 菌株之型態................................................................................90 二、 菌株於重油培養基之生存........................................................92 三、 分離菌株之生理與生化特性....................................................94 四、 DNase test agar測試結果..........................................................95 五、 表面張力測試結果....................................................................99 六、 疏水性指數分析 (Hydrophobicity) .......................................101 七、 菌株對BTEX毒物質之耐受性..............................................103 八、 API ZYM® 酵素反應..............................................................108 九、 代謝活性之二氧化碳產量......................................................110 十、 Lipase脂肪分解酵素活性分析...............................................112 十一、 Catechol dioxygenase鄰苯二酚雙氧化酶活性分析........112 十二、 BOX-PCR 指紋圖譜........................................................113 十三、 擴增核醣體DNA限制性分析 (ARDRA) ...........................117 十四、 16S rDNA菌種鑑定..................................................117 十五、 降解試驗...........................................................................121 十六、 catechol dioxygenase 基因片段分析...............................123 十七、 變性梯度膠體電泳 (DGGE) 監測菌相變化..................127 結論.......................................................................................................132 參考文獻..............................................................................................134 附錄.......................................................................................................145 表次 表一、臺灣重大油污染事件......................................................................8 表二、油污清理方法之描述及其限制與環境影響之比較....................27 表三、分子生物技術與縮寫....................................................................32 表四、苓雅試驗土壤基本性質................................................................40 表五、 BH 無機鹽類培養液配方...........................................................43 表六、 DNase test agar 培養基配方.......................................................47 表七、 API ZYM® 套組中試驗項目與判讀依據..................................52 表八、 DNA片段於不同類型瓊脂醣膠體中分離範圍.........................62 表九a、 BOX-PCR 試劑.........................................................................65 表九b、 BOX-PCR 升降溫條件............................................................65 表十、本實驗室常用之16S rDNA增殖與定序引子..............................67 表十一a、 16S rDNA PCR 試劑............................................................69 表十一b、 16S rDNA PCR 升降溫條件................................................69 表十二a、 PCR for sequencing 試劑......................................................74 表十二b、螢光標定 PCR 升降溫條件..................................................74 表十三a、氣相層析儀使用之氣體及其流速..........................................78 表十三b、氣相層析儀中烘箱溫度及時間設定......................................78 表十四a、變性梯度膠體電泳之聚合酶連鎖反應試劑..........................86 表十四b、變性梯度膠體電泳之PCR 升降溫條件................................86 表十五、分離株之菌落外觀型態特徵....................................................91 表十六、分離株於不同酸鹼度存活之測試結果表................................96 表十七、分離株於含不同濃度氯化鈉培養基存活之測試結果表........97 表十八a、 LSH菌群之API ZYM結果................................................109 表十八b、 SR菌群之API ZYM結果...................................................109 圖次 圖一、疏水性有機污染物之推測宿命與行為模式................................10 圖二、三類型化合物的理論降解曲線....................................................11 圖三、土壤中污染物之物理行為模式圖................................................13 圖四、地下水層對可氧化有機污染物與含氯溶劑之生物復育反應....16 圖五、脂肪代謝........................................................................................17 圖六、β-oxidation....................................................................................18 圖七、多環芳香碳氫化合物之代謝途徑................................................20 圖八、兒茶酚開環之路徑........................................................................21 圖九、常見細菌產出之界面活性劑........................................................25 圖十、微生物降解重油之生物可利用性研究實驗流程圖....................39 圖十一、增殖培養方法示意圖................................................................42 圖十二、聚合酶連鎖反應示意圖............................................................68 圖十三、重油培養基中菌株生長情形....................................................93 圖十四、 分離株於 DNase test agar生長之菌落外表型......................98 圖十五a、 LSH分離株於NB培養基經不同天數之表面張力變化...100 圖十五b、 SR分離株於NB培養基經不同天數之表面張力變化......100 圖十六a、菌株疏水性指數分析............................................................102 圖十六b、分離株CC-JG39之疏水性.................................................102 圖十七a-1、 CC-LSH各菌株於 benzene 中生存趨勢圖...................104 圖十七a-2、 CC-SR各菌株於 benzene 中生存趨勢圖.....................104 圖十七b-1、 CC-LSH各菌株於 toluene 中生存趨勢圖....................105 圖十七b-2、 CC-SR各菌株於 toluene 中生存趨勢圖......................105 圖十七c-1、 CC-LSH各菌株於 ethylbenzene 中生存趨勢圖...........106 圖十七c-2、 CC-SR各菌株於 ethylbenzene 中生存趨勢圖.............106 圖十七d-1、 CC-LSH各菌株於 xylene 中生存趨勢圖.....................107 圖十七d-2、 CC-SR各菌株於 xylene 中生存趨勢圖........................107 圖十八a、 LSH菌群於30天試驗期 CO2 產量..................................111 圖十八b、 SR菌群於30天試驗期 CO2 產量.....................................111 圖十九、土壤中混合菌群lipase活性變化............................................114 圖二十、土壤中添加混和菌群之catechol dioxygenase活性變化....115 圖二十一a、 LSH菌群BOX-PCR圖譜...............................................116 圖二十一b、 SR菌群BOX-PCR圖譜..................................................116 圖二十二a、 EcoRI限制酵素ARDRA圖譜........................................118 圖二十二b、 HaeIII限制酵素ARDRA圖譜........................................118 圖二十二c、 PstI限制酵素ARDRA圖譜............................................119 圖二十二d、 BamHI限制酵素ARDRA圖譜.......................................119 圖二十三a、分離株於30天內降解水樣中重油之百分率...................122 圖二十三b、試驗土壤添加純菌30天TPH濃度圖..............................122 圖二十四、經不同處理後土壤重油殘留濃度......................................124 圖二十五、 C23O 引子增殖catechol 2,3-dioxygenase gene圖譜......125 圖二十六a、 cpC12O引子增殖catechol 1,2-dioxygenase gene圖譜..126 圖二十六b、 cpC23O引子增殖catechol 2,3-dioxygenase gene圖譜..126 圖二十七、八株測試菌株以純菌DNA經DGGE之指紋圖譜............128 圖二十八a、 LSH2、LSH6、LSH7、JG39混菌菌相變化圖..................129 圖二十八b、 SR1、SR5、SR7、PF混菌菌相變化圖..............................130 圖二十九、 DGGE分生監測生物優殖法與土壤中生物多樣性分析.13

鑑定固氮螺旋菌之引子對、套組及其鑑定方法

一種用於鑑定固氮螺旋菌屬之引子對，其係選自下列引子對及彼等之退化性引子(degenerate primer)以進行鑑定：正向引子，其具有5’-GGCCYGWTYAGTCAGRAGTG-3’之序列；反向引子，其具有5’-GCTAGACGYYGGGGTGCATGCACTT-3’之序列；並藉由添加相關試劑即可應用於微生物製劑，如生物肥料產品中固氮螺旋菌屬之快速檢測、碳氫化合物或重金屬汙染土壤生物復育可行性評估之依據

鑑定固氮螺旋菌之引子對、套組及其鑑定方法

一種用於鑑定固氮螺旋菌屬之引子對，其係選自下列引子對及彼等之退化性引子(degenerate primer)以進行鑑定：正向引子，其具有5’-GGCCYGWTYAGTCAGRAGTG-3’之序列；反向引子，其具有5’-GCTAGACGYYGGGGTGCATGCACTT-3’之序列；並藉由添加相關試劑即可應用於微生物製劑，如生物肥料產品中固氮螺旋菌屬之快速檢測、碳氫化合物或重金屬汙染土壤生物復育可行性評估之依據

Castellaniella fermenti sp. nov., isolated from a fermented meal

A polyphasic taxonomic approach was used to characterize a presumably novel bacterium, designated strain CC-YTH191T, isolated from a fermented meal in Taiwan. Cells of strain CC-YTH191T were Gram-stain-negative aerobic rods, which grew at 15-40 °C (optimal 25-30 °C), pH 6.0-9.0 (optimal 7.0) and 1-2 % (w/v) NaCl (optimal 1 %). On the basis of 16S rRNA gene sequence analysis, strain CC-YTH191T appeared to belong to the genus Castellaniella, and was closely related to Castellaniella hirudinis (96.7 % similarity), Castellaniella ginsengisoli (96.7 %) and Castellaniella caeni (96.0 %), while with other related species it shared <96.0 % similarity. The major cellular fatty acids of the isolate were C16 : 0, C17 : 0cyclo, C14 : 0 3OH/C16 : 1iso I and C18 : 1ω7c/C18 : 1ω6c. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, three unidentified phospholipids, an unidentified aminolipid and an unidentified aminophospholpid. Putrescine was the predominant polyamine followed by spermidine. The DNA G+C content was 62.2 mol% and the predominant quinone system was ubiquinone 8 (Q-8). All these features confirmed the placement of the strain CC-YTH191T as a novel species within the genus Castellaniella, for which the name Castellaniella fermenti sp. nov. is proposed. The type strain is CC-YTH191T (=BCRC 81023T=JCM 31755T)

Sphingomonas colocasiae sp. nov., isolated from taro (Colocasia esculanta)

A polyphasic approach was used to characterize an aerobic, Gram-stain-negative, rod-shaped bacterium (designed as strain CC-MHH0539T) isolated from the chopped tuber of taro (Colocasia esculanta) in Taiwan. Strain CC-MHH0539T was able to grow at 15-30 °C (optimum, 25 °C), at pH 6.0-9.0 (optimum, 7.0) and with 0-1 % (w/v) NaCl. Strain CC-MHH0539T showed highest 16S rRNA gene sequence similarity to Sphingomonas laterariae LNB2T (96.8 %), Sphingobium boeckii 469T (96.5 %), Sphingomonas faucium E62-3T (96.4 %) and Sphingosinicella vermicomposti YC7378T (96.2 %) and <96.1 % similarity to other sphingomonads. Strain CC-MHH0539T was found to cluster mainly with the clade that accommodated members of the genus Sphingomonas. The dominant cellular fatty acids were C16 : 0, C16 : 1ω5c, C14 : 0 2-OH, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, two sphingoglycolipids and two unidentified phospholipids were detected in strain CC-MHH0539T. The DNA G+C content was 69.5 mol%. The respiratory quinone system and predominant polyamine was ubiquinone 10 (Q-10) and sym-homospermidine, respectively, which is in line with Sphingomonas representatives. Based on the distinct phylogenetic, phenotypic and chemotaxonomic traits, strain CC-MHH0539T is considered to represent a novel species of the genus Sphingomonas, for which the name Sphingomonas colocasiae sp. nov. is proposed. The type strain is CC-MHH0539T (=BCRC 80933T=JCM 31229T)