臺灣博碩士論文加值系統

　　本實驗針對油污汙染土壤中篩選出來的菌種 (Pseudomonas aeruginosa strain T1、Ochrobactrum sp. strain T2 ) 和對於苯系化合物有降解能力的Enterbacter sp. strain NKNU02 ( E02 )以及對於鎘有耐受性的Enterbacter sp. strain CD01四株菌進行對於降解甲苯( Toluene )化合物之能力探討，同時評估各分離菌株對重金屬鎘( Cd )及苯( Benzene )、甲苯( Toluene )和二甲苯( Xylenes )之耐受能力。本實驗使用兩種不同的培養基( LB培養基和MSB培養基)來探討這些分離菌株對於在含有苯、甲苯或二甲苯等化合物加上鎘( Cd )之耐受度測試。實驗結果在含有苯、甲苯或二甲苯之LB培養基之下，有添加鎘( Cd )的培養基都會抑制菌體的生長，菌株生長時的適應期延長。

　　在分離之菌株中，Ochrobactrum sp. strain T2在500 ppm的甲苯做為單一碳源的環境當中，可生長至OD600值0.3；Pseudomonas aeruginosa strain T1在500 ppm苯、甲苯或二甲苯做為單一碳源的環境當中，都可生長至OD600值0.1，對於甲苯( Toluene ) 500 ppm的降解效率為每小時8.06 ppm；但對於有機化合物加上鎘( Cd )有最好耐受度的則是Enterbacter sp. strain NKNU02 ( E02 )，OD600值皆可達到1左右，但降解效率方面卻低於Pseudomonas aeruginosa strain T1。

In this study, microorganisms isolated from contaminated soil (Pseudomonas aeruginosa strain T1 and Ochrobactrum sp. strain T2), heavy metal (Cd) resistant microorganism Enterbacter sp. strain CD01 and BTEX degradative microorganism Enterbacter sp. strain NKNU02 (E02) were investigated for their ability of toluene degradation and tolerance against benzene, toluene or xylenes (BTX) and cadmium.

The results indicated that when LB medium containing 500 ppm of benzene, toluene or xylenes, and 1 mM Cd, all strains showed to have a prolonged lag growth phase before cell growth.

In a minimal salta (MSB) medium containing 500 ppm toluene, the growth of Ochrobactrum sp. strain T2 could reach 0.3 as measured by optical density at 600 nm (OD600). In MSB medium containing 500 ppm benzene, toluene or xylenes, Pseudomonas aeruginosa strain T1 could grow to OD600 0.1. As for the degradation of toluene, the degradation efficiency is 8.06 ppm per hour for strain T1 when incubated in the medium containing 500 ppm of toluene. On the other hand, Enterbacter sp. strain NKNU02 ( E02 ) was better than other strains in terms of cadmium resistance, which could tolerate up to 500 ppm of BTX in addition of 1mM of cadmium in LB medium. However, the degradation efficiency of strain E02 was still lower than that of strain T1.

中文摘要 i
英文摘要 ii
致謝 iv
目錄 v
表目錄 ix
圖目錄 x
第一章　前言 1
第二章　文獻回顧 2
2-1　苯系化合物 ( BTEX ) 2
2-1-1　苯（Benzene） 2
2-1-2　甲苯（Toluene） 3
2-1-3　二甲苯（Xylenes） 3
2-2　重金屬 3
2-2-1　金屬離子在細胞內的功能 4
2-2-2　微生物抵抗有毒金屬的機制 4
2-3　生物復育的種類 5
2-3-1　生物復育的種類 6
2-4　苯、甲苯、二甲苯化合物之生物降解代謝途徑 6
2-4-1　礦化作用 6
2-4-2　共代謝作用 6
2-4-3　化學自營好氧菌 7
2-4-4　化學自營厭氧菌 10
2-4-5　光合菌 10
2-4-6　發酵作用 10
第三章　實驗架構 11
第四章 材料與方法 13
4-1　實驗材料 13
4-1-1　實驗儀器 13
4-1-2　試劑 14
4-1-3　實驗藥品 14
4-1-4　培養基 17
4-1-5　引子 18
4-1-6　菌種名 18
4-2　實驗方法 19
4-2-1　菌種篩選 19
4-2-2　繼代培養 19
4-2-3　菌種保存與前培養 19
4-2-4　菌體生長情況的測定 20
4-2-5　Genomic DNA 純化方式 20
4-2-6　聚合酶鏈鎖反應 20
4-2-7　16S rDNA洋菜膠體電泳檢測 21
4-2-8　洋菜膠體DNA萃取純化 21
4-2-9　本實驗主要培養方法 22
4-2-10　氣相層析儀前處理步驟 23
4-2-11　氣相層析 23
4-2-12　感應偶合電漿原子發射光譜儀 24
第五章　結果 25
5-1　降解菌株篩選結果 25
5-2　菌株對有機化合物的耐受度測試結果 29
5-3　菌株對重金屬鎘( Cd )的耐受度測試結果 30
5-4　菌株對有機化合物及有無添加鎘之耐受性探討 34
5-5　菌株降解鎘( Cd )能力探討 39
5-6　菌株對於有機化合物甲苯的降解情形 40
5-7　菌株對於有機化合物甲苯的降解速率 41
第六章　討論 42
第七章　參考文獻 44
第八章　附錄 49

1.Smith, M. R., The biodegradation of aromatic hydrocarbons by bacteria. Biodegradation. 1990; 1(2-3):191-206.
2.Jahn, M. K., S. B. Haderlein, and R. U. Meckenstock, Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment- free iron-reducing enrichment cultures. Appl Environ Microbiol. 2005; 71(6):3355-8.
3.Shinoda, Y., Y. Sakai, H. Uenishi, Y. Uchihashi, A. Hiraishi, H. Yukawa, H.Yurimoto, and N. Kato. Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, Appl Environ Microbiol. 2004; 70(3):1385-92.
4.Dean, B. J., Recent findings on the genetic toxicology of benzene, toluene, xylenes and phenols. Mutat Res. 1985; 154(3):153-81.
5.International Agency for Research on Cancer (IARC) Benzo[a]pyrene, Polynuclear Aromatic Compounds, Part 1, Chemical, Environmental and Experimental Data, vol. 32, Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, 1983, pp. 211–224.
6.Zylstra, G. J., W. R. McCombie, D. T. Gibson, and B. A. Finette. Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon, Appl Environ Microbiol. 1988; 54(6):1498-503.
7.Tabak, H.H., et al., "Developments in bioremediation of soils and sediments polluted with metals and radionuclides - 1. Microbial processes and mechanisms affecting bioremediation of metal contamination and influencing metal toxicity and transport." Re-views in Environmental Science and Biotechnology, 2005. 4(3): p. 115-156.
8.Nies, D.H.,"Resistance to cadmium, cobalt, zinc, and nickel in microbes." Plasmid, 1992. 27(1): p. 17-28
9.pLloyd, J.R., "Microbial reduction of metals and radionuclides." Fems Microbiology Reviews, 2003. 27(2-3): p. 411-425.
10.Van Hullebusch, E.D., P.N.L. Lens, and H.H. Tabak, "Developments in bioremediation of soils and sediments polluted with metals and radionuclides. 3. Influence of chemical speciation and bioavailability on contaminants immobilization mobilization bio-processes." Re-views in Environmental Science and Biotechnology, 2005. 4(3): p. 185-212.
11.Guimaraes-Soares, L., et al., "Metal-binding proteins and peptides in the aquatic fungi Fontanospora fusiramosa and Flagellospora curta exposed to severe metal stress." Sci Total Environ, 2006. 372(1): p. 148-56.
12.Narender Reddy, G. and M.N.V. Prasad, "Heavy metal-binding proteins/peptides:Occurrence, structure, synthesis and functions. A review." Environmental and Experimental Botany, 1990. 30(3):p. 251-264.
13.Nies, D.H., "Efflux-mediated heavy metal resistance in prokaryotes." FEMS Microbiol Rev, 2003. 27(2-3): p. 313-39.
14.Franke, S., et al.,"Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli." J Bacteriol, 2003. 185(13):p. 3804-12.
15.Rensing, C., B. Mitra, and B.P. Rosen, "The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase." Proc Natl Acad Sci U S A, 1997. 94(26): p. 14326-31.
16.Borsetti F, M. P.,Casadio R,et al.,"Metals and metalloids in photosynthetic bacteria : interactions,resistance and putative homeostasis revealed by genome analysis. "In the purple phototrophic bacteria. Netherlands : springer science + Business media BV, 2009. 28: p. 655-689.
17.Guerinot, M.L., "The ZIP family of metal transporters." Biochim Biophys Acta, 2000. 1465(1-2): p. 190-8.
18.Munkelt, D., G. Grass, and D.H. Nies, "The chromosomally encoded cation diffusion facilitator proteins DmeF and FieF from Wautersia metallidurans CH34 are transporters of broad metal specificity." J Bacteriol, 2004. 186(23): p. 8036-43.
19.中華民國環境工程學會，「環境微生物」，1997
20.袁紹英，生物復育技術簡介 ，1995
21.王竣弘，土壤及地下水整治技術－生物復育法，2003
22.蔡昀達，中興工程顧問股份有限公司，地下水生物整治技術及案例介紹 ，1997
23.郭書萍，黃嘉嫻，無所不在的微生物，2007
24.陳維婷編譯；蔡麗伶審校，清理多氯聯苯致癌物，研究發現可以細菌取代掏沙法，2007
25.盧至人，葉玉雯，張峻嘉，蘇世昌，邱明良，地下水及土壤污染防治策略，1983
26.「A Citizen’s Guide to Bioremediation」(EPA 542-F-01-001)，April 2001.
27.Kotterman MJJ, Vis EH & Field JA, Successive mineralization detoxification of benzo(a)pyrene by the white rot fungus Bjerkandera sp. strain BOS55 and indigenous microflora, Appl Environ Microbiol. 1998; 64(8):2853-8.
28.Boonchan S Britz Margaret L & Stanley Grant A, Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures, Appl Environ Microbiol. 2000; 66(3):1007-19.
29.Finette BA, Subramanian V, Gibson DT., Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system, J Bacteriol. 1984; 160(3):1003-9.
30.Yeh WK, Gibson DT, Liu T-N. , Toluene dioxygenase: a multicomponent enzyme system, Biochem Biophys Res Commun. 1977; 78(1):401-10.
31.Lau PCK, Bergeron H, Labbe′ D, Wang Y, Brousseau R, Gibson DT., Sequence and expression of the todGIH genes involved in the last three steps of toluene degradation by Pseudomonas putida F1. Gene. 1994; 146(1):7-13.
32.Menn T-M, Zylstra GJ, Gibson DT. , Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1. Gene. 1991; 104(1):91-4.
33.Zylstra GJ, Gibson DT. , Toluene degradation by Pseudomonas putida F1: nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli, J Biol Chem. 1989; 264(25):14940-6.
34.Widdel F, Rabus R, Anaerobic biodegradation of saturated and aromatic hydrocarbons, Curr Opin Biotechnol. 2001; 12(3):259-76.
35.許原彰，黃雪莉，溫度對於Pseudomonas putida 鄰苯二酚加氧酵素之活性與結構的效應，國立中央大學，碩士論文，1990
36.蔡輔育，陳師慶，油污染廠址之菌相分析及分離具分解MTBE潛力之菌種，國立高雄師範大學，碩士論文，2008
37.Chih-Ching, Chien, Yu-Mei, Kuo, Chang-Chieh, Chen, Chun-Wei, Hung, Chih-Wei, Yeh and Wei-Jen, Yeh, Microbial diversity of soil bacteria in agricultural field contaminated with heavy metals, J Environ Sci (China). 2008; 20(3):359-63.
38.Jeong-Myeong, Kim, Ngoc-Thuan, Le, Bok-Sil, Chung, Jin-Ho, Park, Jin-Woo, Bae, Eugene L. Madsen, and Che-Ok, Jeon, Influence of Soil Components on the Biodegradation of Benzene, Toluene, Ethylbenzene, and o-, m-, and p-Xylenes by the Newly Isolated Bacterium Pseudoxanthomonas spadix BD-a59, Applied and Environmental Microbiology, 2008, p.7313–7320