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Title: | 利用穩定同位素探針技術探討北台灣紅樹林土壤活性產甲烷菌群落結構 DNA stable isotope probing reveals the compositions of active methanogens in mangrove forests in northern Taiwan |
Authors: | 曾雅微 Ya-Wei Zeng |
Advisor: | 蕭友晉 Yo-Jin Shiau |
Keyword: | 溫室氣體,海岸型濕地,紅樹林,產甲烷菌,穩定同位素探測,mcrA基因, Greenhouse gas,Coastal wetland,Mangrove,Methanogen,DNA-SIP,mcrA gene, |
Publication Year : | 2023 |
Degree: | 碩士 |
Abstract: | 近年來極端氣候發生頻率增加,造成許多生態環境的問題,也使氣候變遷議題備受關注,其中造成此現象的主要原因為溫室氣體的排放。根據IPCC統計,在2019年排放最多的溫室氣體為二氧化碳(75 %),其次為甲烷(18 %)及氧化亞氮(4 %),且甲烷作為第二大溫室氣體,在100年的全球變暖潛能(GWP)為二氧化碳的26倍,可知控制甲烷排放量為減緩全球暖化的另一大重點。甲烷主要是由產甲烷菌代謝有機質所形成,又以利用碳源不同,分為氫營養型、醋酸分解型及甲基營養型產甲烷菌,這種產甲烷作用僅發生在厭氧條件下,而濕地便是高甲烷排放的來源之一(約70 %)。同時,濕地不僅是甲烷排放源,也是目前非常重要的碳匯資源,特別是高碳儲存能力的海岸型濕地-紅樹林。因此,本論文為了解甲烷排放多寡是否造成紅樹林生態系統作為碳匯的價值,以北台灣淡水河口紅樹林(八里挖仔尾、竹圍、關渡)作為研究地點,探討其產甲烷活性及土壤中產甲烷菌群組成,並針對利用碳源、現地營養鹽不同,討論其甲烷生成的速率及族群差異。本研究主要會進行土壤物理化學參數的分析以及同位素碳源添加的培養實驗,且以發展成熟的分子生物技術及DNA穩定同位素探測技術(DNA-SIP)來識別土壤中具活性的產甲烷菌,最後進行生物資訊分析及統計分析,來討論紅樹林土壤中產甲烷菌群落組成、產甲烷潛力及與環境因子之間的相關性,並探討DNA-SIP應用於本研究之可行性。研究結果發現3個樣點的環境參數沒有顯著差異,僅在竹圍樣點觀察到偏高的氧化還原電位、鹽度、鉀鈉離子及氯化物濃度,這是由於竹圍樣點的高程偏高,僅在滿潮時會被海水淹沒,使這些海水相關的離子長期積累在土壤中,造成濃度高於其他兩個樣點的現象;而我們添加不同碳源(葡萄糖、H2/CO2、甲醇)進行培養實驗發現,甲醇組具有最高的甲烷產率,為1.86-2.78 µg CH4/g soil/hr,其次是H2/CO2組,為0.05-0.38 µg CH4/g soil/hr,葡萄糖組則沒有產甲烷反應,這也表示在紅樹林土壤中產甲烷菌所利用的碳源以甲醇為主,但在關渡樣點的H2/CO2與葡萄糖組的甲烷產率略高於八里挖仔尾及竹圍,推測此樣點的產甲烷菌群可能不同。從現地的產甲烷菌群落組成來看,功能性基因(mcrA)拷貝數僅1.1×10^5 -2.2×10^5,且土壤中存在多比例的硫循環微生物,表示現地土壤中產甲烷菌偏弱勢,並與硫酸鹽還原菌為競爭關係,而產甲烷菌群主要以未知產甲烷菌群(41 %)及已知產甲烷菌群(59 %)所組成,已知產甲烷菌群又以利用多種碳源的多重營養型(Methanosarcinales)比例最多,氫營養型(Methanobacteriales及Methanomicrobiales)產甲烷菌比例略少,其中Methanolobus與Methanosarcina為3個樣點最優勢的產甲烷菌屬;而經過DNA-SIP後的活性產甲烷菌群組成完全以Methanosarcinales的Methanolobus占主導,在八里挖仔尾及竹圍樣點的Methanolobus約占98 %,關渡樣點則是具有差異,以Methanolobus、Methanosarcina及氫營養型的Methanofollis所組成,顯示紅樹林土壤中具有活性的產甲烷菌以甲基營養型(Methanolobus)為主,而關渡樣點具河口遠且有廢水流經,因此活性產甲烷菌群落組成多元,存在利用不同碳源的產甲烷菌。最後在相關性分析發現活性產甲烷菌(Methanolobus)與鹽度、海水相關參數(K+、Na+、Cl-)呈正相關,與硫酸鹽濃度呈負相關,並且Methanolobus與其他活性菌群呈負相關,表示Methanolobus利用了多數的碳源,大量生長造成其他菌群的生長受抑制,且主要影響紅樹林土壤活性產甲烷菌的環境因子為鹽度及海水中K+、Na+、Cl-濃度。本論文顯示淡水河口紅樹林土壤中產甲烷菌主要利用甲醇作為碳源,現地中存在的產甲烷菌偏少,以多重營養型產甲烷菌為主,且真正具有活性的產甲烷菌以甲基營養型(Methanolobus)為主,並在關渡樣點有較多元的菌群結構,也證實在紅樹林土壤中能與硫酸鹽還原菌競爭的產甲烷菌以甲基營養型為主,且主要影響的因子為鹽度。以此論文作為基礎,未來可以針對全台灣紅樹林的產甲烷菌群落差異或是淡水與鹹水系統之間的差異進一步了解。 In recent years, the frequency of extreme weather has increased and caused many ecological and environmental problems. It makes the issue of climate change more and more important, among which the main reason for this problem is the emission of greenhouse gases. According to IPCC statistics, the greenhouse gas emitted the most is carbon dioxide(75 %), followed by methane(18 %) and nitrous oxide(4 %) in 2019. The second largest greenhouse gas(CH4) has a global warming potential(GWP) of 26 times that of CO2 in 100 years, it shows that control CH4 emission is very important. CH4 is mainly formed by methanogens metabolize organic matter, and can be divided into hydrogenotrophic, acetoclastic and methylotrophic methanogens based on different carbon source. The effect only occurs under anaerobic conditions, and one of high methane emissions source is wetlands. Wetlands are not only a source of methane emissions, but also a important carbon sink, especially coastal wetland-mangrove(with high carbon storage capacity). Therefore, in order to know whether the methane flux cause the value of mangrove as a carbon sink. We choose Danshui Estuary mangrove(Bali-Wazihwei, Zhuwei, Guandu) of northern Taiwan to analyze their methane flux and methanogens composition, also we compare the three site’s methane flux and methanogens compositions difference based on different carbon source and environmental factors. By using molecular biotechnology and DNA stable isotope probing to identify active methanogens in mangrove soil. Finally, we discuss the composition of methanogens community, methane flux and environmental factors’s correlation, and to explore the feasibility of applying DNA-SIP in this study. The result shows that three site’s environmental factors is similar. But high ORP, salinity, potassium, sodium and chloride are observed in Zhuwei, which has high altitude. Due to the altitude so high that seawater can’t entry soil, it causes seawater factors(ex: salinity, sodium, chloride) will accumulate in the soil. Thus, we find the high concentration in Zhuwei. In our incubation experiment, we add different carbon source(glucose, H2/CO2, methanol) to observe CH4 production rate. The result shows that methanol is most highest(1.86-2.78 µg CH4/g soil/hr), H2/CO2 is about 0.05-0.38 µg CH4/g soil/hr, and glucose has no CH4 production. It means that methanogens mainly used methanol as carbon source in our mangrove soil. Also, we observe H2/CO2 and glucose product more CH4 in Guandu than Bali-Wazihwei and Zhuwei, so methanogens composition in Guandu is different. Three sites mcrA genes copies is 1.1×10^5 -2.2×10^5 and our mangrove soil exist sulfur cycle microorganisms, so we can know methanogens is relatively weak and complete with sulfate-reducing bacteria. The methanogens are mainly unknown archaea (41 %) and known methanogens (59 %), and largest proportion of Methanosarcinales utilizing multiple carbon source, small proportion is hydrogenotrophic methanogens (Methanobacteriales and Methanomicrobiales). The dominant methanogens is Methanolobus and Methanosarcina. After DNA-SIP, we find active methanogens is Methanolobus in Bali-Wazihwei and Zhuwei, about 98 %. While the dominant methanogens is Methanolobus, Methanosarcina and Methanofollis in Guandu, so we can know methylotrophic methanogens(Methanolobus) is the dominant methanogens in mangrove soil and Guandu site is far from the estuary and has wastewater so have different methanogens composition. Finally, Methanolobus is positively correlated with salinity, K+, Na+, Cl-, negatively correlated with sulfate and other active methanogens, so we can know Methanolobus use most methanol and will cause other methanogens grow. In our study, we find methanol is the main carbon source for methanogens in Danshui Estuary mangrove. The mangrove soil’s methanogens is less, and dominant methanogens is Methanolobus, which is methylotrophic methanogens. We also find different methanogens compositions in Guandu. Moreover, this study proves methylotrophic methanogens can complete with sulfate-reducing bacteria in mangrove soil, and the main factor that causes methanogens is salinity. Base on this study, we can further understand the differences in methanogen community of mangrove in Taiwan or the differences between freshwater and saltwater systems. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88742 |
DOI: | 10.6342/NTU202302662 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生物環境系統工程學系 |
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