利用穩定同位素標定研究台灣西南海域甲烷氧化菌和甲基氧化菌

Abstract

過去研究已指出海水中存在著能夠代謝甲烷或甲醇的微生物，調控著甲烷、甲醇或其他甲基物質在海水中的變動，進而影響海域環境中的碳循環，然而現今對於這些微生物族群的相關研究仍有諸多未明之處，因此本研究選擇台灣西南海域 MV1、MV2、MV5 以及 MV12 泥火山和四方圈合冷泉區作為研究區域，分析其海水中甲烷濃度變化，並利用富化培養和穩定同位素標定 (Stable-isotope Probing, SIP) 探究海水中是否存在甲烷氧化菌 (methanotrophs) 或甲基氧化菌 (methylotrophs) 族群。 海水中溶解甲烷的分析結果顯示在多數海底泥火山的底層水均可測得較高濃度的甲烷，其中又以 MV12 泥火山供應甲烷的情形最劇烈，四方圈合冷泉區具有明顯的冷泉生態系統，底層水之甲烷濃度不高，這些由底層逸出的甲烷最終至表層大多已降為表層海水的背景值，顯見海洋中存在著機制進行甲烷的消耗，大幅降低了海洋輸送至大氣的甲烷逸散量。 選自 MV12 泥火山和四方圈合 TTC61 站位的底層水或中層水，以添加 13C 標定或未標定甲烷或甲醇進行富化培養，培養期間持續對樣本的代謝反應做監測，結果顯示添加甲烷之 TTC61 站位底層水培養的甲烷濃度有顯著下降，應為甲烷氧化菌消耗甲烷所致，而添加甲醇之培養所有樣本的二氧化碳濃度均顯著上升，推測是培養樣本中能夠代謝甲醇的甲基氧化菌或甲烷氧化菌所釋出。藉由 SIP 追蹤並分離出使用甲烷或甲醇之微生物的 DNA，再透過 16S rDNA 選殖基因庫的建立來檢視微生物族群結構，發現主要是由 methylotrophic group 和 Methylophaga sp. 所組成。使用甲烷而被 13C 標定的 DNA，透過即時定量聚合酶鏈鎖反應 (quantitative real-time polymerase chain reaction, qPCR) 可驗證其具有大量屬於甲烷氧化菌的功能性基因 (pmoA)。利用 pmoA 和 mxaF 功能性基因建立選殖基因庫，可進一步確認使用甲烷而被 13C 標定之 DNA 與 Type I 甲烷氧化菌 Methylobacter sp.、Methylovulum sp. 和 Methylomonas sp. 有 72 至 80 % 的相似度；使用甲醇而被 13C 標定之 DNA 與 Methylophaga sp. 有 82 % 以上的相似度，與 Type I 甲烷氧化菌 Methylomonas sp. 和 Methylobacter sp. 也有 88 % 以上的相似度。

Methanotrophs and methylotrophs can utilize methane, methanol or a variety of single carbon compounds as their sole carbon and energy source. They have been found in seawater and play an essential role in carbon cycling, but the details are still less clear. This study investigated methane concentrations in water column above submarine mud volcanoes (MV1, MV2, MV5, MV12) and cold seeps (the Four Way Closure Ridge) offshore southwestern Taiwan and applied cultivation and stable-isotope probing (SIP) to examine the identity of methanotrophys and methylotrophys in these environments. Higher methane concentrations were detected in the bottom water of most submarine mud volcanoes, while MV12 was the most active one during sampling periods. Chemosynthetic communities (shells or mussels) and authigenic minerals are widely observed on the seafloor around the Four Way Closure Ridge, although the methane concentration is low in the bottom water. Because the methane concentration in surface water is closed to background, a certain machanism should be responsible for methane consumption in seawater. Several water samples from MV12 and Four Way Closure Ridge were incubated with additions of normal or 13C-labelled methane or methanol. All incubations with methanol addition showed CO2 increase in headspace, which may infer the growth of methanol oxidizer. CH4 decrease in headspace was only observed in one sample, indicating possible growth of methanotrophs. On the basis of SIP, 13C-DNA was separated from the unlabelled DNA for further examination. The 16S rDNA sequence of 13C-DNA fractions were closely related to the sequence of methylotrophic group and Methylophaga sp. Quantitative real-time PCR assays for the pmoA gene showed highest abundance in 13C-DNA fractions. The pmoA gene of 13C-DNA fractions were closed to that of Type I methanotrophs (similarity of 72-80 %) and the mxaF gene of 13C-DNA fractions was related to that of Type I methanotrophs (similarity > 82 %) and Methylophaga sp. (similarity > 88 %).