甲基叔丁基醚降解培养物特性及降解基因的研究
|
摘要
甲基叔丁基醚(methyl tert-butyl ether，MTBE)是从20世纪90年代以来广泛使用的一种汽油添加剂，以提高汽油发动机运行效率、降低发动机废气造成空气污染和消除含铅汽油导致的铅污染。但由于MTBE在水中溶解度较高，而且很难生物降解，泄漏到环境中后，尤其在地下水中能够长期存留而蓄积，最终造成严重的地下水污染，影响人类健康和生活质量。随着我国MTBE的大量生产及其作为汽油添加剂的应用，大范围的地下水MTBE污染必将成为今后重要的环境生态问题。为了有效地开展地下水MTBE污染进行生物修复，我们在野外富集获得的MTBE降解混合物的基础上，开展了该混合物的MTBE降解效率及影响因素、混合物微生物组成类型及特性、关键酶基因序列及调控等方面的研究，为建立MTBE的生物降解技术提供了储备。
首先建立了检测水溶液中MTBE含量稳定的环境顶空-气相色谱(ambient headspace-gas chromatography)检测方法，分别适用于检测MTBE浓度为1-1000μg／L和1-500mg／L的样品，并进行了环境样品检测验证，为实验室以及环境样品中MTBE含量的测定和MTBE污染监测奠定了基础。
混合培养物降解分析特性表明，该培养物能够降解浓度达500mg／L的MTBE，降解速率为33.14mg／L／h。辅助碳源，如葡萄糖，能够提高培养物降解MTBE的速率。连续培养的培养物降解延迟期缩短，降解速率加快。
在采用传统分离技术对混合培养物中微生物分离鉴定的基础上，通过对混合物中不同组分的16S rDNA PCR及RFLP分析，证明混合物主要是由Terrimonas ferruginea，以及酸杆菌门(Acidobacteria)的细菌组成，而后者是已知的在环境中广泛存在的未培养微生物。这也是首次在MTBE降解培养物中报道酸杆菌门微生物的存在。
同时，本研究通过PCR的方法得到了一个长为260 bp的DNA片段，BLAST结果表明是与alk基因有很高的一致性。随后以此片段开始，应用基因组步行(genome walking)的方法获得了长度为2810 bp DNA片段，经过ORF预测软件分析，其中可能包含3个ORF，依次分别是TetR家族转录调节蛋白、烷烃单加氧酶和phyH双加氧酶基因。为下一步alk基因的克隆表达和活性检测，以及此混合培养物降解MTBE途径的研究提供了良好的基础。
Methyl tert-butyl ether (MTBE) is a broadly used gasoline additive which wasused to replace the traditional gasoline additive tetra-ethyl lead for reducing gasolinelead pollution, improving gasoline engine operating efficiency and decreasing airpollution caused by engine. However MTBE caused serious groundwater pollutionbecause of its high resolution in water and resistance to biodegradation. AlthoughMTBE has been used in China since 1990s, the possible groundwater pollution is stillunknown. With the increasing usage of MTBE, the management of seriousgroundwater pollution by MTBE will be one of the greatest challenges in environmentmanagement program in China.
Considering the future national need for the management of MTBE pollutant, weinitiated the study on biodegradation of MTBE. In this paper, the MTBE degradationability, bacterial composition, and affecting factors, as well as one key gene of MTBEdegrading consortium has been investigated. The data might provide solid informationfor the development of MTBE bioremediation technology. In the first part of thisthesis, an ambient headspace-gas chromatography method was established to measureMTBE in water, which could be used for water solution contained 1-1000μg/L or1-500 mg/L MTBE. This stable MTBE assay method can be used for monitoringMTBE pollution in environments.
The consortium culture degraded MTBE concentration up to 500 mg/L and thedegrading speed was 33.14 mg/L/h. Extra supplement of low concentration of carbon sources, such as glucose, increased consortium degradation speed. After a continuouscultivation, the consortium had an increased MTBE degrading rate.
Several isolates were isolated from the consortium, but none of them has beenproved to have MTBE degradation ability. 16S rDNA sequence and their RFLPanalysis revealed that the consortium, was composed of several bacteria and most ofthem belonged to Terrimonas ferruginea and Acidobacteria. The latter were knownuncultured bacteria. This was the first report on the existence of Acidobacteria inMTBE degradation cultures.
The alkane-1-monooxygenase is the key enzyme in MTBE degradation pathway.A 260 bp DNA fragment was obtained by PCR from the total consortium DNA. Byusing a genome walking method, a 2810 bp DNA fragment was spliced. NCBI ORFprediction indicted there were 3 possible ORFs: a TetR family regulatory factor gene, an alk gene, and a pyhH dioxygenase gene. The data provide a basis for the futureresearch on degradation pathway and the MTBE management.
首先建立了检测水溶液中MTBE含量稳定的环境顶空-气相色谱(ambient headspace-gas chromatography)检测方法，分别适用于检测MTBE浓度为1-1000μg／L和1-500mg／L的样品，并进行了环境样品检测验证，为实验室以及环境样品中MTBE含量的测定和MTBE污染监测奠定了基础。
混合培养物降解分析特性表明，该培养物能够降解浓度达500mg／L的MTBE，降解速率为33.14mg／L／h。辅助碳源，如葡萄糖，能够提高培养物降解MTBE的速率。连续培养的培养物降解延迟期缩短，降解速率加快。
在采用传统分离技术对混合培养物中微生物分离鉴定的基础上，通过对混合物中不同组分的16S rDNA PCR及RFLP分析，证明混合物主要是由Terrimonas ferruginea，以及酸杆菌门(Acidobacteria)的细菌组成，而后者是已知的在环境中广泛存在的未培养微生物。这也是首次在MTBE降解培养物中报道酸杆菌门微生物的存在。
同时，本研究通过PCR的方法得到了一个长为260 bp的DNA片段，BLAST结果表明是与alk基因有很高的一致性。随后以此片段开始，应用基因组步行(genome walking)的方法获得了长度为2810 bp DNA片段，经过ORF预测软件分析，其中可能包含3个ORF，依次分别是TetR家族转录调节蛋白、烷烃单加氧酶和phyH双加氧酶基因。为下一步alk基因的克隆表达和活性检测，以及此混合培养物降解MTBE途径的研究提供了良好的基础。
Methyl tert-butyl ether (MTBE) is a broadly used gasoline additive which wasused to replace the traditional gasoline additive tetra-ethyl lead for reducing gasolinelead pollution, improving gasoline engine operating efficiency and decreasing airpollution caused by engine. However MTBE caused serious groundwater pollutionbecause of its high resolution in water and resistance to biodegradation. AlthoughMTBE has been used in China since 1990s, the possible groundwater pollution is stillunknown. With the increasing usage of MTBE, the management of seriousgroundwater pollution by MTBE will be one of the greatest challenges in environmentmanagement program in China.
Considering the future national need for the management of MTBE pollutant, weinitiated the study on biodegradation of MTBE. In this paper, the MTBE degradationability, bacterial composition, and affecting factors, as well as one key gene of MTBEdegrading consortium has been investigated. The data might provide solid informationfor the development of MTBE bioremediation technology. In the first part of thisthesis, an ambient headspace-gas chromatography method was established to measureMTBE in water, which could be used for water solution contained 1-1000μg/L or1-500 mg/L MTBE. This stable MTBE assay method can be used for monitoringMTBE pollution in environments.
The consortium culture degraded MTBE concentration up to 500 mg/L and thedegrading speed was 33.14 mg/L/h. Extra supplement of low concentration of carbon sources, such as glucose, increased consortium degradation speed. After a continuouscultivation, the consortium had an increased MTBE degrading rate.
Several isolates were isolated from the consortium, but none of them has beenproved to have MTBE degradation ability. 16S rDNA sequence and their RFLPanalysis revealed that the consortium, was composed of several bacteria and most ofthem belonged to Terrimonas ferruginea and Acidobacteria. The latter were knownuncultured bacteria. This was the first report on the existence of Acidobacteria inMTBE degradation cultures.
The alkane-1-monooxygenase is the key enzyme in MTBE degradation pathway.A 260 bp DNA fragment was obtained by PCR from the total consortium DNA. Byusing a genome walking method, a 2810 bp DNA fragment was spliced. NCBI ORFprediction indicted there were 3 possible ORFs: a TetR family regulatory factor gene, an alk gene, and a pyhH dioxygenase gene. The data provide a basis for the futureresearch on degradation pathway and the MTBE management.
引文
1．徐怡珊．甲基叔丁基醚的研究进展．化工环保．2004．24(6)：416-420．
2．张倩．国内外MTBE的生产和供需．石油化工技术经济．1999．15(4)：50-52．
3. Rey M D, Mealey's MTBE Conference. 2000, California.
4. Office of Pollution Prevention and Toxics U. CHEMICAL SUMMARY FOR METHYL-TERT-BUTYL ETHER (EPA 749-F-94-017a). 1994 [cited; Available from: http://www.epa.gov/chemfact/s_mtbe.txt.
5．程伟，江桂斌．无铅汽油添加剂甲基叔丁基醚(MRE)的环境化学行为及其分析方法．环境污染治理技术与设备．2001．2(3)：48-55．
6. Hartley W R, Englande a J and Harrington D J. Health risk assessment of grundwater contaminated with Methyl tert-butyl ether (MTBE). Water Science Research. 1999. 39(10-11): 305-310.
7. USEPA. Drinking water advisory consumer acceptability and health effects alysis on MTBE. Washington. EPA 822-F-97-008. U. S. Government Printing office. 1997: 1-120.
8. Kolb A and Puttmann W. Methyl tert-butyl ether (MTBE) in finished drinking water in Germany. Environmental Pollution. 2006. 140(2): 294-303.
9．贺性鹏，蔡亚平，贺栋梁等．汽油添加剂甲基叔丁基醚的毒性研究．南华大学学报·医学版．2002．30(3)：221-223．
10．袁东，叶舜华，周伟等．无铅汽油新型添加剂甲基叔丁基醚的致突变性研究中国公共卫生．2002．18(2)：129-130．
11. Mcgregor D B, Cruzan G, Callander R D, et al. The mutagenicity testing of tertiary-butyl alcohol, tertiary-butyl acetate and methyl tertiary-butyl ether in Salmonella typhimurium. Mutat Res. 2005. 565(2): 181-9.
12. Joseph P. Illness due to methyl tertiary butyl ether. Arch Environ Health. 1995.50(5): 395-6.
13．陈井影，赵晓松，王玉军．甲基叔丁基醚(MTBE)研究新进展．吉林农业大学学报．2004．26(2)：182-186．
14．顾洁，王艳斌，袁洪福等．甲基叔丁基醚含量对近红外光谱法测定汽油族组成的影响．石油化工．2005(12)：89-92．
15．李添魁．红外光谱法测定汽油中的MTBE含量．石油化工．1995(3)：198-200．
16. Meusinger R. Gasoline analysis by 1H nuclear magnetic resonance spectroscopy. Fuel. 1995.75(10): 1235-1243.
17. Chambers D M, Grace L I and Andresen B D. Development of an Ion Store/Time-of-Flight Mass Spectrometer for the Analysis of Volatile Compounds in Air. Anal. Chem. 1997.69(18): 3780-3790.
18. Halden R U, Happel a M and Schoen S R. Evaluation of Standard Methods for the Analysis of Methyl tert-Butyl Ether and Related Oxygenates in Gasoline-Contaminated Groundwater. Environ. Sci. Technol. 2001.35(7): 1469-1474.
19. Nouda B, Fouillet B, Toussaint G, et al. Complementarity of purge-and-trap and head-space capillary gas chromatographic methods for determination of methyl-tert.-butyl ether in water. Journal of Chromatography A. 1996. 726(1-2): 153-159.
20. Achten C, Kolb A and Puttmann W. Sensitive method for determination of methyl tert-butyl ether (MTBE) in water by use of headspace-SPME/GC-MS. Fresenius J Anal Chem. 2001. 371(4): 519-25.
21. Hong S, Duttweiler C M and Lemley a T. Analysis of methyl tert.-butyl ether and its degradation products by direct aqueous injection onto gas chromatography with mass spectrometry or flame ionization detection systems. Journal of Chromatography A. 1999. 857(1-2): 205-216.
22．周升如，马健，王少波等．密闭空间空气有机物的色／质谱定性分析研究．分析化学．1991(10)：1115-1121．
23. Szelewski M J and Quimby B D. Ambient Headspace GC and GC-MSD Analysis of Non-Polar Volatiles in Water. Agilent Technologies. 2000.
24. Feldman J and Orchin M. Determination of Methyl Tert. Butyl Ether (MTBE) in Gasoline. Analytical Letters. 1993.26(2): 357-365.
25. Hong M S, Farmayan W F, Dortch I J, et al. Phytoremediation of MTBE from a groundwater plume. Environ Sci Technol. 2001.35(6): 1231-9.
26. Fayolle F, Vandecasteele J P and Monot F. Microbial degradation and fate in the environment of methyl tert-butyl ether and related fuel oxygenates. Appl Microbiol Biotechnol. 2001.56(3-4): 339-49.
27．朱琳，刘涉江，黄国强等．甲基叔丁基醚的降解技术研究进展．化工进展．2005．24(10)：1136-1140．
28. Burbano aA, Dionysiou D D, Suidan M T, et al. Oxidation kinetics and effect ofpH on the degradation of MTBE with Fenton reagent. Water Res. 2005.39(1): 107-18.
29. Xu X-R, Zhao Z-Y, Lib X-Y, et al. Chemical oxidative degradation of methyl tert-butyl ether in aqueous solution by Fenton's reagent Chemosphere 2004.55(1): 73-79.
30. Bergendahl J A and Thies T P. Fenton's oxidation of MTBE with zero-valent iron. Water Research 2004.38(2): 327-334.
31. Wagler J L and Malley J P. Removal of methyl tertiary-butyl ether from a model ground water using UV/Peroxide oxidation. Journal of New England Water Works Association. 1994. 108(3): 236-260.
32. Barreto R D, Gray K A and Anders K. Photocatalytic degradation of methyl-tert-butyl ether in TiO2 slurries: a proposed reaction scheme. Water Research. 1995.29(5): 1243-1248.
33. Sahle-Demessie E, Richardson T, Almquist C B, et al. Comparison of Liquid and Gas-Phase Photooxidation of MTBE: Synthetic and Field Samples. Journal of Environmental Engineer. 2002. 128(9): 782-790.
34. Xu X R, Zhao Z Y, Li X Y, et al. Chemical oxidative degradation of methyl tert-butyl ether in aqueous solution by Fenton's reagent. Chemosphere. 2004. 55(1): 73-9.
35. Bergendahl J A and Thies T P. Fenton's oxidation of MTBE with zero-valent iron. Water Res. 2004.38(2): 327-34.
36. Kang J-W and Hoffmann M R. Kinetics and Mechanism of the Sonolytic Destruction of Methyl tert-Butyl Ether by Ultrasonic Irradiation in the Presence of Ozone. Environ. Sci. Technol. 1998.32(20): 3194-3199.
37. Shiha T C, Wangpaichitrb M and Suffet M. Evaluation of granular activated carbon technology for the removal of methyl tertiary butyl ether (MTBE) from drinking water. Water Research 2003.37(2): 375-385.
38. Wilhelm M J, Adams V D, Curtis J G, et al. Carbon Adsorption and Air-Stripping Removal of MTBE from River Water. Journal of Environmental Engineering. 2002. 128(9): 813-823.
39. Cooper W J, Nickelsen M G, Mezyk S P, et al. MTBE and priority contaminant treatment with high energy electron beam injection. Radiation Physics and Chemistry 2002. 65(4-5): 451-460.
40. Zang Y and Famood R. Effects of hydrogen peroxide concentration and ultraviolet light intensity on methyl tert-butyl ether degradation kinetics. Chemical Engineering Science 2005.60(6): 1641-1648.
41. Hsieh L-L, Lin Y-L and Wu C-H. Degradation of MTBE in dilute aqueous solution by gamma radiolysis. Water Research 2004. 38(16): 3627-3633.
42. Wu T, Cruz V, Mezyk S, et al. Gamma radiolysis of methyl t-butyl ether: a study of hydroxyl radical mediated reaction pathways. Radiation Physics and Chemistry 2002. 65(4-5): 335-341.
43. Kang J-W, Hung H-M, Lin A, et al. Sonolytic Destruction of Methyl tert-Butyl Ether by Ultrasonic Irradiation: The Role of O3, H2O2, Frequency, and Power Density. Environmental Science & Technology. 1999.33(18): 3199-3205.
44. Neppolian B, Jung H, Choi H, et al. Sonolytic degradation of methyl tert-butyl ether: the role of coupled Fenton process and persulphate ion. Water Res. 2002.36(19): 4699-708.
45. Bernhardt D and Diekmann H. Degradation of dioxane, tetrahydrofuran and other cyclic ethers by an environmental Rhodococcus strain. Appl Microbiol Biotechnol. 1991.36(1): 120-3.
46. Parales R E, Adamus J E, White N, et al. Degradation of 1, 4-dioxane by an actinomycete in pure culture. Appl Environ Microbiol. 1994. 60(12): 4527-30.
47. Hyman M R and Arp D J. Effects of ammonia on the de novo synthesis of polypeptides in cells of Nitrosomonas europaea denied ammonia as an energy source. J Bacteriol. 1995. 177(17): 4974-9.
48. Hur H, Newman L M, Wackett L P, et al. Toluene 2-Monooxygenase-Dependent Growth of Burkholderia cepacia G4/PR1 on Diethyl Ether. Appl Environ Microbiol. 1997. 63(4): 1606-1609.
49. Salanitro J P, Diaz L A, Williams M P, et al. Isolation of a Bacterial Culture That Degrades Methyl t-Butyl Ether. Appl Environ Microbiol. 1994. 60(7): 2593-2596.
50. Mo K, Lora C O, Wanken a E, et al. Biodegradation of methyl t-butyl ether by pure bacterial cultures. Appl Microbiol Biotechnol. 1997.47(1): 69-72.
51. Hanson J R, Ackerman C E and Scow K M. Biodegradation of methyl tert-butyl ether by a bacterial pure culture. Appl Environ Microbiol. 1999.65(11): 4788-92.
52. Deeb R A, Hu H Y, Hanson J R, et al. Substrate interactions in BTEX and MTBE mixtures by an MTBE-degrading isolate. Environ Sci Technol. 2001.35(2): 312-7.
53. Finneran K T and Lovley D R. Anaerobic degradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA). Environ Sci Technol. 2001.35(9): 1785-90.
54. Bradley P M, Chapelle F H and Landmeyer J E. Effect of redox conditions on MTBE biodegradation in surface water sediments. Environ Sci Technol. 2001.35(23): 4643-7.
55. Bradley P M, Chapelle F H and Landmeyer J E. Methyl t-butyl ether mineralization in surface-water sediment microcosms under denitrifying conditions. Appl Environ Microbiol. 2001.67(4): 1975-8.
56. Pruden A, Sedran M A, Suidan M T, et al. Anaerobic biodegradation of methyl ten-butyl ether under iron-reducing conditions in batch and continuous-flow cultures. Water Environ Res. 2005.77(3): 297-303.
57. Zaitsev G M, Uotila J S and Haggblom M M. Biodegradation of methyl ten-butyl ether by cold-adapted mixed and pure bacterial cultures. Appl Microbiol Biotechnol. 2007. 74(5): 1092-102.
58. Deeb R A, Scow K M and Alvarez-Cohen L. Aerobic MTBE biodegradation: an examination of past studies, current challenges and future research directions. Biodegradation. 2000. 11(2-3): 171-86.
59. Fortin N Y and Deshusses M A. Treatment of Methyl ten-Butyl Ether Vapors in Biotrickling Filters. 1. Reactor Startup, Steady-State Performance, and Culture Characteristics. Environmental Science and Technology 1999. 33(17): 2980-2986.
60. Fortin N Y and Deshusses M A. Treatment of Methyl tert-Butyl Ether Vapors in Biotrickling Filters. 2. Analysis of the Rate-Limiting Step and Behavior under Transient Conditions. Environmental Science and Technology. 1999. 33(17): 2987-2991.
61. Hardison L K, Curry S S, Ciuffetti L M, et al. Metabolism of Diethyl Ether and Cometabolism of Methyl ten-Butyl Ether by a Filamentous Fungus, a Graphium sp. Appl. Environ. Microbiol. 1997. 63(8): 3059-3067.
62. Hyman M and O'reilly K, Physiological and enzymatic features of MTBE-degrading bacteria., in Proceedings of the Fifth International In Situ and On-site Bioremediation Symposium, B.C. Alleman and A. Leeson, Editors. 1999, Battelle Press, Columbus, OH: Monterey, CA,.
63. Steffan R J, Mcclay K, Vainberg S, et al. Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria. Appl Environ Microbiol. 1997. 63(11): 4216-22.
64. Gamier P M, Auria R, Augur C, et al. Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane. Appl Microbiol Biotechnol. 1999. 51 (4): 498-503.
65. Koenigsberg S, Sandefur C, Mahaffey W, et al., Peroxygen mediated bioremediation of MTBE, in Proceedings of the Fifth International In Situ On-Site Bioremediation Symposium, A. BC and A. Leeson, Editors. 1999, Battelle Press, Columbus, OH: Monterey, CA. p. 13-18.
66. Francois A, Mathis H, Godefroy D, et al. Biodegradation of methyl ten-butyl ether and other fuel oxygenates by a new strain, Mycobacterium austroafricanum IFP 2012. Appl Environ Microbiol. 2002.68(6): 2754-62.
67. Okeke B C and Frankenberger W T, Jr. Biodegradation of methyl tertiary butyl ether (MTBE) by a bacterial enrichment consortia and its monoculture isolates. Microbiol Res. 2003. 158(2): 99-106.
68. Smith C A, O'reilly K T and Hyman M R. Cometabolism of methyl tertiary butyl ether and gaseous n-alkanes by Pseudomonas mendocina KR-1 grown on C5 to C8 n-alkanes. Appl Environ Microbiol. 2003.69(12): 7385-94.
69. Magana-Reyes M, Morales M and Revah S. Methyl tert-butyl ether and tert-butyl alcohol degradation by Fusarium solani. Biotechnol Lett. 2005.27(22): 1797-801.
70. Vainberg S, Mcclay K, Masuda H, et al. Biodegradation of ether pollutants by Pseudonocardia sp. strain ENV478. Appl Environ Microbiol. 2006.72(8): 5218-24.
71. Wilson G J, Pruden A, Suidan M T, et al. B iodegradation Kinetics of MTBE in Laboratory Batch and Continuous Flow Reactors. Journal of Environmental Engineering. 2002. 128(9): 824-829.
72. Vainberg S, Togna a P, Sutton P M, et al. Treatment of MTBE-Contaminated Water in Fluidized Bed Bioreactor. Journal of Environmental Engineering. 2002. 128(9): 842-851
73. Morrison J R, Suidan M T and Venosa a D. Use of Membrane Bioreactor for Biodegradation of MTBE in Contaminated Water. Journal of Environmental Engineering. 2002. 128(9): 836-841.
74. Reed T, Allen M H and Peabody J, JP-4/JP-8 BIOREMEDIATION USING ORC PUMPED INTO SEMI-PERMANENT INJECTION WELLS, in Sixth Annual In-Situ and On-Site Bioremediation Conference. 2001, Battelle Press, Columbus, OH.: San Diego, CA.
75. Magar V S, Hartzell K, Burton C, et al. Aerobic and Cometabolic MTBE Biodegradation at Novato and Port Hueneme. Journal of Environmental Engineering. 2002. 128(9): 883-890.
76. Barcelona M J and Jaglowski D R, Subsurface fate and transport of MTBE in a controlled reactive tracer experiment, in Proceedings of the 2000 Petroleum Hydrocarbons and Organic chemicals in Ground Water: Prevention, Detection, and Remediation. 1999, National Ground Water Association, Westerville, OH: Houston. p. 123-137.
77. Salanitro J P, Johnson P C, Spinnler G E, et al. Field-Scale Demonstration of Enhanced MTBE Bioremediation through Aquifer Bioaugrnentation and Oxygenation. Environ Sci Technol. 2000.34(19): 4152-4162.
78．于晓章 and Stefan T．汽油添加剂甲基叔丁基醚(MTBE)污染的植物修复．生态科学．2003．22(2)：109-112，146．
79. Rubin E and Ramaswami A. The potential for phytoremediation of MTBE. Water Res. 2001.35(5): 1348-53.
80. Xingrnao, Richter a R, Albers S, et al. Phytoremediation of MTBE with Hybrid Poplar Trees. International Journal of Phytoremediation. 2004.6(2): 157-167.
81．路争，钟卫鸿，陈建孟，et al．甲基叔丁基醚微生物降解研究进展．微生物学通报．2006．33(1)：122-127．
82. Hatzinger P B, Mcclay K, Vainberg S, et al. Biodegradation of methyl tert-butyl ether by a pure bacterial culture. Appl Environ Microbiol. 2001.67(12): 5601-7.
83. Church C D, Tratnyek P G and Scow K M. Pathways for the degradation of MTBE and other fuel oxygenates by isolate PM1. in 219th ACS National Meeting. 2000. San Fransisco, CA: Division of Environmental Chemistry, American Chemical Society.
84. Piveteau P, Fayolle F, Vandecasteele J P, et al. Biodegradation oftert-butyl alcohol and related xenobiotics by a methylotrophic bacterial isolate. Appl Microbiol Biotechnol. 2001.55(3): 369-73.
85. Chauvaux S, Chevalier F, Le Dantec C, et al. Cloning of a genetically unstable cytochrome P-450 gene cluster involved in degradation of the pollutant ethyl tert-butyl ether by Rhodococcus ruber. J Bacteriol. 2001. 183(22): 6551-7.
86．中华人民共和国国家统计局．中华人民共和国2006年国民经济和社会发展统计公报．2007 [cited; Available from:http://www.stats.gov.cn/tjgb/ndtjgb/qgndtjgb/t20070228_402387821.htm.
87．新华网．2007年国内石油实际消费增速将稳中略降．2007 [cited; Available from:http://www.sinopecnews.com.cn/shnews/2007-01/31/content_424346.htm.
88．新华网．中国水资源现况．2005[cited; Available from:http://news.xinhuanet.com/fortune/2005-09/19/content_3512629.htm.
89．新华每日电讯．京津唐：地下水中有机污染物达百余种．2005．
90．徐志强，宋宪臣，王玉军等．静态顶空-气相色谱法测定土壤中甲基叔丁基醚．吉林农业大学学报．2005．27(3)：310-314．
91. Labhut.Com. What is Headspace Gas Chromatography? [cited; Available from: http://www.labhut.com/education/headspace/index.php.
92. Szelewski Mj Q B. Ambient Headspace GC and GC-MSD Analysis of Non-Polar Volatiles in Water. Agilent Technologies. 2000.
93．陈井影，宋宪臣，孙华等．顶空-气相色谱法测定水体中甲基叔丁基醚．吉林农业大学学报．2006．28(3)：303-307．
94. Rohwerder T, Breuer U, BenndorfD, et al. The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway. Appl Environ Microbiol. 2006.72(6): 4128-35.
95．Sambrook J，分子克隆实验指南．第3版 ed．2005，北京：科学出版社．
96．萨姆布鲁克 J，拉塞尔 D W，SDS碱裂解法制备质粒DNA，in分子克隆实验指南，科学出版社：北京．p．27-36．
97. Jia X, Han S-J, Zhao Y-H, et al. Comparisons of extraction and purification methods of soil microorganism DNA from rhizosphere soil. Journal of Forestry Research. 2006. 17(1): 31-34.
98．萨姆布鲁克 J，拉塞尔 D W，用氯化钙制备和转化大肠杆菌，in分子克隆实验指南．2002，科学出版社：北京．p．96-99．
99. Ncbi. VecScreen. [cited; Available from: http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html.
100. Rice P, Longden I and Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000. 16(6): 276-7.
101. Altschul S F, Madden T L, Schaffer a A, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997. 25(17): 3389-402.
102. Lopes Ferreira N, Malandain C and Fayolle-Guichard F. Enzymes and genes involved in the aerobic biodegradation of methyl tert-butyl ether (MTBE). Appl Microbiol Biotechnol. 2006.72(2): 252-62.
103. Kumar S, Tamura K and Nei M. MEGA 3: Integrated Software for Molecular Evolutionary Genetics Analysis and Sequence Alignment. Briefings in Bioinformatics. 2004. 5: 150-163.
104. Gremion F, Chatzinotas A and Harms H. Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal-contaminated bulk and rhizosphere soil. Environ Microbiol. 2003.5(10): 896-907.
105. Benson D A, Karsch-Mizrach I, Lipman D J, et al. GenBank. Nucleic Acids Res. 2005. 33(Database Issue): D34-D38.
106. Thompson J D, Gibson T J, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997.25(24): 4876-82.
107. Singh V K, Mangalam a K, Dwivedi S, et al. Primer premier: program for design of degenerate primers from a protein sequence. Biotechniques. 1998.24(2): 318-9.
108．宝生物(大连)有限公司．双链DNA的制备方法．2005 [cited; Available from: http://www.takara.com.cn/product/2005/o/O-9FH11001.html.
109. Tatusov T and Tatusov R. ORF Finder. [cited; Available from: http://www.ncbi.nlm.nih.gov/gorf/gorf.html.
110. Shanklin J and Whittle E. Evidence linking the Pseudomonas oleovorans alkane g-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family. FEBS Letters. 2003.545: 188-192.
111. Consortium T I. The InterPro database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Research. 2001.29(1): 37-40.
112. Morales M, Velazquez E, Jan J, et al. Methyl tert-butyl ether biodegradation by microbial consortia obtained from soil samples of gasoline-polluted sites in Mexico. Biotechnol Lett. 2004.26(4): 269-75.
2．张倩．国内外MTBE的生产和供需．石油化工技术经济．1999．15(4)：50-52．
3. Rey M D, Mealey's MTBE Conference. 2000, California.
4. Office of Pollution Prevention and Toxics U. CHEMICAL SUMMARY FOR METHYL-TERT-BUTYL ETHER (EPA 749-F-94-017a). 1994 [cited; Available from: http://www.epa.gov/chemfact/s_mtbe.txt.
5．程伟，江桂斌．无铅汽油添加剂甲基叔丁基醚(MRE)的环境化学行为及其分析方法．环境污染治理技术与设备．2001．2(3)：48-55．
6. Hartley W R, Englande a J and Harrington D J. Health risk assessment of grundwater contaminated with Methyl tert-butyl ether (MTBE). Water Science Research. 1999. 39(10-11): 305-310.
7. USEPA. Drinking water advisory consumer acceptability and health effects alysis on MTBE. Washington. EPA 822-F-97-008. U. S. Government Printing office. 1997: 1-120.
8. Kolb A and Puttmann W. Methyl tert-butyl ether (MTBE) in finished drinking water in Germany. Environmental Pollution. 2006. 140(2): 294-303.
9．贺性鹏，蔡亚平，贺栋梁等．汽油添加剂甲基叔丁基醚的毒性研究．南华大学学报·医学版．2002．30(3)：221-223．
10．袁东，叶舜华，周伟等．无铅汽油新型添加剂甲基叔丁基醚的致突变性研究中国公共卫生．2002．18(2)：129-130．
11. Mcgregor D B, Cruzan G, Callander R D, et al. The mutagenicity testing of tertiary-butyl alcohol, tertiary-butyl acetate and methyl tertiary-butyl ether in Salmonella typhimurium. Mutat Res. 2005. 565(2): 181-9.
12. Joseph P. Illness due to methyl tertiary butyl ether. Arch Environ Health. 1995.50(5): 395-6.
13．陈井影，赵晓松，王玉军．甲基叔丁基醚(MTBE)研究新进展．吉林农业大学学报．2004．26(2)：182-186．
14．顾洁，王艳斌，袁洪福等．甲基叔丁基醚含量对近红外光谱法测定汽油族组成的影响．石油化工．2005(12)：89-92．
15．李添魁．红外光谱法测定汽油中的MTBE含量．石油化工．1995(3)：198-200．
16. Meusinger R. Gasoline analysis by 1H nuclear magnetic resonance spectroscopy. Fuel. 1995.75(10): 1235-1243.
17. Chambers D M, Grace L I and Andresen B D. Development of an Ion Store/Time-of-Flight Mass Spectrometer for the Analysis of Volatile Compounds in Air. Anal. Chem. 1997.69(18): 3780-3790.
18. Halden R U, Happel a M and Schoen S R. Evaluation of Standard Methods for the Analysis of Methyl tert-Butyl Ether and Related Oxygenates in Gasoline-Contaminated Groundwater. Environ. Sci. Technol. 2001.35(7): 1469-1474.
19. Nouda B, Fouillet B, Toussaint G, et al. Complementarity of purge-and-trap and head-space capillary gas chromatographic methods for determination of methyl-tert.-butyl ether in water. Journal of Chromatography A. 1996. 726(1-2): 153-159.
20. Achten C, Kolb A and Puttmann W. Sensitive method for determination of methyl tert-butyl ether (MTBE) in water by use of headspace-SPME/GC-MS. Fresenius J Anal Chem. 2001. 371(4): 519-25.
21. Hong S, Duttweiler C M and Lemley a T. Analysis of methyl tert.-butyl ether and its degradation products by direct aqueous injection onto gas chromatography with mass spectrometry or flame ionization detection systems. Journal of Chromatography A. 1999. 857(1-2): 205-216.
22．周升如，马健，王少波等．密闭空间空气有机物的色／质谱定性分析研究．分析化学．1991(10)：1115-1121．
23. Szelewski M J and Quimby B D. Ambient Headspace GC and GC-MSD Analysis of Non-Polar Volatiles in Water. Agilent Technologies. 2000.
24. Feldman J and Orchin M. Determination of Methyl Tert. Butyl Ether (MTBE) in Gasoline. Analytical Letters. 1993.26(2): 357-365.
25. Hong M S, Farmayan W F, Dortch I J, et al. Phytoremediation of MTBE from a groundwater plume. Environ Sci Technol. 2001.35(6): 1231-9.
26. Fayolle F, Vandecasteele J P and Monot F. Microbial degradation and fate in the environment of methyl tert-butyl ether and related fuel oxygenates. Appl Microbiol Biotechnol. 2001.56(3-4): 339-49.
27．朱琳，刘涉江，黄国强等．甲基叔丁基醚的降解技术研究进展．化工进展．2005．24(10)：1136-1140．
28. Burbano aA, Dionysiou D D, Suidan M T, et al. Oxidation kinetics and effect ofpH on the degradation of MTBE with Fenton reagent. Water Res. 2005.39(1): 107-18.
29. Xu X-R, Zhao Z-Y, Lib X-Y, et al. Chemical oxidative degradation of methyl tert-butyl ether in aqueous solution by Fenton's reagent Chemosphere 2004.55(1): 73-79.
30. Bergendahl J A and Thies T P. Fenton's oxidation of MTBE with zero-valent iron. Water Research 2004.38(2): 327-334.
31. Wagler J L and Malley J P. Removal of methyl tertiary-butyl ether from a model ground water using UV/Peroxide oxidation. Journal of New England Water Works Association. 1994. 108(3): 236-260.
32. Barreto R D, Gray K A and Anders K. Photocatalytic degradation of methyl-tert-butyl ether in TiO2 slurries: a proposed reaction scheme. Water Research. 1995.29(5): 1243-1248.
33. Sahle-Demessie E, Richardson T, Almquist C B, et al. Comparison of Liquid and Gas-Phase Photooxidation of MTBE: Synthetic and Field Samples. Journal of Environmental Engineer. 2002. 128(9): 782-790.
34. Xu X R, Zhao Z Y, Li X Y, et al. Chemical oxidative degradation of methyl tert-butyl ether in aqueous solution by Fenton's reagent. Chemosphere. 2004. 55(1): 73-9.
35. Bergendahl J A and Thies T P. Fenton's oxidation of MTBE with zero-valent iron. Water Res. 2004.38(2): 327-34.
36. Kang J-W and Hoffmann M R. Kinetics and Mechanism of the Sonolytic Destruction of Methyl tert-Butyl Ether by Ultrasonic Irradiation in the Presence of Ozone. Environ. Sci. Technol. 1998.32(20): 3194-3199.
37. Shiha T C, Wangpaichitrb M and Suffet M. Evaluation of granular activated carbon technology for the removal of methyl tertiary butyl ether (MTBE) from drinking water. Water Research 2003.37(2): 375-385.
38. Wilhelm M J, Adams V D, Curtis J G, et al. Carbon Adsorption and Air-Stripping Removal of MTBE from River Water. Journal of Environmental Engineering. 2002. 128(9): 813-823.
39. Cooper W J, Nickelsen M G, Mezyk S P, et al. MTBE and priority contaminant treatment with high energy electron beam injection. Radiation Physics and Chemistry 2002. 65(4-5): 451-460.
40. Zang Y and Famood R. Effects of hydrogen peroxide concentration and ultraviolet light intensity on methyl tert-butyl ether degradation kinetics. Chemical Engineering Science 2005.60(6): 1641-1648.
41. Hsieh L-L, Lin Y-L and Wu C-H. Degradation of MTBE in dilute aqueous solution by gamma radiolysis. Water Research 2004. 38(16): 3627-3633.
42. Wu T, Cruz V, Mezyk S, et al. Gamma radiolysis of methyl t-butyl ether: a study of hydroxyl radical mediated reaction pathways. Radiation Physics and Chemistry 2002. 65(4-5): 335-341.
43. Kang J-W, Hung H-M, Lin A, et al. Sonolytic Destruction of Methyl tert-Butyl Ether by Ultrasonic Irradiation: The Role of O3, H2O2, Frequency, and Power Density. Environmental Science & Technology. 1999.33(18): 3199-3205.
44. Neppolian B, Jung H, Choi H, et al. Sonolytic degradation of methyl tert-butyl ether: the role of coupled Fenton process and persulphate ion. Water Res. 2002.36(19): 4699-708.
45. Bernhardt D and Diekmann H. Degradation of dioxane, tetrahydrofuran and other cyclic ethers by an environmental Rhodococcus strain. Appl Microbiol Biotechnol. 1991.36(1): 120-3.
46. Parales R E, Adamus J E, White N, et al. Degradation of 1, 4-dioxane by an actinomycete in pure culture. Appl Environ Microbiol. 1994. 60(12): 4527-30.
47. Hyman M R and Arp D J. Effects of ammonia on the de novo synthesis of polypeptides in cells of Nitrosomonas europaea denied ammonia as an energy source. J Bacteriol. 1995. 177(17): 4974-9.
48. Hur H, Newman L M, Wackett L P, et al. Toluene 2-Monooxygenase-Dependent Growth of Burkholderia cepacia G4/PR1 on Diethyl Ether. Appl Environ Microbiol. 1997. 63(4): 1606-1609.
49. Salanitro J P, Diaz L A, Williams M P, et al. Isolation of a Bacterial Culture That Degrades Methyl t-Butyl Ether. Appl Environ Microbiol. 1994. 60(7): 2593-2596.
50. Mo K, Lora C O, Wanken a E, et al. Biodegradation of methyl t-butyl ether by pure bacterial cultures. Appl Microbiol Biotechnol. 1997.47(1): 69-72.
51. Hanson J R, Ackerman C E and Scow K M. Biodegradation of methyl tert-butyl ether by a bacterial pure culture. Appl Environ Microbiol. 1999.65(11): 4788-92.
52. Deeb R A, Hu H Y, Hanson J R, et al. Substrate interactions in BTEX and MTBE mixtures by an MTBE-degrading isolate. Environ Sci Technol. 2001.35(2): 312-7.
53. Finneran K T and Lovley D R. Anaerobic degradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA). Environ Sci Technol. 2001.35(9): 1785-90.
54. Bradley P M, Chapelle F H and Landmeyer J E. Effect of redox conditions on MTBE biodegradation in surface water sediments. Environ Sci Technol. 2001.35(23): 4643-7.
55. Bradley P M, Chapelle F H and Landmeyer J E. Methyl t-butyl ether mineralization in surface-water sediment microcosms under denitrifying conditions. Appl Environ Microbiol. 2001.67(4): 1975-8.
56. Pruden A, Sedran M A, Suidan M T, et al. Anaerobic biodegradation of methyl ten-butyl ether under iron-reducing conditions in batch and continuous-flow cultures. Water Environ Res. 2005.77(3): 297-303.
57. Zaitsev G M, Uotila J S and Haggblom M M. Biodegradation of methyl ten-butyl ether by cold-adapted mixed and pure bacterial cultures. Appl Microbiol Biotechnol. 2007. 74(5): 1092-102.
58. Deeb R A, Scow K M and Alvarez-Cohen L. Aerobic MTBE biodegradation: an examination of past studies, current challenges and future research directions. Biodegradation. 2000. 11(2-3): 171-86.
59. Fortin N Y and Deshusses M A. Treatment of Methyl ten-Butyl Ether Vapors in Biotrickling Filters. 1. Reactor Startup, Steady-State Performance, and Culture Characteristics. Environmental Science and Technology 1999. 33(17): 2980-2986.
60. Fortin N Y and Deshusses M A. Treatment of Methyl tert-Butyl Ether Vapors in Biotrickling Filters. 2. Analysis of the Rate-Limiting Step and Behavior under Transient Conditions. Environmental Science and Technology. 1999. 33(17): 2987-2991.
61. Hardison L K, Curry S S, Ciuffetti L M, et al. Metabolism of Diethyl Ether and Cometabolism of Methyl ten-Butyl Ether by a Filamentous Fungus, a Graphium sp. Appl. Environ. Microbiol. 1997. 63(8): 3059-3067.
62. Hyman M and O'reilly K, Physiological and enzymatic features of MTBE-degrading bacteria., in Proceedings of the Fifth International In Situ and On-site Bioremediation Symposium, B.C. Alleman and A. Leeson, Editors. 1999, Battelle Press, Columbus, OH: Monterey, CA,.
63. Steffan R J, Mcclay K, Vainberg S, et al. Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria. Appl Environ Microbiol. 1997. 63(11): 4216-22.
64. Gamier P M, Auria R, Augur C, et al. Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane. Appl Microbiol Biotechnol. 1999. 51 (4): 498-503.
65. Koenigsberg S, Sandefur C, Mahaffey W, et al., Peroxygen mediated bioremediation of MTBE, in Proceedings of the Fifth International In Situ On-Site Bioremediation Symposium, A. BC and A. Leeson, Editors. 1999, Battelle Press, Columbus, OH: Monterey, CA. p. 13-18.
66. Francois A, Mathis H, Godefroy D, et al. Biodegradation of methyl ten-butyl ether and other fuel oxygenates by a new strain, Mycobacterium austroafricanum IFP 2012. Appl Environ Microbiol. 2002.68(6): 2754-62.
67. Okeke B C and Frankenberger W T, Jr. Biodegradation of methyl tertiary butyl ether (MTBE) by a bacterial enrichment consortia and its monoculture isolates. Microbiol Res. 2003. 158(2): 99-106.
68. Smith C A, O'reilly K T and Hyman M R. Cometabolism of methyl tertiary butyl ether and gaseous n-alkanes by Pseudomonas mendocina KR-1 grown on C5 to C8 n-alkanes. Appl Environ Microbiol. 2003.69(12): 7385-94.
69. Magana-Reyes M, Morales M and Revah S. Methyl tert-butyl ether and tert-butyl alcohol degradation by Fusarium solani. Biotechnol Lett. 2005.27(22): 1797-801.
70. Vainberg S, Mcclay K, Masuda H, et al. Biodegradation of ether pollutants by Pseudonocardia sp. strain ENV478. Appl Environ Microbiol. 2006.72(8): 5218-24.
71. Wilson G J, Pruden A, Suidan M T, et al. B iodegradation Kinetics of MTBE in Laboratory Batch and Continuous Flow Reactors. Journal of Environmental Engineering. 2002. 128(9): 824-829.
72. Vainberg S, Togna a P, Sutton P M, et al. Treatment of MTBE-Contaminated Water in Fluidized Bed Bioreactor. Journal of Environmental Engineering. 2002. 128(9): 842-851
73. Morrison J R, Suidan M T and Venosa a D. Use of Membrane Bioreactor for Biodegradation of MTBE in Contaminated Water. Journal of Environmental Engineering. 2002. 128(9): 836-841.
74. Reed T, Allen M H and Peabody J, JP-4/JP-8 BIOREMEDIATION USING ORC PUMPED INTO SEMI-PERMANENT INJECTION WELLS, in Sixth Annual In-Situ and On-Site Bioremediation Conference. 2001, Battelle Press, Columbus, OH.: San Diego, CA.
75. Magar V S, Hartzell K, Burton C, et al. Aerobic and Cometabolic MTBE Biodegradation at Novato and Port Hueneme. Journal of Environmental Engineering. 2002. 128(9): 883-890.
76. Barcelona M J and Jaglowski D R, Subsurface fate and transport of MTBE in a controlled reactive tracer experiment, in Proceedings of the 2000 Petroleum Hydrocarbons and Organic chemicals in Ground Water: Prevention, Detection, and Remediation. 1999, National Ground Water Association, Westerville, OH: Houston. p. 123-137.
77. Salanitro J P, Johnson P C, Spinnler G E, et al. Field-Scale Demonstration of Enhanced MTBE Bioremediation through Aquifer Bioaugrnentation and Oxygenation. Environ Sci Technol. 2000.34(19): 4152-4162.
78．于晓章 and Stefan T．汽油添加剂甲基叔丁基醚(MTBE)污染的植物修复．生态科学．2003．22(2)：109-112，146．
79. Rubin E and Ramaswami A. The potential for phytoremediation of MTBE. Water Res. 2001.35(5): 1348-53.
80. Xingrnao, Richter a R, Albers S, et al. Phytoremediation of MTBE with Hybrid Poplar Trees. International Journal of Phytoremediation. 2004.6(2): 157-167.
81．路争，钟卫鸿，陈建孟，et al．甲基叔丁基醚微生物降解研究进展．微生物学通报．2006．33(1)：122-127．
82. Hatzinger P B, Mcclay K, Vainberg S, et al. Biodegradation of methyl tert-butyl ether by a pure bacterial culture. Appl Environ Microbiol. 2001.67(12): 5601-7.
83. Church C D, Tratnyek P G and Scow K M. Pathways for the degradation of MTBE and other fuel oxygenates by isolate PM1. in 219th ACS National Meeting. 2000. San Fransisco, CA: Division of Environmental Chemistry, American Chemical Society.
84. Piveteau P, Fayolle F, Vandecasteele J P, et al. Biodegradation oftert-butyl alcohol and related xenobiotics by a methylotrophic bacterial isolate. Appl Microbiol Biotechnol. 2001.55(3): 369-73.
85. Chauvaux S, Chevalier F, Le Dantec C, et al. Cloning of a genetically unstable cytochrome P-450 gene cluster involved in degradation of the pollutant ethyl tert-butyl ether by Rhodococcus ruber. J Bacteriol. 2001. 183(22): 6551-7.
86．中华人民共和国国家统计局．中华人民共和国2006年国民经济和社会发展统计公报．2007 [cited; Available from:http://www.stats.gov.cn/tjgb/ndtjgb/qgndtjgb/t20070228_402387821.htm.
87．新华网．2007年国内石油实际消费增速将稳中略降．2007 [cited; Available from:http://www.sinopecnews.com.cn/shnews/2007-01/31/content_424346.htm.
88．新华网．中国水资源现况．2005[cited; Available from:http://news.xinhuanet.com/fortune/2005-09/19/content_3512629.htm.
89．新华每日电讯．京津唐：地下水中有机污染物达百余种．2005．
90．徐志强，宋宪臣，王玉军等．静态顶空-气相色谱法测定土壤中甲基叔丁基醚．吉林农业大学学报．2005．27(3)：310-314．
91. Labhut.Com. What is Headspace Gas Chromatography? [cited; Available from: http://www.labhut.com/education/headspace/index.php.
92. Szelewski Mj Q B. Ambient Headspace GC and GC-MSD Analysis of Non-Polar Volatiles in Water. Agilent Technologies. 2000.
93．陈井影，宋宪臣，孙华等．顶空-气相色谱法测定水体中甲基叔丁基醚．吉林农业大学学报．2006．28(3)：303-307．
94. Rohwerder T, Breuer U, BenndorfD, et al. The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway. Appl Environ Microbiol. 2006.72(6): 4128-35.
95．Sambrook J，分子克隆实验指南．第3版 ed．2005，北京：科学出版社．
96．萨姆布鲁克 J，拉塞尔 D W，SDS碱裂解法制备质粒DNA，in分子克隆实验指南，科学出版社：北京．p．27-36．
97. Jia X, Han S-J, Zhao Y-H, et al. Comparisons of extraction and purification methods of soil microorganism DNA from rhizosphere soil. Journal of Forestry Research. 2006. 17(1): 31-34.
98．萨姆布鲁克 J，拉塞尔 D W，用氯化钙制备和转化大肠杆菌，in分子克隆实验指南．2002，科学出版社：北京．p．96-99．
99. Ncbi. VecScreen. [cited; Available from: http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html.
100. Rice P, Longden I and Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000. 16(6): 276-7.
101. Altschul S F, Madden T L, Schaffer a A, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997. 25(17): 3389-402.
102. Lopes Ferreira N, Malandain C and Fayolle-Guichard F. Enzymes and genes involved in the aerobic biodegradation of methyl tert-butyl ether (MTBE). Appl Microbiol Biotechnol. 2006.72(2): 252-62.
103. Kumar S, Tamura K and Nei M. MEGA 3: Integrated Software for Molecular Evolutionary Genetics Analysis and Sequence Alignment. Briefings in Bioinformatics. 2004. 5: 150-163.
104. Gremion F, Chatzinotas A and Harms H. Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal-contaminated bulk and rhizosphere soil. Environ Microbiol. 2003.5(10): 896-907.
105. Benson D A, Karsch-Mizrach I, Lipman D J, et al. GenBank. Nucleic Acids Res. 2005. 33(Database Issue): D34-D38.
106. Thompson J D, Gibson T J, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997.25(24): 4876-82.
107. Singh V K, Mangalam a K, Dwivedi S, et al. Primer premier: program for design of degenerate primers from a protein sequence. Biotechniques. 1998.24(2): 318-9.
108．宝生物(大连)有限公司．双链DNA的制备方法．2005 [cited; Available from: http://www.takara.com.cn/product/2005/o/O-9FH11001.html.
109. Tatusov T and Tatusov R. ORF Finder. [cited; Available from: http://www.ncbi.nlm.nih.gov/gorf/gorf.html.
110. Shanklin J and Whittle E. Evidence linking the Pseudomonas oleovorans alkane g-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family. FEBS Letters. 2003.545: 188-192.
111. Consortium T I. The InterPro database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Research. 2001.29(1): 37-40.
112. Morales M, Velazquez E, Jan J, et al. Methyl tert-butyl ether biodegradation by microbial consortia obtained from soil samples of gasoline-polluted sites in Mexico. Biotechnol Lett. 2004.26(4): 269-75.