苯酚降解菌的筛选、鉴定及其降解特性的研究

上海师范大学

硕士学位论文

苯酚降解菌的筛选、鉴定及其降解特性的研究

姓名：何小丽

申请学位级别：硕士

专业：微生物学

指导教师：肖明

20090501

上海师范大学硕士学位论文摘要论文题目：苯酚降解菌的筛选、鉴定及其降解特性的研究

学校专业：微生物学

学位申请人：何小丽

指导教师：肖明

摘要

酚类化合物为细胞原浆毒物，属高毒性物质。这类物质来源广泛，通常污染水源，毒死鱼虾，危害农作物，并严重威胁人类的健康。含酚有机物的毒性还在于其只能被少数的微生物分解。从自然界中筛选分离出能够降解特定污染物的高效菌种，有针对性的投加到已有的污水处理系统中的生物强化技术，能够快速提供大量具有特殊作用的微生物，在有毒有害污染物治理中显示出巨大的潜力。

1、本研究从胜利油田河口采油厂的飞雁滩油田土壤样品中分离得到10株能够利用并降解苯酚的菌株P1-P4、P7、P9-P13。该10株苯酚降解菌能够在以苯酚为唯一碳源和能源的培养基上生长，经16S rDNA分子鉴定和生理生化检测，该10株降酚菌分别被鉴定到属或种。其中降酚菌株P1、P3和P4这3株菌株分别属于劳尔氏菌属(Ralstonia)、贪噬菌属（Variovorax）和节杆菌属（Arthrobacter）里的种。其它7株降酚菌株P

2、P7、P9-P13都属于假单胞菌属（Pseudomonas）里的种。这4个属里的细菌在国内外都已被报道有降解苯酚的特性，其中有关假单胞菌降解环境有机物的报道较多。

2、培养液中的苯酚含量通过4-氨基安替比啉分光光度法测定，通过苯酚降解效率的比较，菌株P2降解苯酚的能力较其它9株菌株要强。于是将菌株P2作为本研究中进一步研究的对象，研究了不同的环境条件下该菌株降解苯酚和菌体生长的情况。

3、通过苯酚羟化酶特异性引物的设计，从菌株P2扩增出苯酚羟化酶大亚基基因，该基因片段编码对苯酚有催化活性的多肽，催化苯酚代谢的第一步反应；表明菌株P2能降解苯酚是由于细胞具有降解苯酚的遗传基础。

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摘要上海师范大学硕士学位论文关键词：苯酚；生物降解；类产碱假单胞菌；苯酚羟化酶；16S rDNA；水平基因转移（HGT）；

论文类型：a.理论研究

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上海师范大学硕士学位论文Abstract

Abstract

Phenol is a strongly toxical subsubstance and is very harmful to cytoplasm. Its origin existed wildly in our life. It can pollute water, kill fish, do farm to crops and even heavily threat our health. The phenolic compound can only be degraded by several kinds of microbe. Bacteria that can effectively degrade phenolic compound had been isolated from natural environment, they can be poured into waste water treat system and obviously enhance the effect, therefore, they have extroordinary potential in bioremediation.

1. Ten bacterial strains P1-P4, P7, P9-P13 with phenol-degradating ability were isolated from a site contaminated with petroleum at Feiyan Beach Oilfield near Shengli Oilfield. They grew in basal medium with phenol as sole carbon and energy source. Through 16S ribosomal DNA (16S-rDNA) sequence analysis and determination of physiological and biochemistry parameters these ten strains respectively were classified and identification . The strian P1, P3, P4 belong to species of the genus Ralstonia,Variovorax and Arthrobacter respectively.The strian P2, P7, P9-P13 belong to species of the genus Pseudomonas.

2. Determination of phenol contents in culture medium was finished by spectrophotometric method of 4-aminoantipyrin. Comparing with phenol removal efficiency among these ten strians , strain P2 could degrade phenol better than other nine strains. so we made a further research on strian P2. Defferent environmental factors have defferent effect on the phenol-degradating of strain P2.

3. We designed a pair of special primer and gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified in strian P2 by PCR. This gene codes enzyme catalyzing the first step of phenol-degradating metabolism. It

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