本研究目的在探討受五氯酚(PCP)污染之土壤利用植物修復之可行性，首先，採用韭菜(Allium tuberosum)、綠豆(Vigna radiat(L.)Wi-l-czek)、狼尾草(Pennisetum alopecuroides)、紫花苜宿(Medicago sativa)等四種植物進行對PCP抗逆境能力之測試。結果顯示韭菜具有最好的降解率，在含PCP濃度為20mg/kg的土壤中，經過35天實驗期後，其去除率可達76%。
第二階段實驗是利用韭菜觀察隨時間變化PCP的降解效果。經330天的溫室盆栽實驗。結果顯示韭菜的種植有助於土壤中微生物及去氫酵素活性的增加，而營養鹽的添加亦可幫助韭菜抵抗土壤中PCP污染所造成的逆境。實驗至第330天，土壤的PCP污染的去除率，以有種植韭菜及添加營養鹽的實驗組最高可達98.4%，土壤中的PCP濃度由原先的42mg kg-1降解至0.68 mg kg-1。
至於以分子生物技術PCR-DGGE分析土壤中微生物的菌相。發現韭菜的栽種及營養鹽的添加可提高微生物的多樣性。而所有添加PCP污染土壤的菌相皆比空白土壤的菌相豐富，推論PCP的添加可刺激土壤微生物的活性。另外，比較根際微生物與非根際的菌相，發現在植物根分泌物的作用下，可提高根際微生物多樣性。

In this study, the phytoremediation techniques are used to treat the soil contaminated by pentachlorophenol(PCP).First, four plants species were selected,including Allium tuberosum, Vigna radiata (L.) Wilczek, Pennisetum alopecuroides, and Medicago sativa to compare their treatment efficiencies for PCP in soil.The experimental results showed that the species of Allium tuberosum presented the highest degradation rate 76% after 35-day test run with the initial concentration of 20mg/kg in soil.
In the second stage,the species of Allium tuberosum was thus selected to run the tests of feasibility of using phytoremediayion to treat the soils contaminated byPCP.During the e xperiment,the pot tests inside a greenhouse were run for 330 days.The result indicated that the species of Allium tuberosum contributed to the increase of microorganism and dehydrogenase activity in the soil. Bisides,we also found that adding with nutrients could help Allium tuberosum to depress the PCP stress.The test with vegetation of Allium tuberosum and addition of nutrients showed that the PCP degradation rate was measured equal to 98.4% with the concentration of PCP degraded from 42mgkg-1 to 0.68mgkg-1 after 330days.
Finally, molecule biotechnology of PCR-DGGE was applied to the test of observing the microbiota in the soils.According to the test results,we found that the diversity of microorganisms could be raised through planting the species of Allium tuberosum. The microbiota in the soils with PCP pollutant have more varieties than the microbiota in soils without vegetation, which was infered that the addition of PCP might stimulate the vitality of microbes in the soils. Moreover, comparing the microbiota on rhizosphere of the plant species and in the bulk soils, it was found that the actitivies of root exudates might be able to increase the varieties of rhizospheric microorganisms.

中文摘要 Ⅰ
英文摘要 Ⅱ
表目錄 Ⅵ
圖目錄 Ⅶ
第一章 前言 1
1.1研究動機 1
1.2研究目的 2
第二章 文獻回顧 3
2.1五氯酚化合物之特性 3
2.2五氯酚對生物的危害 4
2.3環境中五氯酚的來源 5
2.3.1自然污染源 5
2.3.2人為污染源 5
2.4五氯酚在環境中的傳輸及分佈 8
2.5五氯酚化合物之管制標準 13
2.6植物介紹 14
2.7植物修復 15
2.7.1植物修復機制 16
2.7.2影響植物修復的因素 19
2.7.3植物與微生物交互作用 20
2.7.4植物根分泌物 21
2.7.5去氫酵素活性 22
2.7.6植物復育的優缺點 22
2.7.7處理成本 23
2.7.8應用案例 23
2.8分子生物技術應用於土壤微生物分析的重要性 24
2.8.1聚合酶連鎖反應 24
2.8.2變性梯度膠體電泳 25
第三章 實驗材料與方法 27
3.1實驗流程 27
3.2植物培養 28
3.3土壤來源與前處理 28
3.4土壤的基本性質 29
3.4.1土壤酸鹼值 29
3.4.2含水率 30
3.4.3有機物含量 30
3.4.4土壤有機碳 30
3.4.5土壤有機氮 31
3.4.6粒徑分析 31
3.5污染土壤配置 32
3.6土壤採樣與樣品保存 33
3.7植物初步篩選 33
3.8不同五氯酚濃度土壞試驗 33
3.9 五氯酚分析方法 33
3.10土壤分析方法 34
3.10.1 五氯酚土壤萃取 34
3.10.2去氫酵素活性 35
3.10.3土壤總菌落數 35
3.11植體分析 36
3.11.1植體葉綠素 36
3.11.2生物質量 36
3.12菌相分析 36
3.12.1土壤中DNA萃取 36
3.12.2 DNA瓊脂膠體電泳檢視 37
3.12.3 DNA濃度與純度測定 38
3.12.4 DNA純化 38
3.12.5聚合酶連鎖反應 39
3.12.6 PCR-16SrDNA純化 39
3.12.7 PCR-16S rDNA片段瓊脂膠體電泳檢視 40
3.12.8變性梯度膠體電泳(DGGE) 40
3.12.9以Quantity One Version 4.5軟體分析DGGE電泳圖譜 41
3.13實驗之QA/QC 42
3.14實驗相關器材 43
第四章 結果與討論 44
4.1土壤基本性質分析 44
4.2耐五氯酚植物初步篩選 45
4.3耐五氯酚植物不同濃度試驗 46
4.4植物生長受五氯酚的影響 48
4.5去氫酵素活性 50
4.6土壤總菌落數 51
4.7菌相分析 53
4.8根際土壤與非根際土壤之菌相比較 61
4.9以Quantity One Version 4.5軟體分析DGGE電泳圖譜 63
4.10五氯酚隨時間降解試驗 68
第五章 結論與建議 71
5.1結論 71
5.2建議 72
參考文獻 74
附錄 80























表目錄
表2-1 五氯酚的物化特性 4
表2-2五氯酚主要用途 7
表2-3環保署公告五氯酚毒性化學物質一覽表 13
表2-4現行環保署規定五氯酚各項管制標準 14
表2-5植物復育應用機制 18
表2-6植物復育的優缺點 22
表3-1 Hoagland solution營養鹽成份 28
表3-2 PCR反應試劑比例 39
表3-3 16SrDNA primers 39
表3-4 Touchdown PCR反應條件 40
表3-5 DGGE變性膠配製成份 41
表3-6 實驗相關器材及型號 43
表4-1土壤基本性質分析 45











圖目錄
圖2-1 五氯酚的結構 3
圖2-2五氯酚在環境中的命運 9
圖2-3五氯酚之光解反應途徑 10
圖2-4五氯酚生物分解反應途徑 12
圖2-5植物修復示意圖 18
圖3-1實驗規劃流程圖 27
圖3-2紫花苜蓿 29
圖3-3綠豆 29
圖3-4韭菜 29
圖3-5狼尾草 29
圖3-6五氯酚污染土壤配置圖 32
圖4-1耐五氯酚植物初步篩選 46
圖4-2耐五氯酚植物不同濃度試驗 47
圖4-3植物受不同五氯酚污染土壤之葉綠素a含量變化 47
圖4-4植物生長高度比較 49
圖4-5植物葉片質量比較 49
圖4-6土壤中去氫酵素活性變化 51
圖4-7土壤中總菌落數含量 52
圖4-8原始土壤DGGE剖面圖 55
圖4-9第30天土壤DGGE剖面圖 56
圖4-10第60天土壤DGGE剖面圖 57
圖4-11第90天土壤DGGE剖面圖 58
圖4-12第150天土壤DGGE剖面圖 59
圖4-13第330天土壤DGGE剖面圖 60
圖4-14第330天根際土壤與非根際土壤之DGGE剖面比較圖 62
圖4-15原始土壤DGGE電泳圖的UPGMA歸群分析樹狀圖 64
圖4-16第30天DGGE電泳圖的UPGMA歸群分析樹狀圖 64
圖4-17第60天DGGE電泳圖的UPGMA歸群分析樹狀圖 65
圖4-18第90天DGGE電泳圖的UPGMA歸群分析樹狀圖 65
圖4-19第150天DGGE電泳圖的UPGMA歸群分析樹狀圖 66
圖4-20第330天DGGE電泳圖的UPGMA歸群分析樹狀圖 66
圖4-21根際土壤與非根際土壤DGGE電泳圖UPGMA歸群分析樹狀圖67
圖4-22土壤中五氯酚去除率 70
圖4-22土壤中五氯酚殘留量 70

王一雄，1997，“土壤環境污染與農藥”，台北市，明文書局，177~187頁。
王月雲、陳是瑩、童武夫，1994，“植物生理學實驗”，台北市，藝軒出版社，93~95頁。
林良平，1987，“土壤微生物學(上/下冊)初版”，台北市，南山堂出版社，533~550頁。
林畢修平，2000，“環境中含氯有機污染源生物復育之可行性介紹”，中華民國微生物學會微生物與環境賀爾蒙研討會，56-69頁。
林宏達，2004，“以植物修復技術處理遭受溢油污染濕地土壤之研究”，國立中山大學海洋環境及工程學系碩士論文。
陳俊位，2003，“台中區農業專訊”，第四十期，4-11頁。
郭雅惠，2003，“玉米水稻輪種系統下不同施肥管理對根圈土壤酵素活性之影響”，台灣大學農業化學研究所碩士論文。
喬云發，2004，“植物根系分泌物研究發展”，中國科學院東北地理與農業生態研究所研究論文。
游勝傑，1998，“分子生物學應用於營養鹽去除程序中微生物族群的判定”，國立中央大學環境工程研究所博士論文。
廖文彬，1997，“砂質土壤中黏土礦物與含水勢能對酚生物分解之影響”，國立中興大學環境工程學系國科會研究報告。
蔡啟堂，2002，“五氯酚分解菌之生理特性探討”，國立中山大學生物科學學系碩士論文。
謝昶毅，2004，“以PCR-DGGE技術分析石油碳氫化合物污染地下水之微生物相”，國立中山大學生物科學學系碩士論文。
行政院農委會，民眾專區:http://www.tlri.gov.tw/book/Issue/issue16.asp#1
行政院環保署網站，
土壤及地下水整治法令規章:http://ww2.epa.gov.tw/SoilGW/index.asp
毒性化學物質: http://flora2.epa.gov.tw/prog/database/7333.htm

Alker,G.R. and Cardellino,R.W.2001,“Phytoremediation of Contaminated Sites UsingWood Biomass.”The Florida Center for Solid and Hazardous Waste
Management,Report:01-03.

Anderson,T.A.,Kruger,E.L. and Coats,J.R.1994,“Enhanced degradation of a mixture of three herbicides in the rhizosphere of a herbicide-tolerant plant.”
.Chemosphere, 28:1551-1557.

Anne,K.,Kamma,M.W.,Sorenm,C. and Soren J.S.2001,“The effect of long-term mercury pollution on soil microbial community.”FEMS Microbiology Ecological
,36:11-9.

Arsenault,R.D.1976,“Pentachlorophenol and containedchlorinated in the: enviro-
-nment A study of environmental fate, stability, and significance when used in
wood preservation.”Am. Wood Preservation,20:122-148.

Assmuth,T. and Vartiainen,T.1994,“Concentrations of 2,3,7,8-Chlorinated
-p-Dioxins and Dibenzofurans at Landfills and Disposal Sites for Chlorophenolic Wood Preservative Wastes.”Chemosphere,28(5):971-979.

Betts,K.1997,“Phytoremediation project taking up TCE.”Environmental Science and Technology,31(8):347A.
Boyajian,G.E. and Carreira,L.H.1997,“Phytoremediation:a clean transition from laboratory to marketplace.”Na.Biotechnology,15:127-128.

Burken,J.G. and Schnoor,J.L.1996,“Phytoremediation : plant uptake of atrazine and role of root exudates.”Environmental Engineering,1222:958-963.

Crosby,D.G.1981,“Environmental chemistry of pentachlorphenol.” Chemosph-
-ere,53:1051-1080.

Ferro, A.M., Sims, R.C. and Bugsbee,B.1994,“Hycrest Crested WheatgrassAccelerates the Degradation of Pentachlorophenol in Soil.”Journal of Environm-
-ental Qulity,23:272-279.

Fletcher,J.S. and Hedge,R.S.1995,“Release of phenols by perennial plant root and their potential importance in bioremediation.”Chemosphere,31:3000-3016.

Fortina,N.,Beaumiera,D.,Leeb,K. and Greera,C.W.2004,“Soil washing impro-
-ves the recovery of total community DNA from polluted and high organic content sediments.”Journal of Microbiological Methods,56:181-191.

Hans-Holger L. and Martin A.1999,“Plant-promoted pyrene degradation insoil.”Chemosphere,40:7-10.

Hurst,C.J.,Sorensen,D.L.,Sims,R.C.,Mclean,J.E.,Sims,J.L. and Huling,S.1997,
“Soil Gas Oxygen Tension and Pentachlorophenol Biodegradation.”Journal of Environmental Engineering,123(4):364-370.

Itziar A. and Carlos G.2001,“Phytoremediation of organic contaminants in soils.
”Bioresource Technology, 79:273-276.

Jennifer L. K.,John N. K.,Hung L. and Jack T.T.2005,“The effects of perenni-
-al ryegrass and alfalfa on microbial abundance and diversity in petroleum contam-
-inated soil.”Environmental Pollution,133:455–465.

Katayama A. and Matsumura F.1993,“Degradation of organochlorine pesdcid-
-es particularly endosulfun by Trichoderma harzianum.”Environmental Toxicology and Chemistry, 12:1059-1065.

Lamparski, L.L., Stehl, R.H. and Johnson, R.L.1980,“Photolysis of Penatchlo-
-rphenol-treated wood. Chlorinated dibenzo- p-dioxin formation.”EnvironmentalScience Technology,14:196-200.

Lee,A.N. and Charles,M.R.2004,“Phytodegradation of organic compounds.”
Current Opinion in Biotechnology,5:225–230.


Macek,T.,Macková,M. and Ká.J.2000,“Exploitation of plants for the removal of organics in environmental remediation.”Biotechnology Advances,18:23–34.

MacFarlane,J.C.,Pfleeger,T. and Fletcher,J.1990,“Effect uptake and distribute-
-on of nitrobenzene in several terresrtial plants.”Environmental Toxicology and Chemistry,9:513-520.

Mileski,G.J.,Bumpus,J.A.,Jurek,M.A. and Aust,S.D.1988,“Biodegradation ofpentachlorophenol by the white-rot fungus Phanerochaete chrysosporium.”
Applied and Environmental Microbioogyl,54(12):2885–2889.

Namkoong,W.,Hwang,E.Y.,Park,J.S. and Choi,J.Y.2002,“Bioremediation ofdiesel-contaminated soil with composting.”Environmental Pollution,119:23-31.

Niemi, R.M.,Heiskanen, I.,Wallenius, K. and Lindström,K.2001,“Extractionand purification of DNA in rhizosphere soil samples for PCR- DGGE analysis ofbacterial consortia.”Journal of Microbiological Methods,45:155–165.

.Ogram,A.,Sayler,G.S.,Barkay,T.1987,“The extraction and purification of mic-
-robial DNA from sediment.”Journal of Microbiological Methods,7:57-66.

Page,A.L.,Miller,R.H. and Keeney,D.R.1982,“Methods of soil analysis.”Part II, 2nd ed., ASA-SSSA, Wescosin.

Pradhan,S.P.,Conrad,J.R.,Paterek,J.R. and Srivastava,V.J.1998,“Potential ofphytoremediation for treatment of PAHs in soil at MGP sites.”Journal of SoilContamination,7:467-480.

Ruckdeschel G. and Renner G.1987,“Effects of pentachlorophenol and some of its known and possible metabolites on different species of bacteria.”Applied and Environmental Microbioogyl,53(11):2689-2692.

Salt,D.E.,Smith,R.D. and Raskin,I.1998,“Phytoremediation.Annu Review.”
Plant Physiologists,49:643-668.

Schnoor,J.L.,Licht,L.A.,McCutcheon,S.C.,Wolfe,N.L.,Carreira,L.H.1995,“Phytoremediation of organic and nutrient contaminants.”Environmental Toxico-
-logy and Chemistry,29:318-323.

Schroll,R.,Bierling,B.,Cao,G.,Dorfler,U.and Lahaniati,M.1994,“Uptake path-
-ways of organic chemicals from soil by agricultural plants.”Chemosphere,28:
297-303.

Seigle-Murandi F.,Steiman R.,Benoit-Guyod J.L.and Guiaud P.1993,“Fungal degradation of pentachlorophenol by micromycetes.”Journal of Biotechnology,30
:27-35.

Sridhar S.,Victor F.M. and Steven C.M.1999,“Phytomediation:An ecological solution to organic chemical contamination.”Ecological Engineering,18:647-658.

SUZUKI, T.1983,“Metabolism of pentachlorophenol (PCP) by soil microorgani-
sms.”Nippon Noyaku Gakkaishi,8:385-394.

Takashi N.,Takayuki M.,Yoshikatsu S. and Isamu Y.2004,“Biotransformation of pentachlorophenol by Chinese chive and a recombineant derivative of its rhizos-
-pherecompent microorganism, Pseudomonas gladioli M-2196.”Soil Biology and Biochemistry,36:787-795.

Trevors,J.T.1982,“Effect of temperature on the degradation ofpentachlorophen-
-ol by Pseudomonas spp.”Chemosphere,11:471-475.

Tuomela,M.,Lyytika¨inen,M.,Oivanen,P.and Hatakka,A.1999,“Mineralization and conversion of pentachlorophenol (PCP) in soil inoculated with the white-rot fungus Trametes versicolor.” Soil Biology and Biochemistry ,31:65–74.

Waltera,M.,Boulb,L. and Chongc,R.C.2004,“Forda Growth substrate selection and biodegradation of PCP by New Zealand white-rot fungi.”Journal of Environ-
mental Management,71:361–369

Wong, A.S. and Crosby, D.G.1978,“Pentachlorophenol:chemistry pharmacolog-
-y and environmental toxicology.”New York, London, Plenum Press,19-25.

Wylie,G.D.,Finger,S.E. and Grawford,R.W.1990,“Toxicity of Municipal Waste-
-water Effluents Contaminated by Pentachlorophenol in Southwest Missouri.” Environmental Pollution,64(1):43-53.

Yan H., Jianming X., Caixian T. and Yuping W.2005,“Facilitation of pentach-
lorophenol degradation in the rhizosphereof regrass (Lolium perenne L.).”Soil
Biology and Biochemistry,37:2017–2024

Yeates C,Gillings M.R.,Davison A.D.,Altavilla N.and Veal D.A.1998,“Methods
for microbial DNA extraction from soil for PCR amplification.”Biological Proced-
-ures Online,1:40-47.

IPCS INCHEM.2005,International Programe on Chemical Safety:
http://www.inchem.org/documents/ehc/ehc/ehc71.htm#SubSectionNumber:2.1.1

NAVFAC,Environmentalservices:
http://enviro.nfesc.navy.mil/scripts/WebObjects.exe/erbweb
Phytokinetics:www.phytokinetics.com/techs.htm