原油微生物群落构成及降解菌降解特性的研究

生物技术通报 ›› 2013, Vol. 0 ›› Issue (1): 178-185.

原油微生物群落构成及降解菌降解特性的研究

王中华¹, 梁静儿¹ ,杨建强² ,周君¹ ,李成华¹, 李太武^1,3

1. 宁波大学海洋学院，宁波 315211 ；2. 国家海洋局北海分局，青岛 266033 ；3. 宁波城市职业技术学院，宁波 315100

收稿日期:2012-09-24 修回日期:2013-01-31 出版日期:2013-01-30 发布日期:2013-01-30
作者简介:王中华，男，博士研究生，研究方向：海洋生态环境；E-mail: wzhxinxiang76@sina.com
基金资助:
浙江省重大科技攻关项目（2006C13089），宁波市科技局资助项目（2008C50027）

Microbial Diversity of the Crude Oil and Characteristics of a Hydrocarbon-degrading Bacterium

Wang Zhonghua¹, Liang Jinger¹ ,Yang Jianqiang²,Zhou Jun¹ ,Li Chenghua¹ ,Li Taiwu^1,3

1. Faculty of Life Science and Biotechnology，Ningbo University，Ningbo 315211;
2. North China Sea Branch of The State Oceanic Administration，Qingdao 266033;
3. Ningbo City College of Vocational Technology，Ningbo 315100

Received:2012-09-24 Revised:2013-01-31 Published:2013-01-30 Online:2013-01-30

摘要/Abstract

摘要： 为分离得到高效原油降解菌，直接向原油中加入营养物刺激，培养一段时间后原油乳化降解。气相色谱法、红外光谱法和紫外吸收均表明降解菌体系具有较强的降解原油烃的能力。采用细菌 16S rDNA 通用引物和 PCR 扩增等方法，构建原油降解菌体系 16S rDNA 克隆文库，并对所构建的文库进行分析。同时，从降解菌体系中分离得到一株降解菌，鉴定结果表明所分离的细菌为短小芽孢杆菌（Bacillus pumilus）。采用气相色谱技术对降解过程中的原油烃的变化进行分析，结果显示原油烃类组分会随着降解时间的变化而发生变化，降解菌表现出其对烷烃类物质的降解能力。

关键词: 原油 , 红外光谱 , 16S , rDNA , 生物降解 , 气相色谱

Abstract: The biodegradation bacterium system was obtained through adding nutrition, and the IR Technology, GC analysis and UVmethond were used. The analyse showed high degradation ability for crude oil. The diversity of bacterial communities in crude oil was researchedusing 16S rDNA library approach. A high efficiency bacterium was obtained from the biodegradation bacterium system, which was identified asBacillus pumilus by automated cellular fatty acid identification system and phylogenetic analyses based on the 16S rDNA sequence. GC analysisshowed that the low-carbon chain alkanes and high-carbon-chain alkanes as well as aromatic hydrocarbons can be degraded.

Key words: Crude oil , IR technology , 16S rDNA , Biodegradation , Gas chromatography

王中华, 梁静儿, 杨建强, 周君, 李成华, 李太武. 原油微生物群落构成及降解菌降解特性的研究[J]. 生物技术通报, 2013, 0(1): 178-185.

Wang Zhonghua, Liang Jinger, Yang Jianqiang, Zhou Jun, Li Chenghua, Li Taiwu. Microbial Diversity of the Crude Oil and Characteristics of a Hydrocarbon-degrading Bacterium[J]. Biotechnology Bulletin, 2013, 0(1): 178-185.

参考文献

[1] Balba MT, Awadhi NA, Daher RA. Bioremediation of oil contaminatedsoil microbiological methods for feasibility assessment and fieldevaluation[J]. Journal of Microbiological Methods, 1998, 32 ：155-164.
[2] 丁明宇, 黄健, 李永祺. 海洋微生物降解石油的研究[J]. 环境科学学报, 2001, 21（1）：84-88.
[3] Maneerat S, Bamba T, Harada K, et al. A novel crude oil emulsifierexcreted in the culture supernatant of a marine bacterium, Myroidessp. strain SM1[J]. Appl Microbiol Biotechnol, 2006, 70 ：254-259.
[4] Mimmi TH, Alexander W, Ellingsen ET, et al Identification of novelgenes involved in long-chain n-Alkane degradation by Acinetobactersp. strain DSM 17874[J]. Appled Environmental Microbiology,2007, 73（10）：3327-3332
[5] Kishore D, Ashis KM. Crude petroleum-oil biodegradation eYciency2013年第1期185 王中华等：原油微生物群落构成及降解菌降解特性的研究of Bacillus subtilis and Pseudomonas aeruginosa strains isolated froma petroleum-oil contaminated soil from North-East India[J].BioresourceTechnology, 2007, 98 ：1339-1345.
[6] 于晓丽. 油气田废水中石油类物质测定问题的探讨[J]. 油气田环境保护, 1994, 4（2）：34-37.
[7] Zhou JL. Distribution of polycyclic aromatic hydrocarbons in waterand surface sediments from Data Bay[J]. China EnvironmentalPollution, 2003, 12（1）：269-281.
[8] Rahman MH, Suzuki S, Kawai K. Formation of viable but nonculturablestate（VBNC）of Aeromonas hydrophila and its virulencein Goldfish, Carassius auratus[J]. Microbiological Research,2001, 156（1）：103-106.
[9] Dianne KN, Jillian FB. Geomicrobiology ：howmolecular-scaleinteractions underpin biogeochemical systems[J]. Science, 2002,296（5570）：10-71.
[10] Tarasov AL, Borzenkov IA, Milekhina EI. Dynamics of microbialprocesses in the stratal waters of the Romashkinskoe oil field[J].Microbiology, 2002, 71（6）：735-742.
[11] Nazina TN, Grigoryan AA, Xue YF, et al. Phylogenetic diversity ofaerobic saprotrophic bacteria isolated from the Daqing oil field[J].Microbiology, 2002. 71（1）：91-97.
[12] Liu JF, Mu BZ. Extreme environment of oil reservoir and associatedmicroorganisms[J]. Microbiology, 2004, 24（4）：31-34.
[13] Darby AC, Birkle LM, Tumer SI, et al. An aphidbome bacterium alliedto the secondary symbionts of whitefly[J]. Microbial Ecological,2001, 36 ：43-50.
[14] Katsivela E, Edward RB, et al. Biodegradation of aliphatic and aromatichydrocarbons ：specificity among bacteria isolated from refinerywastesludge[J]. Water, Air, and Soil Pollution, 2003, 3 ：103-115.
[15] 王靖, 刘元琴, 姜凯, 等. 嗜蜡菌R 的代谢特性及其摄取烷烃的机理研究[J]. 中国石油大学学报, 2007, 31（3）：143-147.
[16] Kemp PF, Aller JY. Bacterial diversity in aquatic and other environments：what 16S rDNA libraries can tell us[J]. MicrobiologyEcology, 2004, 47 ：161-177.
[17] Kotani T, Kawashima Y, Yurimoto H, et al. Gene structure andregulation of alkane monooxygenases in propane-utilizing Mycobacteriumsp. TY-6 and Pseudonocardia sp. TY-7 The Society forBiotechnology[J]. 2006, 102（3）：184-192.
[18] van Beilen JB, Smits THM, Whyte LG, et al. Alkane hydroxylase homologuesin Gram-positive strains[J]. Environmental Microbiology,2002, 4（11）：676-682.
[19] Smits TH, Rothlisberger M, Witholt B, van Beilen JB. Molecular screeningfor alkane hydroxylase genes in Gram-negative and Grampositivestrains[J]. Environmental Microbiology, 1999, 1（4）：307-317.
[20] Whyte LG, Smits THM, Labbe D, et al. Gene cloning and characterizationof multiple alkane hydroxylase systems in Rhodococcusstrains Q15 and NRRL B-16531[J]. Appled And EnvironmentalMicrobiology, 2002, 68（12）5933-5942.
[21] Kahng HY, Malinverni JC, Majko MM, et al. Genetic and functionalanalysis of the tbc operons for catabolism of alkyl and chloroaromaticcompounds in Burkholderia sp. strain JS150[J]. Appl EnvironMicrobiol, 2001, 67 ：4805-4816.