廖秀娟Liao, Vivian Hsiu-Chuan臺灣大學:生物環境系統工程學研究所魏嘉徵Wei, Chia-ChengChia-ChengWei2010-05-052018-06-292010-05-052018-06-292008U0001-1807200815465300http://ntur.lib.ntu.edu.tw//handle/246246/181111砷為一自然產生的元素且廣泛地存在於環境中。飲用含砷的地下水對於人類健康所造成的危害，已經是個全球性的議題。在六零年代的台灣，砷首度被視為與烏腳病疫情有直接相關的物質，隨之而來的更是被認定為相關癌症病變的致癌物。然而在台灣關於存在礦物中的砷以及砷在地下水中的生物地質循環等過程卻還沒被深入的研究與探討。越來越多的研究結果顯示，微生物在砷的地化循環扮演重要的角色，並影響到物種砷在環境中的分佈以及流動性。已知研究結果顯示，微生物可藉由三價砷的氧化以及五價砷還原的過程中，對砷產生抵抗作用並從中獲得生長所需的能量。雖然至今有很多在演化分類上對砷有還原作用的菌種被分離出來，但是對於藉砷產生呼吸作用的菌種其分子機制的探討卻是非常有限的。本研究中，我們分離出三株對砷具有高度抗性且能進行呼吸作用的菌種，其中針對菌種W2做了更進一步的研究。菌種W2為一兼養性微生物，其最佳的生長溫度為37℃，在好氧及厭氧的條件下皆可生長，且其對五價砷以及三價砷都有很高的抗性。菌種W2於LB (Luria-Bertani)的厭氧培養下，可以在42小時之內將2 mM的五價砷全部還原成三價砷，也可以在含有10 mM葡萄糖的MSM(minimal salts medium)的厭氧培養下，於十天之內將2 mM的五價砷全部還原成三價砷。另外，在砷還原的過程中並伴隨菌種W2的生長。利用PCR亦將PhsA (thiosulfate reductase)與ArsR 兩段基因從菌種W2的基因序列中成功的找到。因此實驗結果顯示，菌種W2同時含有異化砷還原作用(dissimilatory arsenate reduction)及砷解毒機制(arsenic detoxification mechanism)。在本研究中，也利用變性梯度凝膠電泳（DGGE）來探討及比較在台灣地區不同水井之含砷地下水樣中微生物族群的組成與分佈。結果顯示，不同的含砷地下水樣存在有不同的微生物。Arsenic is a naturally occurring element that is found in many environments. The contamination of groundwater with arsenic is a major concern to public health from local to international. Arsenic groundwater problems in Taiwan were first recognized during the 1960s and then Taiwan becomes the classic area for the study of Blackfoot disease and a variety of other typical problems including cancers. However, both the mineral sources of arsenic and the biogeochemistry of the groundwater of arsenic are poorly defined in Taiwan. 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. In this study, we isolated and molecular characterized the relationship between arsenate respiration and arsenic resistance bacterial strains. Of these isolated strains, strain W2 was selected for further investigation. Strain W2 is a mixotrophic microbe, and the optimal temperature for growth was 37℃. Strain W2 was able to grow both under aerobic and anaerobic conditions. Resistance tests showed that strain W2 could resist high concentrations of arsenate and arsenite when grown in LB medium. Arsenic transformation analysis showed that strain W2 could completely reduced 2 mM (150 ppm) of arsenate to arsenite within 42 hours in LB under anaerobic condition, and within 10 days in MSM (minimal salts medium) medium supplemented with 10 mM glucose under anaerobic condition. In addition, strain W2 growth was accompanied with arsenate reduction. Moreover, thiosulfate reductase PhsA and ArsR genes were amplified by PCR reaction, suggesting that strain W2 contains both dissimilatory arsenate reduction and arsenic detoxification mechanisms. Furthermore, denaturing gradient gel electrophoresis analysis showed that diversed microbial was present in arsenic-contaminated groundwater samples of different wells in Taiwan.誌謝.................i文摘要...........iiiBSTRACT............ivABLE OF CONTENT....viIST OF TABLES....viiiIST OF FIGURES.....ixHAPTER 1 INTRODUCTION....................................1.1 Arsenic toxicology....................................1.2 Arsenic geochemical cycle and environmemtal impacts of microbial arsenic transformation..........................2.3 Arsenic microbial mechanisms..........................3.3.1 Arsenic resistance and detoxification microorganisms5.3.2 Arsenite-oxidizing microbes.........................8.3.3 Arsenate-reducing microbes.........................11.4 Denaturing gradient gel electrophoresis (DGGE).......17.5 Purpose of study.....................................18HAPTER 2 MATERIALS & METHODS............................22.1 Chemicals............................................22.2 Growth condition and medium composition..............22.3 Arsenic-resistant bacterial strains isolation procedures...............................................22.4 Microplate color screening assay.....................25.5 Toxicity test for bacterial strains..................27.6 PCR amplification of 16S rRNA, ArsR, and thiosulfate reductase (PhsA) genes...................................27.6.1 16S rRNA...........................................27.6.2 ArsR...............................................28.6.3 PhsA...............................................28.7 Substrate test for growth of strain W2...............29.8 Arsenate transformation test for strain W2...........29.9 Analytical methods...................................30.10 Phylogenetic analysis...............................30.11 Denaturing gradient gel electrophoresis (DGGE)......30.11.1 PCR for DGGE......................................30.11.2 DGGE condition....................................31.12 Anaerobic technique.................................32HAPTER 3 RESULTS........................................33.1 Isolation of arsenate reduction bacterial strains....33.2 Characterization of the arsenate reducer strain W2...33.2.1 General description of bacterial strain W2.........33.2.2 Sequence analysis of strain W2.....................34.2.3 Substrate test for growth of strain W2.............34.2.4 Growth curve of strain W2..........................38.2.5 Toxicity test for strain W2........................40.2.6 Arsenate transformation for strain W2..............44.2.7 Cloning of arsenate reductase gene in strain W2....48.2.8 Denaturing Gradient Gel Electrophoresis of groundwater samples......................................51HAPTER 4 DISCUSSION & CONCLUSION........................54EFERENCES...............................................62application/pdf1270308 bytesapplication/pdfen-US五價砷還原地下水菌種W2變性梯度凝膠電泳groundwaterstrain W2arsenate reductiondenaturing gradient gel electrophoresis[SDGs]SDG3[SDGs]SDG14thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/181111/1/ntu-97-R95622011-1.pdf