含氯乙烯類化合物為國內外土壤與地下水中常見的污染物質，而厭氧脫鹵程序為目前所推崇的生物復育法之一。 於此程序中，Dehalococcoides屬在還原四氯乙烯 (PCE) 至無害的乙烯 (ETH) 中扮演關鍵性的角色，而還原脫鹵酵素 (RDase) 的分析則有助於近一步地了解氯乙烯類化合物的降解路徑。本研究目的為利用SybrGreen-qPCR技術分別結合DNA和RNA生物標記法，定量經進行1.4年生物復育的污染場址中不同時期地下水樣本內，Dehalococcoides屬與其脫鹵酵素的豐富度和活性。
首先，本研究發展一個最適的RNA萃取法- TRIzol® MaxTM Bacterial RNA Isolation Method，並應用於萃取受氯乙烯類污染的地下水樣本。 根據DNA定量結果顯示，67口監測井中有5口井有足夠的Dehalococcoides屬 (> 107 copies/L groundwater) 進行生物降解，約91%的監測井則存在適當數量的Dehalococcoides屬 (103 - 107 copies/L groundwater) 有利於進行生物刺激法的復育手段，而只有一口井因微生物數量太低 (<103 copies/L groundwater)，需藉由生物添加來進行生物整治。 此外，在注入糖蜜和松下藥劑於污染場址後，兩種還原脫鹵基因 (tceA和bvcA) 能被優化，進而取代土生土長具有vcrA基因的Dehalococcoides屬。
在RNA定量方面，相較於添加糖蜜或乳化油的生物刺激法，在松下藥劑的添加期間，可觀察到高活性的Dehalococcoides屬與氯乙烯 (VC) 的形成間有很高的相關性，推測松下藥劑的成分有助於誘發VC還原酵素的活性；當井9、13和15觀察到VC的形成時，可檢測到vcrA和bvcA (兩種VC還原酵素) 基因的表現。 進一步說，存在污染熱源井1中的高濃度順-1,2-二氯乙烯 (cis-1,2-DCE，0.153 mg/L)，可能促使啟動vcrA基因的大量複製，可達到每單位基因轉錄4.8複製數的RNA表現最大值，高於污染場址中其他存在低濃度順-1,2-二氯乙烯或是未檢測到的觀測井。
統計分析結果可彙整出以下幾點的重要資訊：一、生物刺激法的應用能夠牽動還原脫鹵基因數量與含氯乙烯類物種的改變；二、於生物整治後，主要的污染物種類將明顯地由三氯乙烯 (TCE) 轉化至低氯數鍵結的cis-1,2-DCE 和VC；三、在藥劑添加的選擇方面，相較於糖蜜和乳化油，松下藥劑的添加能有效地將TCE降解至低氯數的乙烯化合物；四、在評估整治場址的環境條件方面，還原態環境的形成有助於活化具有催化脫鹵反應的功能性酵素所對應的目標基因；五、在目標DNA基因分析方面，具有vcrA基因的Dehalococcoides屬，其豐富度與所有氯乙烯類化合物的濃度均有正相關；六、在RNA基因表現方面，tceA和vcrA基因分別與TCE和生物降解產物 (cis-1,2-DCE和VC)具有正相關；七、一個存在豐富電子提供者 (如各種氯乙烯類化合物) 的厭氧還原環境，有利於藉由增加微生物活性，而獲得成功的氯乙烯類化合物的生物降解。
根據上述結果，SybrGreen-qPCR結合DNA/RNA生物標記法為一個有用的分析平台，用於監測含氯乙烯類污染場址中目標基因的動態變化。此外，松下藥劑的添加能成功地誘發VC還原酵素的活性，進而轉化高氯數乙烯化合物至低氯數物種。

Chloroethenes are an important class of contaminants present in the soil and groundwater sites. Bioremediation with anaerobic dehalogenation is a promising method for chloroethenes degradation. Genus Dehalococcoides plays a critical role in reduction of tetrachloroethene (PCE) to harmless ethene (ETH). Furthermore, several reductive dehalogenases (RDase) extracted from genus Dehalococcoides may provide the important information for chloroethenes degradation pathway. This study aimed to quantify the abundance and activity of genus Dehalococcoides and RDase genes in a contaminated groundwater site with 1.4 year and different stages of bioremediation, using the analytic platform of SybrGreen-qPCR method with DNA/RNA biomarkers.
First, an optimal approach to extract RNA was developed using TRIzol® MaxTM Bacterial RNA Isolation Method, and then applied for the analysis of chloroethene-contaminated groundwater samples. DNA quantification results showed that 5 of 67 monitoring wells had high Dehalococcoides population (> 107 copies/L groundwater), which is suitable for natural attenuation. Ninety one percent (61 of 67) of the wells had moderate abundance of Dehalococcoides population, which may be enhanced for bioremediation if using biostimulation approach. Only 1 monitoring well has low Dehalococcoides population (<103 copies/L groundwater). When applying bioremediation agents in the site, two RDase genes (tceA and bvcA) were enhanced to replace vcrA-carried Dehalococcoides in the subsurface.
In comparison with molasses and emulsified oil injections, RNA quantification showed high Dehalococcoides 16S rRNA expression in August 2016 after injection of Panasonic chemical and the expression was related to VC formation, suggesting that the composition of Panasonic chemical might induce the activity of VC-reductases. The observation was further confirmed, by the fact that the expression of vcrA or bvcA transcripts were activated while VC formed from cis-1,2-DCE in the wells 9, 13 and 15. Moreover, the highest cis-1,2-DCE concentration of 0.153 mg/L in the hot spot (well 1) also induced large amounts of transcripts from vcrA gene, with 4.8 copies of transcripts per copies of genes, compared to other monitoring wells.
Statistical analyses of the observed data provide a few important results and were summarized as follows. First, chemical injection (also called biostimulation) could draw the changes of reductive dehalogenases amounts and chloroethene species. Second, the main chloroethene species were transformed from TCE to both cis-1,2-DCE and VC after bioremediation. Third, for the selection of injecting chemicals, compared to molasses and emulsified oil, Panasonic chemical could enhance in effective degradation of polychlorinated TCE to daughter compounds. Fourth, for environmental condition of the bioremediation region, formation of reducing environment may promote activation of the targeted genes related to reductive dechlorination. Fifth, for targeted DNA analysis, vcrA-carried Dehalococcoides was positively correlated with all chloroethene species. Sixth, tceA and vcrA genes were positively correlated with TCE and both degrading products, cis-1,2-DCE and VC respectively, for RNA expression. Seventh, an anaerobic and reductive region with sufficient amounts of electron donor (i.e. chloroethene species) would be beneficial to the increment of microbial activity to achieve the success bioremediation of chloroethenes.
In summary, the approach of SybrGreen-qPCR with DNA/RNA biomarkers was a useful analytic platform to monitor the dynamic changes of targeted genes in the chloroethene-contaminated site. Bioremediation with Panasonic chemical could successfully promote the activity of VC-reductases, enhancing the biodegradation of TCE in the groundwater.

Abrahamsson, K., Ekdahl, A., Collén, J., Fahlström, E., & Pedersén, M. (1995). The natural formation of trichloroethylene and perchloroethylene in sea water. Naturally-Produced Organohalogens (pp. 327-331). Springer Netherlands.
Adrian, L., Szewzyk, U., Wecke, J., & Görisch, H. (2000). Bacterial dehalorespiration with chlorinated benzenes. Nature, 408(6812), 580-583.
Adrian, L., & Löffler, F. E. (2016). Organohalide Respiring Bacteria. Springer, Berlin.
Akladiss, N., Faris, B., Hadley, P., Hausamann, E., Shirazi, G. A., & Syverson, L. (2005). Overview of in situ bioremediation of chlorinated ethene DNAPL source zones. The Interstate Technology and Regulatory Council, Washington, DC.
Alliance, H. S. I. (2008). Perchloroethylene white paper.
Amann, R. I., Ludwig, W., & Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological reviews, 59(1), 143-169.
ATSDR (1996). Agency for Toxic Substances and Disease Registry. Toxicological profile for 1,2-dichloroethene. Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA.
ATSDR (2006). Agency for Toxic Substances and Disease Registry. Toxicological profile for Vinyl Chloride. Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA.
ATSDR (2011). Agency for Toxic Substances and Disease Registry. Toxicological profile for Trichloroethylene. Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA.
ATSDR (2012). Agency for Toxic Substances and Disease Registry. Toxicological profile for Tetrachloroethylene. Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA.
Auer, H., Lyianarachchi, S., Newsom, D., Klisovic, M. I., & Kornacker, K. (2003). Chipping away at the chip bias: RNA degradation in microarray analysis. Nature genetics, 35(4), 292-293.
Aulenta, F., Majone, M., & Tandoi, V. (2006). Enhanced anaerobic bioremediation of chlorinated solvents: environmental factors influencing microbial activity and their relevance under field conditions. Journal of Chemical Technology and Biotechnology, 81(9), 1463-1474.
Badin, A., Broholm, M. M., Jacobsen, C. S., Palau, J., Dennis, P., & Hunkeler, D. (2016). Identification of abiotic and biotic reductive dechlorination in a chlorinated ethene plume after thermal source remediation by means of isotopic and molecular biology tools. Journal of Contaminant Hydrology, 192, 1-19.
Bass, D. H., Hastings, N. A., & Brown, R. A. (2000). Performance of air sparging systems: a review of case studies. Journal of Hazardous Materials, 72(2), 101-119.
Bastian, M., Heymann, S., & Jacomy, M. (2009). Gephi: an open source software for exploring and manipulating networks. Proc Third Int ICWSM Conf (ICWSM) 8: 361–362.
Behrens, S., Azizian, M. F., McMurdie, P. J., Sabalowsky, A., Dolan, M. E., Semprini, L., & Spormann, A. M. (2008). Monitoring abundance and expression of “Dehalococcoides” species chloroethene-reductive dehalogenases in a tetrachloroethene-dechlorinating flow column. Applied and environmental microbiology, 74(18), 5695-5703.
Beretta, G. P. (2017). Point and nonpoint pollution and restoring groundwater quality in Italy: 30 years of experience. Rendiconti Lincei, 1-10.
Bhagwat, A. A., Ying, Z. I., Karns, J., & Smith, A. (2013). Determining RNA quality for NextGen sequencing: some exceptions to the gold standard rule of 23S to 16S rRNA ratio §. Microbiology Discovery, 1(1), 10.
Borden, R. C. (2006). Protocol for enhanced in situ bioremediation using emulsified edible oil. Solutions Industrial and Environmental Services Raleigh NC.
Bouwer, E. J. (1994). Bioremediation of chlorinated solvents using alternate electron acceptors. Handbook of bioremediation, 149-175.
Bradley, P. M., Landmeyer, J. E., & Dinicola, R. S. (1998a). Anaerobic oxidation of [1, 2-14C] dichloroethene under Mn (IV)-reducing conditions. Applied and environmental microbiology, 64(4), 1560-1562.
Bradley, P. M., & Chapelle, F. H. (1998b). Microbial mineralization of VC and DCE under different terminal electron accepting conditions. Anaerobe, 4(2), 81-87.
Bradley, P. M. (2003). History and ecology of chloroethene biodegradation: a review. Bioremediation Journal, 7(2), 81-109.
Chen W-F, Lu H-Y, & Liu T-K (2000). The Redox Condition and Arsenic Concentration in Groundwater of Taiwan. Journal of Taiwan Agricultural Engineering, 56(2), P57-70.
Cheng, D., & He, J. (2009). Isolation and characterization of “Dehalococcoides” sp. strain MB, which dechlorinates tetrachloroethene to trans-1, 2-dichloroethene. Applied and environmental microbiology, 75(18), 5910-5918.
Chem. Eng. News (1963, Nov 25). New dry-cleaning system under field test. 41(57).
Chem. Eng. News (1967, Dec 11). Wide scope seen for dry-cleaning chemicals. 45(30).
Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162(1), 156-159.
Chow, W. L., Cheng, D., Wang, S., & He, J. (2010). Identification and transcriptional analysis of trans-DCE-producing reductive dehalogenases in Dehalococcoides species. The ISME journal, 4(8), 1020-1030.
CMR. (2003). Vinyl chloride. Chemical Market Reporter.
Cohen, R. M., & Mercer, J. W. (1993). DNAPL Site Investigation. CK Smoley, Boca Raton, Florida.
Cupples, A. M., Spormann, A. M., & McCarty, P. L. (2003). Growth of a Dehalococcoides-like microorganism on vinyl chloride and cis-dichloroethene as electron acceptors as determined by competitive PCR. Applied and environmental microbiology, 69(2), 953-959.
Doherty, R. E. (2000). A history of the production and use of carbon tetrachloride, tetrachloroethylene, trichloroethylene and 1, 1, 1-trichloroethane in the United States: Part 1-historical background; carbon tetrachloride and tetrachloroethylene. Environmental forensics, 1(2), 69-81.
Dolfing, J., & Tiedje, J. M. (1986). Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzoate. FEMS Microbiology Ecology, 2(5), 293-298.
Dolfing, J., & Tiedje, J. M. (1987). Growth yield increase linked to reductive dechlorination in a defined 3-chlorobenzoate degrading methanogenic coculture. Archives of microbiology, 149(2), 102-105.
Dolfing, J. (2003). Thermodynamic considerations for dehalogenation. Dehalogenation: Microbial processes and environmental applications, 89-114.
Duhamel, M., Mo, K., & Edwards, E. A. (2004). Characterization of a highly enriched Dehalococcoides-containing culture that grows on vinyl chloride and trichloroethene. Applied and Environmental Microbiology, 70(9), 5538-5545.
Duhamel, M., & Edwards, E. A. (2006). Microbial composition of chlorinated ethene-degrading cultures dominated by Dehalococcoides. FEMS Microbiology Ecology, 58(3), 538-549.
Eisen, J. A. (2007). Environmental shotgun sequencing: its potential and challenges for studying the hidden world of microbes. PLoS Biol, 5(3), e82.
Ensign, S. A., Hyman, M. R., & Arp, D. J. (1992). Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain. Applied and environmental microbiology, 58(9), 3038-3046.
Evguenieva-Hackenberg, E. (2005). Bacterial ribosomal RNA in pieces. Molecular microbiology, 57(2), 318-325.
Fazi, S., Amalfitano, S., Pizzetti, I., & Pernthaler, J. (2007). Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content. Systematic and applied microbiology, 30(6), 463-470.
Fazi, S., Aulenta, F., Majone, M., & Rossetti, S. (2008). Improved quantification of Dehalococcoides species by fluorescence in situ hybridization and catalyzed reporter deposition. Systematic and applied microbiology, 31(1), 62-67.
Fennell, D. E., Nijenhuis, I., Wilson, S. F., Zinder, S. H., & Häggblom, M. M. (2004). Dehalococcoides ethenogenes strain 195 reductively dechlorinates diverse chlorinated aromatic pollutants. Environmental science & technology, 38(7), 2075-2081.
Fleige, S., & Pfaffl, M. W. (2006). RNA integrity and the effect on the real-time qRT-PCR performance. Molecular aspects of medicine, 27(2), 126-13.
Frascari, D., Zanaroli, G., & Danko, A. S. (2015). In situ aerobic cometabolism of chlorinated solvents: A review. Journal of hazardous materials, 283, 382-399.
Freedman, D. L., & Gossett, J. M. (1991). Biodegradation of dichloromethane and its utilization as a growth substrate under methanogenic conditions. Applied and environmental microbiology, 57(10), 2847-2857.
Futagami, T., Goto, M., & Furukawa, K. (2008). Biochemical and genetic bases of dehalorespiration. The chemical record, 8(1), 1-12.
George, I., Stenuit, B., Agathos, S., & Marco, D. (2010). Application of metagenomics to bioremediation. Metagenomics: Theory, Methods and Applications, 1, 119-140.
Gerritse, J., Drzyzga, O., Kloetstra, G., Keijmel, M., Wiersum, L. P., Hutson, R., ... & Gottschal, J. C. (1999). Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1. Applied and Environmental Microbiology, 65(12), 5212-5221.
Gribble, G. W. (1992). Naturally occurring organohalogen compounds--a survey. Journal of natural products, 55(10), 1353-1395.
Gribble, G. W. (1994). The natural production of chlorinated compounds. Environmental science & technology, 28(7), 310A-319A.
Haber, C. L., Allen, L. N., Zhao, S., & Hanson, R. S. (1983). Methylotrophic bacteria: biochemical diversity and genetics. Science, 221(4616), 1147-1153.
Hartmans, S. (1995). Microbial degradation of vinyl chloride. Progress in Industrial Microbiology, 32, 239-248.
He, J., Sung, Y., Dollhopf, M. E., Fathepure, B. Z., Tiedje, J. M., & Löffler, F. E. (2002). Acetate versus hydrogen as direct electron donors to stimulate the microbial reductive dechlorination process at chloroethene-contaminated sites. Environmental Science & Technology, 36(18), 3945-3952.
He, J., Ritalahti, K. M., Aiello, M. R., & Löffler, F. E. (2003a). Complete detoxification of vinyl chloride by an anaerobic enrichment culture and identification of the reductively dechlorinating population as a Dehalococcoides species. Applied and Environmental Microbiology, 69(2), 996-1003.
He, J., Ritalahti, K. M., Yang, K. L., Koenigsberg, S. S., & Löffler, F. E. (2003b). Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium. Nature, 424(6944), 62-65.
He, J., Sung, Y., Krajmalnik‐Brown, R., Ritalahti, K. M., & Löffler, F. E. (2005). Isolation and characterization of Dehalococcoides sp. strain FL2, a trichloroethene (TCE) and 1,2‐dichloroethene‐respiring anaerobe. Environmental Microbiology, 7(9), 1442-1450.
Holliger, C., Schraa, G., Stams, A. J., & Zehnder, A. J. (1993). A highly purified enrichment culture couples the reductive dechlorination of tetrachloroethene to growth. Applied and Environmental Microbiology, 59(9), 2991-2997.
Holliger, C., Wohlfarth, G., & Diekert, G. (1998a). Reductive dechlorination in the energy metabolism of anaerobic bacteria. FEMS Microbiology Reviews, 22(5), 383-398.
Holliger, C., Hahn, D., Harmsen, H., Ludwig, W., Schumacher, W., Tindall, B., Vazquez, F., Weiss, N., & Zehnder, A. J. (1998b). Dehalobacter restrictus gen. nov. and sp. nov., a strictly anaerobic bacterium that reductively dechlorinates tetra-and trichloroethene in an anaerobic respiration. Archives of Microbiology, 169(4), 313-321.
Holmes, V. F., He, J., Lee, P. K., & Alvarez-Cohen, L. (2006). Discrimination of multiple Dehalococcoides strains in a trichloroethene enrichment by quantification of their reductive dehalogenase genes. Applied and Environmental Microbiology, 72(9), 5877-5883.
Hopkins, G. D., Semprini, L., & McCarty, P. L. (1993). Microcosm and in situ field studies of enhanced biotransformation of trichloroethylene by phenol-utilizing microorganisms. Applied and Environmental Microbiology, 59(7), 2277-2285.
HSDB (2017a). Hazardous Substances Data Bank. Vinyl chloride. Environmental standards and regulations. https://toxnet.nlm.nih.gov/cgi-bin/sis/search2/f?./temp/~WB5HhM:3.
HSDB (2017b). Hazardous Substances Data Bank. Cis-1,2-Dichloroethylene. Environmental standards and regulations. https://toxnet.nlm.nih.gov/cgi-bin/sis/search2/f?./temp/~eoeHHP:3
Hsueh, H-T (2016). Trend study of academic research on soil and groundwater remediation by co-word analysis. 3rd International conference on contaminated land, ecological assessment and remediation.
Hug, L. A., Maphosa, F., Leys, D., Löffler, F. E., Smidt, H., Edwards, E. A., & Adrian, L. (2013). Overview of organohalide-respiring bacteria and a proposal for a classification system for reductive dehalogenases. Phil. Trans. R. Soc. B, 368(1616), 20120322.
IARC (1979). International Agency for Research on Cancer. Vinyl chloride, polyvinyl chloride and vinyl chloride-vinyl acetate copolymers. IARC Monogr Eval Carcinog Risk Chem Hum19:377-438.
Innis, M. A., Gelfand, D. H., Sninsky, J. J., & White, T. S. (1990). PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.
ITRC (2005). Interstate Technology & Regulatory Council. Technical and Regulatory Guidance for In Situ Chemical Oxidation of Contaminated Soil and Groundwater.
ITRC (2008). Interstate Technology and Regulatory Council. In Situ Bioremediation of Chlorinated Ethene: DNAPL Source Zones.
ITRC (2011). Interstate Technology & Regulatory Council. Permeable Reactive Barrier: Technology Update, PRB-5.
Johnson, R. A., & Wichern, D. W. (2002). Applied multivariate statistical analysis (Vol. 5, No. 8). Upper Saddle River, NJ: Prentice hall.
Johnson, D. R., Lee, P. K., Holmes, V. F., & Alvarez-Cohen, L. (2005a). An internal reference technique for accurately quantifying specific mRNAs by real-time PCR with application to the tceA reductive dehalogenase gene. Applied and environmental microbiology, 71(7), 3866-3871.
Johnson, D. R., Lee, P. K., Holmes, V. F., Fortin, A. C., & Alvarez-Cohen, L. (2005b). Transcriptional expression of the tceA gene in a Dehalococcoides-containing microbial enrichment. Applied and environmental microbiology, 71(11), 7145-7151.
Kanitkar, Y. H., Stedtfeld, R. D., Steffan, R. J., Hashsham, S. A., & Cupples, A. M. (2016). Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9. Applied and environmental microbiology, 82(6), 1799-1806.
Kanitkar, Y. H., Stedtfeld, R. D., Hatzinger, P. B., Hashsham, S. A., & Cupples, A. M. (2017). Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from groundwater. Applied Microbiology and Biotechnology, 1-9.
Kao, C. M., Liao, H. Y., Chien, C. C., Tseng, Y. K., Tang, P., Lin, C. E., & Chen, S. C. (2016). The change of microbial community from chlorinated solvent-contaminated groundwater after biostimulation using the metagenome analysis. Journal of hazardous materials, 302, 144-150.
Keppler, F., Borchers, R., Pracht, J., Rheinberger, S., & Schöler, H. F. (2002). Natural formation of vinyl chloride in the terrestrial environment. Environmental science & technology, 36(11), 2479-2483.
Krajmalnik-Brown, R., Hölscher, T., Thomson, I. N., Saunders, F. M., Ritalahti, K. M., & Löffler, F. E. (2004). Genetic identification of a putative vinyl chloride reductase in Dehalococcoides sp. strain BAV1. Applied and Environmental Microbiology, 70(10), 6347-6351.
Kranzioch, I., Ganz, S., & Tiehm, A. (2015). Chloroethene degradation and expression of Dehalococcoides dehalogenase genes in cultures originating from Yangtze sediments. Environmental Science and Pollution Research, 22(4), 3138-3148.
Krumme, M. L., Timmis, K. N., & Dwyer, D. F. (1993). Degradation of trichloroethylene by Pseudomonas cepacia G4 and the constitutive mutant strain G4 5223 PR1 in aquifer microcosms. Applied and environmental microbiology, 59(8), 2746-2749.
Kuo, M. C., Chen, C. M., Lin, C. H., Fang, H. C., & Lee, C. H. (2000). Surveys of volatile organic compounds in soil and groundwater at industrial sites in Taiwan. Bulletin of environmental contamination and toxicology, 65(5), 654-659.
Lai, Y., & Becker, J. G. (2013). Compounded effects of chlorinated ethene inhibition on ecological interactions and population abundance in a Dehalococcoides-Dehalobacter co-culture. Environmental science & technology, 47(3), 1518-1525.
Lawrence, S. J. (2006). Description, Properties, and Degradation of Selected Volatile Organic Compounds Detected in Ground Water--A Review of Selected Literature (No. 2006-1338).
Lebrón, C. A., Petrovskis, E., Löffler, F., & Henn, K. (2011). Application of nucleic acid-based tools for monitoring monitored natural attenuation (MNA), biostimulation and bioaugmentation at chlorinated solvent sites (No. NFESC-CR-11-028-ENV). Naval Facilities Engineering Command Port Hueneme CA Engineering Service Center.
Lee, M. D., Odom, J. M., & Buchanan Jr, R. J. (1998). New perspectives on microbial dehalogenation of chlorinated solvents: insights from the field. Annual Reviews in Microbiology, 52(1), 423-452.
Lee, P. K., Johnson, D. R., Holmes, V. F., He, J., & Alvarez-Cohen, L. (2006). Reductive dehalogenase gene expression as a biomarker for physiological activity of Dehalococcoides spp. Applied and Environmental Microbiology, 72(9), 6161-6168.
Lee, P. K., Macbeth, T. W., Sorenson, K. S., Deeb, R. A., & Alvarez-Cohen, L. (2008). Quantifying genes and transcripts to assess the in situ physiology of “Dehalococcoides” spp. in a trichloroethene-contaminated groundwater site. Applied and Environmental Microbiology, 74(9), 2728-2739.
Lee, P. K., Cheng, D., Hu, P., West, K. A., Dick, G. J., Brodie, E. L., Andersen, G.L., Zinder, S. H., He, J., & Alvarez-Cohen, L. (2011). Comparative genomics of two newly isolated Dehalococcoides strains and an enrichment using a genus microarray. The ISME journal, 5(6), 1014-1024.
Lee, P. K., Cheng, D., West, K. A., Alvarez‐Cohen, L., & He, J. (2013). Isolation of two new Dehalococcoides mccartyi strains with dissimilar dechlorination functions and their characterization by comparative genomics via microarray analysis. Environmental microbiology, 15(8), 2293-2305.
Leeson, A., Beevar, E., Henry, B., Fortenberry, J., & Coyle, C. (2004). Principles and practices of enhanced anaerobic bioremediation of chlorinated solvents (No. NFESC-TR-2250-ENV). Naval Facilities Engineering Service Center Port Hueneme CA.
Löffler, F. E., Yan, J., Ritalahti, K. M., Adrian, L., Edwards, E. A., Konstantinidis, K. T., Muller, J. A., Fullerton, H., Zinder, S. H., & Spormann, A. M. (2013). Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi. International journal of systematic and evolutionary microbiology, 63(2), 625-635.
Lowenheim, F., & Moran, M. (Eds.) (1975). Faith, Keyes and Clark's Industrial Chemicals, 4th edition. New York, Wiley & Sons.
Lu, X., Wilson, J. T., & Kampbell, D. H. (2006). Relationship between Dehalococcoides DNA in ground water and rates of reductive dechlorination at field scale. Water research, 40(16), 3131-3140.
Magnuson, J. K., Stern, R. V., Gossett, J. M., Zinder, S. H., & Burris, D. R. (1998). Reductive dechlorination of tetrachloroethene to ethene by a two-component enzyme pathway. Applied and Environmental Microbiology, 64(4), 1270-1275.
Major, D. W., McMaster, M. L., Cox, E. E., Edwards, E. A., Dworatzek, S. M., Hendrickson, E. R., Starr, M. G., Payne, J. A., & Buonamici, L. W. (2002). Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene. Environmental Science & Technology, 36(23), 5106-5116.
Maness, A. D., Bowman, K. S., Yan, J., Rainey, F. A., & Moe, W. M. (2012). Dehalogenimonas spp. can reductively dehalogenate high concentration of 1, 2-dichloroethane, 1, 2-dichloropropane, and 1, 1, 2-trichloroethane. AMB Express, 2(1), 54.
Maphosa, F., de Vos, W. M., & Smidt, H. (2010). Exploiting the ecogenomics toolbox for environmental diagnostics of organohalide-respiring bacteria. Trends in biotechnology, 28(6), 308-316.
Marco-Urrea, E., Nijenhuis, I., & Adrian, L. (2011). Transformation and carbon isotope fractionation of tetra-and trichloroethene to trans-dichloroethene by Dehalococcoides sp. strain CBDB1. Environmental science & technology, 45(4), 1555-1562.
Mattes, T. E., Alexander, A. K., & Coleman, N. V. (2010). Aerobic biodegradation of the chloroethene: pathways, enzymes, ecology, and evolution. FEMS microbiology reviews, 34(4), 445-475.
Mattes, T. E., Jin, Y. O., Livermore, J., Pearl, M., & Liu, X. (2015). Abundance and activity of vinyl chloride (VC)-oxidizing bacteria in a dilute groundwater VC plume biostimulated with oxygen and ethene. Applied microbiology and biotechnology, 99(21), 9267-9276.
Maymo-Gatell, X., Tandoi, V., Gossett, J. M., & Zinder, S. H. (1995). Characterization of an H2-utilizing enrichment culture that reductively dechlorinates tetrachloroethene to vinyl chloride and ethene in the absence of methanogenesis and acetogenesis. Applied and Environmental Microbiology, 61(11), 3928-3933.
Maymo-Gatell, X., Chien, Y. T., Gossett, J. M., & Zinder, S. H. (1997). Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science, 276(5318), 1568-1571.
Maymo-Gatell, X., Anguish, T., & Zinder, S. H. (1999). Reductive dechlorination of chlorinated ethenes and 1, 2-dichloroethane by “Dehalococcoides ethenogenes” 195. Applied and Environmental Microbiology, 65(7), 3108-3113.
Maymo-Gatell, X., Nijenhuis, I., & Zinder, S. H. (2001). Reductive dechlorination of cis-1, 2-dichloroethene and vinyl chloride by “Dehalococcoides ethenogenes”. Environmental science & technology, 35(3), 516-521.
McCarty, P. L., & Semprini, L. (1994). Ground-water treatment for chlorinated solvents. Handbook of bioremediation, 87-116.
McCarty, P. L., Goltz, M. N., Hopkins, G. D., Dolan, M. E., Allan, J. P., Kawakami, B. T., & Carrothers, T. J. (1998). Full-scale evaluation of in situ cometabolic degradation of trichloroethylene in groundwater through toluene injection. Environmental Science & Technology, 32(1), 88-100.
Mercer, J. W., & Cohen, R. M. (1990). A review of immiscible fluids in the subsurface: properties, models, characterization and remediation. Journal of Contaminant Hydrology, 6(2), 107-163.
Miller, E., Wohlfarth, G., & Diekert, G. (1997). Comparative studies on tetrachloroethene reductive dechlorination mediated by Desulfitobacterium sp. strain PCE-S. Archives of Microbiology, 168(6), 513-519.
Moore, S. (1981). 15 Pancreatic DNase. The enzymes, 14, 281-296.
Moran, M. J., Zogorski, J. S., & Squillace, P. J. (2007). Chlorinated solvents in groundwater of the United States. Environmental Science & Technology, 41(1), 74-81.
Muller, J. A., Rosner, B. M., Von Abendroth, G., Meshulam-Simon, G., McCarty, P. L., & Spormann, A. M. (2004). Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp. strain VS and its environmental distribution. Applied and Environmental Microbiology, 70(8), 4880-4888.
Nelson, M. J., Montgomery, S. O., O'neill, E. J., & Pritchard, P. H. (1986). Aerobic metabolism of trichloroethylene by a bacterial isolate. Applied and Environmental Microbiology, 52(2), 383-384.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic acids research, 28(12), e63-e63.
Oolman, T., Godard, S. T., Pope, G. A., Jin, M., & Kirchner, K. (1995). DNAPL flow behavior in a contaminated aquifer: Evaluation of field data. Groundwater Monitoring & Remediation, 15(4), 125-137.
Opel, K. L., Chung, D., & McCord, B. R. (2010). A study of PCR inhibition mechanisms using real time PCR. Journal of forensic sciences, 55(1), 25-33.
Poritz, M., Goris, T., Wubet, T., Tarkka, M. T., Buscot, F., Nijenhuis, I., Lechner, U. & Adrian, L. (2013). Genome sequences of two dehalogenation specialists–Dehalococcoides mccartyi strains BTF08 and DCMB5 enriched from the highly polluted Bitterfeld region.
Rahm, B. G., & Richardson, R. E. (2007). Correlation of respiratory gene expression levels and pseudo-steady-state PCE respiration rates in Dehalococcoides ethenogenes. Environmental science & technology, 42(2), 416-421.
Ritalahti, K. M., Amos, B. K., Sung, Y., Wu, Q., Koenigsberg, S. S., & Löffler, F. E. (2006). Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains. Applied and Environmental Microbiology, 72(4), 2765-2774.
Rivett, M. O., Chapman, S. W., Allen-King, R. M., Feenstra, S., & Cherry, J. A. (2006). Pump-and-treat remediation of chlorinated solvent contamination at a controlled field-experiment site. Environmental science & technology, 40(21), 6770-6781.
Rosner, B. M., McCarty, P. L., & Spormann, A. M. (1997). In vitro studies on reductive vinyl chloride dehalogenation by an anaerobic mixed culture. Applied and environmental microbiology, 63(11), 4139-4144.
Rowe, A. R., Heavner, G. L., Mansfeldt, C. B., Werner, J. J., & Richardson, R. E. (2012). Relating chloroethene respiration rates in Dehalococcoides to protein and mRNA biomarkers. Environmental science & technology, 46(17), 9388-9397.
Rubin, C. M., & Schmid, C. W. (1980). Pyrimidine-specific chemical reactions useful for DNA sequencing. Nucleic acids research, 8(20), 4613-4620.
Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
Schaefer, C. E., Condee, C. W., Vainberg, S., & Steffan, R. J. (2009). Bioaugmentation for chlorinated ethenes using Dehalococcoides sp.: Comparison between batch and column experiments. Chemosphere, 75(2), 141-148.
Scholz-Muramatsu, H., Neumann, A., Meßmer, M., Moore, E., & Diekert, G. (1995). Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing, strictly anaerobic bacterium. Archives of Microbiology, 163(1), 48-56.
Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M., & Ragg, T. (2006). The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology, 7, 3. http://doi.org/10.1186/1471-2199-7-3.
Sharma, P. K., & McCarty, P. L. (1996). Isolation and characterization of a facultatively aerobic bacterium that reductively dehalogenates tetrachloroethene to cis-1, 2-dichloroethene. Applied and Environmental Microbiology, 62(3), 761-765.
Smits, T. H., Devenoges, C., Szynalski, K., Maillard, J., & Holliger, C. (2004). Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides, and Desulfitobacterium in microbial communities. Journal of Microbiological Methods, 57(3), 369-378.
Sung, Y., Ritalahti, K. M., Apkarian, R. P., & Löffler, F. E. (2006). Quantitative PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring Dehalococcoides isolate. Applied and Environmental Microbiology, 72(3), 1980-1987.
Suthersan, S. S., Lutes, C. C., Palmer, P. L., Lenzo, F., Payne, F. C., Liles, D. S., & Burdick, J. (2002). Technical protocol for using soluble carbohydrates to enhance reductive dechlorination of chlorinated aliphatic hydrocarbons. Arcadis Geraghty and Miller Inc Durham NC.
Tee J. J., Chuang H. P., Whang L. M., & Lin T. F. (2017). Bioremediation Effect on Key Microorganisms and Targeted Pollutants in the Chlorinated Ethene Contaminated Groundwater in South Taiwan. Journal of Taiwan Agricultural Engineering, 63(1), P42-49.
Uchino, Y., Miura, T., Hosoyama, A., Ohji, S., Yamazoe, A., Ito, M., Takahata, Y., Suzuki, K. I., & Fujita, N. (2015). Complete genome sequencing of Dehalococcoides sp. strain UCH007 using a differential reads picking method. Standards in genomic sciences, 10(1), 102.
USEPA (1979). United States Environmental Protection Agency. Federal Register. March 15, 1979. 44. 15926.
USEPA (1991). United States Environmental Protection Agency. Federal Register. Jan. 30, 1991. 56 (20). 3536.
USEPA (2000). United States Environmental Protection Agency. Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents: Fundamentals and Field Applications. EPA 542-R-00-008
USEPA (2001). United States Environmental Protection Agency. Toxics Release Inventory Public Data Release Report; 260-R-03-001; Office of Environmental Information: Washington, DC, 2003.
USEPA (2005). United States Environmental Protection Agency. Common Chemicals Found at Superfund Sites; http://www.epa.gov/superfund/ resources/chemicals.htm (accessed January 2005).
Utkin, I., Woese C., & Wiegel, J. (1994). Isolation and Characterization of Desulfitobacterium dehalogenans gen. nov., sp. Nov., an Anaerobic Bacterium Which Reductively Dechlorinates Chlorophenolic Compounds. International Journal of Systematic Bacteriology, 44(4), 612-620.
van der Zaan, B., Hannes, F., Hoekstra, N., Rijnaarts, H., de Vos, W. M., Smidt, H., & Gerritse, J. (2010). Correlation of Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenase genes with geochemical conditions in chloroethene-contaminated groundwater. Applied and environmental microbiology, 76(3), 843-850.
Vogel, T. M., & McCARTY, P. L. (1985). Biotransformation of tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl chloride, and carbon dioxide under methanogenic conditions. Applied and Environmental Microbiology, 49(5), 1080-1083.
Vogel, T. M., Criddle, C. S., & McCarty, P. L. (1987). ES&T critical reviews: transformations of halogenated aliphatic compounds. Environmental Science & Technology, 21(8), 722-736.
Von Ahlfen, S., & Schlumpberger, M. (2010). Effects of low A260/A230 ratios in RNA preparations on downstream applications. QIAGEN Gene Expression Newsletter, 15, 6-7.
Wang, S., & He, J. (2013). Dechlorination of commercial PCBs and other multiple halogenated compounds by a sediment-free culture containing Dehalococcoides and Dehalobacter. Environmental science & technology, 47(18), 10526-10534.
Wang, S., Chng, K. R., Wilm, A., Zhao, S., Yang, K. L., Nagarajan, N., & He, J. (2014). Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls. Proceedings of the National Academy of Sciences, 111(33), 12103-12108.
Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of bacteriology, 173(2), 697-703.
Wiedemeier, T. H., Swanson, M. A., Moutoux, D. E., Gordon, E. K., Wilson, J. T., Wilson, B. H., Kampbell, D. H., Haas, P. E., Miller, R. N., Hanson, J. E., & Chapelle, F. H. (1998). Technical protocol for evaluating natural attenuation of chlorinated solvents in groundwater. USEPA Office of Research and Development, Washington, DC.
Wilson, J. T., & Wilson, B. H. (1985). Biotransformation of trichloroethylene in soil. Applied and Environmental Microbiology, 49(1), 242.
Woese, C. R. (1987). Bacterial evolution. Microbiological reviews, 51(2), 221.
Yang, Y., & Zeyer, J. (2003). Specific detection of Dehalococcoides species by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes. Applied and environmental microbiology, 69(5), 2879-2883.
Yew, H., Chuang, H. P., Chein, I. C., Dong, P. L., Whang, L. M., & Lin, T. F. (2015). Application of SybrGreen-qPCR method to quantify the key microbes and functional genes in the chloroethene-contaminated site. Soil and Groundwater Conference in CIENVE, Taoyuan, Taiwan (In Chinese).
Yohda, M., Ikegami, K., Aita, Y., Kitajima, M., Takechi, A., Iwamoto, M., Fukuda, T., Tamura, N., Shibasaki, J., Koike, S., & Komatsu, D. (2017). Isolation and genomic characterization of a Dehalococcoides strain suggests genomic rearrangement during culture. Scientific Reports, 7.
Yoshida, N., Takahashi, N., & Hiraishi, A. (2005). Phylogenetic characterization of a polychlorinated-dioxin-dechlorinating microbial community by use of microcosm studies. Applied and environmental microbiology, 71(8), 4325-4334.
Yu, Y., Lee, C., Kim, J., & Hwang, S. (2005). Group specific primer and probe sets to detect methanogenic communities using quantitative real time polymerase chain reaction. Biotechnology and bioengineering, 89(6), 670-679.
Zhang, S., Hou, Z., Du, X. M., Li, D. M., & Lu, X. X. (2016). Assessment of biostimulation and bioaugmentation for removing chlorinated volatile organic compounds from groundwater at a former manufacture plant. Biodegradation, 27(4-6), 223-236.
Zhao, S., Ding, C., & He, J. (2016). Genomic characterization of Dehalococcoides mccartyi strain 11a5 reveals a circular extrachromosomal genetic element and a new tetrachloroethene reductive dehalogenase gene. FEMS Microbiology Ecology, fiw235.