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系統識別號 U0026-2608202011354300
論文名稱(中文) 建立總體基因體學方法以輔助分離與鑑定環境中硫氧化菌—以臺灣南部濕地為例
論文名稱(英文) Establishing a metagenomics-assisted approach for the isolation and identification of sulfur-oxidizing bacteria—A case study of the wetlands in Southern Taiwan
校院名稱 成功大學
系所名稱(中) 熱帶植物與微生物科學研究所
系所名稱(英) Institute of Tropical Plant Sciences and Microbiology
學年度 108
學期 2
出版年 109
研究生(中文) 陳胤丞
研究生(英文) Yin-Cheng Chen
學號 Z36071023
學位類別 碩士
語文別 中文
論文頁數 73頁
口試委員 指導教授-黃兆立
口試委員-蔣鎮宇
口試委員-黃浩仁
口試委員-江友中
口試委員-劉世慧
中文關鍵字 硫氧化菌  富集培養  多源基因體  濕地 
英文關鍵字 sulfur-oxidizing bacteria  enrichment culture  metagenomics  wetland 
學科別分類
中文摘要 畜牧業所排出的廢棄物通常伴隨嚴重惡臭,究其源頭是含硫分子,尤以硫化氫為主。,為了建立環境友善的永續產業,在廢水管理去除還原態硫化物成為人們重視的焦點。過去研究中嘗試利用硫氧化菌 (sulfur-oxidizing bacteria) 處理廢水中的硫化氫等臭味氣體,藉此代替物理化學除臭方法以降低成本。此外近年來發現沿海的溼地中硫還原反應較淡水濕地旺盛,抑制著產甲烷菌的生長以減緩溫室效應,同時生產大量的硫化氫,因此推斷該地涵養大量硫氧化菌。本研究使用限制培養基成份為策略,針對硫氧化菌,於七股、四草、茄萣、永安四處沿海濕地採集土壤作為材料,進行富集培養並分離菌株,同步以16S rRNA 擴增子觀察原始土壤與培養過程的菌相組成變動。土壤的菌相明顯以鹽度為界區分茄萣與四草、七股與永安的樣本,而代謝體預測中顯示低鹽度的茄萣地區硫還原與硫氧化較其他高鹽度的濕地旺盛,與過去對於鹽水濕地硫循環之了解相左。富集培養的菌相以及代謝型態依循著原始土壤菌相的差異可以分為茄萣型與其他濕地型。由代謝體預測推測茄萣地區所培養出之菌群傾向互利,使其富集群聚的硫氧化效應高於單一菌株。另外四草、七股與永安的富集培養中多數的菌群可能依賴著少數能夠代謝無機物的硫氧化菌維持生長,因此從中所得的單一分離株多顯現高且齊一的硫氧化能力。而本研究中所富集出的優勢分類群為 Pseudomonas、Halomonas、Marinobacter、Thioalkalimicrobium 與 Mycobacterium,多數的分類群並未被用於處理畜牧廢棄物,惟 Pseudomonas 一類可能具有廢水去硝化的應用潛力,而其他分離株可能具初級純化礦物之潛力,其去除硫化氫或除臭能力須進一步實驗證實。
英文摘要 Sulfide is a common contaminant in livestock waste. It not only causes problems for odor nuisance but leads poisoning in to human. Removing sulfide from wastewater has great importance of livestock management. As sulfur-oxidizing bacteria (SOB) utilize sulfide as energy and convert it to sulfate, the isolates, mainly from wetlands, have been applied in wastewater treatments. This study aims to establish an enrichment process to isolate local strains of SOB with monitoring by 16S-metagenomics. The soil from four coastal wetlands around Tainan, Qigu (QG), Sicao (SC), Jiading (JD), and Yongan (YA), was collected for initiating enrichment culture. In wetland microbial community, the salinity was a major determinant to distinguish the JD samples which had low salinity from the other higher salt content location. Such effect also extended to the enrichment culture. In JD sample, the sulfur-oxidizing activity of enrichment was higher than single strain, and the sample from the other locations showed an opposite pattern. The metabolism prediction indicated that bacteria in JD samples were favored to cooperate for coexistence; most bacteria from enrichment culture from the other locations may not utilize thiosulfate and were supported by a small set of SOB. Notedly, the isolates from high-salinity samples had strong and uniform sulfur-oxidizing ability. Gammaproteobacteria, including Pseudomonas, Halomonas, Marinobacter, Thioalkalimicrobium and Mycobacterium, were more likely to be enriched from these wetlands. Of them, Pseudomonas had the highest potential for denitrification in wastewater treatment,. In conclusion, this study provided the information of microbial dynamics during SOB enrichment process.
論文目次 摘要......................................I
Extended abstract........................II
致謝.....................................VI
目錄....................................VII
表目錄.................................VIII
圖目錄...................................IX
壹、緒論..................................1
一、畜牧場的臭氣的問題....................1
二、生物性除臭方法........................2
三、硫氧化菌介紹..........................2
四、化學自營硫氧化菌之代謝途徑............4
五、濕地中的硫循環與採集地介紹............5
六、DNA條碼與多源基因體學.................7
七、DADA2 擴增子演算法....................8
八、研究動機與目的........................9
貳、材料與方法...........................11
一、實驗材料.............................11
二、實驗方法.............................11
三、資料分析.............................15
參、結果.................................19
一、濕地細菌菌相的分布與多樣性...........19
二、富集培養過程與篩選分離株.............22
三、富集培養的菌相分析...................24
肆、討論.................................27
一、鹽度濃度對於濕地土壤菌相的選擇強度...27
二、濕地中的微生物的代謝預測.............27
三、維繫富集培養的不同策略...............28
四、濕地的硫氧化菌豐度很低...............29
五、潛在硫氧化菌屬.......................31
六、本研究的富集培養的偏好變形菌門.......32
七、菌株應用的可能性.....................32
伍、結論.................................34
陸、參考文獻.............................35
表附錄...................................43
圖附錄...................................50
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