台灣西南部關仔嶺溫泉微生物生成甲烷作用造成之同位素分化與族群結構關係

Abstract

本研究以關子嶺高溫泥泉為素材，利用地球化學與分子生物的方法，探討於關子嶺地下環境的微生物生成甲烷途徑以及微生物族群的結構是否存在異質性的分布。從關仔嶺地區的兩個不同地點(泥池與古井)所採集的沉積物，在無氧環境中，分別外加與無外加醋酸鹽類於不同溫度之下進行培養。在培養過程中定時測量甲烷、二氧化碳和醋酸鹽類的濃度，以及甲烷和二氧化碳的碳同位素值。並利用分子生物方法，分析包括 mcrA 基因和 16S rDNA 序列，以建立甲烷菌族 群種類與結構。 本研究結果顯示，不論是哪一個採樣點的沉積物培養，在不同溫度下，皆呈現醋酸鹽濃度下降並伴隨著甲烷產量的增加。然而甲烷的碳同位素分析卻發現，在不同樣本中出現了不同的趨勢。在泥池所採集的樣本，於 40 oC 到 60 oC 的培養中，甲烷的 δ13C 值隨著培養的時間增加而上升，然而在高溫的培養中，甲烷的 δ13C 值則是隨著時間呈現下降的狀況。由於使用醋酸鹽的甲烷菌所造成的碳同位素分化量通常比使用氫氣/二氧化碳和甲基類者所造成的分化量來得小，因此根據同位素的證據指出，隨著溫度上升，甲烷菌的種類逐漸由使用醋酸鹽類的甲烷菌轉變為使用氫氣和二氧化碳的甲烷菌。而取自可能來自比較深部區域之古井的樣本，在所有的培養溫度底下，甲烷的 δ13C 值皆是隨時間而下降。同位素的變化指示著，所提供的醋酸鹽類在被轉換成甲烷之前，先被分解為氫氣和二氧化碳。 mcrA 基因序列與基因庫比對，最相近的物種為使用氫氣和二氧化碳的甲烷菌種，然而， 16S rDNA 序列則與使用醋酸鹽類的甲烷菌和使用氫氣和二氧化碳的甲烷菌相近。地球化學結合分子生物的資料指示，這兩種不同的甲烷生成途徑是同時存在於關子嶺滲油氣區中較深處的區域內，而這兩個採樣點所採集到的沉積物與流體可能源自於不同深度的微生物群落。

The aim of this study was to investigate whether methanogenic pathways and community structures are heterogeneously distributed in the Kuan-Tzu-Ling high temperature seepage using geochemical and molecular approaches. Sediments collected from two sites, the mud pool and the borehole, in the Kuan-Tzu-Ling region were incubated with and without exogenous acetate at different temperatures under anoxic conditions. Concentrations of CH4, CO2 and acetate, and δ13C of methane and CO2 were monitored through time. Analyses of mcrA gene that catalyzes the final step of methanogenesis and 16S rRNA gene sequences were also carried out to explore the assemblages of methanogenic populations. Our results showed that methane yields increased along with the acetate consumption at different rates at different temperatures regardless of sediments inoculated. Analyses of carbon isotopic compositions of methane yielded different patterns for different samples. For the samples collected from a mud pool, the δ13C values of methane increased through time at 40 to 60oC. In contrast, the δ13C values decreased through time at high temperatures. Since acetoclastic methanogenesis generally fractionates carbon isotopes at a magnitude smaller than hydrogenotrophic and methylotrophic methanogenesis does, evidence based on the isotopic compositions suggests a shift from acetoclastic to hydrogenotrophic methanogensis with the increasing temperatures. For the sample collected from a borehole that potentially penetrates to a deeper region, the δ13C values of methane decreased through time at all investigated temperatures. The variation in isotopic compositions suggests that the provided acetate was decomposed into hydrogen and carbon dioxide prior to being converted into methane. Analyses of mcrA gene yielded the sequences related to those of hydrogenotrophic methanaogens. The detected 16S rDNA sequences were related to acetoclastic and hydrogenotrophic methanogens. These observations suggestthat the two methanogenic pathways may coexist in the deeper region of the Kuan-Tzu-Ling hydrocarbon seepage, and sediments and fluids collected from two sites might entrain different microbial communities at different depths.