台灣與菲律賓地熱溫泉中可培養嗜熱砷轉化菌之多樣性與生理代謝特性

Abstract

地熱溫泉中常含有高濃度的砷，被認為對微生物是具有毒性。瞭解微生物催化反應如何轉化砷價態，有助於探索自然環境中微生物和砷之間的交互作用。本研究第一個目的是純化並探討嗜熱砷轉化菌的生理特性，以擴充目前我們已知全球嗜熱砷轉換菌之多樣性及微生物在地熱環境中砷循環可能扮演的角色。到目前為止，沒有任何文獻總結砷轉換機制和親緣關係之間的連結，本研究第二個目的是系統性總結目前已發表文獻中，可培養菌株之親緣與砷代謝之關係。 本研究將採集自台灣與菲律賓的地熱流體樣本接種入含砷培養基，並培養於40至90oC，利用塗盤或系列稀釋的方式，從大屯火山群地區與關仔嶺溫泉純化分離出五隻菌株；從龜山島淺海熱液溫泉中純化分離出三十五隻菌株；另外結合自本實驗室過去純化分離或由陳俊堯博士提供共二十隻菌株，共挑選其中九隻嗜熱菌株進一步探討其砷轉換能力之特性，在這些菌株中 Anoxybacillus 和 Alicyclobacillus 相關菌株被確認可氧化三價砷；相對的， Acidianu 和 Geobacillus 相關菌株可還原五價砷； Meiothermus 和 Thermus 相關菌株則可雙向轉化砷。三隻純化分離自龜山島的嗜中溫菌株則為化學自營亞砷酸鹽氧化菌，隸屬於菌屬 Marinobacter 與 Alcanivorax。這是第一次報導 Alicyclobacillus 、 Anoxybacillus 、 Acidianus 、 Geobacillus 、 Meiothermus 、 和 Alcanivorax 屬中菌株具有轉換物種砷的能力。這個結果不只擴大了目前對地熱環境中砷轉換菌多樣性的瞭解，同樣也利於建立微生物活動和現地砷轉化過程之關係。 親緣和砷生理代謝之間的關係總結如下：異營亞砷酸鹽氧化菌主要分佈在Deinococcus-Thermus 、 Alphaproteobacteria 、 Betaproteobacteria 和 Gammaproteobacteria 中的 Psudomonas ；化學自營亞砷酸鹽氧化菌則主要為 Alphaproteobacteria 和 Betaproteobacteria 之成員，或 Gammaproteobacteria 和 Aquificae 中一些菌株；砷酸鹽異化還原菌則住要隸屬於 Pyrobaculum 、 Chrysiogenetes 、 Firmicutes 、 Deltaproteobacteria 、 Epsilonproteobacteria 分類中占優勢，亦有一些 Aquificae 和 Gammaproteobacteria 之成員；異營砷酸鹽還原菌則零散分佈於許多分類群，包含：Crenarchaeota 、 Actinobacteria 、 Bacteroidetes 、 Deinococcus-Thermus 、 Firmicutes 、 Alphaproteobacteria 和 Gammaproteobacteria。這樣的結果顯示砷轉換機制僅主要分佈於某些親緣分支之中。

Geothermal springs typically contain high arsenic concentrations that are considered to be toxic to microorganisms. Understanding how microbially-catalytic reactions are involved in the changes of the redox state of arsenic would provide important constraints on the interaction between arsenic and microorganisms in natural occurrences. The first aim of this study was to isolate and characterize the physiological properties of thermophilic arsenic transformers in order to enhance our understanding about the global diversity and microbial role in the arsenic cycling in geothermal environments. To date, there is no literature that summarizes arsenic transforming mechanisms in connection with phylogenetic relationships. Therefore, the second aim of this study was to synthesize and summarize the relationships between the phylogeny and arsenic metabolism for cultivable strains previously published in liteatures. Geothermal fluid samples collected from Taiwan and Philippines were inoculated into arsenite-containing media and incubated at 40 to 90oC. Five strains were isolated through plating or series dilution from the Ta-Tun volcanic area and Kuan-Tzu-Ling mud spring; thirty-five strains from the shallow submarine hydrothermal vent in the Kuei-Shan Island. Twenty strains were isolated from previous studies or provided by Dr. Chun-Yao Chen. Of these strains, nine thermophilic strains were further subject to the characterization of their arsenic transforming capabilities. Among these strains, Alicyclobacillus- and Anoxybacillus- related strains were identified to oxidize arsenite. In contrast, Acidianus- and Geobacillus-related strains were capable of reducing arsenate. Meiothermus- and Thermus-related strains were able to dually transform arsenic species. Three mesophilic strains obtained from the Kuei-Shan Island were chemolithoautotrophic arsenite oxidizers. The capability of transforming arsenic redox states by strains of Alicyclobacillus-, Anoxybacillus-, Acidianus-, Geobacillus-, Meiothermus-, and Alcanivorax- genus is first reported. These results not only expand the current view about the diversity of arsenic transformers in geothermal environments, but also facilitate to establish the linkages between microbial activities and in situ arsenic transforming processes. The relationships between the phylogeny and arsenic metabolism was summarized as followed: heterotrophic arsenite oxidizers predominantly appear in Deinococcus-Thermus, Alphaproteobacteria, Betaproteobacteria and Psudomonas of Gammaproteobacteria; chemoautotrophic arsenite oxidizers were composed of members related with Alphaproteobacteria and Betaproteobacteria, and some strains in Gammaproteobacteria and Aquificae; dissimilatory arsenate-reducing prokaryotes were primarily affiliated with Pyrobaculum, Chrysiogenetes, Firmicutes, Deltaproteobacteria, Epsilonproteobacteria, and some Aquificae and Gammaproteobacteria members; heterotrophic arsenate reducers erratically scatter in almost all taxa, including Crenarchaeota, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Firmicutes, Alphaproteobacteria, and Gammaproteobacteria. The results show that arsenic transforming mechanisms could be correlated with certain phylogenetic clades.