排放至環境中的戴奧辛污染物以高氯數的同源物為主，由於其化學結構穩定且水溶性極低，故難以被微生物降解，並易經由食物鏈累積於高等生物體中，而八氯二聯苯呋喃則是眾多戴奧辛同源物中結合氯數最多的二聯苯呋喃。本研究利用傳統培養方法從受戴奧辛污染的土壤中分離菌株，希望能發展出於好氧環境下可有效降解八氯二聯苯呋喃之菌劑，進一步應用於實際污染土壤中，以發揮生物整治土壤戴奧辛污染之效能。
本研究所使用的土壤樣本來自於受五氯酚與戴奧辛污染的土壤，用以分離菌株之土壤戴奧辛濃度為6.4 µg I-TEQ/kg，進行土壤降解實驗的戴奧辛毒性當量為90～230 µg I-TEQ/kg，而土壤中八氯二聯苯戴奧辛與八氯二聯苯呋喃兩者的莫爾比例共占90%以上。將污染土壤進行優渥培養後，以塗抹八氯二聯苯呋喃的基礎培養基與海水培養基共分離出102個菌落，並利用RFLP技術分類出40個OTUs，以16S rRNA基因進行親緣分析顯示，這些菌株分佈範圍可達三個不同的門，十三個不同的屬，表示土壤中可忍受高濃度戴奧辛污染的菌株多樣性豐富。
根據PCR偵測戴奧辛雙氧氧化酵素之基因實驗結果，進一步篩選出具有潛力的菌株，並進行八氯二聯苯呋喃降解測試，得知Bacillus, Rhodococcus, Micrococcus, Sphingomonadaceae, 以及Mesorhizobium等五個屬的六株純菌有降解八氯二聯苯呋喃的能力，經過21天後的平均降解率在26%-43%左右。此結果為首次發現八氯二聯苯呋喃可被好氧菌降解。然後，挑選Bacillus, Rhodococcus, Micrococcus, Pseudomonas屬的菌株，添加至土壤中進行降解測試，培養21天後雙重複試驗結果發現土壤戴奧辛毒性分別減少了17%、15%、17%以及42%，表示這些菌株皆可去除土壤中戴奧辛。以混合菌劑添加於不同環境土壤，測試之最佳操作條件為土壤pH 8.1、粒徑93.5 µm，戴奧辛毒性去除率分別達34%以及26%。本研究利用土壤中DNA含量表示微生物之數量與活性，結果顯示在培養21天後表現出最佳活性的土壤條件是pH 6.3與粒徑31 µm。
根據本研究結果可得知受戴奧辛污染之土壤中，確實存在好氧性降解高氯數戴奧辛之多樣化菌群，進一步由菌株生理特性、土壤環境與微生物數量探討之結果得知，土壤環境中的汞、pH以及土壤粒徑與有機質對於微生物之生長有極大的影響，可能直接或間接影響到菌群結構與戴奧辛降解之效率，此結果可用於未來生物復育整治之參考。

Highly chlorinated dioxins contribute mainly to total toxicity and mass concentration of dioxin congeners emitted to the environments. These persistent organic pollutants are chemically stable, low soluble, high hydrophobic, and recalcitrant to microbial degradations, so as to be easily accumulated in higher organisms via food chains, which in turn greatly affect health and ecology. Octachlorodibenzofuran (OCDF) fully substituted with chlorine as a model congener was studied. The aims of this study are to develop the bacteria agents capable of degrading OCDF aerobically and apply them to effectively remedy dioxin-contaminated soil.
The contaminated soil, in which more than 90% of total toxic dioxins (toxicity level, 6.4 µg I-TEQ/kg) were octa-chlorinated dioxins was used for isolating potential OCDF degraders. Totally, 40 aerobic cultures from 102 bacterial colonies were obtained with OCDF as the substrate, and phylogenetically classified over three phyla and thirteen genera. This result demonstrated that the diversified functional microbes can tolerate the high concentrations of dioxin in soil. With high potential of those isolates capable of dioxin degradation, six cultures from the genera Bacillus, Rhodococcus, Micrococcus and Mesorhizobium and from Sphingomonadaceae were demonstrated with excellent degradation capacities of OCDF. The average degradation efficiency were 26%-43% within a 21-d incubation. This is the first report, showing diversified bacteria for aerobic degradation of OCDF. Further, the specialists of Bacillus, Rhodococcus, Micrococcus and Pseudomonas were applied to actual dioxin-contaminated soil to study the effectiveness of dioxin degradations. After 21 days of incubation, the total toxicity level was decreased by 17%, 15%, 17% and 42%, respectively. The result showed that the soil dioxins can be detoxified by these bacteria under aerobic conditions. The mixed bacterial agents were then used for degradation of soil dioxins under various conditions. The results suggested that with the conditions at pH 8.1 and soil size of 93.5 µm, the removal of soil dioxins achieved the highest, with average toxicity loss of 34% and 26%. By measuring the amount of bacterial DNA in soil, it was suggested that microbial quantity can substantially increase at the conditions of soil size at 31µm and pH 6.3.
In conclusion, this study uncovered that the OCDF degraders could be developed within complex microbial community in the contaminated soil. The factors such as bioavailable mercury, pH and size of soil particle (or organic matter) were determinant for the microbial growth of OCDF degraders in soil and hence the degradation efficiency. The overall results will be valuable for operating bioremediation and bioaugmentation on soil dioxins.

參考文獻
1. Agriculture, U. C. f. 2011, posting date. Nutrient Management for Ornamentals. [Online.]
2. Aranda, C., F. Godoy, J. Becerra, R. Barra, and M. Martinez. 2003. Aerobic secondary utilization of a non-growth and inhibitory substrate 2,4,6-trichlorophenol by Sphingopyxis chilensis S37 and sphingopyxis-like strain S32. Biodegradation 14:265-274.
3. Armengaud, J., B. Happe, and K. N. Timmis. 1998. Genetic analysis of dioxin dioxygenase of Sphingomonas sp. Strain RW1: catabolic genes dispersed on the genome. J Bacteriol 180:3954-3966.
4. Ashrafosadat Hatamian-Zarmia, S. A. S., Ebrahim Vasheghani-Farahania, Saman Hosseinkhanib, Abdolrahman Emamzadehb. 2009. Extensive biodegradation of highly chlorinated biphenyl and Aroclor 1242 by Pseudomonas aeruginosa TMU56 isolated from contaminated soils. International Biodegradation & Biodegradation 63:788-794.
5. Bernhardt, D., and H. Diekmann. 1991. Degradation of dioxane, tetrahydrofuran and other cyclic ethers by an environmental Rhodococcus strain. Appl Microbiol Biotechnol 36:120-123.
6. Boopathy, R. 2000. Factors limiting bioremediation technologies. Bioresource Technol 74:63-67.
7. Boopathy, R., and J. Manning. 1999. Surfactant-enhanced bioremediation of soil contaminated with 2,4,6-trinitrotoluene in soil slurry reactors. Water Environment Research 71:119-124.
8. Borodina, E., M. J. Cox, I. R. McDonald, and J. C. Murrell. 2005. Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil. Environ Microbiol 7:1318-1328.
9. Bunge, M., L. Adrian, A. Kraus, M. Opel, W. G. Lorenz, J. R. Andreesen, H. Gorisch, and U. Lechner. 2003. Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium. Nature 421:357-360.
10. Bunge, M., and U. Lechner. 2009. Anaerobic reductive dehalogenation of polychlorinated dioxins. Appl Microbiol Biotechnol 84:429-444.
11. Cao, J., H. Guo, H. M. Zhu, L. Jiang, and H. Yang. 2008. Effects of SOM, surfactant and pH on the sorption-desorption and mobility of prometryne in soils. Chemosphere 70:2127-2134.
12. Chang, J. S., and W. S. Law. 1998. Development of microbial mercury detoxification processes using mercury-hyperresistant strain of Pseudomonas aeruginosa PU21. Biotechnol Bioeng 57:462-470.
13. Cho, Y. G., S. K. Rhee, and S. T. Lee. 2000. Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41. Biodegradation 11:21-28.
14. Correa, O. S., E. A. Rivas, and A. J. Barneix. 1999. Cellular envelopes and tolerance to acid pH in Mesorhizobium loti. Current microbiology 38:329-334.
15. Dabrock, B., M. Kesseler, B. Averhoff, and G. Gottschalk. 1994. Identification and characterization of a transmissible linear plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism. Appl Environ Microbiol 60:853-860.
16. de Carvalho, C. C. 2012. Adaptation of Rhodococcus erythropolis cells for growth and bioremediation under extreme conditions. Research in microbiology 163:125-136.
17. Dodge, A. G., L. P. Wackett, and M. J. Sadowsky. 2012. Plasmid localization and organization of melamine degradation genes in Rhodococcus sp. strain Mel. Appl Environ Microbiol 78:1397-1403.
18. Evans, C. S., and B. Dellinger. 2005. Mechanisms of dioxin formation from the high-temperature oxidation of 2-chlorophenol. Environmental Science & Technology 39:122-127.
19. Feng, Y. S., P. C. Chen, F. S. Wen, W. Y. Hsiao, and C. M. Lee. 2008. Nitrile hydratase from Mesorhizobium sp F28 and its potential for nitrile biotransformation. Process Biochem 43:1391-1397.
20. Fetzner, S. 1998. Bacterial degradation of pyridine, indole, quinoline, and their derivatives under different redox conditions. Appl Microbiol Biot 49:237-250.
21. Fukuda, K., S. Nagata, and H. Taniguchi. 2002. Isolation and characterization of dibenzofuran-degrading bacteria. FEMS Microbiol Lett 208:179-185.
22. Futamata H., U. T., Yoshida N., Yonemitsu Y., Hirashi A. 2004. Distribution of dibenzofuran-degrading bacteria in soils polluted with different levels of polychlorinated Dioxins. Microbes Environ 19:172-177.
23. Godoy, F. A., M. Bunster, V. Matus, C. Aranda, B. Gonzalez, and M. A. Martinez. 2003. Poly-beta-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37. Letters in applied microbiology 36:315-320.
24. Hoekstra, E. J., H. De Weerd, E. W. B. De Leer, and U. A. T. Brinkman. 1999. Natural formation of chlorinated phenols, dibenzo-p-dioxins, and dibenzofurans in soil of a Douglas fir forest. Environmental Science & Technology 33:2543-2549.
25. Hong, H. B., I. H. Nam, K. Murugesan, Y. M. Kim, and Y. S. Chang. 2004. Biodegradation of dibenzo-p-dioxin, dibenzofuran, and chlorodibenzo-p-dioxins by Pseudomonas veronii PH-03. Biodegradation 15:303-313.
26. Howard, G. T., C. Ruiz, and N. P. Hilliard. 1999. Growth of Pseudomonas chlororaphis on apolyester–polyurethane and the purification andcharacterization of a polyurethanase–esterase enzyme. International Biodeterioration & Biodegradation 43:7-12.
27. Kasuga, K., H. Habe, J. S. Chung, T. Yoshida, H. Nojiri, H. Yamane, and T. Omori. 2001. Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63. Biochem Biophys Res Commun 283:195-204.
28. Kearney, P. C., E. A. Woolson, and Ellingto.Cp. 1972. Persistence and Metabolism of Chlorodioxins in Soils. Environmental Science & Technology 6:1017-&.
29. Kimura, N., and Y. Kamagata. 2009. Impact of dibenzofuran/dibenzo-p-dioxin amendment on bacterial community from forest soil and ring-hydroxylating dioxygenase gene populations. Appl Microbiol Biotechnol 84:365-373.
30. Kimura, N., W. Kitagawa, T. Mori, N. Nakashima, T. Tamura, and Y. Kamagata. 2006. Genetic and biochemical characterization of the dioxygenase involved in lateral dioxygenation of dibenzofuran from Rhodococcus opacus strain SAO101. Appl Microbiol Biotechnol 73:474-484.
31. Kimura, N., and Y. Urushigawa. 2001. Metabolism of dibenzo-p-dioxin and chlorinated dibenzo-p-dioxin by a gram-positive bacterium, Rhodococcus opacus SAO101. Journal of bioscience and bioengineering 92:138-143.
32. Kutz, F. W., D. G. Barnes, D. P. Bottimore, H. Greim, and E. W. Bretthauer. 1990. The International Toxicity Equivalency Factor (I-Tef) Method of Risk Assessment for Complex-Mixtures of Dioxins and Related-Compounds. Chemosphere 20:751-757.
33. Lane, D. J. 1991. 16S/23S rRNA sequencing. In M. G. E. Stackebrandt (ed.), Nucleic Acid Techniques in Bacterial Systematics. Wiley.
34. LaRoe Sarah L., B. W., Jong-In Han. 2010. Isolation and Characterization of a Novel Polycyclic Aromatic Hydrocarbon–Degrading Bacterium, Sphingopyxis sp. strain M2R2, Capable of Passive Spreading Motility Through Soil. Environ Engin Sci 27:505-512.
35. Li, H., J. Chen, W. Wu, and X. Piao. 2010. Distribution of polycyclic aromatic hydrocarbons in different size fractions of soil from a coke oven plant and its relationship to organic carbon content. J Hazard Mater 176:729-734.
36. Liang, B., Y. K. Zhao, P. Lu, S. P. Li, and X. Huang. 2010. Biotransformation of the diphenyl ether herbicide lactofen and purification of a lactofen esterase from Brevundimonas sp. LY-2. Journal of agricultural and food chemistry 58:9711-9715.
37. Lin, C., L. Gan, and Z. L. Chen. 2010. Biodegradation of naphthalene by strain Bacillus fusiformis (BFN). J Hazard Mater.
38. Liu, F., and D. E. Fennell. 2008. Dechlorination and detoxification of 1,2,3,4,7,8-hexachlorodibenzofuran by a mixed culture containing Dehalococcoides ethenogenes Strain 195. Environ Sci Technol 42:602-607.
39. Masunaga, S., T. Takasuga, and J. Nakanishi. 2001. Dioxin and dioxin-like PCB impurities in some Japanese agrochemical formulations. Chemosphere 44:873-885.
40. McKay, G. 2002. Dioxin characterisation, formation and minimisation during municipal solid waste (MSW) incineration: review. Chem Eng J 86:343-368.
41. Mimura, J., and Y. Fujii-Kuriyama. 2003. Functional role of AhR in the expression of toxic effects by TCDD. Biochim Biophys Acta 1619:263-268.
42. Mukerjee-Dhar, G., M. Shimura, D. Miyazawa, K. Kimbara, and T. Hatta. 2005. bph genes of the thermophilic PCB degrader, Bacillus sp. JF8: characterization of the divergent ring-hydroxylating dioxygenase and hydrolase genes upstream of the Mn-dependent BphC. Microbiology 151:4139-4151.
43. Muthukumar, K., C. Bharath, V. Pugalenthi, and M. Velan. 2009. Biodegradation kinetics of benzoic and anthranilic by Micrococcus sp. Jornal of Scientific & Industrial Research 68:900-903.
44. Nakayama, M., N. Fujita, T. Ohama, S. Osawa, and A. Ishihama. 1989. Micrococcus luteus, a bacterium with a high genomic G + C content, contains Escherichia coli-type promoters. Mol Gen Genet 218:384-389.
45. Nam, I. H., Y. M. Kim, S. Schmidt, and Y. S. Chang. 2006. Biotransformation of 1,2,3-tri- and 1,2,3,4,7,8-hexachlorodibenzo-p- dioxin by Sphingomonas wittichii strain RW1. Appl Environ Microbiol 72:112-116.
46. Potrawfke, T., K. N. Timmis, and R. M. Wittich. 1998. Degradation of 1,2,3,4-tetrachlorobenzene by pseudomonas chlororaphis RW71. Appl Environ Microbiol 64:3798-3806.
47. Reda, A. B., and T. A.-H. Ashraf. 2010. Optimization of Bacterial Biodegradation of Toluene and Phenol Under Different Nutritional and Environmental Conditions. Journal of Applied Sciences 8:1086-1095.
48. Saito, A., T. Iwabuchi, and S. Harayama. 2000. A novel phenanthrene dioxygenase from Nocardioides sp. Strain KP7: expression in Escherichia coli. J Bacteriol 182:2134-2141.
49. Sinkkonen, S., and J. Paasivirta. 2000. Degradation half-life times of PCDDs, PCDFs and PCBs for environmental fate modeling. Chemosphere 40:943-949.
50. Sulistyaningdyah, W. T., J. Ogawa, Q. S. Li, R. Shinkyo, T. Sakaki, K. Inouye, R. D. Schmid, and S. Shimizu. 2004. Metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 BM-3 and its mutant. Biotechnol Lett 26:1857-1860.
51. Valero, M., S. Leontidis, P. S. Fernandez, A. Martinez, and M. C. Salmeron. 2000. Growth of Bacillus cereus in natural and acidified carrot substrates over the temperature range 5-30 degrees C. Food Microbiol 17:605-612.
52. Widada, J., H. Nojiri, T. Yoshida, H. Habe, and T. Omori. 2002. Enhanced degradation of carbazole and 2,3-dichlorodibenzo-p-dioxin in soils by Pseudomonas resinovorans strain CA10. Chemosphere 49:485-491.
53. Yoshida, N., N. Takahashi, and A. Hiraishi. 2005. Phylogenetic characterization of a polychlorinated-dioxin-dechlorinating microbial community by use of microcosm studies. Appl Environ Microb 71:4325-4334.
54. Zamil, S. S., M. H. Choi, J. H. Song, H. Park, J. Xu, K. W. Chi, and S. C. Yoon. 2008. Enhanced biosorption of mercury(II) and cadmium(II) by cold-induced hydrophobic exobiopolymer secreted from the psychrotroph Pseudomonas fluorescens BM07. Appl Microbiol Biotechnol 80:531-544.
55. 蔡子衿、吳清吉、許武榮. 2008. 台灣土壤溫度變化和土壤熱擴散係數推估. 大氣科學 36:83-100.