臺灣博碩士論文加值系統

近年來，高度的工業發展導致許多環境汙染物排放到環境中，包括重金屬、農藥、新興汙染物等，且已影響世界眾多人口的健康。化學檢測法雖然具有高靈敏度及準確度，然而其價格高以及檢測耗時，難以應用於大規模的樣品篩測。因此本研究利用硫氧化細菌作為生物檢測器檢測環境中的環境汙染物，以期能和現有的化學分析方法互補。本研究於陽明山硫磺谷的土壤中篩選出一株嗜酸性硫氧化菌，並利用環境汙染物的毒性，因而使得在有氧的環境下，抑制硫氧化酵素將元素硫氧化成硫酸的能力，進而造成pH值無法下降及EC值無法上升的原理，建構出以檢測pH值及EC值變化為基礎的生物感測器。本研究篩選出的現地硫氧化菌SV5經16S rDNA定序並鑑定發現其序列與Acidicaldus organivorans strain Y008最為相近，序列相似度達99%。研究結果顯示SV5最佳生長環境為FYM培養基，需大量培養時可加入葡萄糖。最佳生長條件為pH值2.5、溫度37 ℃。此外，菌株SV5雖不能檢測塑化劑 (DEHP)、三唑醇 (triadimenol)，但能檢測超過環保署放流水標準10倍的砷及100倍的鎘，也能檢測環保署放流水標準濃度的鉻，檢測時間為4天。另外本研究也發現六價鉻能有效抑制SV5硫氧化蛋白的活性。總的來說，本研究建構之硫氧化生物感測器具有操作簡單、成本低的優勢，可與化學分析方法進行互補，提升大量篩檢重金屬樣品檢測效率。

In recent years, highly industrial development causes many environmental pollutants releasing into the environment, such as heavy metals, agricultural pesticides and emerging contaminants, thereby threaten people’s health around the world. Despite chemical analysis has been proven highly sensitive and accuracy, it still has limitation for large scale screening due to the cost and time consuming. The goal of this study is to use a sulfur-oxidizing bacterium as a biosensor to detect environmental pollutants.in order to current complement chemical analysis. In this study, an acidophilic sulfur-oxidizing bacterium was isolated from the soil of the Sulfur Valley near Yangmingshan. We developed the biosensor based on the bacterium’s sulfur-oxidizing ability to H2SO4 is inhibited by the environmental pollutants toxicity in the presence of O2 and S0, thereby preventing the decrease in pH and the increase in EC. Based on the 16S rDNA sequence analysis, the bacterium SV5 has 99% sequence similarity to Acidicaldus organivorans strain Y008. The optimal growth medium for SV5 is ferrous iron/yeast extract liquid medium (FYM), and the optimal temperature and pH is 37 ℃ and pH 2.5, respectively. The results showed that although SV5 could not effectively detect DEHP and triadimenol, it was able to detect 10-fold EPA effluent standard concentration of arsenic, 100-fold EPA effluent standard concentration of cadmium, and 1-fold EPA effluent standard concentration of chromium in 4 days. Finally, this study demonstrated that chromium inhibits sulfur-oxidizing protein activity of SV5. In conclusion, the sulfur-oxidizing biosensor in this study is easy to operate and cost-effective. It can not only compromise current chemical analysis but also increase the efficiency of large scale screening of environmental pollutants.

誌謝 I
摘要 II
ABSTRACT III
Graphical Abstract V
Highlights VI
目錄 VII
圖次 X
表次 XI
縮寫表 XII
一、研究動機 1
二、文獻回顧 3
2.1 環境汙染物 3
2.1.1 鄰苯二甲酸二(2-乙基己基)酯 (DEHP) 3
2.1.2 三唑醇 (Triadimenol) 4
2.1.3 重金屬及類金屬 5
2.2 環境汙染物的檢測方法 8
2.2.1化學分析法 8
2.2.2生物感測方法 8
2.2.3 以硫氧化細菌作為生物感測器 9
三、研究目的 11
3.1 篩選與鑑定現地之嗜酸性硫氧化菌 11
3.2 嗜酸性硫氧化菌SV5之基本特性分析 11
3.3 嗜酸性硫氧化菌SV5於廣效型生物感測器之應用 11
四、材料與方法 12
4.1 實驗架構流程圖 12
4.2 實驗藥品 13
4.3 土壤採樣與現地菌株之分離 13
4.3.1 硫磺谷土壤採樣 13
4.3.2 生長條件與培養基之組成 13
4.3.3 嗜酸菌富化培養及分離純化 13
4.3.4 嗜酸菌之菌種鑑定 14
4.4 嗜酸性硫氧化菌SV5之基本特性分析 14
4.4.1 生長曲線試驗 14
4.4.2 硫氧化試驗 14
4.4.3 生長溫度容許範圍 15
4.4.4 生長酸鹼度容許範圍 15
4.4.5 碳源與硫源之利用情形 16
4.4.6 環境汙染物對硫氧化功能的影響 16
4.4.7 六價鉻對SV5之毒性機制探討 16
4.5 SV5硫氧化蛋白質測試 17
4.5.1 SV5蛋白質萃取 17
4.5.2 SV5硫氧化蛋白質試驗 18
4.5.3 SV5硫氧化蛋白質重金屬試驗 18
4.6 統計分析 19
五、結果與討論 20
5.1 土壤採樣與現地菌株之分離 20
5.1.1 鑑定與分離嗜酸菌 20
5.1.2菌株SV5之特性分析 22
5.1.2.1生長曲線 22
5.1.2.2 溫度及酸鹼度適性 22
5.1.2.3 SV5之碳源及硫源利用情形 26
5.1.2.4 SV5之硫氧化功能 28
5.1.2.5環境汙染物對SV5硫氧化功能的影響 30
5.1.2.6 六價鉻對SV5之毒性機制探討 40
5.2 SV5蛋白質活性測試 45
5.2.1 SV5硫氧化蛋白質試驗 45
5.2.2 重金屬及類金屬對SV5硫氧化蛋白質的影響 47
5.3 探討SV5作為生物感測器之潛力 51
5.3.1菌株SV5生物感測器與其他研究之比較 51
5.3.2菌株SV5生物感測器之成本分析 52
六、結論 56
七、建議 57
八、參考文獻 58
九、附錄 64
附錄一、陽明山硫磺谷土壤採樣地點分布圖。 64
附錄二、Nutrient mineral buffer (NMB) 溶液配方表。 65
附錄三、PCR反應試劑濃度及作用條件。 66
附錄四、國立台灣大學生物資源暨農業學院感應偶合電漿質譜儀(ICP-MS)收費標準。 67

Abernathy, C.O., Liu, Y.P., Longfellow, D., Aposhian, H.V., Beck, B., Fowler, B., Goyer, R., Menzer, R., Rossman, T., Thompson, C., Waalkes, M., 1999. Arsenic: health effects, mechanisms of actions, and research issues. Environ Health Perspect 107, 593-597.
Akesson, A., Julin, B., Wolk, A., 2008. Long-term dietary cadmium intake and postmenopausal endometrial cancer incidence: a population-based prospective cohort study. Cancer Res 68, 6435-6441.
Avudainayagam, S., Megharaj, A., Owens, G., Kookana, R.S., Chittleborough, D., Naidu, R., 2003. Chemistry of chromium in soils with emphasis on tannery waste sites. Rev Environ Contam T 178, 53-91.
Behera, B.C., Patra, M., Dutta, S.K., Thatoi, H.N., 2013. Isolation and Characterisation of Sulphur Oxidising Bacteria from Mangrove Soil of Mahanadi River Delta and Their Sulphur Oxidising Ability. Appl Environ Microbiol 2, 1-5.
Biemann, R., Navarrete Santos, A., Navarrete Santos, A., Riemann, D., Knelangen, J., Bluher, M., Koch, H., Fischer, B., 2012. Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows. Biochem Biophys Res Commun 417, 747-752.
Chakraborti, D., Rahman, M.M., Paul, K., Chowdhury, U.K., Sengupta, M.K., Lodh, D., Chanda, C.R., Saha, K.C., Mukherjee, S.C., 2002. Arsenic calamity in the Indian subcontinent What lessons have been learned? Talanta 58, 3-22.
Daunert, S., Barrett, G., Feliciano, J.S., Shetty, R.S., Shrestha, S., Smith-Spencer, W., 2000. Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev 100, 2705-2738.
Delnomdedieu, M., Basti, M.M., Otvos, J.D., Thomas, D.J., 1994. Reduction and binding of arsenate and dimethylarsinate by glutathione: a magnetic resonance study. Chem Biol Interact 90, 139-155.
Dhal, B., Thatoi, H.N., Das, N.N., Pandey, B.D., 2013. Chemical and microbial remediation of hexavalent chromium from contaminated soil and mining/metallurgical solid waste: a review. J Hazard Mater 250-251, 272-291.
Dixon, H.B.F., 1997. The biochemical action of arsonic acids especially as phosphate analogues. Adv Inorg Chem 44, 191-227.
Erkekoglu, P., Kocer-Gumusel, B., 2014. Genotoxicity of phthalates. Toxicol Mech Methods 24, 616-626.
Fasanya-Odewumi, C., Latinwo, L.M., Ikediobi, C.O., Gilliard, L., Sponholtz, G., Nwoga, J., Stino, F., Hamilton, N., Erdos, G.W., 1998. The genotoxicity and cytotoxicity of dermally-administered cadmium: effects of dermal cadmium administration. Int J Mol Med 1, 1001-1006.
Fendorf, S., Wielinga, B.W., Hansel, C.M., 2000. Chromium transformations in natural environments: The role of biological and abiological. processes in chromium(VI) reduction. Int Geol Rev 42, 691-701.
Guha Mazumder, D.N., 2008. Chronic arsenic toxicity & human health. Indian J Med Res 128, 436-447.
Gurung, A., Van Ginkel, S.W., Kang, W.C., Qambrani, N.A., Oh, S.E., 2012. Evaluation of marine biomass as a source of methane in batch tests: A lab-scale study. Energy 43, 396-401.
Hassan, S.H., Van Ginkel, S.W., Kim, S.M., Yoon, S.H., Joo, J.H., Shin, B.S., Jeon, B.H., Bae, W., Oh, S.E., 2010. Isolation and characterization of Acidithiobacillus caldus from a sulfur-oxidizing bacterial biosensor and its role in detection of toxic chemicals. J Microbiol Methods 82, 151-155.
Hauser, R., Calafat, A.M., 2005. Phthalates and human health. Occup Environ Med 62, 806-818.
Hiraishi, A., Matsuzawa, Y., Kanbe, T., Wakao, N., 2000. Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments. Int J Syst Evol Microbiol 50 Pt 4, 1539-1546.
HSDB, 2009. Triadimenol.
Ishaque, A.B., Johnson, L., Gerald, T., Boucaud, D., Okoh, J., Tchounwou, P.B., 2006. Assessment of individual and combined toxicities of four non-essential metals (As, Cd, Hg and Pb) in the microtox assay. Int J Environ Res Public Health 3, 118-120.
Jarup, L., 2003. Hazards of heavy metal contamination. Br Med Bull 68, 167-182.
Johnson, D.B., Stallwood, B., Kimura, S., Hallberg, K.B., 2006. Isolation and characterization of Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo-acidophilic heterotrophic Proteobacterium. Arch Microbiol 185, 212-221.
Kajikawa, K., Kitagawa, M., Nakanishi, I., Ueshima, H., Katsuda, S., Kuroda, K., 1974. A pathological study of itai-itai disease. Zasshi Kanazawa Daigaku Igakubu Juzen Igakkai 83, 309-347.
Kara, H., Cevik, A., Konar, V., Dayangac, A., Yilmaz, M., 2007. Protective effects of antioxidants against cadmium-induced oxidative damage in rat testes. Biol Trace Elem Res 120, 205-211.
Khan, S., Cao, Q., Zheng, Y.M., Huang, Y.Z., Zhu, Y.G., 2008. Health risks of heavy metals in contaminated soils and food crops irrigated with wastewater in Beijing, China. Environ Pollut 152, 686-692.
Kim, J.H., Park, H.Y., Bae, S., Lim, Y.H., Hong, Y.C., 2013. Diethylhexyl phthalates is associated with insulin resistance via oxidative stress in the elderly: a panel study. PLoS One 8, e71392.
Kishimoto, T., Oguri, T., Yamabe, S., Tada, M., 1996. Effect of cadmium injury on growth and migration of cultured human vascular endothelial cells. Hum Cell 9, 43-48.
Kreuger, J., 1998. Pesticides in stream water within an agricultural catchment in southern Sweden, 1990-1996. Sci Total Environ 216, 227-251.
Kriegel, A.M., Soliman, A.S., Zhang, Q., El-Ghawalby, N., Ezzat, F., Soultan, A., Abdel-Wahab, M., Fathy, O., Ebidi, G., Bassiouni, N., Hamilton, S.R., Abbruzzese, J.L., Lacey, M.R., Blake, D.A., 2006. Serum cadmium levels in pancreatic cancer patients from the East Nile Delta region of Egypt. Environ Health Perspect 114, 113-119.
Ku, H.Y., Su, P.H., Wen, H.J., Sun, H.L., Wang, C.J., Chen, H.Y., Jaakkola, J.J., Wang, S.L., Group, T., 2015. Prenatal and postnatal exposure to phthalate esters and asthma: a 9-year follow-up study of a taiwanese birth cohort. PLoS One 10, e0123309.
Kueseng, P., Thavarungkul, P., Kanatharana, P., 2007. Trace phthalate and adipate esters contaminated in packaged food. J Environ Sci Health B 42, 569-576.
Lyche, J.L., Gutleb, A.C., Bergman, A., Eriksen, G.S., Murk, A.J., Ropstad, E., Saunders, M., Skaare, J.U., 2009. Reproductive and developmental toxicity of phthalates. J Toxicol Environ Health B Crit Rev 12, 225-249.
Mangold, S., Valdes, J., Holmes, D.S., Dopson, M., 2011. Sulfur metabolism in the extreme acidophile acidithiobacillus caldus. Front Microbiol 2, 17.
McLaughlin, M.J., Whatmuff, M., Warne, M., Heemsbergen, D., Barry, G., Bell, M., Nash, D., Pritchard, D., 2006. A field investigation of solubility and food chain accumulation of biosolid-cadmium across diverse soil types. Environ Chem 3, 428-432.
Mehrotra, P., 2016. Biosensors and their applications - A review. J Oral Biol Craniofac Res 6, 153-159.
Menegola, E., Broccia, M.L., Di Renzo, F., Prati, M., Giavini, E., 2000. In vitro teratogenic potential of two antifungal triazoles: triadimefon and triadimenol. In Vitro Cell Dev Biol Anim 36, 88-95.
Mertz, W., 1992. Chromium. History and nutritional importance. Biol Trace Elem Res 32, 3-8.
Min, J., Kim, E.J., LaRossa, R.A., Gu, M.B., 1999. Distinct responses of a recA::luxCDABE Escherichia coli strain to direct and indirect DNA damaging agents. Mutat Res 442, 61-68.
O''Brien, T.J., Ceryak, S., Patierno, S.R., 2003. Complexities of chromium carcinogenesis: role of cellular response, repair and recovery mechanisms. Mutat Res 533, 3-36.
Oh, S.E., Hassan, S.H.A., Van Ginkel, S.W., 2011. A novel biosensor for detecting toxicity in water using sulfur-oxidizing bacteria. Sensors and Actuators B-Chemical 154, 17-21.
Okamura, K., Hisada, T., Kanbe, T., Hiraishi, A., 2009. Rhodovastum atsumiense gen. nov., sp. nov., a phototrophic alphaproteobacterium isolated from paddy soil. J Gen Appl Microbiol 55, 43-50.
Oremland, R.S., Stolz, J.F., 2003. The ecology of arsenic. Science 300, 939-944.
Pesch, B., Haerting, J., Ranft, U., Klimpel, A., Oelschlagel, B., Schill, W., 2000. Occupational risk factors for renal cell carcinoma: agent-specific results from a case-control study in Germany. MURC Study Group. Multicenter urothelial and renal cancer study. Int J Epidemiol 29, 1014-1024.
Petrick, J.S., Ayala-Fierro, F., Cullen, W.R., Carter, D.E., Vasken Aposhian, H., 2000. Monomethylarsonous acid (MMA(III)) is more toxic than arsenite in Chang human hepatocytes. Toxicol Appl Pharmacol 163, 203-207.
Pose-Juan, E., Sanchez-Martin, M.J., Andrades, M.S., Rodriguez-Cruz, M.S., Herrero-Hernandez, E., 2015. Pesticide residues in vineyard soils from Spain: Spatial and temporal distributions. Sci Total Environ 514, 351-358.
Qambrani, N.A., Hwang, J.H., Oh, S.E., 2016. Comparison of chromium III and VI toxicities in water using sulfur-oxidizing bacterial bioassays. Chemosphere 160, 342-348.
Ratnaike, R.N., 2003. Acute and chronic arsenic toxicity. Postgrad Med J 79, 391-396.
Richard, F.C., Bourg, A.C.M., 1991. Aqueous Geochemistry of Chromium - a Review. Water Res 25, 807-816.
Schettler, T., 2006. Human exposure to phthalates via consumer products. Int J Androl 29, 134-139; discussion 181-135.
Scott, N., Hatlelid, K.M., MacKenzie, N.E., Carter, D.E., 1993. Reactions of arsenic(III) and arsenic(V) species with glutathione. Chem Res Toxicol 6, 102-106.
Sedman, R.M., Beaumont, J., McDonald, T.A., Reynolds, S., Krowech, G., Howd, R., 2006. Review of the evidence regarding the carcinogenicity of hexavalent chromium in drinking water. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 24, 155-182.
SenGupta, R., Kim, J., Gomes, C., Oh, S., Park, J., Im, W.B., Seong, J.Y., Ahn, R.S., Kwon, H.B., Soh, J., 2004. Effect of ascorbic acid supplementation on testicular steroidogenesis and germ cell death in cadmium-treated male rats. Mol Cell Endocrinol 221, 57-66.
Sharma, P., Bihari, V., Agarwal, S.K., Verma, V., Kesavachandran, C.N., Pangtey, B.S., Mathur, N., Singh, K.P., Srivastava, M., Goel, S.K., 2012. Groundwater contaminated with hexavalent chromium [Cr (VI)]: a health survey and clinical examination of community inhabitants (Kanpur, India). PLoS One 7, e47877.
Sharma, R.K., Agrawal, M., Marshall, F.M., 2008. Heavy metal (Cu, Zn, Cd and Pb) contamination of vegetables in urban India: a case study in Varanasi. Environ Pollut 154, 254-263.
Shetty, R.S., Deo, S.K., Shah, P., Sun, Y., Rosen, B.P., Daunert, S., 2003. Luminescence-based whole-cell-sensing systems for cadmium and lead using genetically engineered bacteria. Anal Bioanal Chem 376, 11-17.
Singh, N., 2005. Factors affecting triadimefon degradation in soils. J Agric Food Chem 53, 70-75.
Smith, A.H., 2008. Hexavalent chromium, yellow water, and cancer: a convoluted saga. Epidemiology 19, 24-26.
Smith, E., Naidu, R., Alston, A.M., 1998. Arsenic in the soil environment: A review. Adv Agro 64, 149-195.
Strakovsky, R.S., Lezmi, S., Shkoda, I., Flaws, J.A., Helferich, W.G., Pan, Y.X., 2015. In utero growth restriction and catch-up adipogenesis after developmental di (2-ethylhexyl) phthalate exposure cause glucose intolerance in adult male rats following a high-fat dietary challenge. J Nutr Biochem 26, 1208-1220.
Tauriainen, S., Karp, M., Chang, W., Virta, M., 1998. Luminescent bacterial sensor for cadmium and lead. Biosens Bioelectron 13, 931-938.
Templeton, D.M., Liu, Y., 2010. Multiple roles of cadmium in cell death and survival. Chem Biol Interact 188, 267-275.
Thompson, J., Bannigan, J., 2008. Cadmium: toxic effects on the reproductive system and the embryo. Reprod Toxicol 25, 304-315.
Tseng, I.L., Yang, Y.F., Yu, C.W., Li, W.H., Liao, V.H., 2013. Phthalates induce neurotoxicity affecting locomotor and thermotactic behaviors and AFD neurons through oxidative stress in Caenorhabditis elegans. PLoS One 8, e82657.
Tseng, W.P., 1977. Effects and dose--response relationships of skin cancer and blackfoot disease with arsenic. Environ Health Perspect 19, 109-119.
USEPA, 2006. Triadimefon Reregistration Eligibility Decision (RED) and Triadimenol Tolerance Reassessment and Risk Management Decision (TRED) Fact Sheet (U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances).
Van Ginkel, S.W., Hassan, S.H.A., Oh, S.E., 2010. Detecting endocrine disrupting compounds in water using sulfur-oxidizing bacteria. Chemosphere 81, 294-297.
Waalkes, M.P., Klaassen, C.D., 1985. Concentration of metallothionein in major organs of rats after administration of various metals. Fundam Appl Toxicol 5, 473-477.
Walker, Q.D., Mailman, R.B., 1996. Triadimefon and triadimenol: effects on monoamine uptake and release. Toxicol Appl Pharmacol 139, 227-233.
World Health Organization, 1981. Environmental Health Criteira 18; Arsenic. World Health Organization Geneva.
Zayed, A.M., Terry, N., 2003. Chromium in the environment: factors affecting biological remediation. Plant Soil 249, 139-156.
Zhang, Y., Zhang, G., Fu, P., Ma, Y., Zhou, J., 2012. Study on the interaction of triadimenol with calf thymus DNA by multispectroscopic methods and molecular modeling. Spectrochim Acta A Mol Biomol Spectrosc 96, 1012-1019.
Zhitkovich, A., 2005. Importance of chromium-DNA adducts in mutagenicity and toxicity of chromium(VI). Chem Res Toxicol 18, 3-11.
Zhu, Y.G., Williams, P.N., Meharg, A.A., 2008. Exposure to inorganic arsenic from rice: a global health issue? Environ Pollut 154, 169-171.
Zota, A.R., Calafat, A.M., Woodruff, T.J., 2014. Temporal trends in phthalate exposures: findings from the National Health and Nutrition Examination Survey, 2001-2010. Environ Health Perspect 122, 235-241.
行政院環境保護署(2008)。 水中有機磷農藥檢測方法－氣相層析儀／火焰光度偵測器法，NIEA W610.52B。
行政院環境保護署(2012)。 重金屬檢測方法總則，NIEA M103.02C。