臺灣博碩士論文加值系統

摘要
本研究為自台灣淡水環境中找出具有新種潛力的菌株，並進行分類與鑑定。實驗樣品分別採集自高雄市仁武區觀音湖水、台南市官田區葫蘆埤、高雄市左營區半屏山濕地、台東縣鹿野鄉空心菜田、高雄市燕巢區阿公店水庫 、 台南市佳里區台北小城社區旁魚塭 、台南市佳里區北門農工水池及屏東縣大津瀑布。從樣品中共分離出256株細菌，其中包含了53株革蘭氏陽性菌和203株革蘭氏陰性菌，經16S rRNA基因序列分析，初步鑑定發現為Acinetobacter、Acidovorax 、Aquincola、Aquitalea、Azohydromonas、Azonexus、 Brevundimonas、Caulobacter、Catellibacterium、Comamonas、Chromobacterium、Curvibacter、Delftia、Endophytic、Enterobacter、Flavobacterium 、 Flectobacillus 、 Fluviimonas、Grimontella、Hydrogenophaga、Hymenobacter、Ideonella、Leclercia、Leptothrix、Methylobacterium、Microbacterium、Novosphingobium、Paludibacterium、Pelomonas、Porphyrobacter Pseudomonas、Pseudacidovorax、Ralstonia、Rheinheimera、Rhodobacter、Rubellimicrobium、Siphonobacter、Sphaerotilus、Uliginosibacterium、Yokenella等菌屬。其中，菌株shin9-1、Thu-47、KBP-13、KBP-14、KBP-21、KBP-30、KBP-31、SLH-16、SLH-24、SLH-25及Agd-32具有成為新種細菌之潛力，本實驗選用shin9-1、KBP-21、KBP-30、KBP-31、SLH-16進行進一步的分類及鑑定。

菌株shin9-1T從台東縣鹿野鄉空心菜田所分離，屬於革蘭氏陰性桿菌，具有運動性，有鞭毛，不產孢子，有莢膜，不產色素。菌落為白色，菌落形狀呈圓形、邊緣呈規則狀。生長條件為10-45 oC (最適為30 oC)， pH 7.0-9.0 (最適為pH 7.0)及0~3.0 % NaCl (最適為0.5 %)。菌株shin9-1T之DNA G+C含量為58.27 mol%，極性脂肪為phosphatidylethanolamine、 phosphatidylglycerol、diphosphatidylglycerol、phosphatidylcholine及aminoglycolipid。，主要脂肪酸組成為C18 :1 ω7c。菌株shin9-1T經16S rRNA基因序列分析，最相近模式菌株為Rhizobium daejeonense KCTC 12121T，其菌種間相似度為97.57 %，菌株shin9-1T與已知相近模式菌株進行DNA-DNA雜合反應，結果顯示與Rhizobium daejeonense KCTC 12121T 、 Rhizobium rosettiformans W3T 及 Rhizobium selenireducens ATCC BAA-1503T的雜交相似度分別為47.65 ± 15.01 %、43.12 ± 5.45 % 及 54.78 ± 10.62 % 。經由形態、生理生化分析及遺傳特徵之鑑定，證實菌株shin9-1T為Rhizobium屬中的新種細菌，命名為Rhizobium ipomoeae，模式菌株為shin9-1T (=BCRC 80495T=KCTC 32148T = LMG27163T)。
菌株KBP-21T從高雄市左營區半屏山濕地所分離，屬於革蘭氏陰性桿菌，兼性厭氧菌，具有運動性，有鞭毛，不產孢子，無莢膜，不產色素。菌落為乳黃色，菌落形狀不規則狀、凸起、表面皺褶，菌落直徑為1.2-1.4 mm。生長條件為15-40 oC (最適為30 oC)，pH 5.0-8.0 (最適為pH 7.0)及0~1.0 % NaCl (最適為0 %)，有PHB累積能力。菌株KBP-21T DNA G+C含量為62.1 mol%，極性脂肪為phosphatidylethanolamine、phosphatidylglycerol、diphosphatidylglycerol、unknown aminophospholipid及unknown polar lipids，主要脂肪酸組成為C16:0、C18 :1 ω7c、summed feature 3 (C16:1 ω7c / C16:1 ω6c)。菌株KBP-21T經16S rRNA基因序列分析，最相近模式菌株為Paludibacterium yongneupense 5YN8-15T，其菌種間相似度為96.4%。經由形態、生理生化分析及遺傳特徵之鑑定，證實菌株KBP-21T為Paludibacterium屬中的新種細菌，命名為Paludibacterium paludies，模式菌株為KBP-21T(=BCRC 80514T=KCTC 32182T=LMG 27230T)。

菌株KBP-30T為革蘭氏陰性桿菌，好氧菌，無鞭毛但具有滑行運動性，具莢膜，不產孢子，菌落為粉紅色，菌落形狀呈不規則狀圓形，表面光滑、半透明狀、菌落直徑為2.2-3.5 mm。生長條件為10~37 oC (最適為20 oC)，pH 6.0~8.0 (最適為pH 6.0) 及0~1.0% NaCl (最適為0 %)。經16S rRNA基因序列分析，菌株KBP-30T與Hymenobacter ocellatus Myx 2105T有最高相似度97.68%。菌株KBP-30T 之DNA G+C含量為60.31 mol%，極性脂肪為phosphatidylethanolamine、unknown polar lipids、unknown aminophospholipids、unknown glycolipid及unknown aminolipid，主要多胺為sym-homospermidine (HSPD)及次要的spermidine (SPD) 和putrescin (PUT)，主要脂肪酸組成為C16:0、 summed feature 3 (C16:1 ω7c and/or C16:1 ω6c)、 C17:0 cyclo、 C18:1 ω7c、C17:0、C18:0。經由形態、生理生化分析及遺傳特徵之鑑定，證實菌株KBP-30T為Hymenobacter屬中的新種細菌，命名為Hymenobacter paludies，模式菌株為KBP-30T (=BCRC 80523T= KCTC 32237T)。

菌株KBP-31T為革蘭氏陰性桿菌，兼性厭氧菌，無鞭毛且不具有運動性，不產孢子，菌落為奶黃色，菌落凸起、表面光滑、黏質狀。生長條件為10~37 oC (最適為25 oC)，pH 6.0~9.0 (最適為pH 6.0~7.0)及0 % NaCl (最適為0 %)。經16S rRNA基因序列分析，菌株KBP-31T與Ideonella dechloratans CCUG 30898T有最高相似度98.15 %。菌株KBP-31T DNA G+C含量為67.94 mol%，極性脂肪為phosphatidylethanolamine、phosphatidylglycerol、diphosphatidylglycerol 、unknown aminophospholipid及unknown phospholipids ，主要脂肪酸組成為C16:0、summed feature 3 (C16:1 ω7c / C16:1 ω6c)。經由形態、生理生化分析及遺傳特徵之鑑定，證實菌株KBP-31T為Ideonella屬中的新種細菌，命名為Ideonella paludies，模式菌株為KBP-31T (= BCRC 80524T = KCTC 32238T )。

菌株SLH-16T為革蘭氏陰性桿菌，有鞭毛，具有運動性，有莢膜，不產孢子，菌落形狀呈圓形規則狀、邊緣規則狀、黃色菌落、表面光滑。生長條件為20-40 oC (最適40 oC)，pH 4.0~9.0 (最適為pH 5.0~6.0)及0 % NaCl (最適為0 %)。經16S rRNA基因序列分析，菌株SLH-16T與Novosphingobium taihuense T3-B9T有最高相似度97.31 %。菌株SLH-16T 之DNA G+C含量為65.18 mol%，主要脂肪酸組成為C18:1 ω7c，summed feature 3 (C16:1 ω7c and/or C16:1 ω6c)和 C16:0。經由形態、生理生化分析及遺傳特徵之鑑定，證實菌株SLH-16T為Novosphingobium屬中的新種細菌，命名為Novosphingobium piscinae，模式菌株為SLH-16T (=BCRC 80888T=KCTC 42194T=LMG 28418T)。

乳化力測試顯示KBP-21對礦物油、氯仿、Hexane和Xylene有較好的乳化力，KBP-30對礦物油有較好的乳化力，KBP-31對柴油、煤油、礦物油、氯仿、Hexane和Xylene有很好的乳化能力。

Abstract
In this study, the novel bacterial species isolated from freshwater in Taiwan were subjected to identification and classification. The freshwater samples were collected from freshwater in Guanyin lake ( Renwu District., Kaohsiung city), in hulu lake (Guantian District., Tainan city), in Banping Lake Wetland Park, (Zuoying District., Kaohsiung city), in farmland water of Ipomoea (Luye Township., Taitung city), in Agongdian Reservoir, (Yanchao District., Kaohsiung city), in a pond of National Pei-Men Senior Agricultural and Industrial Vocational School, (Jiali District, Tainan city), in a fish farm (Jiali District, Tainan city) and in Dajin Waterfall, (Sandimen Township., Pingtung city). In total 256 bacterial strains were isolated including 53 Gram-positive strains and 203 Gram-negative strains. Phylogenetic analyses based on 16S rRNA gene sequences indicates that these strains belong to bacterial genera, including Acinetobacter, Acidovorax, Aquincola, Aquitalea, Azohydromonas, Azonexus, Brevundimonas, Caulobacter, Catellibacterium, Comamonas, Chromobacterium, Curvibacter, Delftia, Endophytic, Enterobacter, Flavobacterium, Flectobacillus, Fluviimonas, Grimontella, Hydrogenophaga, Hymenobacter, Ideonella, Leclercia, Leptothrix, Methylobacterium, Microbacterium, Novosphingobium, Paludibacterium, Pelomonas, Porphyrobacter, Pseudomonas, Pseudacidovorax, Ralstonia, Rheinheimera, Rhodobacter, Rubellimicrobium, Siphonobacter, Sphaerotilus, Uliginosibacterium and Yokenella. Among them, strains shin9-1, Thu-47, KBP-13, KBP-14, KBP-21, KBP-30, KBP-31, SLH-16, SLH-24, SLH-25 and Agd-32 may is potential to be the novel species, and strains shin9-1, KBP-21, KBP-30, KBP-31 and SLH-16 were subjected to further indetification and classification.

Strain shin9-1T was isolated from the farmland water of Ipomoea in Luye Township., Taitung city. Cell of strain shin9-1T were Gram-negative, aerobic, motile by means of a polar single flagellum, non-spore forming, rods surrounded by a thick capsule and formed circular, entire white colonies. Growth occurred at 10-45 oC (optimum, 30 oC), at pH 7.0-9.0 (optimum, pH 7.0) and with 0-3.0 % NaCl (optimum, 0.5 %). The predominant fatty acid was C18 :1 ω7c. The DNA G+C content was 58.27 mol%. The mean level of DNA-DNA relatedness between the novel strain and Rhizobium daejeonense KCTC 12121T, Rhizobium rosettiformans W3T and Rhizobium selenireducens ATCC BAA-1503T was 47.65 ± 15.01 %, 43.12 ± 5.45% and 54.78 ± 10.62 %. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and aminoglycolipid. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain shin9-1T belonged to the genus Rhizobium and its closest neighbour was Rhizobium daejeonense KCTC 12121T with sequence similarities of 97.6 %. On the basis of the genotypic and phenotypic data, strain shin9-1T represents a novel species in the genus Rhizobium, for which the name Rhizobium ipomoeae sp. nov. is proposed. The type strain is shin9-1T (=BCRC 80495T=KCTC 32148T = LMG 27163T).

Strain KBP-21T was isolated from the Banping Lake Wetland Park in Zuoying District., Kaohsiung city. Strain KBP-21T was Gram negative, facultatively anaerobic, poly-β-hydroxybutyrate-accumulating, motile rods that formed yellow colonies. Growth occurred at 15-40 oC (optimum, 30 oC), with 0-2.0 % NaCl (optimum, 0 %) and at pH 5.0-8.0 (optimum, pH 7.0). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain KBP-21T belonged to the genus Paludibacterium within the family Neisseriaceae of the class Betaproteobacteria and the closest related neighbour was Paludibacterium yongneupense 5YN8-15 T with a 16S rRNA gene sequence similarity value of 96.4%. Strain KBP-21T contained summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C18 : 1ω7c as the predominant fatty acids. The major respiratory quinone was Q-8. The DNA G+C content of the genomic DNA was 62.1 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unknown aminophospholipid and several unknown phospholipids. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain KBP-21T represents a novel species in the genus Paludibacterium, for which the name Paludibacterium paludis sp. nov. is proposed. The type strain is KBP-21 T ( = BCRC 80514 T = LMG 27230 T  = KCTC 32182 T).

Strain KBP-30T was isolated from the Banping Lake Wetland Park in Taiwan. Strain KBP-30T is Gram negative, aerobic, motile by means of gliding , rods surrounded by a thick capsule and formed irregular, circular pink colonies. Growth occurred at 10-37 oC (optimum, 20 oC), with 0-1.0 % NaCl (optimum, 0 %) and at pH 6.0-8.0 (optimum, pH 6.0). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain KBP-30T belonged to the genus Hymenobacter within the family Cytophagaceae of the class Bacteroidetes and the closest related neighbour was Hymenobacter ocellatus Myx 2105T with a 16S rRNA gene sequence similarity value of 97.68%. Strain KBP-30T contained iso-C15 : 0、Summed feature 4 (C17:1 iso I / anteiso B )、anteiso-C15 : 0、iso-C17 : 0 3OH、Summed feature 3 (C16:1ω7c/ C16:1ω6c)、iso-C15 : 0 3OH、C16:1 ω5c、C16:0、iso-C17 : 0、C18:0、C14:0 anteiso、Summed feature 1 (C15:1 iso H/ C13:0 3OH)、C12:0、C15:0 2OH、iso-C16 : 1H、C17 : 0 2OH and C18:1ω9c as the predominant fatty acids. The major polyamine was sym-homospermidine. The DNA G+C content of the genomic DNA was 60.31 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, unknown polarlipid, unknown aminophospholipids, unknown glycolip and unknown amino lipid. The mean level of DNA-DNA relatedness between the novel strain and Hymenobacter ocellatus Myx 2105T was 41.84%. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain KBP-30T represents a novel species in the genus Hymenobacter, for which the name Hymenobacter paludies sp. nov. is proposed. The type strain is KBP-30T ( =BCRC 80523T= KCTC 32237T).

Strain KBP-31T is isolated from the Banping Lake Wetland Park in Taiwan. Strain KBP-31T was Gram negative, facultatively anaerobic, no motile rods that formed cream yellow colonies. Growth occurred at 10-37 oC (optimum, 25 oC), with 0 % NaCl (optimum, 0 %) and at pH 6.0-9.0 (optimum, pH 6.0-7.0). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain KBP-31T belonged to the genus Ideonella within the order Burkholderiales of the class Betaproteobacteria and the closest related neighbour was Ideonella dechloratans CCUG 30898T with a 16S rRNA gene sequence similarity value of 98.15 %. Strain KBP-31T contained C16:0, summed feature 3(C16:1ω7c / C16:1ω6c) as the predominant fatty acids. The DNA G+C content of the genomic DNA was 67.94 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, unknown aminophospholipid and unknown phospholipids. The mean level of DNA-DNA relatedness between the novel strain and Ideonella dechloratans CCUG 30898Tand Ideonella azotifigens 1a22T was 49.92 and 50.01%, respectively. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain KBP-31T represents a novel species in the genus Ideonella, for which the name Ideonella paludis sp. nov. is proposed. The type strain is KBP-31T (=BCRC 80524T = KCTC 32238T ).

Strain SLH-16T is isolated from a pond in the National Pei-Men Senior Agricultural and Industrial Vocational School, Jiali District, Tainan city. Strain SLH-16T was Gram negative, non-spore forming, motile rods surrounded by a thick capsule and formed yellow colonies. Growth occurred at 20-40 oC (optimum, 40 oC), with 0 % NaCl (optimum, 0 %) and at pH 4.0-9.0 (optimum, pH 5.0-6.0). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain SLH-16T belonged to the genus Novosphingobium within the order Sphingomonadales of the class Alphaproteobacteria and the closest related neighbour was Novosphingobium taihuense T3-B9T with a 16S rRNA gene sequence similarity value of 97.31%. Strain SLH-16T contained C18 :1 ω7c and C16:0 as the predominant fatty acids. The DNA G+C content of the genomic DNA was 65.18 mol%. The mean level of DNA-DNA relatedness between the novel strain and Novosphingobium taihuense T3-B9T is 54.64 ± 8.70 %. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain SLH-16T represents a novel species in the genus Novosphingobium, for which the name Novosphingobium piscinae sp. nov. is proposed. The type strain is SLH-16T (=BCRC 80888T=KCTC 42194T=LMG 28418T).

摘要
Abstract
目錄
圖目錄
表目錄
第壹章 前言
一、前言
二、研究目的
第貳章 文獻回顧
一、細菌分類學
(一) 簡介
(二) 新菌種的定義和標準
(三) 新菌種發表
二、Hymenobacter屬簡介
三、Ideonella屬簡介
四、Novosphingobium屬簡介
五、Paludibacterium屬簡介
六、Rhizobium屬簡介
七、生物界面活性劑介紹
(一)定義
(二)應用
第參章 材料與方法0
一、實驗架構與流程
二、菌株分離和純化
(一) 樣品來源
(二) 菌株分離及純化
三、菌種保存
四、細菌細胞蛋白質SDS-PAGE電泳圖譜分析與分群
五、遺傳特徵分析
(一) DNA分離
(二) 16S rRNA gene序列分析
(三) 演化樹建構
(四) DNA-DNA 雜合相似度分析
(五) DNA G+C mol %
六、形態學分析
(一) 菌落形態觀察
(二) 菌體形態觀察
1. 簡單染色
2. 革蘭氏染色
3. KOH反應
4. 芽孢染色 (Spore staining)
5. 莢膜染色法 (Capsule staining)
6. 運動性觀察
7. 鞭毛染色法 (Flagella staining)
七、生理生化特性
(一) 最適生長條件 (溫度、鹽度、pH值)
(二) 生長氧氣需求測定
(三) 抗生素感受性測試 (Antibiotic sensitivity test)
(四) 極性脂肪組成分析
(五) 脂肪酸組成分析
(六) 醌分析
(七) 多胺 (polyamine)分析
(八) 生化代謝
1.氧化酶試驗 (Oxidase test)
2.觸媒試驗 (Catalase test)
(九) 水解能力
1. 澱粉水解
2. 核酸水解
3. 酪蛋白水解
4. 脂肪水解
5. 幾丁質水解
6. Tweens 20、40、60、80水解
7. 纖維素水解
8. 藻酸水解
9. 卵磷脂水解
(十) BIOLOG GN II系統鑑定
(十一) API ZYM 系統之鑑定
(十二) API 20NE 系統之鑑定
(十三) API 50 CH系統鑑定
(十四) 色素分析
(十五) 乳化測試
第肆章 實驗結果
一、樣品採集
二、菌株分離純化與初步鑑定
三、菌株shin9-1之分類鑑定
(一) 菌株shin9-1之形態特徵
(二) 菌株shin9-1之生理生化特性
(三) 菌株shin9-1之遺傳特徵
(四) 菌種寄存
(五) 菌株shin9-1菌種命名
(六) 菌株shin9-1與鄰近之已知模式菌株差異性比較
四、菌株KBP-21之分類鑑定
(一) 菌株KBP-21之形態特徵
(二) 菌株KBP-21之生理生化特性
(三) 菌株KBP-21之遺傳特徵
(四) 菌種寄存
(五) 菌株KBP-21菌種命名
(六) 菌株KBP-21與鄰近之已知模式菌株差異性比較
五、菌株KBP-30之分類鑑定
(一) 菌株KBP-30之形態特徵
(二) 菌株KBP-30之生理生化特性
(三) 菌株KBP-30之遺傳特徵
(四) 菌種寄存
(五) 菌株KBP-30菌種命名
(六) 菌株KBP-30與鄰近之已知模式菌株差異性比較
六、菌株KBP-31之分類鑑定
(一) 菌株KBP-31之形態特徵
(二) 菌株KBP-31之生理生化特性
(三) 菌株KBP-31之遺傳特徵
(四) 菌種寄存
(五) 菌株KBP-31菌種命名
(六) 菌株KBP-31與鄰近之已知模式菌株差異性比較
七、菌株SLH-16之分類鑑定
(一) 菌株SLH-16之形態特徵
(二) 菌株SLH-16之生理生化特性
(三) 菌株SLH-16之遺傳特徵
(四) 菌種寄存
(五) 菌株SLH-16菌種命名
(六) 菌株SLH-16與鄰近之已知模式菌株差異性比較
八、乳化能力測試
第伍章 結論
第陸章 參考文獻

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