臺灣博碩士論文加值系統

藍菌是一大量且分布廣泛的光合自營微生物，生存環境遍及海陸和極端環境，像是寒冷南極的湖或是溫泉，而可生存在上述環境的微生物又可稱為極端微生物。極端微生物當中的嗜熱性微生物為了生存於高溫的環境，常會發展出特異的生理特性與細胞結構，以應付極端環境，如耐熱的細胞膜、DNA修補系統、具熱穩定性的酵素系統等，在溫泉、海底熱泉、火山硫氣孔，都可以發現此微生物的生長。因具有與真核生物相當類似之光合作用系統的photosystem Ⅰ和photosystem Ⅱ，實驗借由改變生長環境條件，例如溫度、光照、酸鹼度、鹽類、氮源等變化促進生長以及影響色素蛋白的含量。
在台灣地區具有陸生及海水溫泉等多樣化的地熱溫泉，針對台灣溫泉進行嗜熱性微生物之研究，本研究所使用的菌株為實驗室前人於台灣苗栗泰安溫泉採樣取得之高溫藍菌標記為TA01，適合生長溫度在40~60℃，探討其鑑定、生理和生化以及色素蛋白的分析。實驗結果發現與嗜熱性藍菌Thermosynechococcus elongatus BP-1之16S rRNA相似度為99％，高溫藍菌TA01菌株呈現桿狀、為格蘭氏陽性細菌；強光照環境下（光照6000lux）高溫藍菌TA01生長快速但也很快達到生長巔峰；生長酸鹼值範圍為pH 5-10，最利其生長酸鹼值為 pH9；耐鹽度最高可達50mM NaCl，在1M NaCl培養下則無生長現象；以利用NaNO3作為生長氮源生長狀況最良好；螢光光譜分析高溫藍菌的藻藍蛋白，以580nm為最適合的激發光源，並可以觀測到在654nm有一高峰，即顯示高溫藍藻有大量的phycocyanin存在。初步推測本研究的高溫藍菌TA01可能成為Thermosynechococcus 菌屬中的新菌株。
未來可以針對其單一碳源的利用，進而研究固碳的效應；粗蛋白、粗脂肪分析，初步了解其組成分；藻藍素蛋白的純化，可應用在醫藥生技研發。

Cyanobacteria are large and diverse group of obligate photoautotrophs which inhabit most of Earth’s environments, even though there is a lake in the South Pole or a hot spring. The bacteria which live in extreme environments are called extremophiles, one of them are thermophiles. In order to exist in hot air environment, extremophiles develop distinctive physiological character and cell structure such as heat-resistant cell membrane, DNA repair system, heat-stable enzyme system, etc. They inhabit various hot springs, hydrothermal vents and continental solfataras. The photosynthetic apparatus of cyanobacteria is practically the same as those of higher plants, including both photosystem Ⅰ and photosystem Ⅱ. Various parameters influence generation time and protein content in different ways, a few selected parameters are presented such as temperature, light intensity, pH, salinity, nutrient availability, etc.
There are many hot springs which include variable microorganisms in Taiwan. The study is aimed to resaerch the thermophile called TA01 which is isolated from the sample of hot spring on Miaoli country and growth occurs between 40~60℃ by forefathers in our laboratory. and to analyze its species identification, physiology and biochemistry.
According to the result of 16S rDNA sequence analysis, the strain shows 99％ sequence similarity to Thermosynechococcus elongatus BP-1. The strain is a short gram-positive rod. The strain grows fast in high light intensity. The pH range for growth is 5-10, with an optimal at pH9. Growth occurs in BGⅡ medium which is contained 50mM NaCl but doesn’t occur in the presence of 1M NaCl. It utilizes NaNO3 for growing. Aanlysis of thermophile cyanobacteria of phycocyanin by photoluminescence,it has an optimal fluorescence excitation at 580 nm with emission at 654 nm. The result shows there is a lots of phycocyanin in this strain.
As a result, the strain TA01 might be a novel strain in the genus of Thermosynechococcus.
it can also be studied on utilizing carbon source to find more efficient ways of carbon fixation, analyzing crude protein and crude fat to realize its preliminary composition and purifying the protein of phycocyanin to apply to bio-research for biopharmaceutical research and development in the future.

中文摘要…………………………………………………………………i
英文摘要…………………………………………………………………ii
縮寫對照表………………………………………………………………iv
圖目錄……………………………………………………………………v
表目錄…………………………………………………………………vii
培養基…………………………………………………………………viii


第一章 緒言…………………………………………………………1
一、研究動機……………………………………………………………1
二、研究目的……………………………………………………………2

第二章 文獻回顧……………………………………………………3
一、嗜熱性微生物（thermophiles）…………………………………………4
二、藍菌（cyanobacteria）…………………………………………………5
三、光合作用的光捕捉器-藻膽體（phycobilisome，簡稱PBS）…………………7
(1)藻藍素（phycocyanin，PC）…………………………………………8
(2)藻紅素（phycoerythrin，PE）…………………………………………8
(3)異藻藍素（allophycocyanin，APC）…………………………………9
四、影響藍菌生長的條件…………………………………………………9
(1)溫度………………………………………………………………9
(2)酸鹼度（pH值）……………………………………………………10
(3)氧氣……………………………………………………………11
(4)氮源……………………………………………………………11
(5)光照強度…………………………………………………………12
(6)鹽濃度…………………………………………………………12
五、光激螢光光譜分析藻藍素……………………………………………13

第三章 材料與方法…………………………………………………14
一、實驗架構……………………………………………………………14
二、實驗材料……………………………………………………………14
1 菌種…………………………………………………………………14
2 藍菌菌株來源-泰安溫泉………………………………………………14
3 試劑…………………………………………………………………15
4 儀器設備……………………………………………………………18
三、實驗方法……………………………………………………………20
1 高溫藍菌TA01保存…………………………………………………20
2 高溫藍菌TA01菌量分析……………………………………………20
3 高溫藍菌TA01菌種遺傳分析…………………………………………20
3.1高溫藍菌TA01染色體DNA之萃取………………………………20
3.2 16S rDNA序列分析和cpc-IGS序列分析……………………………23
3.3 高溫藍菌TA01型態分析…………………………………………24
3.3.1 菌株型態觀察…………………………………………………24
3.3.2 格蘭氏染色……………………………………………………24
3.3.3 螢光顯微攝影…………………………………………………25
3.3.4 掃描式電子顯微攝影…………………………………………25
3.3.5 移動性測驗……………………………………………………26
4 高溫藍菌TA01生理生化特性分析……………………………………26
4.1 光照生長範圍測試………………………………………………27
4.2 初始生長環境酸鹼度範圍測試……………………………………27
4.3 耐鹽度測試………………………………………………………27
4.4 氮源生長範圍測試……………………………………………28
4.4.1 單一氮源對高溫藍菌TA01生長的影響………………………28
4.4.2 不同硝基氮源對高溫藍菌TA01生長的影響……………………28
4.5 氧化酶（oxidase）活性測試………………………………………29
4.6 觸酶（catalase）活性測試………………………………………29
5 儀器分析……………………………………………………………29

第四章 結果…………………………………………………………30
1高溫藍菌TA01菌量分析………………………………………………30
2高溫藍菌TA01菌種遺傳分析…………………………………………30
2.1 16S rDNA序列分析………………………………………………30
2.2 cpc-IGS序列分析…………………………………………………30
2.3 高溫藍菌TA01型態分析…………………………………………31
2.3.1 菌株型態觀察…………………………………………………31
2.3.2 格蘭氏染色……………………………………………………31
2.3.3 螢光顯微攝影…………………………………………………31
2.3.4 掃描式電子顯微攝影…………………………………………31
2.3.5 移動性測驗……………………………………………………31
3 高溫藍菌TA01生理生化特性分析……………………………………32
3.1 光照生長範圍測試………………………………………………32
3.2 酸鹼度生長範圍測試……………………………………………32
3.3 耐鹽度測試………………………………………………………33
3.4 氮源生長範圍測試………………………………………………34
3.4.1 單一氮源對高溫藍菌TA01生長的影響…………………………34
3.4.2 不同硝基氮源對高溫藍菌TA01生長的影響……………………34
3.5 氧化酶（oxidase）活性測試………………………………………35
3.6 觸酶（catalase）活性測試…………………………………………35
4 儀器分析……………………………………………………………35

第五章 討論…………………………………………………………37

參考文獻………………………………………………………………41

圖表……………………………………………………………………51

附錄……………………………………………………………………81

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