台灣烏腳病地區含砷地下水菌種Bosea sp. str. L7506的分離與分析

Abstract

砷為已知的人類致癌物質。雖然砷對大多數的生物體具有毒性，但已有證據顯示微生物能夠調控砷在環境中的轉化、循環與分佈。本研究旨在探討台灣烏腳病地區受砷汗染之地下水中微生物的分離及調控砷循環的機制。本研究一共分離出345株和砷的抗性相關的菌株，根據16S rDNA的次序鑑定比對判定其環境水樣中的優勢菌種為Pseudomonas屬。於好氧的培養條件下，利用microplate color screening方法篩選出其中87菌株對五價砷具有還原功能以及一菌株對三價砷具有氧化功能。本研究更進一步對三價砷氧化菌，Bosea sp. str. L7506，進行更深入的探討與研究。 結果顯示Bosea sp. str. L7506為一革蘭氏陰性桿菌 (Gram-negative)，最適的生長條件為pH值7.4-8.4，37℃於LB (Luria-Bertani) 培養基中培養。其對砷的抗性極高，能存活於含有100mM的五價砷環境中。Bosea sp. str. L7506為一好氧自營菌，但在適當的培養基質及碳源的供應下能刺激其生長，甚至能抵抗2mM三價砷的毒性。另外，結果顯示Bosea sp. str. L7506在完全不提供營養源的狀況下，仍能在三天之內就將2mM的三價砷完全氧化成五價砷，而且若於原生環境地下水樣中培養，則可在二天之內完全氧化2mM的三價砷，此結果在未來環境復育的利用上佔有相當大的優勢。此外，本研究亦clone出三價砷氧化酶 (arsenite oxidase) 之large subunit，並在菌體的periplasm 及spheroplast內皆發現受三價砷誘導之三價砷氧化酶活性。本研究除了發現在台灣烏腳病地區受砷汗染之地下水中菌種的分布，並且對三價砷之氧化菌Bosea sp. str. L7506做進一步的研究與分析，期能應用於未來復育環境中的砷汙染。

Arsenic is an environmental carcinogen of toxicological concern. Although arsenic is generally toxic to life, it has been demonstrated that some microorganisms can use arsenic compounds as electron donors, electron acceptors, or possess arsenic detoxification mechanisms. Increasing evidences suggest that the biogeochemical cycle of arsenic is significantly dependent on microbial transformations which affect the distribution and the mobility of arsenic species in the environment. However, the roles of the bacteria in the arsenic cycles are yet to be fully elucidated. In this study, we isolate arsenite-arsenate redox bacteria using known arsenic-contaminated groundwater in Blackfoot disease region in Taiwan under oxic condition. Three hundred and forty-five arsenic-resistant bacterial strains were isolated. A microplate color screening assay with addition of AgNO3 was used to initially characterize the ability of oxidation and reduction of those bacteria. Eighty-seven bacteria were capable of arsenate reduction, whereas only one bacterial strain L7506 was characterized as arsenite oxidizer. Analysis of the 16S rRNA gene sequence of the isolated bacteria revealed that some of the bacteria have been indicated involving in arsenic transformation, while others have not been reported to be associated with arsenic transformation. The designated strain L7506 was selected for further investigation. It is a Gram-negative, straight to curved rod, and motile bacteria. It belongs to genus Bosea based on 16S rRNA sequence analysis. The optimal growth condition was at pH 7.4-8.4, 37℃ in LB medium. Moreover, it was able to grow in the presence of 100mM arsenate. Substrate test showed that Bosea sp. str. L7506 is an autotrophic bacterium. However, the addition of appropriate substrate and carbon sources enhance its growth rate even in the presence of 2mM arsenite. Arsenite transformation analysis showed that Bosea sp. str. L7506 completely oxidized 2mM (150ppm) of arsenite to arsenate within 3 days without the presence of any nutrient, and within 2 days when cultured in groundwater samples. Furthermore, the large subunit of the arsenite oxidase gene was cloned using a pair of degenerate primers. Arsenite oxidase activity was stimulated by 2mM arsenite and was present in both periplasm and spheroplast of the bacteria. Taken together, results from this study showed that diverse bacteria were present in arsenic-contaminated groundwater in Blackfoot disease region in Taiwan. The identified arsenite-oxidizing bacteria may be potentially used for bioremediation of arsenic-contaminated groundwater.