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標題: | 利用DNA穩定同位素技術探討人工濕地產甲烷菌族群與活性 Composition and Activity of Methanogens in the Constructed Wetland Soils by DNA-Stable Isotope Probing |
作者: | 黃玟婷 Wen-Ting Huang |
指導教授: | 蕭友晉 Yo-Jin Shiau |
關鍵字: | 人工濕地,甲烷,產甲烷菌,DNA 穩定同位素探測,次世代定序, constructed wetlands,methane,methanogens,DNA-stable isotope probing,next generation sequencing, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 人工濕地提供各種生態系統服務,在調節全球碳和氮循環方面具有重要意義,但它們也是甲烷 (CH4) 排放的重要來源。 由於 CH4 估計在 100 年內具有 27-30 的全球變暖潛能值,因此了解濕地生態系統中土壤理化特性如何調節排放量相當重要。
因此,在本研究中,嘗試使用基於 DNA 的穩定同位素 (DNA-SIP) 技術確定台灣三個不同年齡的人工濕地中活性產甲烷菌的組成。 在確立不同碳源對現地土壤產甲烷率,接著通過用13C甲醇對野外採集的土壤樣品進行培養實驗,並通過等密度梯度離心對重層中的mcrA基因進行次世代定序,確定濕地建成後活性產甲烷菌的變化。 結果表明,用甲醇、葡萄糖和H2/CO2培養時,潛在的CH4產甲烷率分別為7.19 mg CH4/g soil-day、2.62 mg CH4/g soil-day和0.16 mg CH4/ g soil-day,不同碳源培養實驗甲烷排放潛力分別為0.12 Tg CO2-eq/m2-year、0.04 Tg CO2-eq/m2-year、0.00 Tg CO2-eq/m2-year,甲醇組別顯著高於其他兩組碳源。 不僅如此,mcrA 基因在新鮮和培養濕地土壤中的拷貝數分別為 3.12-6.24×108 和 0.81-8.88×108 拷貝。 且在不同年齡的人工濕地中,經親緣樹分析代表OTU,主要的優勢活性產甲烷菌是Methanosarcina、Methanosarseta、Methanolinea等產甲烷菌,並且發現Acetobacterium在上述不同營養型產甲烷菌群交互作用中可能扮演重要的角色。 在環境因子與活性產甲烷菌經多元迴歸分析(MLR),結果顯示電導度、TC、TN是主要調節活性產甲烷菌群的前三個變量。在活性產甲烷菌及其他土壤微生物spearman相關性分析,顯示活性產甲烷菌與Desulfosporosinus有顯著正相關,與Thiobacillus亦有顯著正相關。不僅如此,亦發現產甲烷率可顯著調控有許多未分類的細菌,而這些細菌與mcrA基因拷貝數有顯著正相關。 本研究提供利用調控濕地中硫化物濃度對於減排溫室氣體的可能性,對於淡水人工濕地永續綠色管理做出貢獻。 Constructed wetlands provide various ecosystem services and are important in regulating the global C and N cycles, but they are also important sources for methane (CH4) emissions. Because CH4 is estimated to have a global warming potential of 27-30 over 100 years, it is important to know how the emissions are regulated by soil physiochemical properties in wetland ecosystems. Therefore, in this study, we attempted to determine the composition of active methanogens in three constructed wetlands of different ages in Taiwan using the DNA-based stable isotope (DNA-SIP) technique. After establishing the methane production rate of different carbon sources on the soil, and then cultivating soil samples collected in the field with 13C methanol, and sequencing the mcrA gene in the heavy layer by isopycnic density gradient centrifugation, we were able to determine that the wetland was built Changes in post-active methanogens. The results showed that the potential CH4 methanogenesis rates were 7.19 mg CH4/g soil-day, 2.62 mg CH4/g soil-day, and 0.16 mg CH4/g soil-day when incubated with methanol, glucose, and H2/CO2, respectively. The methane flux in the different carbon source cultivation experiment was 0.1195 Tg CO2-eq/m2-year, 0.0428 Tg CO2-eq/m2-year, and 0.0003 Tg CO2-eq/m2-year, respectively, and the methanol group was significantly higher than the other two carbon sources. Also, the copy numbers of the mcrA gene in fresh and cultivated wetland soils were 3.12-6.24×108 and 0.81-8.88×108 copies, respectively. Furthermore, the phylogenetic tree analysis represents the OTU in the constructed wetlands of different ages. The dominant active methanogens are Methanosarcina, Methanosarseta, Methanolinea. Acetobacterium may plays a vital role in interacting with the different nutritional methanogens mentioned. The multiple regression analysis (MLR) results on environmental factors and active methanogens showed that electrical conductivity(EC), total carbon(TC), and total nitrogen(TN) were the first three variables that mainly regulated active methanogens. The Spearman correlation analysis between active methanogens and other soil microorganisms showed that active methanogens significantly correlated with Desulfosporosinus and Thiobacillus. It also found that many unclassified bacteria can significantly regulate the rate of methanogenesis, and these bacteria have a significant positive correlation with the copy number of the mcrA gene. This study provides the possibility of reducing greenhouse gas emissions by regulating sulfide concentration in wetlands and contributes to the sustainable green management of freshwater constructed wetlands. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88975 |
DOI: | 10.6342/NTU202302689 |
全文授權: | 未授權 |
顯示於系所單位: | 生物環境系統工程學系 |
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