长庆油田石油污染土壤的原位修复微生物的筛选和鉴定
摘要
本论文论述了石油土壤污染的起因、危害及当前国际国内对于土壤石油污染的最新修复技术。通过对采集于长庆油田地区的石油污染土壤微生物的分离筛选,初步获得了182株具有石油降解能力的菌株,选取其中20株降解能力较好的菌株进行了降解特性分析,并选取其中最高效的菌株组合成高效降解菌群C1。通过对土壤背景值的测定,找出最合适的营养物质配比。采用单因素和正交设计的方法,得到最佳的外界环境因素。最后,利用分子生物学的手段,初步分析土壤中石油降解菌的多样性,获得组成C1的4株菌16S rDNA的序列,并与相关菌株共同构建系统发育树,获得他们在分类学上的地位。
(1)实验选用以石油为碳源的选择性培养基筛选出具有石油降解能力的菌株,初筛得到单菌182株。复筛分析了初筛菌株对正十六烷、环己烷和菲的降解能力,进而得到降解效果比较好的单菌4株,组合出一组高效菌群C1。
(2)通过实验,分析了采样土壤的部分理化性质。其中土壤样品中的磷含量为0.7652 g·kg~(-1),氮含量为0.5748 g·kg~(-1),有机质含量为169.73 g·kg~(-1),pH值为8.12。根据生物降解过程中对氮、磷营养盐的要求大约为mC:mN:mP=100:5:l,则土壤中的氮含量应大于4.9225 g·kg~(-1),土壤中的磷含量应大于0.9845 g·kg~(-1)。因此,对微生物降解石油的生化过程而言,营养状况很不平衡,特别是氮素,缺量很大,为了使降解率提高,我们必须添加额外的营养物质,主要是N源和P源。
(3)通过正交设计实验,我们得到:在1000 mL的培养基中应加入KNO_3 6g,K_2HPO_4 2g,在37℃,pH为7,转速为180 rpm时,C1对石油的降解率可以到达78.5%。通过气相色谱分析,从质上进一步验证了C1的石油降解能力。
(4)利用分子生物学的方法获得了土壤样本中20株石油降解菌的DNA,酶切后,分析了它们种群的多样性。通过PCR扩增,获得了组成C1的4株菌的16S rDNA全序列,通过系统发育树,获得了他们在分类学上的基本地位,通过GeneBank的比对,结果CQ~(-1)为芽孢杆菌属(Bacillus),CQ-2和CQ-9均为不动杆菌属(Acinetobacter),CQ~(-1)3为威廉土氏菌属(Williamsia)。
The paper discussed the origin and harm of oil pollution in soil and also included the newest bioremediation technology in the world wide. Screening soil microorganism through the soil which were collected from ChangQing oil field areas, we got 182 strains with oil degradation ability, choose 20 strains which were outstanding on the degradation characteristics, and combined one bacterium group (C1) with high efficiency of degradation. We identified the most appropriate proportion of nutrients by analyzing the soil background values. Furthermore, we determined the best environmental factors by single factor and orthogonal design method. Finally, using the method of molecular biology, we had a preliminary understanding on the diversity of soil degradation bacterium and obtained 16S rDNA gene sequences of four strains composing C1 group. On the basis of 16S rDNA gene sequences of four and other related strains, we build the phylogenetic tree in order to identify these strains taxonomical status.
(1) By using the selective medium which carbon source was oil, we initially got 182 strains with the ability of oil degradation. After analyzing the ability of hexadecane, cyclohexane and phenanthrene degradation of 182 strains, we got four stains which degradation ability were remarkable and combined one bacterium group (C1) with high efficiency of degradation.
(2) Through analysis of the physical and chemical properties of soil, we knew the phosphorus content of soil samples was 0.7652 g·kg~(-1), nitrogen content was 0.5748 g·kg~(-1), organic matter content was 169.73 g·kg~(-1) and pH value was 8.12. According to the nitrogen, phosphorus, nutrient requirement during biodegradation (mC:mN:mP = 100:5:1), the soil nitrogen content and phosphorus content should be more than 4.9225 g·kg~(-1) and 0.9845 g·kg~(-1) respectively. Therefore, during the biochemical process of oil degradation, the nutrition was uneven, especially on the nitrogen element which were very rare. In order to improve this problem, we must add extra nutrients such as N and P.
(3) Through orthogonal experiment design, we got the results that every 1000 mL medium should be added KNO_3 6 g, K_2HPO_4 2 g and when the temperature was 37 degrees, pH was 7, speed was 180 rpm, the degradation rate of C1 could reach 78.5%. Through the meteorological chromatographic analysis, we confirmed the ability of oil degradation of C1 group.
(4) We got the DNA of 20 strains with ability of oil degradation by using the method of molecular biology, and analyzed the diversity of these strains after RFLP method. After the PCR amplification, we obtained 16S rDNA whole gene sequences of four strains composing C1 group. Through comparison in Genebank and phylogenetic tree of 16S rDNA gene sequences, the taxonomical statuses of these strains were determined that CQ-1 was Bacillus, CQ-2 and CQ-9 were Acinetobacter , CQ– 13 was Williamsia.
(1)实验选用以石油为碳源的选择性培养基筛选出具有石油降解能力的菌株,初筛得到单菌182株。复筛分析了初筛菌株对正十六烷、环己烷和菲的降解能力,进而得到降解效果比较好的单菌4株,组合出一组高效菌群C1。
(2)通过实验,分析了采样土壤的部分理化性质。其中土壤样品中的磷含量为0.7652 g·kg~(-1),氮含量为0.5748 g·kg~(-1),有机质含量为169.73 g·kg~(-1),pH值为8.12。根据生物降解过程中对氮、磷营养盐的要求大约为mC:mN:mP=100:5:l,则土壤中的氮含量应大于4.9225 g·kg~(-1),土壤中的磷含量应大于0.9845 g·kg~(-1)。因此,对微生物降解石油的生化过程而言,营养状况很不平衡,特别是氮素,缺量很大,为了使降解率提高,我们必须添加额外的营养物质,主要是N源和P源。
(3)通过正交设计实验,我们得到:在1000 mL的培养基中应加入KNO_3 6g,K_2HPO_4 2g,在37℃,pH为7,转速为180 rpm时,C1对石油的降解率可以到达78.5%。通过气相色谱分析,从质上进一步验证了C1的石油降解能力。
(4)利用分子生物学的方法获得了土壤样本中20株石油降解菌的DNA,酶切后,分析了它们种群的多样性。通过PCR扩增,获得了组成C1的4株菌的16S rDNA全序列,通过系统发育树,获得了他们在分类学上的基本地位,通过GeneBank的比对,结果CQ~(-1)为芽孢杆菌属(Bacillus),CQ-2和CQ-9均为不动杆菌属(Acinetobacter),CQ~(-1)3为威廉土氏菌属(Williamsia)。
The paper discussed the origin and harm of oil pollution in soil and also included the newest bioremediation technology in the world wide. Screening soil microorganism through the soil which were collected from ChangQing oil field areas, we got 182 strains with oil degradation ability, choose 20 strains which were outstanding on the degradation characteristics, and combined one bacterium group (C1) with high efficiency of degradation. We identified the most appropriate proportion of nutrients by analyzing the soil background values. Furthermore, we determined the best environmental factors by single factor and orthogonal design method. Finally, using the method of molecular biology, we had a preliminary understanding on the diversity of soil degradation bacterium and obtained 16S rDNA gene sequences of four strains composing C1 group. On the basis of 16S rDNA gene sequences of four and other related strains, we build the phylogenetic tree in order to identify these strains taxonomical status.
(1) By using the selective medium which carbon source was oil, we initially got 182 strains with the ability of oil degradation. After analyzing the ability of hexadecane, cyclohexane and phenanthrene degradation of 182 strains, we got four stains which degradation ability were remarkable and combined one bacterium group (C1) with high efficiency of degradation.
(2) Through analysis of the physical and chemical properties of soil, we knew the phosphorus content of soil samples was 0.7652 g·kg~(-1), nitrogen content was 0.5748 g·kg~(-1), organic matter content was 169.73 g·kg~(-1) and pH value was 8.12. According to the nitrogen, phosphorus, nutrient requirement during biodegradation (mC:mN:mP = 100:5:1), the soil nitrogen content and phosphorus content should be more than 4.9225 g·kg~(-1) and 0.9845 g·kg~(-1) respectively. Therefore, during the biochemical process of oil degradation, the nutrition was uneven, especially on the nitrogen element which were very rare. In order to improve this problem, we must add extra nutrients such as N and P.
(3) Through orthogonal experiment design, we got the results that every 1000 mL medium should be added KNO_3 6 g, K_2HPO_4 2 g and when the temperature was 37 degrees, pH was 7, speed was 180 rpm, the degradation rate of C1 could reach 78.5%. Through the meteorological chromatographic analysis, we confirmed the ability of oil degradation of C1 group.
(4) We got the DNA of 20 strains with ability of oil degradation by using the method of molecular biology, and analyzed the diversity of these strains after RFLP method. After the PCR amplification, we obtained 16S rDNA whole gene sequences of four strains composing C1 group. Through comparison in Genebank and phylogenetic tree of 16S rDNA gene sequences, the taxonomical statuses of these strains were determined that CQ-1 was Bacillus, CQ-2 and CQ-9 were Acinetobacter , CQ– 13 was Williamsia.
引文
白洁,崔爱玲,吕艳华.石油降解菌对石油烃的降解能力及影响因素研究.海洋湖沼通报, 2007, 3: 41～46.
鲍士旦.土壤农化分析.中国农业出版社, 2000. 12,北京.
程国玲,李培军.石油污染土壤的植物与微生物修复技术.环境工程学报, 2007, l(6): 91.
程丽娟,薛泉宏.微生物学实验技术.世界图书出版社, 2000,西安.
都芳兰,史权,冀海南.冀东油田主力区原油组分分析.石油钻采工艺, 2007, 29: 88～90.
郭军康.铅锌尾矿抗重金属放线菌的分离筛选鉴定.西北农林科技大学硕士论文, 2008.
韩萍萍,朱虎,魏东芝,沈亚领.石油降解茵的筛选、鉴定及降解石油的初步研究.化学与生物工程, 2008, 9: 34～38.
赫荣乔.一种低温石油烃降解菌产生的烃类乳化剂.微生物学通报, 2008, 35(5): 836.
贺永华,胡立芳,沈东升,朱荫湄.污染环境生物修复技术研究进展.科技通报, 2007, 23(2): 271～275.
侯发可.论影响微生物降解石油烃类物质的因素.湖南农机, 2007, 9: 158～159.
胡晓芳,夏福军,朱南文,张之崟,陈佳一.原油污染土壤的生物法修复效果研究.环境化学, 2006, 25(5): 593～596.
金文标,王宝贞,宋莉晖.油污土壤堆制处理实验研究.环境科学学报, 2008, 22(3): 405～407.
李大平,王春明,田崇民,何晓红,王晓梅,董微.高温原油降解菌处理油田采油废水的研究.应用与环境生物学报, 2005, 11(6): 738～741.
李辉,牟伯中.油藏微生物多样性的分子生态学研究进展.微生物学通报, 2008, 35(5): 805～808.
李红权,李红梅,蒋继志,杨雪丽,郭荣君,平淑珍,张维.一株DDT降解菌的筛选、鉴定及降解特性的初步研究.微生物学通报, 2008, 35(5): 696～699.
李培军,郭书海,孙铁珩,台培东,张春桂,白玉兴,孙强,生平.不同类型原油污染土壤生物修复技术研究.应用生态学报, 2002, 13(11): 1455～1458.
刘媚媚,金腊华,李文松,贾雅漪.一株石油降解菌的活性炭纤维固定化研究.环境污染与防治, 2009, 3l(10): 48～51.
刘晓艳,李兴伟,纪学雁,刘晓.油田城市地表土壤石油污染特点及其防治对策.国土与自然资源研究, 2004, 4: 68～69.
孔繁翔.环境生物学.高等教育出版社, 2001.12,北京.
倪怀英,缪明富,方娅.钻井液润滑及抗磨处理剂.钻井液处理剂进展评价情报调研文集, 1991, 5.
任随周,郭俊,邓穗儿,岑英华,孙永革,孙国萍.石油降解茵的分离鉴定及石油污染土壤的细菌多样性.生态学报, 2005, 25(12): 3314～3322.
苏增建,李敏,王颖.土壤石油污染的生物修复原理及研究进展.安徽农业科学, 2007, 35(6): 1742～1744.
孙晓媛,李影,钱爱东.开发未可培养微生物技术的研究进展.微生物学通报, 2008, 35(5): 798～802.
谭丽泉,周焕清.表面活性剂在微生物降解石油中的应用研究进展.化学与生物工程, 2007, 10:9～11.
滕应,骆永明,李振高.污染土壤的微生物修复原理与技术进展.土壤, 2007, 39 (4): 497～502.
王伟平,李伟,邱雁临,张华山,彭晓斌.高效石油降解茵的筛选及初步鉴定.化学与生物工程, 2009, 7: 64～66.
万春黎,杜茂安,杨巧利,高种,施雪华.一株石油降解菌的生长特性.石油学报(石油加工), 2009, 25(1): 134～138.
王爱杰,阚洪晶,于振国,任南琪,刘春爽,张运晴,赵阳国. SSCP技术分析活性污泥微生物群落结构的条件优化及检验.微生物学通报, 2008, 35(7): 1164～1169.
吴新世,叶锋,张金涛,王茜,刁虎欣.新型润滑油生物降解性及其分析方法改进探讨.微生物学通报, 2008, 35(6): 872～875.
辛玉峰,曲晓华.一株石油降解菌的筛选、分离纯化及降解性能分析.黑龙江农业科学, 2008, (1):17～18.
徐金兰,黄廷林,唐智新,肖洲强.石油污染土壤生物修复高效菌的降解特性.石油学报(石油加工), 2009, 25(4): 570～575.
杨乐,鲁建江,王开勇,庞玮,李春.石油降解菌的筛选及其产表面活性剂的研究.石河子大学学报(自然科学版), 2009, 27(2): 221～224.
杨雪莲,李凤梅,刘婉婷,李刚.高效石油降解菌的筛选及其降解特性.农业环境科学学报, 2008, 27(1): 0230～0233.
张胜,陈立,崔晓梅,马彦超,么红超,张翠云,李政红,张发旺,殷密英,马琳娜.西北黄土区石油污染土壤原位微生物生态修复试验研究.微生物学通报, 2008, 35(5): 765～771.
张旭,李广贺,黄巍.石油烃污染土层生物修复模拟实验研究.清华大学学报(自然科学版), 2000, 40(11): 106～108.
张妍.黄土区石油污染土壤的现状及其修复.商洛学院学报, 2008, 22(2): 56～58.
张逸飞,钟文辉,王国祥.微生物在污染环境生物修复中的应用.中国生态农业学报, 2007, 15(3): 198～201.
周海霞,单爱琴,王莉淋,李海花.石油降解菌的筛选及其降解效率的研究.环境科学与技术, 2008, 31(10): 56～58.
A. Husaini H, A.Roslan K, S.Y.Hii. Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites. World J Microbiol Biotechnol, 2008, 24: 2789–2797.
A. Rajasekar S. Bacterial Degradation and Corrosion of Naphtha in Transporting Pipeline. Curr Microbiol, 2007, 55: 374–381.
Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. 1990. Basic local alignment search tool. J Mol Biol, 215(3): 403-410.
Ania C, U Lrich , Selma E,Guigard. Effect of salt on aerobic biodegradation of petroleum hydrocarbons in contaminated groundwater. Biodegradation, 2009, 20: 27–38.
Aruliah Rajasekar, Thambidurai Ganesh Babu. Biodegradation and corrosion behaviour of Serratia marcescens ACE2 is olated from an Indian diesel-transporting pipeline. World J Microbiol Biotechnol, 2007, 23: 1065–1074.
D. Kotresha G, M. Vidyasagar. Isolation and characterisation of phenol-degrading Pseudomonas aeruginosa MTCC 4996. World J Microbiol Biotechnol, 2008, 24: 541–547.
Daisuke Sugimori. Edible oil degradation by using yeast coculture of Rhodotorula pacifica ST3411 and Cryptococcus laurentii ST3412. Appl Microbiol Biotechnol, 2009, 82: 351–357.
Frederic Coulon, Daniel Delille. Influence of substratum on the degradation processes in diesel polluted sub-Antarctic soils (Crozet Archipelago). Polar Biol, 2006, 29: 806–812.
Ji Hye Hong, Jaisoo Kim. Characterization of a diesel-degrading bacterium, Pseudomonas aeruginosa IU5,isolated from oil-contaminated soil in Korea. World Journal of Microbiology & Biotechnology, 2005, 21: 381–384.
Manoj Kumar,Vladimir Leon. Biosurfactant production and hydrocarbon-degradation by halotolerantand thermotolerant Pseudomonas sp. World J Microbiol Biotechnol, 2008, 24: 1047–1057.
Meixiang Xu, Xiang Xiao, Fengping Wang. Isolation and characterization of alkane hydroxylases from a metagenomic library of Pacific deep-sea sediment. Extremophiles, 2008, 12: 255–262.
Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 4(4): 406-425.
Sunday A. Adebusoye. Microbial degradation of petroleum hydrocarbons in a polluted tropical stream. World J Microbiol Biotechnol, 2007, 23: 1149–1159.
Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, Higgins D G. 1997. The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25(24): 4876-4882.
Uta Deppe,Hans-Hermann Richnow. Degradation of crude oil by an arctic microbial consortium. Extremophiles, 2005, 9: 461–470.
Van de Peer Y, De Wachter R. 1994. TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Applic Biosci, 10(5): 569-570.
Weisburg W G, Barns S M, Pelletier D A, Lane D J. 1991. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol, 173(2): 697-703.
鲍士旦.土壤农化分析.中国农业出版社, 2000. 12,北京.
程国玲,李培军.石油污染土壤的植物与微生物修复技术.环境工程学报, 2007, l(6): 91.
程丽娟,薛泉宏.微生物学实验技术.世界图书出版社, 2000,西安.
都芳兰,史权,冀海南.冀东油田主力区原油组分分析.石油钻采工艺, 2007, 29: 88～90.
郭军康.铅锌尾矿抗重金属放线菌的分离筛选鉴定.西北农林科技大学硕士论文, 2008.
韩萍萍,朱虎,魏东芝,沈亚领.石油降解茵的筛选、鉴定及降解石油的初步研究.化学与生物工程, 2008, 9: 34～38.
赫荣乔.一种低温石油烃降解菌产生的烃类乳化剂.微生物学通报, 2008, 35(5): 836.
贺永华,胡立芳,沈东升,朱荫湄.污染环境生物修复技术研究进展.科技通报, 2007, 23(2): 271～275.
侯发可.论影响微生物降解石油烃类物质的因素.湖南农机, 2007, 9: 158～159.
胡晓芳,夏福军,朱南文,张之崟,陈佳一.原油污染土壤的生物法修复效果研究.环境化学, 2006, 25(5): 593～596.
金文标,王宝贞,宋莉晖.油污土壤堆制处理实验研究.环境科学学报, 2008, 22(3): 405～407.
李大平,王春明,田崇民,何晓红,王晓梅,董微.高温原油降解菌处理油田采油废水的研究.应用与环境生物学报, 2005, 11(6): 738～741.
李辉,牟伯中.油藏微生物多样性的分子生态学研究进展.微生物学通报, 2008, 35(5): 805～808.
李红权,李红梅,蒋继志,杨雪丽,郭荣君,平淑珍,张维.一株DDT降解菌的筛选、鉴定及降解特性的初步研究.微生物学通报, 2008, 35(5): 696～699.
李培军,郭书海,孙铁珩,台培东,张春桂,白玉兴,孙强,生平.不同类型原油污染土壤生物修复技术研究.应用生态学报, 2002, 13(11): 1455～1458.
刘媚媚,金腊华,李文松,贾雅漪.一株石油降解菌的活性炭纤维固定化研究.环境污染与防治, 2009, 3l(10): 48～51.
刘晓艳,李兴伟,纪学雁,刘晓.油田城市地表土壤石油污染特点及其防治对策.国土与自然资源研究, 2004, 4: 68～69.
孔繁翔.环境生物学.高等教育出版社, 2001.12,北京.
倪怀英,缪明富,方娅.钻井液润滑及抗磨处理剂.钻井液处理剂进展评价情报调研文集, 1991, 5.
任随周,郭俊,邓穗儿,岑英华,孙永革,孙国萍.石油降解茵的分离鉴定及石油污染土壤的细菌多样性.生态学报, 2005, 25(12): 3314～3322.
苏增建,李敏,王颖.土壤石油污染的生物修复原理及研究进展.安徽农业科学, 2007, 35(6): 1742～1744.
孙晓媛,李影,钱爱东.开发未可培养微生物技术的研究进展.微生物学通报, 2008, 35(5): 798～802.
谭丽泉,周焕清.表面活性剂在微生物降解石油中的应用研究进展.化学与生物工程, 2007, 10:9～11.
滕应,骆永明,李振高.污染土壤的微生物修复原理与技术进展.土壤, 2007, 39 (4): 497～502.
王伟平,李伟,邱雁临,张华山,彭晓斌.高效石油降解茵的筛选及初步鉴定.化学与生物工程, 2009, 7: 64～66.
万春黎,杜茂安,杨巧利,高种,施雪华.一株石油降解菌的生长特性.石油学报(石油加工), 2009, 25(1): 134～138.
王爱杰,阚洪晶,于振国,任南琪,刘春爽,张运晴,赵阳国. SSCP技术分析活性污泥微生物群落结构的条件优化及检验.微生物学通报, 2008, 35(7): 1164～1169.
吴新世,叶锋,张金涛,王茜,刁虎欣.新型润滑油生物降解性及其分析方法改进探讨.微生物学通报, 2008, 35(6): 872～875.
辛玉峰,曲晓华.一株石油降解菌的筛选、分离纯化及降解性能分析.黑龙江农业科学, 2008, (1):17～18.
徐金兰,黄廷林,唐智新,肖洲强.石油污染土壤生物修复高效菌的降解特性.石油学报(石油加工), 2009, 25(4): 570～575.
杨乐,鲁建江,王开勇,庞玮,李春.石油降解菌的筛选及其产表面活性剂的研究.石河子大学学报(自然科学版), 2009, 27(2): 221～224.
杨雪莲,李凤梅,刘婉婷,李刚.高效石油降解菌的筛选及其降解特性.农业环境科学学报, 2008, 27(1): 0230～0233.
张胜,陈立,崔晓梅,马彦超,么红超,张翠云,李政红,张发旺,殷密英,马琳娜.西北黄土区石油污染土壤原位微生物生态修复试验研究.微生物学通报, 2008, 35(5): 765～771.
张旭,李广贺,黄巍.石油烃污染土层生物修复模拟实验研究.清华大学学报(自然科学版), 2000, 40(11): 106～108.
张妍.黄土区石油污染土壤的现状及其修复.商洛学院学报, 2008, 22(2): 56～58.
张逸飞,钟文辉,王国祥.微生物在污染环境生物修复中的应用.中国生态农业学报, 2007, 15(3): 198～201.
周海霞,单爱琴,王莉淋,李海花.石油降解菌的筛选及其降解效率的研究.环境科学与技术, 2008, 31(10): 56～58.
A. Husaini H, A.Roslan K, S.Y.Hii. Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites. World J Microbiol Biotechnol, 2008, 24: 2789–2797.
A. Rajasekar S. Bacterial Degradation and Corrosion of Naphtha in Transporting Pipeline. Curr Microbiol, 2007, 55: 374–381.
Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. 1990. Basic local alignment search tool. J Mol Biol, 215(3): 403-410.
Ania C, U Lrich , Selma E,Guigard. Effect of salt on aerobic biodegradation of petroleum hydrocarbons in contaminated groundwater. Biodegradation, 2009, 20: 27–38.
Aruliah Rajasekar, Thambidurai Ganesh Babu. Biodegradation and corrosion behaviour of Serratia marcescens ACE2 is olated from an Indian diesel-transporting pipeline. World J Microbiol Biotechnol, 2007, 23: 1065–1074.
D. Kotresha G, M. Vidyasagar. Isolation and characterisation of phenol-degrading Pseudomonas aeruginosa MTCC 4996. World J Microbiol Biotechnol, 2008, 24: 541–547.
Daisuke Sugimori. Edible oil degradation by using yeast coculture of Rhodotorula pacifica ST3411 and Cryptococcus laurentii ST3412. Appl Microbiol Biotechnol, 2009, 82: 351–357.
Frederic Coulon, Daniel Delille. Influence of substratum on the degradation processes in diesel polluted sub-Antarctic soils (Crozet Archipelago). Polar Biol, 2006, 29: 806–812.
Ji Hye Hong, Jaisoo Kim. Characterization of a diesel-degrading bacterium, Pseudomonas aeruginosa IU5,isolated from oil-contaminated soil in Korea. World Journal of Microbiology & Biotechnology, 2005, 21: 381–384.
Manoj Kumar,Vladimir Leon. Biosurfactant production and hydrocarbon-degradation by halotolerantand thermotolerant Pseudomonas sp. World J Microbiol Biotechnol, 2008, 24: 1047–1057.
Meixiang Xu, Xiang Xiao, Fengping Wang. Isolation and characterization of alkane hydroxylases from a metagenomic library of Pacific deep-sea sediment. Extremophiles, 2008, 12: 255–262.
Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 4(4): 406-425.
Sunday A. Adebusoye. Microbial degradation of petroleum hydrocarbons in a polluted tropical stream. World J Microbiol Biotechnol, 2007, 23: 1149–1159.
Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, Higgins D G. 1997. The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25(24): 4876-4882.
Uta Deppe,Hans-Hermann Richnow. Degradation of crude oil by an arctic microbial consortium. Extremophiles, 2005, 9: 461–470.
Van de Peer Y, De Wachter R. 1994. TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Applic Biosci, 10(5): 569-570.
Weisburg W G, Barns S M, Pelletier D A, Lane D J. 1991. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol, 173(2): 697-703.