隨著採油廢水和含油固體廢物的排放，土壤中的石油污染越來越嚴重，至今在台灣，公告為污染控制場址就高達642處。生物復育是一種經濟而且無二次污染的整治技術，具有其他復育技術難以比擬的優勢，並且在先前實驗室之研究，乃利用低污染之土壤額外添加油污的方式來模擬實場TPH降解情形。因此，為了了解以往之實驗設計是否能反應出自然環境中之TPH降解情形，故本研究以污染年限長之原址污染土與污染年限短之低污染土中額外添加油污此兩組土樣進行比較，透過批次實驗模擬離場土耕法以探討污染年限在TPH降解中所扮演的角色。由實驗結果中發現，以批次實驗操作118天後，原址污染土之平均總去除率達57％，而額外添加油污土之平均總去除率達76％，兩者之第一階段之反應速率常數分別為-0.0196 day-1及-0.0253 day-1。因此污染年限愈久之TPH其生物降解效果越差，而額外添加油污土之實驗結果未能完全模擬原址污染土之降解情形。
因此，為了模擬原址污染土之TPH降解情形，我們針對整治前調查結果取得另一批菌量較低之原址污染土，以生物添加及生物刺激法觀察TPH之降解情形，經批次實驗操作126天後，生物刺激法中之添加營養鹽組別產生最佳之去除率達81％且其第一階段反應速率常數達-0.0414day-1，而第二階段反應速率常數最高之組別各為添加油分解菌KH1及添加廚餘組，其數值分別為-0.0124day-1及-0.0119day-1，以此批次實驗結果用於原址同性質污染之土壤，推測於第一階段添加優勢菌種或適當營養鹽，配合第二階段添加油分解菌KH1或廚餘，將可縮短TPH降解之整治時程。
同時為了了解批次實驗中微生物族群之變化，我們利用末端限制酶片段長度多型性分析方法來監測各復育試驗中菌群之變化。在T-RFLP圖譜上，發現優勢菌種之片段長度均落在123bp、213bp及514bp，且其共通點為其操作時程處於TPH降解快速期，同批批次實驗中其優勢菌種皆相同，故由T-RFLP圖譜分析可得知在TPH降解過程中之菌群變化以找出當中之優勢菌種。
最後，為了了解批次實驗中各變數與菌群變化間之相關性，透過統計軟體對T-RFLP之數據進行多變量分析，以期能夠提供幫助生物復育進行之相關資訊。在多變量分析結果中，在各變數中我們發現污染年限、降解天數及污染物性質之差異與TPH的降解具有相當之關聯性，故當對未知土壤進行整治時，可由針對上述之三項變數進行調查，以提供評估污染整治時程之重要資訊。

Oil pollution in soil became more and more seriously due to the discharge of oil extraction wastewater and oil-contained solid wastes, and there were 642 pollution sites announced nowadays in Taiwan. Bioremediation is an economical and ‘green’ solution to the problem of oil pollution. In previous studies in our lab, we utilized low polluted soil with adding extra oil to mimic the condition of TPH degradation in nature. In this study, to investigate that whether past experiments could response to the real condition of TPH degradation, we used batch tests to simulate ex situ land farming for TPH degradation in polluted long soil and low polluted soil added extra oil to find the role of time as soil polluted in TPH degradation. In our results, as operating batch tests for 118 days the average total removal up to 57% in polluted long soil and 76% in low polluted soil with adding extra oil, and the first stage reaction rate constant was -0.0196 day-1 and -0.0253 day-1. Consequently, the efficiency of TPH degradation decreased with time after the soil was polluted and the results in experiment of low polluted soil with adding extra oil were not enough to stimulate the actual condition of TPH degradation.
In order to mimic the condition of TPH degradation in polluted soil naturally, we tried to utilize another soil sample with low concentration of microbes to find the efficiency of TPH degradation in that after the treatment of biostimulation and bioaugmentation. As operating batch tests for 126 days the average total removal up to 81% in soil after treatment of biostimulation , and the first stage reaction rate constant was -0.0414 day-1. After the treatment of bioaugmentation by adding diesel-eliminating strains KH1 and kitchen wastes performed the best removal of TPH and the second stage reaction rate constant of them was -0.0124 day-1 and -0.0119 day-1. Therefore we supposed that the operating process in TPH polluted soil would be biostimulation of adding suitable nutrients in the first and subsequently the treatment of bioaugmentation by adding diesel-eliminating strains KH1 and kitchen wastes could reduce the required time of biodegradation. Simultaneously we tried to detect the changes of microorganisms in batch tests by terminal restriction fragment length polymorphism (T-RFLP).We found that the major fragments were at 123bp, 213bp, and 514bp in the TPH rapid degradation phase, and corresponding with other batch tests. Thus, we could understand the changes of microorganisms in polluted soil as degradation of TPH by T-RFLP diagram to find the dominant strains in biodegradation process.
Finally, in order to understand the relationship between the variables of batch and microbial population variation, we carried multivariate statistical analysis out to analyze the data of T-RFLP diagram. We considered that there results would provide us the related information to help the efficiency of TPH degradation. We demonstrated that the relationship in time as soil polluted in TPH degradation, the time of the biodegradation, pollutants properties and the efficiency of TPH degradation in the results of multivariate statistical analysis. While we proceeded to choose the treatment of unknown polluted soils in the future, we should investigate into these three variables to provide us the important information of estimating the time required for treatment.

丁克強，駱永明，生物修復石油污染土壤，土壤，33(4):179~184 (2001)
毛麗華，呂華，李子君，石油污染土壤生物強化修復的機制與實施途徑，有色金屬，58(1):92~96 (2006)
台灣中油股份有限公司，石油教室，四通八達輸油網—油管與泵站，http://www.cpc.com.tw/big5/content/index01.asp?sno=198&pno=108 (2008)
行政院環保署，土壤中總石油碳氫化合物(TPHs)檢測之研究 (2003)
行政院環保署，全國十年以上加油站及大型儲槽潛在污染源調查計畫 (2001)
馬文漪，楊柳燕，環境微生物工程，南京:南京大學出版社，250~267 (1998)
陳姍玗，生物界面活性劑-鼠李糖酯之醱酵策略開發，國立成功大學化學工程所博士論文 (2007)
陳順宇，多變量分析(四版)，華泰書局，台北 (2005)
楊明潔，柴油分解菌與生物界面活性劑應用於土壤地下水復育之研究，國立成功大學環境工程所碩士論文 (2006)
經濟部九五年度學界開發產業技術細部計畫書，系統化環境分子生物技術之應用三年計畫，53~54 (2006)
經濟部工業局，石油碳氫化合物土壤及地下水污染預防與整治技術手冊 (2007)
廖翊廷，應用生物刺激及菌種添加之離場土耕法整治總石油碳氫化物污染土壤之模場研究，國立成功大學環境工程所碩士論文 (2007)
蔡見能，地面儲槽洩漏污染潛勢評估及傳輸模式研究，朝陽科技大學環境工程與管理所碩士論文 (2005)
環保署土壤及地下水污染整治基金管理委員會，場址資訊，土壤及地下水污染整治網，http://sgw.epa.gov.tw/public/site_chart.asp (2008)

Antizar-Ladislao, B., Beck, A. J., Spanova, K., Lopez-Real, J., and Russell, N. J. The influence of different temperature programmes on the bioremediation of polycyclic aromatic hydrocarbons (PAHs) in a coal-tar contaminated soil by in-vessel composting. Journal of Hazardous Materials. 144:340~347 (2007)
Banat, I.M. Characteristics of biosurfactants and their use in pollution removal-state of art. Acta Biotechnol. 15:251~267 (1995)
Blackwood, C. B., and Buyer J.S. Evaluating the physical capture method of terminal restriction fragment length polymorphism for comparison of soil microbial communities. Soil Biology and Biochemistry. 39:590~599 (2007)
Blackwood, C. B., Marsh, T. L., and Kim, S. H. Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities. Applied and Environmental Microbiology. 69(2):926~932 (2003)
Boszczyk-Maleszak, H., Zabost, A., Wolicka, D., and Kacieszczenko, J. Effectiveness of biodegradation of petroleum products by mixed bacterial populations in liquid medium at different pH values. Polish Journal of Microbiology. 55:69~73 (2006)
Burlage, R. S. Emerging technologies：Bioreporters, biosensors and microprobes. Washington D C (1997)
Carmody O., Frost R., Xi Y., and Kokot S. Surface characterization of selected sorbent materials for common hydrocarbon fuels. Surface Science. 601:2066~2076 (2007)
Chen, C. L., Macarie, H., Ramirez, I., Olmos, A., Ong, S. L., Monroy, O., and Liu, W. T. Microbial community structure in a thermophilic anaerobic hybrid reactor degrading terephthalate. Microbiology. 150:3429~3440 (2004)
Chiou, C. T., Kile, D. E., and Malcolm, R. L. Sorption of Vapors of Some Organic Liquids on Soil Humic-Acid And Its Relation to Partitioning of Organic-Compounds in Soil Organic-Matter. Environmental Science and Technology. 22:298~303 (1988)

Coates, J. D., and Woodward, J. Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments. Applied and Environmental Microbiology. 63:3589~3593 (1997)
Cookson, E. W. Policing waterways for hydrocarbons. Sensor Review. 3:21~23 (1995)
Deziel, E., Paquette, G., and Villemur, R. Biosurfactant production by a soil pseudomonas strain growing on polycyclic aromatic hydrocarbons. Applied and Environmental Microbiology. 62:1908~l912 (1996)
Dollhopf, S.L., Hashsham, S.A., and Tiedje, J.M. Interpreting 16S rDNA T-RFLP Data: Application of Self-Organizing Maps and Principal Component Analysis to Describe Community Dynamics and Convergence. Microbial Ecology. 42:495~505 (2001)
Dunbar, J., Ticknor, L. O., and Kuske, C. R. Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities. Applied and Environmental Microbiology. 67(1):190~197 (2001)
Dunber, J., Ticknor, L. O., and Kuske, C. R. Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Applied and Environmental Microbiology. 66(7):2943~2950 (2000)
Fetter, C. W. Contaminant Hydrogeology, 2nd ed., Prentice-Hall, Inc (1998)
Franco, I., Contin, M., and Bragato, G. Microbiological resilience of soils contaminated with crude oil. Geoderma. 121:17~30 (2004)
Frankenberger Jr., W.T. The need for a laboratory feasibility study in bioremediation of petroleum hydrocarbons, In:Calabrese, E.J., Kostecki, P.T.(Eds.), Hydrocarbon contaminated soils and groundwater . Lewis, Boca Raton, FL. 237~293 (1992)

Fusey, P., and Oudot, J. Relative influence of physical removal and biodegradation in the depuration of petroleum-contaminated seashore sediments. Marine Pollution Bulletin. 15:136~141 (1984)
Gallego, J. L.R., Loredo, J., Llamas, J. F., V´azquez, F., and S´anchez, J. Bioremediation of diesel-contaminated soils: Evaluation of potential in situ techniques by study of bacterial degradation, Biodegradation. 12:325~335 (2001)
Giri, A. V., Anandkumar, N., Muthukumaran, G., and Pennathur, G. A novel medium for the enhanced cell growth and production of prodigiosin from Serratia marcescens isolated from soil. BMC Microbiology. 4:11 (2004)
Harvey, S.,Elashvili, I., and Valdes, J. Enhanced removal of Exxon Valdez spilled oil form Alaskan gravel by a microbial surfactant. Biotechnology. 8:228～230 (1990)
Heider, J., Spormann, A. M., Beller, H. R. Anaerobic bacterial metabolism of hydrocarbons. FEMS Microbial Reviews. 22:459~473 (1998)
Johnson, P. C., Johnson, R. L., Bruce, C. L., and Leeson, A. Advancesin In Situ Air Sparging/Biosparging. Bioremediation Journal. 5(4):251~266 (2001)
Johnson R. A., and Wichern D. W. Applied Multivariate Statistical Analysis. 5th ed. Prentice-Hall (2002)
Joseph, G. L. Microbial degradation of hydrocarbon in the environment. Microbial Reviews. 54(3):305~315 (1990)
Juan, C. C., Marta, C., and Angelica, M. A. Biodegradation of aliphatic and aromatic hydrocarbons by natural soil microflora and pure cultures of imperfect and lignolitic fungi. Environmental Pollution. 94:355~362 (1996)
Juan, C. C., Marta, C., and Angelica, M. A. Biodegradation of aliphatic and aromatic hydrocarbons by natural soil microflora and pure cultures of imperfect and lignolitic fungi. Environmental Pollution. 94:355~362 (1996)
Kapley, A., Purohit, H. J., and Chatre, S. Osmotolerance and hydrocarbon degradation by a genetically engineered microbial consortium. Bioresource Technology. 67:241~245 (1999)
Laha, S., and Luthy, R. G. Inhibition of phenanthrene mineralization by nonionic surfactants in soil-water systems. Environmental Science and Technology. 25:1920~1930 (1991)
Lazar, I., Dobrota, S., and Voicu, A. Microbial degradation of waste hydrocarbons in oily sludge from some Romanian oil fields. Journal of Petroleum Science and Engineering. 22:151~160 (1999)
Leahy, J. G., and Colwell, R. R. Microbial Degradation of Hydrocarbons in the Environment. Microbiological Reviews. 54:305～315 (1990)
Li, C., Wang, W., Cao, Y., and Wang, L. Petroleum pollutants degradation by microorganisms. Ecology and Environment. 17(1):113~116 (2008)
Li, J. L., and Chen, B. H. Solubilization of model polycyclic aromatic hydrocarbons by nonionic surfactants. Chemical Engineering Science. 57:2825~2835 (2002)
Lukow, T., Dunfield, P. F., and Liesack, W. Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants. FEMS Microbiology Ecology. 32(3):241~247 (2000)
Moeseneder, M. M., Winter, C., and Arrieta, J. M. Terminal restriction fragment length polymorphism(T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes. Journal of Microbiological Methods. 44(2):159~172 (2001)
Oudot, J., Fusey, P., Praët, M. V., Féral, J., and Gaill, F. Hydrocarbon weathering in seashore invertebrates and sediments over a two-year period following the Amoco Cadiz oil spill: influence of microbial metabolism. Environmental Pollution. 26:93~110 (1981)
Palmer, C. J., and Johnson, R. L. Physical Processes Controlling the Transport of Non-Aqueous Phase Liquids in the Surface, Seminar Publication：Transport and Fate of Contaminants in the Subsurface,Chapter 3. EPA/625/4-89/019. 23~28 (1989)
Pelletier, E., Delille, D., and Delille, B. Crude oil bioremediation in sub-Antarctic intertidal sediments: chemistry and toxicity of oiled residues. Marine Environmental Research. 57:311~327 (2004)
Pruthi, V., and Cameotra, S.S. Short Communication: Production of a biosurfactant exhibiting excellent emulsification and surface active properties by Serratia marcescens. World Journal of Microbiology and Biotechnology. 13:133~135 (1997)
Rehm, H. J., and Reiff, I. Mechanism and occurrence of microbial oxidation of long-chain alkanes. Adv. Bioeng. 24:1241~1269 (1981)
Rittman, B. E., Valocchi, A. J., and Seagren, E. A Critical Review of In-Situ Bioremediation. University of Illinois at Urbana-Champaign (1991)
Roling, W. F. M., Milner, M. G., Jones, D. M., Lee, K., Daniel, F., Swannell, R. J. P., and Head, I. M. Robust Hydrocarbon Degradation and Dynamics of Bacterial Communities during Nutrient-Enhanced Oil Spill Bioremediation. Applied and Environmental Microbiology. 68(11):5537~5548 (2002)
Sarkar, D., Ferguson, M., Datta, R., and Birnbaum, S. Bioremediation of petroleum hydrocarbons in contaminated soils: Comparison of biosolids addition, carbon supplementation, and monitored natural attenuation. Environmental Pollution. 136:187~195 (2005)
Schwarzenbach, R. P., and Westall. Transport of Nonpolar Organic Compounds from Surface Water to Groundwater, Environmental Science and Technology. 15:1360~1367 (1981)
Singer, M. E., and Finnerty, W. R. Microbial metabolism of straight-chain and branched alkanes, Petroleum microbiology. Macmillan Publishing Co., New York (1984)
Troquet, J., Laroche, C., and Dussap, C. G. Evidence for the occurrence of an oxygen limitation during soil bioremediation by solid- state fermentation. Biochemical Engineering Journal. 13(3):103~1121 ( 2003)

U.S.EPA. How to Evaluate Alternative Cleanup Technologies for Underground Storage Tank Sites-A Guide for Corrective Action Plan Reviewers. EPA/510/B/94/003 (1994)
U.S.EPA. Monitored Natural Attenuation of Petroleum Hydrocarbons, U.S.EPA REMEDIAL TECHNOLOGY FACT SHEET. EPA/600/F-98/021 (1999)　
Van-Hamme, J. D., Singh, A., and Ward, O. P. Recent Advances in Petroleum Microbiology. Microbiology and Molecular Biology Reviews. 67(4):503~549 (2003)
Von Wedel, R. J., Mosquera, J. F., Goldsmith, C. D., Hater G. R., Wong, A., Fox, T. A., Hunt, W. T., Paules, M. S., Quiros, J. M., and Wiegand, J. W. Bacterial biodegradation of petroleum hydrocarbons in groundwater：in situ augmented bioreclamation with enrichment isolates in California, Water Science and Technology. 20:501~503 (1988)
Watts, J. E. M., Wu, Q., Schreier, S. B. et al. Comparative analysis of polychlorinated biphenyl-dechlorinating communities in enrichment cultures using three different molecular screeningtechniques. Environmental Microbiology. 3(11):710~719 (2001)
Widdle. The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium strain EbN1. Archives of Microbiology. 183:27~36 (2005)
Williams, C.M., Grimes, J.L., and Mikkelsen, R.L. The use of poultry litter as co-substrate and source of inorganic nutrients and microorganisms for the ex situ biodegradation of petroleum compounds. Poultry Science. 78:956~964 (1999)
Zitrides, T. Biodecontamination of spill site. Pollution Engineering. 15:25~27 (1983)