實驗控制前驅物與溫度於無氧沈積物中微生物來源甲烷之動力學與同位素分化

Abstract

在地下環境中，微生物催化之甲烷合成作用是有機物礦化作用的終端代謝反應，而於反應路徑最常使用之前驅物包括了氫氣（與二氧化碳）、醋酸、甲酸、甲基類化合物（如甲醇與甲胺），皆可由複雜的有機碳經過發酵作用產生。雖然過去已有許多研究針對單一甲烷菌進行甲烷合成機制上的探討，但是對於自然環境下的甲烷菌族群，其所使用的主要前驅物為何仍然尚未明瞭。除了甲烷合成的前驅物種類之外，另一個影響甲烷合成的關鍵因素為溫度的效應。這樣溫度的效應，在海洋沈積物中可能特別顯著，主要原因在於當地的地溫梯度控制於深度增加時增溫的現象，一般而言微生物甲烷合成的溫度範圍在現地溫度至90℃之間，則與大部份成岩作用的溫度區間是相符的。 為了瞭解不同的前驅物，在不同的溫度底下如何影響微生物催化甲烷的形成，本研究利用關子嶺溫泉區的沈積物，於六個溫度（25、40、50、60、70、80℃）進行孵育試驗。為了刺激甲烷合成作用，同時抑制上游的發酵作用產生適合甲烷合成作用所需之前驅物，於孵育試驗中將沈積物與基礎鹽類溶液的混合物，分別添加五種前驅物包括醋酸、甲酸、甲醇、甲胺、氫氣（與二氧化碳），於無氧條件下，觀測甲烷的產生與前驅物的消耗，並量測部份樣本中的甲烷碳同位素比值，進而推衍於現地環境中，微生物的甲烷合成作用對甲烷總儲量的貢獻。 本研究分析結果顯示所有的前驅物皆會刺激甲烷合成，然而每種前驅物在不同的溫度仍產生相異的合成速率。在加入氫氣（與二氧化碳）以及甲酸的實驗中，所有的溫度皆有快速的甲烷合成速率。而於加入醋酸的實驗，最大的甲烷合成速率發生於40~60℃，加入甲醇的實驗則在40~50℃有最大的甲烷合成速率，加入甲胺的實驗，其最大甲烷合成速率落在50℃。甲烷碳同位素值則隨時間的改變分別有增加、減少，以及持平的趨勢，顯示有特定的優勢甲烷合成途徑發生，亦有多重甲烷合成的途徑共存於試驗中。碳同位素的分化係數（ε）範圍落在-3.9至-115.0‰之間，其中加入醋酸的甲烷合成試驗有最小的分化係數（-11.9至-3.9‰）。加入醋酸的試驗所得到的碳同位素結果相當於控制組所得到的結果，顯示使用醋酸的甲烷菌較其他代謝途徑而言，在關子嶺地區更占優勢。結合了野外觀察後，最終推斷利用醋酸的甲烷合成作用可能對於關子領地區的總甲烷儲量提供了重要的貢獻。

Methanogenesis is the terminal metabolism during mineralization of organic carbon in subsurface environments. The precursors of methanogenesis include hydrogen (carbon dioxide), acetate, formate, and methyl-group compounds (e.g. methanol and methylamine), all of which could be derived from fermentation of complex organic carbon. Although lots of studies have been investigating the mechanisms responsible for methanogenesis by pure cultures, it still remains obscure with regard to which precursors are predominantly utilized by methanogens in natural settings. Despite the precursors for methanogenesis, one of the other critical factors governing the methane production would be temperature. This is especially true for marine sediments within which the temperature increases with depth in accordance with the local geothermal. Commonly observed temperatures for methanogenesis span from ambient temperature to 90℃, a temperature range for most diagenetic reactions. In order to address how different precursors would be activated for microbially catalytic methane formation upon different temperatures, we incubated the sediments collected from Kuan-Tzu-Ling hot spring at six different temperatures (25, 40, 50, 60, 70, 80℃). Five precursors including acetate, formate, methanol, methylamine, and hydrogen were added with the inocula to stimulate methanogenesis and inhibit the fermentation, and were monitored together with methane production through time. Results indicated that although the presence of all precursors stimulated methanogenesis, each precursor yielded various rates at different temperatures. In the experiment supplied with hydrogen (plus carbon dioxide) and formate, methanogenic rates were rapid at all temperatures. Maximum methane production rates occurred at 40~60℃ for incubations with acetate, and 40~50℃ for those with methanol and 50℃ for those with methylamine. The δ13C values of methane varied either toward greater values, less values or remained invariant through time, suggesting either a predominant methanogenic pathway or complex interactions of multiple pathways occurred during the incubations. The ε values for carbon isotope fractionation ranged from -3.9 to -115.0‰ with acetoclastic methanogenesis possessing the least negative values (-11.9 to -3.9‰). The isotopic patterns observed in incubations amended with acetate were comparable with those in positive controls, suggesting that acetoclastic methanogenesis was predominant over the other pathways in the Kuan-Tzu-Ling area. This when combined with the field observations lead to the conclusive interpretation that acetoclastic methanogenesis would constitute an essential proportion to the total methane inventory in the Kuan-Tzu-Ling area. Identification of microbial end member signature would appear to be pivotal for natural gas exploration in the future.