臺灣博碩士論文加值系統

台灣西南部泥質溫泉中常富含有機碳、硫酸根、甲烷及氫氣等化學成分，可提供許多微生物族群進行多樣化的代謝活動。關子嶺地區噴出的泥泉溫度相對西南部其它泥火山水溫高，水溫可達 80℃ 左右，適合做為研究嗜熱菌與陸域熱泉生態系統的題材，本研究即是運用微生物富化培養方法，探討關子嶺泥泉中實際存在的微生物代謝，並透過分離泥泉中的微生物，及其生理測試，了解泥泉中各種微生物在碳循環中所扮演的角色，討論各種微生物之間競爭或互利的關係。
本研究根據關子嶺泥泉的水化學及氣體化學分析資料，設計泥泉中可能存在的各種代謝反應之培養基。使用的培養基種類包括厭氧條件下使用醋酸、甲醇、甲胺、氫氣與二氧化碳等四種甲烷菌培養基、使用葡萄糖的硫代硫酸鹽還原菌培養基、發酵菌培養基，及好氧條件下異營菌和甲烷消耗菌等培養基。確認使用特定代謝微生物能夠適應培養基後，再進行純菌分離的工作，以得到單一菌株，並測試生理特性。
上述八類培養基均可富化出使用該代謝反應的微生物群落，但並非所有培養溫度均可見微生物群落的持續生長，厭氧條件下使用醋酸鹽、甲醇、氫氣和二氧化碳的產甲烷菌、使用葡萄糖之硫代硫酸鹽還原菌、發酵菌、好氧異營菌在50℃以上培養中可被富化，其中大部分的代謝在 80℃ 仍能被富化而持續進行，只有使用甲胺之產甲烷菌及好氧甲烷消耗菌僅在50℃以下的培養中被富化而生長。研究證實關子嶺泥泉中微生物具有多樣化的代謝方式，加上前人研究可富化培養之
使用乳酸鹽之硫酸鹽還原菌共為九種代謝反應，與前人之代謝通量計算和分子生物分析結果一致。利用富化培養結果顯示關子嶺泥泉中微生物參與環境中的有機碳降解和碳循環，彼此之間產生互相競爭或是互利的關係。
由好氧異營菌培養基中，純化出二株嗜熱好氧異營性細菌，編號分別為KTL01-S-121106-I1 與 KTL01-S-121106-I2。I1 菌株與Meiothermus ruber strain SPS242 之 16S rDNA 序列的相似度達 99%，可生長在酸鹼值 5~9 之間，可生長溫度範圍在 20~70℃ 之間，最適合生長溫度則為 60℃，其最適生長條件與關子嶺泥泉的地化條件相符； I2 菌株與 Rhodobacter vinaykumarii JA123 之16S rDNA序列的相似度達 95%，但此菌株並非光合作用菌，該菌株生長在酸鹼值 6~9 之間，可生長的溫度範圍在 20~60℃ 之間，最適合生長溫度則為 50℃，為一新菌種。

Organic carbon, sulfate, methane, and hydrogen are abundant in mud springs,southwestern Taiwan. These compounds can provide the microbial community to carry on diverse metabolisms. The temperature of the Kuantzuling mud spring could be as high as 80℃, thus the mud spring may offer a great opportunity to study thermophilic microorganisms and terrestrial hot spring ecosystems. This study performed a set of enrichment experiments, stain isolations and physiological tests to understand the interaction among different microorganisms.
According to the aqueous and gas chemistry data of the Kuantzuling mud spring,we designed eight types of cultural media to enrich different kinds of microbial metabolism in the slurry. They are 6 types of anaerobic media for methanogens using acetate, methanol, methylamine or hydrogen and carbon dioxide to produce methane, thiosulfate reducers using glucose, fermenters and 2 types of aerobic media for heterotrophs and methanotrophs. Once the microorganisms could adapt to the certain types of media, pure stains were tend to be isolated and tested their physiological characteristics.
Microorganisms can be enriched in all types of media, but not in all cultivated temperatures. Methanogens using acetate, methanol or hydrogen and carbon dioxide ,
thiosulfate reducter, fermenter, aerobic heterotroph could be enriched at temperatures higher than 50℃. Mostly of them can also be enriched at 80℃. Methanogen using
methylamine and methanotroph can only be enriched at temperature of lower than 50 ℃. Total nine types of microbial metabolisms, including sulfate reducer using lactate confirmed by previous studies, can be enriched in the Kuantzuling mud spring. This result is consistent with previous energetic evaluation of microbial metabolisms and
molecular analysis of the slurry. It also inferred that the microorganisms exhibited a great diversity of metabolic pathways and were either competitive or beneficial to each
others while processing organic carbon degradation or carbon cycling.
Strain KTL01-S-121106-I1 and KTL01-S-121106-I2 were isolated from the Kuantzuling mud spring with heterotrophy media. The 16S rDNA gene sequence of strain I1 exhibited a very close affiliation (at a similarity of 99%) with Meiothermus ruber strain SPS242. It can grow at the pH between 5 and 9, in the temperature ranging from 20 to 70℃ with the optimal growth temperature at 60℃. The 16S rDNA
gene sequence of strain I2 was close to that of Rhodobacter vinaykumarii JA123 at the similarity of 95%, but stain I2 is not phototroph and probably is a new species. Strain
I2 can grow at the pH between 6 and 9, in the temperature ranging from 20 to 60℃ with the optimal growth temperature at 50℃.

目錄
國立臺灣大學碩士學位論文 ................................................................................... i
口試委員會審定書 ................................................................................................... i
致謝 .......................................................................................................................... ii
摘要 ......................................................................................................................... iii
Abstract......................................................................................................................v
目錄 ........................................................................................................................ vii
圖目錄 .......................................................................................................................x
表目錄 ..................................................................................................................... xi
附表目錄 ................................................................................................................ xii
第一章 緒論 .......................................................................................................1
1-1 前言 .......................................................1
1-1-1 極端環境微生物研究的重要性 ............................1
1-1-2 嗜熱微生物研究的重要性 ................................2
1-1-3 泥火山微生物生態系統研究的重要性 ......................3
1-1-4 泥火山之微生物相關研究 ................................4
1-2 研究動機 ...................................................5
1-2-1 研究微生物多樣性與及代謝能力重要性 ....................5
1-2-2 研究熱泉微生物多樣性的方法 ............................5
1-2-3 分離新菌株的重要性 ....................................7
1-3 研究背景 ...................................................8
1-3-1 台灣泥火山之地質背景介紹 ..............................8
1-3-2 台灣泥火山之地質微生物研究 ...........................10
1-3-3 關子嶺泥泉之地球化學特徵與地質微生物研究 .............10
1-4 研究目的 ..................................................11
第二章 研究方法 ...................................................................................................12
2-1 採樣 ......................................................12
2-1-1 採樣地點 .............................................12
2-1-2 採樣準備與培養基設計 .................................13
2-1-3 採樣方式 .............................................15
2-2 富化培養 ..................................................15
2-2-1 特定代謝富化培養 .....................................15
2-2-2 使用儀器簡介 ..........................................16
2-2-3 生長觀察及代謝反應物產物分析 .........................17
2-3 菌株分離與純化 ............................................18
2-4 16S RDNA 鑑種 ..............................................20
2-5 菌株生理測試 ..............................................22
2-5-1 生長曲線與細胞增代時間計算 ...........................22
2-5-2 最適溫度 .............................................26
2-5-3 最適酸鹼度 ...........................................26
2-5-4 最適鹽度 .............................................26
2-5-5 單一碳源使用測試 .....................................27
2-5-6 厭氧發酵測試 .........................................27
2-5-7 抗生素抗性測試 .......................................27
2-5-8 革蘭氏染色 ...........................................28
2-5-9 穿透式電子顯微鏡負染色觀察 ...........................28
2-5-10 掃描式電子顯微鏡觀察 ................................28
2-5-11 菌株中、長期保存 ....................................29
第三章 結果 ...........................................................................................................30
3-1 代謝富化培養結果 ..........................................30
3-2 菌株分離與純化結果 ........................................31
3-2-1 菌株分離與純化 .......................................31
3-2-2 16S RDNA 序列分析 .....................................31
3-2-3 細菌形態觀察 .........................................31
3-3 菌株之生理特性測試結果 ....................................33
3-3-1 生長溫度範圍測試 .....................................33
3-3-2 生長酸鹼度範圍測試 ...................................36
3-3-3 鹽度生長範圍測試 .....................................39
3-3-4 單一碳源利用測試 .....................................41
3-3-5 厭氧發酵測試 .........................................44
3-3-6 抗生素抗性測試 .......................................44
3-3-7 革蘭氏染色 ...........................................44
3-3-8 穿透式電子顯微鏡負染色觀察 ............................45
3-3-9 掃描式電子顯微鏡觀察 .................................47
第四章 討論 ...........................................................................................................48
4-1 微生物代謝多樣性 ..........................................48
4-2 微生物所參與的有機碳降解 ..................................55
4-3 KTL01-S-121106-I1 菌株特性討論和與其它同屬菌種之比較 ......57
4-4 KTL01-S-121106-I2 菌株特性討論和與其它接近菌種之比較 ......62
第五章 結論 ...........................................................................................................64
參考文獻 .................................................................................................................65
附表 .........................................................................................................................70

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