台灣東部雷公火泥火山噴泥中微生物產甲烷作用與鹽度及溫度變化之關係

Abstract

陸域泥火山被認為是甲烷排放的最重要的天然來源之一，前人研究顯示在表層陸域泥火山的甲烷循環來自生物與微生物的交互作用，在現地環境中的甲烷產生量可能遠超過地下深部熱裂解由泥火山釋出的甲烷，因而增加了甲烷的排放。由於噴泥在地表堆積之後，地表不同程度的蒸發作用使得氯離子濃度提高，有利於較耐鹽或嗜鹽的甲烷產生菌的生存，隨著季節變化與曝曬，溫度也又所變化，因此本研究利用雷公火泥火山的噴泥泉進行甲烷產生菌的富化培養實驗，使用不同種類的前驅物 (precursors) (氫氣/二氧化碳、醋酸、甲醇與甲胺)與不同的鹽度 (從現地鹽度至最高 2000 mM) 以及溫度範圍 (從現地溫度最高至 50 oC)，並隨著培養 時間進行甲烷濃度的監測，以瞭解噴泥中甲烷產生菌隨環境變化的甲烷產生潛力。 富化培養結果顯示，培養於室溫的所有添加前驅物之樣品皆能產生甲烷，甲烷產生速率隨著鹽度提高而明顯趨緩。添加氫氣/二氧化碳之樣品最耐鹽 (氯離子濃度為 2040 mM)，其次是添加甲醇與甲胺之樣品 (氯離子濃度分別為 1824 及 1593 mM)，而添加醋酸樣品可適應生長的鹽度較低 (氯離子濃度為 1168 mM)。培養於 40 oC 現地鹽度 (氯離子濃度約 300 mM) 下，所有添加前驅物之樣品皆能被快速富化而產生甲烷，而於 50 oC 培養中，只有添加氫氣/二氧化碳之樣品能被刺 激而產生甲烷。 分子生物分析方面，利用末端螢光標定限制酵素片段長度多型性 (Terminal restriction fragment length polymorphism, T-RFLP) 方法，區分不同前驅物與鹽度下樣品中的甲烷產生菌基因之異同處。將 T-RFLP 與 16S rDNA 定序分析結果對應下，培養於室溫與 40 oC 的樣品最接近的序列有Methanococcus spp.、Methanosarcina spp.、Methanocalculus spp.、Methanolobus spp. 以及 Methanococcoides spp.，除了Methanococcus spp. 外，其餘菌種被報導過皆能適應高於或等於氯離子濃度 1000 mM 或是可生存於 40 oC。 整體而言，在較高鹽度下添加氫氣/二氧化碳或甲基類之樣品比起添加醋酸之樣品，能更耐鹽而活躍的生長產生甲烷。這些較耐鹽的產甲烷菌於鹽度變化下或於蒸發作用旺盛的表層陸域泥火山中，能更能適應環境而生存著。當考量到陸域泥火山中整體的甲烷逸散量時，這些耐鹽的產甲烷菌成為一個重要的考量因素。

Terrestrial mud volcano is thought to be one of the most important natural sources of methane emission. Previous studies have shown that methane cycling in terrestrial mud volcanoes involves a complex reaction network driven by the interactions between subsurface and surface abiotic and microbial processes.In situ methanogenesis appears to produce methane at quantities exceeding those of deeply-sourced thermogenic methane and the capacities of anaerobic methanotrophy at shallow depth levels, thereby contributing significantly to the methane emission. Various degrees of evaporation at surface also lead to the enhancement of chloride concentrations in pore water, favoring the proliferation of halo-tolerant and/or halophilic methanogens. The goal of this study is to investigate the extent of methanogenesis in terrestrial mud volcanoes by incubating mud slurries with various precursors (H2/CO2,acetate, methanol, and methylamine) at different salinities (up to 2000 mM) and temperatures (up to 50 oC). Methane concentrations were monitored through time and molecular analyses were applied to investigate the changes of methanogenic communities. Growth of methanogenic enrichment cultures was bserved for all investigated precursors at room temperature. The methane production rates and yields declined significantly at higher salinities. Methanogens utilizing H2/CO2 could tolerate highest chlorite concentration (2040 mM). Methyl-compounds (methanol and methylamine) could be used for methane production with the chlorite concentration up to 1824 and 1593 mM. Acetate-utilizing methanogenesis proceeded at chlorite concentration less than 1168 mM. At 40 oC, methanogenesis was performed with all kinds of precursors at the in situ salinity, but only H2-utilizing methanogenesis was observed at 50 oC. Analyses of terminal restriction fragment length polymorphism (T-RFLP) for 16S rDNA genes revealed various patterns upon different precursors and salinities. The T-RFLP results combined with clone library analyses indicated that major RFs recovered from incubations at room temperature and 40 oC were represented by sequences affiliated with Methanococcus spp., Methanosarcina spp., Methanocalculus spp., Methanolobus spp. and Methanococcoides spp., and except to Methanococcus-related members, all above were capable to growth at salinities greater than 1000 mM or at 40 oC. Overall, methanogens utilizing H2/CO2 or methyl-compounds appear to be capable of actively producing methane at salinities greater than that for acetate-utilizing methanogens. These methanogens derived from muddy fluids might adapt to the fluctuation of salinity or extremely high salinity induced by the surface evaporation in terrestrial mud volcanoes. While considering the overall methane emission from terrestrial mud volcanoes, we have to consider the role of these halo-tolerant methanogens.