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研究生:許復先
研究生(外文):Sheu, Fusian
論文名稱:分離自海水與淡水中新種細菌之鑑定與應用
論文名稱(外文):Identification and application of novel species bacteria isolated from freshwater and seawater
指導教授:許世宜
指導教授(外文):Sheu, Shihyi
口試委員:陳文明張淑貞
口試委員(外文):Chen, WenmingChang, Shuchen
口試日期:2011-07-27
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:海洋生物技術研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:166
中文關鍵詞:細菌分類學新種細菌溶藻微囊藻
外文關鍵詞:Bacterial taxonomynovel speciesalgicidalMicrocystis
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本研究自海水與淡水樣本中,共分離88株菌株,包含80株革蘭氏陰性菌及8株革蘭氏陽性菌;經由16S rRNA基因序列之演化分類學分析,顯示88株菌株分別為Hydrogenophaga 、 Psedomonas 、 Bacillus 、 Catellibacterium 、 Rheinheimera、Pelomonas、 Rhodoferax 、 Flectobacillus 、 Azospirillum 、 Delftia 、 Aeromonas 、 Curvibacter 、 Flavobacterium 、 Deinococcus 、 Acinetobacter、 Flectobacillus、 Acidovoraz 、Limnobacter 、 Microbacterium 、 Agrobacterium 、 Duganella 、Rubrivivax 、 Cellvibrio 、 Aquimarina 、 Stappia 、 Inhella等26屬的細菌。其中菌株SPIO-19、TNR-25與Ruye-53具有成為新種的潛力,因此將菌株做進一步的鑑定與分類,而菌株antisso-27具有溶藻潛能,故進行對藍綠藻和綠藻的溶藻能力測試以評估其藻華控制之可行性。
菌株SPIO-19T 從台灣海水所篩選,菌落呈淺黃色,為革蘭氏陰性菌,絕對好氧,桿狀,不產孢子,有單一鞭毛,因此有運動性。可生長在15-42 oC (最適生長為 30-40 °C) , pH 5.0-9.0 (最適生長為pH 8.0) 以及 0.5-9.0 % NaCl (最適生長為1.0-5.0 %)。經16S rRNA基因序列分析最相近的兩株模式菌株分別為Stappia indica LMG 24625T 以及 Stappia stellulata JCM 20692T ,序列相似度為98.0 % 及 97.9 %。主要脂肪酸為C18:1 7c (>70 %)。主要醌型為Q10,DNA G+C含量組成為64.5 mol%。極性脂肪組成分析有phosphatidylglycerol (PG) ,diphosphatidylglycerol (DPG) ,phosphatidylethanolamine (PE) ,phosphatidylmonomethylethanolamine (PMME) ,phosphatidylcholine (PC) ,未知的胺脂和未知的磷脂。菌株SPIO-19T 與兩株相近的模式菌株Stappia indica LMG 24625T 以及 Stappia stellulata JCM 20692T 。DNA-DNA核酸雜合反應結果顯示SPIO-19T與Stappia indica LMG 24625T以及Stappia stellulata JCM 20692T的DNA相似度分別為22.6 ± 2.8 % 及 38.9 ± 3.8 %。依據以上遺傳型及表現型分析結果顯示SPIO-19為Stappia菌屬,並命名為Stappia aquimarina,模式菌株為SPIO-19T (= BCRC 80170T = LMG 25532T) 。
菌株TNR-25T從台灣泉水所篩選,菌落呈黃色,為革蘭氏陰性菌,絕對好氧,桿狀,沒有運動性,不產孢子。可生長在15-40 oC (最適生長為 25 °C) , pH 6.0-10.0 (最適生長為pH 7.0) 以及 0-0.5 % NaCl (最適生長為0 %)。經16S rRNA基因序列分析,最相近模式菌株為Inhella inkyongensis IMCC1713T ,序列相似度為98.1 %。主要脂肪酸 ( >10 %) 為summed feature 3 (包含C16:1 7c 和 C16:1 6c) 、 C16:0 。而主要的羥基脂肪酸是C10:0 3-OH 和 C12:0 3-OH。主要醌型為Q8,DNA G+C含量組成為69.6 mol%。極性脂肪組成分析有phosphatidylethanolamine (PE),phosphatidylglycerol (PG), phosphatidylserine (PS),diphosphatidylglycerol (DPG) ,及幾個未知的磷脂。DNA-DNA雜合反應表示菌株TNR-25T與最相近模式菌株Inhella inkyongensis CCUG54308T的DNA相似度為35.5 ± 1.8 %。依據以上遺傳型及表現型分析結果顯示TNR-25為Inhella菌屬,並命名為Inhella fonticola,模式菌株為TNR-25T (= BCRC 80211T = LMG25721T) 。
菌株Ruye-53T從台灣養殖池所篩選,菌落呈白色,為革蘭氏陰性菌,絕對好氧,桿狀,沒有運動性,不產孢子,可累積PHB (poly-β-hydroxybutyrate)。可生長在10-37 oC (最適生長為 20-25 °C) , pH 6.0-10.0 (最適生長為pH 8.0-9.0) 以及 0-1.0 % NaCl (最適生長為0-0.1 %)。經16S rRNA基因序列分析,最相近模式菌株為Catellibacterium changlense JA139T,序列相似度為96.6 %。主要脂肪酸為summed feature 8 (包含C18:1 6c 和 C18:1 7c ﹔>70 %)。主要醌型為Q10,DNA G+C含量組成為61.2 mol%。極性脂肪組成分析有phosphatidylglycerol (PG)、 phosphatidylcholine (PC)、phosphatidylethanolamine (PE) 以及幾個未知的磷脂和未知的胺脂。依據以上遺傳型及表現型分析結果顯示Ruye-53為Catellibacterium菌屬,並命名為Catellibacterium tilapiae,模式菌株為Ruye-53T (=BCRC 80261T = KCTC 23310T) 。
為了利用生物性的方法有效的控制藻華,因此我們篩選了菌株antisso-27T,並發現它有很強的溶藻活性可以去對抗有毒的微囊藻。菌株antisso-27T是從台灣南部鹽場區域所篩選,菌落呈棕黃色。經16S rRNA基因序列分析,結果顯示菌株antisso-27T為Flavobacteriacea科,Aquimarina屬,與Aquimarina spongiae相似度為96.6 %。依據遺傳型及表現型分析,顯示antisso-27T為新種細菌,並命名為Aquimarina salinaria。本研究中溶藻活性測試結果顯示菌株antisso-27T可以利用直接接觸或間接釋放溶藻物質的方式將Cyanothece sp. SC-2 、 Synechococcus sp. DJN-01 、 Chlamydomonas raudensis L4-2 以及Chlorella pyrenoidosa FIG-14藻株溶解進而達到殺藻的效果,菌株antisso-27T亦會以間接釋放溶藻物質的方式溶解Microcystis aeruginosa MTY01。

In this study, we isolated 88 strains including 80 Gram-negative bacteria and 8 Gram-positive bacteria, collected from freshwater and seawater sample. Phylogenetic analyses based on 16S rRNA gene sequences indicated that these strains belonged to twenty-six genera, including Hydrogenophaga, Psedomonas, Bacillus, Catellibacterium, Rheinheimera, Pelomonas, Rhodoferax, Flectobacillus, Azospirillum, Delftia, Aeromonas, Curvibacter, Flavobacterium, Deinococcus, Acinetobacter, Flectobacillus, Acidovoraz, Limnobacter, Microbacterium, Agrobacterium, Duganella, Rubrivivax, Cellvibrio, Aquimarina, Stappia and Inhella. The results suggested that strains SPIO-19, TNR-25 and Ruye-53 might be the novel species, and they were subjected to further identification and classification. Strain antisso-27 exhibiting algicidal activity was subjected to algicidal activity analyses against cyanobacteria and green algae to evaluate the application of controlling algal blooms.
A light-yellow pigmented bacterial strain designated SPIO-19T was isolated from seawater in Taiwan and was characterized using the polyphasic taxonomy approach. Strain SPIO-19T was Gram-negative, obligately aerobic, rod-shaped, motile by means of one polar flagellum and non-spore forming. Growth occurred at 15-42 oC (optimum, 30-40 °C), at pH 5.0-9.0 (optimum, pH 8.0) and with 0.5-9.0 % NaCl (optimum, 1.0-5.0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain SPIO-19T belonged to the genus Stappia and its closest neighbours were Stappia indica LMG 24625T and Stappia stellulata JCM 20692T with sequence similarities of 98.0 % and 97.9 %, respectively. Major fatty acid was C18:1 7c (>70 %). The major isoprenoid quinone was Q10 and the DNA G+C content was 64.5 mol%. The polar lipid profile consisted of a mixture of phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PMME), phosphatidylcholine (PC), an uncharacterized aminolipid and an uncharacterized phospholipid. The level of DNA-DNA relatedness of strain SPIO-19T to Stappia indica LMG 24625T and to Stappia stellulata JCM 20692T was 22.6 ± 2.8 % and 38.9 ± 3.8 %, respectively. On the basis of the genotypic and phenotypic data, strain SPIO-19T represents a new species in the genus Stappia, for which the name Stappia aquimarina sp. nov. is proposed. The type strain is SPIO-19T (= BCRC 80170T = LMG 25532T).
A yellow pigmented bacterial strain designated TNR-25T was isolated from spring water in Taiwan and was characterized using the polyphasic taxonomy approach. Strain TNR-25T was Gram-negative, obligately aerobic, rod-shaped, non-motile and non-spore forming. Growth occurred at 15-40 oC (optimum, 25 °C), at pH 6.0-10.0 (optimum, pH 7.0) and with 0-0.5 % NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TNR-25T belonged to the genus Inhella and its closest neighbour was Inhella inkyongensis IMCC1713T with sequence similarity of 98.1 %. The major fatty acids (>10 %) of strain TNR-25T were summed feature 3 (comprising C16:1 7c and/or C16:1 6c) and C16:0. The major cellular hydroxy fatty acids were C10:0 3-OH and C12:0 3-OH. The isoprenoid quinone was Q8 and the DNA G+C content was 69.6 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and several uncharacterized phospholipids. The DNA-DNA relatedness between strain TNR-25T and Inhella inkyongensis CCUG54308T was about 35.5 ± 1.8 %. On the basis of the genotypic and phenotypic data, strain TNR-25T represents a new species in the genus Inhella, for which the name Inhella fonticola sp. nov. is proposed. The type strain is TNR-25T (= BCRC 80211T = LMG 25721T).
A white pigmented bacterial strain designated Ruye-53T was isolated from culture pond in Taiwan and was characterized using the polyphasic taxonomy approach. Strain Ruye-53T was Gram-negative, obligately aerobic, rod-shaped, non-motile, non-spore forming and accumulation poly-β-hydroxybutyrate accumulating. Growth occurred at 10-37 oC (optimum, 20-25 °C), at pH 6.0-10.0 (optimum, pH 8.0-9.0) and with 0-1.0 % NaCl (optimum, 0-0.1 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Ruye-53T belonged to the genus Catellibacterium and its closest neighbour was Catellibacterium changlense JA139T with sequence similarity of 96.6 %. The major fatty acid of strain Ruye-53T was summed Summed feature 8 (comprising C18:1 6c and/or C18:1 7c;>70 %). The isoprenoid quinone was Q-10 and the DNA G+C content was 61.2 mol%. The polar lipid profile consisted of a mixture of phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), several uncharacterized phospholipids and an uncharacterized aminolipid. On the basis of the genotypic and phenotypic data, strain Ruye-53T represents a new species in the genus Catellibacterium, for which the name Catellibacterium tilapiae sp. nov. is proposed. The type strain is Ruye-53T (=BCRC 80261T = KCTC 23310T).
In order to develop a biological agent for controlling the algal blooms, we isolated a bacterial strain antisso-27T that exhibited strong algicidal activity against the toxic Microcystis aeruginosa. Strain antisso-27T with brownish yellow pigmented was recently isolated from a water area of saltpan in Southern Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain antisso-27T belongs to the genus Aquimarina in the family Flavobacteriacea and its only closest neighbor is Aquimarina spongiae (96.6 %). On the basis of the genotypic and phenotypic data, strain antisso-27T represents a new species in the genus Aquimarina, for which the name Aquimarina salinaria sp. nov. is proposed. In this study, algicidal activity analyses suggested that strain antisso-27T employed direct or indirect mode of algicidal lysis to Cyanothece sp. SC-2, Synechococcus sp. DJN-01, Chlamydomonas raudensis L4-2 and Chlorella pyrenoidosa FIG-14, nevertheless it released an algicidal substance against Microcystis aeruginosa MTY01.

摘要 I
Abstract IV
誌謝 VIII
表目錄 XVII
圖目錄 XX
第壹章、緒論 1
一、 前言 1
二、 研究目的 2
第貳章、文獻回顧 3
一、 微生物的多樣性 3
(一)海水微生物的多樣性 3
(二)淡水微生物的多樣性 4
二、 細菌分類學 5
(一)分類學簡介 5
(二)新種細菌的定義 6
(三)新種細菌的發表 7
二、 Stappia菌屬簡介 7
三、 Inhella 菌屬簡介 8
四、 Catellibacterium 菌屬簡介 8
五、 Aquimarina 菌屬簡介 9
六、 溶藻細菌 10
七、 Microcystis 藻屬簡介 11
第參章、材料與方法 13
一、 實驗流程與架構 13
(一)新種菌株鑑定流程 13
(二)溶藻能力試驗流程 14
二、 菌種採集地點、分離、純化與保存 14
(一)菌種採集地點 14
(二)分離與純化 15
(三)菌種保存 17
三、 細菌細胞蛋白質SDS-PAGE電泳圖譜分析與分群 17
(一)樣品處理 17
(二)SDS-PAGE 電泳 18
(三)電泳膠片保存 20
四、 菌株形態學分析 20
(一)菌落形態特徵 20
(二)菌體形態特徵 23
1. 革蘭氏染色 24
2. 簡單染色 25
3. KOH試驗 26
4. 鞭毛染色 26
5. 聚羥基丁酯 (poly-hydroxybutyrate ) 顆粒染色法 26
6. 莢膜染色 27
7. 運動性觀察 27
8. 孢子染色 28
9. 尼羅紅 (Nile Red) 染色試驗 28
五、 菌株遺傳特徵分析 29
1. 抽DNA方法之一 29
2. 抽DNA方法之二 29
3. 16S rRNA基因聚合酶連鎖反應 30
4. 洋菜膠體電泳 31
5. DND-DNA 雜合反應 32
6. DNA G+C mol% 35
7. 演化樹的建構 36
六、 菌株生理特徵分析 37
1. 生長溫度範圍測試 37
2. 生長鹽度範圍測試 37
3. 生長pH值範圍測試 37
4. 生長氧氣需求測試 38
5. 抗生素感受性試驗 38
七、 菌株生化特徵分析 39
1. 氧化酶試驗 (Oxidase test) 39
2. 觸酶試驗 (Catalase test) 39
3. 水解能力測試 ( Hydrolysis test ) 40
4. API 20 NE 系統鑑定 42
5. API ZYM 系統鑑定 43
6. BIOLOG GN II系統鑑定 45
7. Microgen GNID A+B鑑定 46
8. 脂肪酸組成分析 48
9. 極性脂肪組成分析 49
10. 醌分析 53
11. 色素分析 54
八、 與已知的模式菌株進行比對 55
九、 溶藻細菌之溶藻活性試驗 55
第肆章、結果與討論 58
一、 樣品來源 58
二、 菌株分離純化與初步鑑定 58
三、 新種菌株SPIO-19 之特性描述 64
1. 形態特徵 64
2. 生理特徵 66
3. 生化特徵 67
4. 遺傳特徵分析 75
5. 菌株SPIO-19與Stappia屬的已知模式菌株之比較 77
6. 菌株SPIO-19新種命名 81
7. 菌株SPIO-19寄存 81
四、 新種菌株 TNR-25 之特性描述 82
1. 形態特徵 82
2. 生理特徵 83
3. 生化特徵 85
4. 遺傳特徵分析 93
5. 菌株TNR-25與Inhella屬的已知模式菌株之比較 95
6. 菌株TNR-25新種命名 98
7. 菌株TNR-25寄存 98
五、 新種菌株Ruye-53之特性描述 98
1. 形態特徵 98
2. 生理特徵 100
3. 生化特徵 103
4. 遺傳特徵分析 112
5. 菌株Ruye-53與Catellibacterium屬的已知模式菌株之比較 114
6. 菌株Ruye-53新種命名 117
7. 菌株Ruye-53寄存 118
六、 溶藻細菌antisso-27之溶藻能力分析 118
1. 溶藻細菌 antisso-27 118
2. 藻株來源 118
3. 溶藻活性試驗 119
第伍章、結論 126
一、 新種細菌之分類與鑑定 126
二、 溶藻細菌之溶藻能力試驗 128
第陸章、參考文獻 129
附錄 140


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